Psyche: A Journal of Entomology Psyche: A Journal of Entomology Volume 2012, Part II ISSN: 0033-2615 (Print), ISSN: 1687-7438 (Online), DOI: 10.1155/6152 Copyright © 2012 Hindawi Publishing Corporation. All rights reserved. This is an issue published in volume 2012 of “Psyche: A Journal of Entomology.” All articles are open access articles distributed under the Cre- ative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents Effective Larval Foraging in Large, Low-Diet Environments by Anopheles gambiae A. C. Sutcliffe and M. Q. Benedict Volume 2012, Article ID 480483, 8 pages Nematode Parasites and Associates of Ants: Past and Present George Poinar Jr. Volume 2012, Article ID 192017, 13 pages Survival of Wild Adults of Ceratitis capitata (Wiedemann) under Natural Winter Conditions in North East Spain E. Penarrubia-Maria, J. Avilla, and L. A. Escudero-Colomar Volume 2012, Article ID 497087, 6 pages Antitermite Activities of C. decidua Extracts and Pure Compounds against Indian White Termite Odontotermes obesus (Isoptera; Odontotermitidae) Ravi Kant Upadhyay, Gayatri Jaiswal, Shoeb Ahmad, Leena Khanna, and Subhash Ghand Jain Volume 2012, Article ID 820245, 9 pages Evolutionary Perspectives on Myrmecophily in Ladybirds Amelie Vantaux, Olivier Roux, Alexandra Magro, and Jerome Orivel Volume 2012, Article ID 591570, 7 pages Effect of Habitat Type on Parasitism of Ectatomma ruidum by Eucharitid Wasps Aymer Andres Vasquez-Ordohez, Inge Armbrecht, and Gabriela Perez-Lachaud Volume 2012, Article ID 170483, 7 pages Diversity of Fungi Associated with Atta bisphaerica (Hymenoptera; Formicidae): The Activity of Aspergillus ochraceus and Beauveria bassiana Myriam M. R. Ribeiro, Karina D. Amaral, Vanessa E. Seide, Bressane M. R. Souza, Terezinha M. G. Della Lucia, Maria Gatarina M. Kasuya, and Danival J. de Souza Volume 2012, Article ID 389806, 6 pages Fire Ants {Solenopsis spp.) and Their Natural Enemies in Southern South America Juan Briano, Luis Galcaterra, and Laura Varone Volume 2012, Article ID 198084, 19 pages Herbivore Larval Development at Low Springtime Temperatures: The Importance of Short Periods of Heating in the Field Esther Muller and Elisabeth Obermaier Volume 2012, Article ID 345932, 7 pages On the Use of Adaptive Resemblance Terms in Chemical Ecology Christoph von Beeren, Sebastian Pohl, and Volker Witte Volume 2012, Article ID 635761, 7 pages Exploitative Competition and Risk of Parasitism in Two Host Ant Species: The Roles of Habitat Complexity, Body Size, and Behavioral Dominance Elliot B. Wilkinson and Donald H. Feener Jr. Volume 2012, Article ID 238959, 8 pages Termiticidal Activity of Parkia biglobosa (Jacq) Benth Seed Extracts on the Termite Coptotermes intermedins Silvestri (Isoptera: Rhinotermitidae) Bolarinwa Olugbemi Volume 2012, Article ID 869415, 5 pages Incorporating a Sorghum Habitat for Enhancing Lady Beetles (Coleoptera: Coccinellidae) in Cotton P. G. Tillman and T. E. Cottrell Volume 2012, Article ID 150418, 6 pages Eggs of the Blind Snake, Liotyphlops albirostrisy Are Incubated in a Nest of the Lower Eungus-Growing Ant, Apterostigma cf. goniodes Gaspar Bruner, Hermogenes Fernandez-Marin, Justin G. Touchon, and William T. Wcislo Volume 2012, Article ID 532314, 5 pages Feeding Preferences of the Endangered Diving Beetle Cybister tripunctatus orientalis Gschwendtner (Coleoptera: Dytiscidae) Shin-ya Ohba and Yoshinori Inatani Volume 2012, Article ID 139714, 3 pages Structure Determination of a Natural Juvenile Hormone Isolated from a Heteropteran Insect Toyomi Kotaki, Tetsuro Shinada, and Hideharu Numata Volume 2012, Article ID 924256, 7 pages The Biology and Natural History of Aphaenogaster rudis David Lubertazzi Volume 2012, Article ID 752815, 11 pages Coexistence and Competition between Tomicus yunnanensis and T. minor (Coleoptera: Scolytinae) in Yunnan Pine Rong Chun Lu, Hong Bin Wang, Zhen Zhang, John A. Byers, You Ju Jin, Hai Feng Wen, and Wen Jian Shi Volume 2012, Article ID 185312, 6 pages Gerris spinolae Lethierry and Severin (Hemiptera: Gerridae) and Brachydeutera longipes Hendel (Diptera: Ephydridae): Two Effective Insect Bioindicators to Monitor Pollution in Some Tropical Freshwater Ponds under Anthropogenic Stress Arijit Pal, Devashish Ghandra Sinha, and Neelkamal Rastogi Volume 2012, Article ID 818490, 10 pages Effect of Larval Density on Development of the Coconut Hispine Beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) Mika Murata, Dang Thi Dung, Shun-ichiro Takano, Ryoko Tabata Ichiki, and Satoshi Nakamura Volume 2012, Article ID 981475, 6 pages Tatuidris kapasi sp. nov.: A New Armadillo Ant from French Guiana (Formicidae: Agroecomyrmecinae) Sebastian Lacan, Sarah Groc, Alain Dejean, Muriel L. de Oliveira, and Jacques H. C. Delabie Volume 2012, Article ID 926089, 6 pages Abundance of Sesamia nonagrioides (Lef.) (Lepidoptera: Noctuidae) on the Edges of the Mediterranean Basin Matilda Eizaguirre and Argyro A. Fantinou Volume 2012, Article ID 854045, 7 pages Hylastes ater (Curculionidae: Scolytinae) Affecting Pinus radiata Seedling Establishment in New Zealand Stephen D. Reay, Travis R. Glare, and Michael Brownbridge Volume 2012, Article ID 590619, 9 pages Properties of Arboreal Ant and Ground-Termite Nests in relation to Their Nesting Sites and Location in a Tropical-Derived Savanna B. C. Echezona, C. A. Igwe, and L. A. Attama Volume 2012, Article ID 235840, 11 pages Effect of Plant Characteristics and Within-Plant Distribution of Prey on Colonization Efficiency of Cryptolaemus montrouzieri (Coleoptera: Coccinellidae) Adults Folukemi Adedipe and Yong-Lak Park Volume 2012, Article ID 503543, 5 pages Attraction of Tomicus yunnanensis (Coleoptera: Scolytidae) to Yunnan Pine Logs with and without Periderm or Phloem: An Effective Monitoring Bait Rong Chun Lu, Hong Bin Wang, Zhen Zhang, John A. Byers, You Ju Jin, Hai Feng Wen, and Wen Jian Shi Volume 2012, Article ID 794683, 5 pages Case Study: Trap Crop with Pheromone Traps for Suppressing Euschistus servus (Heteroptera: Pentatomidae) in Cotton P. G. Tillman and T. E. Cottrell Volume 2012, Article ID 401703, 10 pages Insects of the Subfamily Scolytinae (Insecta: Coleoptera, Curculionidae) Collected with Pitfall and Ethanol Traps in Primary Forests of Central Amazonia Raimunda Liege Souza de Abreu, Greicilany de Araujo Ribeiro, Bazilio Frasco Vianez, and Ceci Sales- Campos Volume 2012, Article ID 480520, 8 pages Galling Aphids (Hemiptera: Aphidoidea) in China: Diversity and Host Specificity Jing Chen and Ge-Xia Qiao Volume 2012, Article ID 621934, 11 pages Specialized Fungal Parasites and Opportunistic Fungi in Gardens of Attine Ants Fernando C. Pagnocca, Virginia E. Masiulionis, and Andre Rodrigues Volume 2012, Article ID 905109, 9 pages Behavior of Paussus favieri (Coleoptera, Carabidae, Paussini): A Myrmecophilous Beetle Associated with Pheidole pallidula (Hymenoptera, Formicidae) Emanuela Maurizi, Simone Fattorini, Wendy Moore, and Andrea Di Giulio Volume 2012, Article ID 940315, 9 pages Improved Visualization of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) — Part I: Morphological Features for Sex Determination of Multiple Stadia J. R Esquivel, T. L. Crippen, and L. A. Ward Volume 2012, Article ID 328478, 7 pages Mechanisms of Odor Coding in Coniferous Bark Beetles: From Neuron to Behavior and Application Martin N. Andersson Volume 2012, Article ID 149572, 14 pages Life Table of Spodoptera exigua (Lepidoptera: Noctuidae) on Five Soybean Cultivars Samira Farahani, Ali Asghar Talebi, and Yaghoub Fathipour Volume 2012, Article ID 513824, 7 pages Cleptobiosis in Social Insects Michael D. Breed, Chelsea Cook, and Michelle O. 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C. Sutcliffe'’^ and M. Q. Benedict^’ ^ ^ Centers for Disease Control and Prevention (CDC), Atlanta, GA 30333, USA ^Atlanta Research & Education Foundation (AREF), 1670 Clairmont Road (151F), Decatur, GA 30033, USA ^ Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universitd Degli Studi di Perugia, Via del Giochetto, 06122 Perugia, Italy Correspondence should be addressed to M. Q. Benedict, mqbenedict@yahoo.com Received 11 August 2011; Accepted 17 October 2011 Academic Editor: G. B. Dunphy Copyright © 2012 A. C. Sutcliffe andM. Q. Benedict. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Adult mosquito size is constrained by conditions experienced in the larval stage including the amount and quality of diet. The energy expended collecting diet depends partly on its concentration, the water depth, and the mosquito species. In order to better understand these interactions, individual Anopheles gambiae s.s. Giles were cultured to the adult stage in three types of experiments in which one of the following conditions was fixed and the other two were varied: water volume, diet amount, and diet concentration. In addition to survival, days of development to pupation and wing length were determined. The same outcomes were measured in experiments for which special containers were constructed that allowed the detection of chemical and tactile interactions. Larvae were able to develop to adulthood in volumes as great as 30 mL/larva when diet was added at an average rate of only 7 /rg/mL/day. The results demonstrate effective foraging in large low- diet volumes far above what had previously been estimated. 1. Introduction Anopheline larvae develop in habitats that are often tran- sient, poor in organic matter, and which vary greatly in size (e.g., [I, 2]). Because the density of larvae also varies greatly, competition for the most limiting factor in habitats — the amount of food — has a strong effect on larval survival [3- 6]. In addition to the limits placed on numerical habitat productivity by diet abundance and quality variation, the size of larval and adult mosquitoes is also irreversibly affected [7, 8]. Size in turn affects vector life history: larger female mosquitoes are more fecund [7, 9], longer lived [10], and more likely to develop eggs from one blood meal [11]. Size is also related to the likelihood of developing infectious viruses [12, 13], though the effect on malaria parasite abundance is less clear [14, 15]. Larger females are also more likely to be mated [16]. Moreover, the physical characteristics of how food is made available have measurable effects: water volume, surface area, and depth [8]. Intraspecific competition during the larval stages also influences adult mosquito body size, at least in part due to effects on diet abundance. It has been shown that increasing larval densities lead to longer development and reduced body size in certain Anopheles species [8, 17-19]. These interactions have been divided into physical and chemical interference [20]. How various kinds of interference individ- ually influence larval development and adult size is poorly understood in spite of numerous studies, for example, [21- 23]. Chemical growth-retarding factors have been identified in Aedes and Culex spp. (discussed in [24]), though only one instance of these in an anopheline has been observed [18]. In order to better understand feeding and development dynamics of Anopheles gambiae, we used a simplified system to determine the effects of diet amount, concentration, and water volume on growth rate and size while considering survival as a secondary objective. By measuring growth out- comes with a reduced number of independent variables in the absence of intraspecific interaction, we determined 2 Psyche Table 1: Daily rate of diet in three experiment types with “Day 1” being when L3s were transferred to dishes. Day Fixed water volume (|Wg/larva in Relative amount of diet 0.1 0.5 1 2 30 mL) 3 Fixed diet concentration (^wg/larva) Water volume (mL) 0.2 1.0 5.0 11.0 30 Fixed diet amount (|Wg/mL) Water volume (mL) 1.0 5.0 11.0 30.0 1 60 300 600 1200 1800 8 40 200 440 1200 440 88 40 15 2 80 400 800 1600 2400 11 53 267 587 1600 580 116 53 19 3 100 500 1000 2000 3000 13 67 333 733 2000 740 148 67 25 4 120 600 1200 2400 3600 16 80 400 880 2400 880 176 80 29 5 140 700 1400 2800 4200 19 93 467 1027 2800 1020 204 93 34 6 160 800 1600 3200 4800 21 107 533 1173 3200 1180 236 107 39 7 180 900 1800 3600 5400 24 120 600 1320 3600 1320 264 120 44 8 200 1000 2000 4000 6000 27 133 667 1467 4000 1460 292 133 49 9 220 1100 2200 4400 6600 29 147 733 1613 4400 1620 324 147 54 the potential responses of individuals to conditions ranging from low to extremely high diet amounts and concentrations. We also devised and tested an experimental system to distin- guish the effects of chemical and physical larval interactions. 2. Materials and Methods The mosquito stock used for this study was obtained from the Malaria Research and Reference Reagent Resource Center (MR4) vector activity at the CDC in Atlanta, GA, USA: Anopheles gamhiae G3 (MRA-112). All stages were reared at 27 ± 2°C using typical methods [25] on a diet of Koi Floating Blend (Aquaricare, Victor, NY. product no longer available). Except for the baker’s yeast fed to hatching larvae, this diet was used in all experiments. Three types of larval culture experiments were conducted by fixing one of the three factors of interest in each type of experiment successively: (1) water volume, (2) diet con- centration/mL, and (3) total diet amount/larva. The order of experiments presented here reflects the order in which the tests were conducted. Outcomes from a given experi- ment determined the fixed parameter implemented in the subsequent experiment. Because we manipulated the diet concentration and amount by changing the volume of water, we will describe the outcomes as a function of water volume in those experiments. On the first day of each trial, approximately 900 eggs were placed in 300 mL of water containing 3 mL of 2% w/v baker’s yeast in a 34 x 25 cm plastic tray. On day three, 300 larvae were counted into a new pan containing 300 mL of 0.3% w/v artificial sea salt (Instant Ocean, Aquarium Systems, Mentor, OH, USA) in reverse-osmosis/deionized (RODI) purified water and 3 mL of 0.2% w/v suspension of finely ground Koi diet in RODI water. Trays were monitored and fed with 3mL of 0.2% w/v finely ground diet daily. On the first day that the third stage larvae (L3s) appeared, they were transferred to polystyrene dishes according to details of each experiment (see Section 3). Water in all dishes was 5 mm deep, and the dishes were covered with the supplied transparent lid to reduce evaporation. Sub- sequently, larvae were fed with various volumes of 0.2% w/v finely ground diet in water (Table 1), the concentration of which was maintained uniform by vigorous manual shaking between each pipetting. Dishes were monitored every morning at which time the dish, water, and diet were changed to eliminate waste and diet accumulation. Each experiment was performed three times, and in each, 10 individuals were usually observed. Pupae were transferred to an emergence container containing 30 mL of RODI water. Approximately, two days after emergence, adults were killed by freezing, sex was determined, and one wing was removed and photographed after which length was measured using size-calibrated ImageJ software [26]. Length was measured from the base of the radial vein to the most distant portion of the wing tip, excluding setae. The larval duration and wing length were analyzed using a general linear model with Minitab (State College, PA, USA). For the interaction experiments, three dish types were used and, for reasons that will be evident, were named “undivided,” “porous,” and “divided.” All were modifications of standard 90 mm diameter polystyrene Petri dishes (the bottom of which is actually 88 mm diameter) described above which for these experiments were called “undivided.” The porous dish was constructed by gluing strips of 2 mm thick hydrophilic, porous polyethylene (Small Parts Inc., Miami, EL, USA) with a pore size of 90-130 jW to the bottom and sides of the undivided dish to create four equal sized compartments. The pore size is sufficiently large that water can pass through the plastic sheet. The glue used for all construction was PVC “hot glue.” The divided dish was of the same overall dimensions and material but manufactured to have four equal sized compartments divided by solid partitions. Both the divided and undivided dishes had a piece of the same porous polyethylene hot- glued to the bottom of the dish to preserve a uniform volume and materials content with the porous dish. In the divided and porous dishes, one larva was added to each compartment and four larvae were added to the undivided dish. Water was not changed throughout the trial, and food was added every other morning in equal volumes to each chamber of the divided dishes or 4- fold as much to the undivided dish. Water volume (30mL/dish), food amount Psyche 3 0.1 0.5 2 3 Relative diet rate/larva Figure 1; Graphical description of the experimental conditions and survival data. The three fixed conditions (water volume, diet concentration, and diet amount) are indicated on the axis corresponding to the experimental values. The Z axis presents the volumes of water used and crosses the XY plane at the diet amount used. Values in the boxes and ovals are the number of larvae that successfully pupated divided by the total number in the experiment. Table 2: Analysis by GLM of three experiments with a single-fixed variable (type III values). df Wing length F P df Larval duration F P Fixed variable water volume (n = 96) Model (error df = 89) 6 100.36 <0.0001 6 36.01 <0.0001 Sex 1 63.88 <0.0001 1 0.19 0.6658 Diet rate 4 121.02 <0.0001 3 34.41 <0.0001 Trial 2 60.72 <0.0001 2 43.79 <0.0001 Fixed variable diet concentration (n = 74) Model (error df = 67) 6 30.62 <0.0001 6 82.65 <0.0001 Sex 1 30.42 <0.0001 1 4.86 0.0309 Volume 3 46.14 <0.0001 3 133.53 <0.0001 Trial 2 8.91 0.0004 2 17.26 <0.0001 Fixed variable diet amount (n = 95) Model (error df = 88) 6 20.40 <0.0001 6 18.01 <0.0001 Sex 1 37.43 <0.0001 1 6.01 0.0162 Volume 3 28.97 <0.0001 3 19.94 <0.0001 Trial 2 3.99 0.0220 2 20.85 <0.0001 (1600jWg/larva/alternating day) and number of larvae per dish remained constant throughout the experiment. Only dishes in which all four larvae pupated were included in the analysis. Three trials were conducted with 20 individuals (five dishes) in each treatment group. Data was analyzed using SAS software (Cary, NC, USA) and Minitab using an alpha of 0.05. Pairwise comparisons of general linear model (GLM) outcomes were performed using Tukey’s method. 3. Results The conditions that were chosen for the experiments and their relationship to one another is shown in Figure 1. With few exceptions, sex and the independent variable had high- ly significant effects on both wing length and larval duration (Table 2). Therefore, we analyzed and graphed sex separately regardless of whether both were significant in the overall 4 Psyche Relative diet rate O Female X Male Figure 2: Mean mosquito wing lengths in a constant water volume of 30 mL. Larvae were provided diet in multiples of the standard diet rate. Dotted lines are for clarity of data groupings and are not intended to represent intermediate values in this or in other figures. Letters above and below data indicate significantly different values for females and males when analyzed by sex. Error bars are 95% Cl of the mean. In this and all other graphs, male data are shifted rightward for clarity. model. All models were highly significant. Trial was signifi- cant; however, the culture condition trends are so strong that this does not obscure the conclusions. Survival in most experiments was high (Figure 1). Of 384 individual larvae used in these experiments, 76% (289) pupated. Those that did not were clustered in specific exper- imental conditions which we will expand upon in context below. 3.1. Constant Water Volume. In the first set of experiments, diet concentration was varied (concomitantly with diet amount), while the dish type (standard 90 mm polystyrene Petri dishes) contained a constant water volume of 30 mL. The relative amount of diet was increased daily according to a scale suggested previously [25]. Because the amount of diet fed daily accelerated in all experiments (Table 1 ), we will refer to the amount of diet as rates relative to those described in that table, that is, 0.1-0. 5-, 1-, 2-, and 3-fold. Of 125 larvae placed individually in dishes, 12 did not survive to pupation, eight of which were in the lowest (0.1- fold) diet-level dishes. Of the 11 larvae tested under the 0.1 -fold conditions, only two females developed, so this condition was removed from analysis of duration and wing length. Until the day of death, larvae in this group survived from 11-15 days. Male wing length increased significantly as the diet increased from 2- to 3-fold, whereas maximum female wing length was reached at 2-fold (Figure 2). Maximum wing length was reached at lower diet concentrations for females than for males indicating that they are able to forage and/or Relative diet rate O Female X Male Figure 3: Mean larval durations in a constant water volume of 30 mL. Larvae were provided diet in multiples of the standard diet rate. Letters above and below indicate significantly different values for females and males when analyzed by sex. Error bars are 95% Cl of the mean. assimilate diet more efficiently. In contrast, minimal larval duration was achieved for both sexes at the 2 -fold level (Figure 3). Based on these experiments, the 2-fold diet level was considered nonrestrictive and was chosen for the next stage of experiments. 3.2. Constant Diet Concentration. In these experiments, the diet concentration/mL water was held constant while the water volume was diminished. Volumes were manipulated to maintain the constant depth of 5 mm by using dishes of various inside diameters: 3, 16, 35, 53 mm in addition to the 88 mm dish used previously. The water volumes were 0.2, 1.0, 5.0, 11.0, and 30.0 mL. The diet rates are listed in Table 1. Because “volume” is more easily visualized, we will refer to the outcomes according to this variable. Pupae formed in all volumes except 0.2 mL {n = 30). In this volume, many lived longer than 20 days though the average was 12,2 days. This demonstrates that diet was not adequate to allow pupation or that the physical size re- striction in some way prevented normal development. Of the 30 larvae cultured in 1 mL (2cm^ surface area), only four pupated (2 males, 2 females), and those that did not died at 1 1,7 days on average, but none lived longer than 15 days. Due to the small number, the four survivors were eliminated from further analysis. The individuals that died in the specific cases described in the first two experiments above account for 65 of 95 of the total that failed to pupate in all experiments. In the remaining treatments, sex, volume, and trial af- fected wing length and larval duration (Table 2, Figures 4 and 5). Increases in wing length and development rates were observed with volume increases from 5.0 to ll.OmL. While the differences in these outcomes between the 11.0 and 30.0 mL volume were not significant, the trends suggest Psyche 5 O Female X Male Figure 4: Mean mosquito wing lengths when provided the 2- fold diet schedule (Table 1) but in reduced amounts to maintain a constant diet concentration per mL in the various dishes. Letters above and below indicate significantly different values when analyzed by sex. Error bars are 95% Cl of the mean. O Female X Male Figure 6: Mean mosquito wing lengths by sex when given a constant diet weight per dish. Water volume and diet concentration varied. Letters above and below indicate significantly different values when analyzed by sex. Error bars are 95% Cl of the mean. O Female X Male Figure 5; Mean larval durations when provided the 2-fold diet schedule (Table 1) but in reduced amounts to maintain a constant diet concentration per mL in the various dishes. Letters above and below indicate significantly different values when analyzed by sex. Error bars are 95% Cl of the mean. that a small increase may be correlated with larger volume that was not detected in our experiments. Based on these experiments, the amount of diet in the ll.OmL treatment was chosen for the next series. 3.3. Constant Diet Amount. The diet weight fed in the ll.OmL treatment was provided in higher and lower con- centrations by using the various dish sizes described above, with the exception that the 0.2 mL dish was not included because no larvae had survived previously in that volume. In these experiments, a trend of mortality as a function of diet concentration existed. There were 11 deaths in the 1.0 mL dishes, five in the 5.0 mL, 3 in the 11, and 0 in the 30 mL. This association (ANOVA F = 78.64, 3 df, P = 0.012) and strong correlation of mortality with increased diet concentration and diminished water volume (S = 0.8961, = 96.3%) indicates a negative effect on survival of a small volume and/or a toxic effect of the diet itself. This negative trend on mortality was not reflected in reduced wing length and increased larval duration of the survivors (Figures 6 and 7, resp.), characters for which positive effects of diminishing the volume, and increasing the concentration of diet were observed. 3.4. Tactile and Chemical Interactions. In order to detect interaction effects of larvae, we cultured individuals in containers which would allow or preclude either chemical and/or tactile effects. Both sexes were pooled for analysis by GLM initially. Dish type and sex both had significant effects on wing length (F = 3.77, 2df, P = 0.025 and F = 8.25, I df, P = 0.005, resp.) but not larval duration. When females and males were pooled, the average wing length of adults from larvae cultured in the divided dish was significantly shorter than of those cultured in either the porous or open dish (Figure 8). When wing length and larval duration were analyzed by sex, no significant differences in wing length or duration as a function of dish type were observed. 4. Discussion Numerous methods have been used to determine the feeding rates of larvae: suspended particle removal [27], gut bolus 6 Psyche 9.5 3 i... Dh 3 Dh I ^ 8 7.5 Figure 7: Mean larval duration when given a constant diet weight of diet per dish. Water volume and diet concentration varied. Letters above and below indicate significantly different values when analyzed by sex. All males (n = 7) cultured in 1.0 mL pupated on the eighth day. Error bars are 95% Cl of the mean. O Female X Male O Female X Male Figure 8: Mean mosquito wing lengths for three different dish types with the same water volume and food amount per larva. Symbols for the three dish types are shown from left to right: open, solid divided, and porous divided. Error bars are 95% Cl of the mean. displacement [28] and mouthpart activity [29] , The methods of presenting the diet (and surrogate indicators such as charcoal and latex beads) have included in solution [30], on the surface, and in slurries [8]. All are appropriate presentations for anophelines which feed on the surface bacterioneuston, drink, graze on the bottom and scrape particles (discussed extensively in [24]). We do not know how much of the diet we provided was deposited in and collected from these different locations, though sedimented particles were visually most apparent. All dishes contained water of the same shallow depth, and no change occurred as water volume and food concentration were varied. There- fore, the diet could be easily obtained by the larvae which are often observed feeding on the surface in the stereotypical anopheline fashion, but which also readily reached the bot- tom. Changing the diet daily reduced the nutritional contri- bution of microorganisms, prevented diet accumulation, and reduced the effects of diet degradation. In all experiments, there was a strong positive correlation between development rate and wing length, but we did not explore this in detail. This relationship has been reported previously (e.g., [19, 31, 32]). Briefly, faster development is correlated with increased wing length. The first three experiments reported here are unlike many (e.g., [3, 8, 23]) in which larvae were allowed to interact and in which diet and water were not replaced on a daily basis. By adjusting the volumes and amounts of diet, we detected growth-limiting effects of extremely low diet concentrations, that is, the concentration of diet at which expenditure of energy required for homeostasis and foraging for food outweighed its nutritional benefits. When cultured in 30 mL, survival was extremely low unless larvae were provided at least 7 fig/mL of diet per day (equal to 220 |Wg/larva/day based on the amount provided on day 9 and thereafter). It is possible that the low amount of diet provided in the earlier development stages (2 |Ug/mL/day) precluded later survival. Nonetheless, it is remarkable that any development occurred at all when diet was provided in such low concentrations and demonstrates that larvae can assimilate diet effectively even when it is very dilute. Because all motion expends energy, foraging activity does not come without cost in size and growth rate. This is demonstrated by significant increases in size that were observed up to diet concentrations near 150 |Ug/mL above which point little benefit of increasing the concentration was observed. We chose to begin the experiments with the early third instar larvae. In our experience, survival under a wide range of conditions after the L3 stage is high — in contrast to rela- tively delicate Lis — thus increasing the probability of obtain- ing useful larval development duration and wing length data. Most of the mass of larval mosquitoes accumulates during the L3 and L4 stages. This choice may have influenced the outcomes we observed, and that of the minimum diet required for development is probably the most sensitive. The minimum amount of diet necessary for survival that we estimated here (220 fig) reflects only what was assimilated after the beginning of the third stage. However, in separate estimates using two different methods with a sibling species of An. gambiae. An. arabiensis, estimates of the minimum amount of the same diet required for development from hatching were 202 and 263 fig per larva [ 19] . Considering the amount of diet required for survival in this study, it is possible to obtain indirect estimates of the volume of water that larvae can filter. Aly [27] esti- mated anopheline filtration rates of 33-55 fiL/larva/h for An. albimanus and An. quadrimaculatus, respectively. If the rate on the high end of this estimate were applied to our Psyche 7 experimental system and species, larvae could process only I. 3 mL of water per day, meaning that only approximately, 10|Wg of diet/day could be consumed in our low concentra- tion experiments — an unrealistically small amount that is not consistent with survival. Our results demonstrate that the method used to determine filtration volume estimates must have resulted in gross underestimates, possibly due to a low efficiency of latex bead collection per volume of water actually processed. We suggest that the volume of water that larvae can process is not a very realistic indicator of feeding efficiency: larvae certainly reduce overall effort by focusing on high-value particles that are encountered rather than randomly processing water. The second set of experiments, in which the diet con- centration was fixed, determined the amount of diet that is necessary when provided at a saturating concentration (determined in the fixed volume experiments). Consistent with the first experiments, we observed almost no survival below 150/rg/larva/day (based on day 9 values) even when larvae were provided this amount in one mL which would require little foraging. The similarity between the minimum amount of diet necessary for survival in the first experi- ments (220 f/g/larva in 30 mL) and those in the second set (150/^g/iarva in 1 mL) demonstrates that foraging is ex- tremely efficient with regard to survival to pupation. The difference between these values provides an estimate of the nutrition required for foraging in 30 mL, approximately 70 jWg of diet per day per larva. On the other end of the spectrum of conditions tested in these experiments, maximal wing length and most rapid development occurred when larvae were each provided II. OmL of water containing 1600 f/g of diet- conditions that are approximately 10-fold more spacious and diet-rich than the minimum required for survival. Volumes and diet levels greater than this provided no measurable benefit on growth. The fixed diet amount experiments returned to the question of foraging efficiency developed in the fixed water volume experiments. In these, diet was provided in the 1 mL volume at a concentration more than seven times as high as the highest feeding rate provided in the first (constant volume) experiments. A negative effect of increased diet concentration on survival was observed suggesting either a toxic effect of the diet, harmful effects of waste concentration, or an inability to utilize extremely high concentrations. It has been established that culicine chemical factors are produced during the larval stage that can inhibit larval development (see [24]), and there is some support for this in An. stephensi [18]. The undivided and the porous dish both allowed chemical interaction, while the undivided dish also allowed tactile interaction. Under these conditions, chemical interaction resulted in larger size. When neither interaction was possible (divided dishes), adults were smaller. We can imagine only one reason: sensing the presence of potential competitors may stimulate more active foraging. Such experiments are admittedly open to criticism for several reasons: some differences existed between the dish volumes or materials, the diet concentration was inappropriate, and so forth. Every effort was made to match the materials and volumes though the geometry could admittedly have an effect. The experiments do provide provocative preliminary results and a method for developing systems for testing such phenomena further. While natural production of healthy mosquitoes is unde- sirable for public health, artificial production is beneficial for laboratory experiments and repositories such as the Ma- laria Research and Reference Reagent Resource Center (MR4, http://www.mr4.org/), genetic control efforts such as the sterile insect technique [33], and for meaningful compar- isons between wild and laboratory- reared specimens, for ex- ample, [34, 35] . The information we have developed here will provide guidance for optimal conditions for such activities, though, to the best of our knowledge, typical densities in production facilities will be much higher than the optimal ones we observed. Consistent with their “clean water” reputation, these experiments reveal that An. gambiae larval foraging activity is extremely efficient and spatially extensive, and that they are able to utilize even very low concentrations of diet. The extended development times that we observed when diet was limiting also mean that surveys of larval habitats may overestimate the standing population as many such sites will contain very long-lived larvae, few of which survive to adulthood, a suspicion observed in some field studies [36]. Acknowledgments The authors appreciate the support of the Malaria Research and Reference Reagent Resource Center (MR4) for the An. gambiae stock. They also appreciate the helpful advice and statistical analysis performed by Jacqueline Roberts of the CDC. References [1] T. B. Ageep, J. Cox, M. M. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 192017, 13 pages doi:10.1155/2012/192017 Review Article Nematode Parasites and Associates of Ants: Past and Present George Poinar Jr. Department of Zoology, Oregon State University, Corvallis, OR 97331, USA Correspondence should be addressed to George Poinar Jr., poinarg@science.oregonstate.edu Received 14 August 2011; Accepted 9 October 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 George Poinar Jr. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ants can serve as developmental, definitive, intermediate, or carrier hosts of a variety of nematodes. Parasitic ant nematodes in- clude members of the families Mermithidae, Tetradonematidae, Allantonematidae, Seuratidae, Physalopteridae, Steinernematidae, and Heterorhabditidae. Those nematodes that are phoretically associated with ants, internally or externally, are represented by the Rhabditidae, Diplogastridae, and Panagrolaimidae. Fossils of mermithids, tetradonematids, allantonematids, and diplogastrids as- sociated with ants show the evolutionary history of these relationships, some of which date back to the Eocene (40 mya). 1. Introduction Nematodes are one of the most abundant groups of animals known. Studies on their evolutionary history suggest that they probably arose in the Precambrian, which explains their wide abundance today in the terrestrial and marine environ- ments. While only some 20,000 have been described, their species diversity has been estimated to be as high as 10 mil- lion [1]. One would assume that with their strict housekeeping habits, ants would not tolerate nematodes in or around their nests and would quickly dispose of any nest mates that might have become infected. However nematodes have been able to use some astonishingly sophisticated tactics to suc- cessfully parasitize these social insects. The present work covers the systematics, life history, pathology, and records of all described extant and fossil nematodes associated with for- micids. This includes representatives of the nematode fami- lies Mermithidae, Tetradonematidae, Allantonematidae, Se- uratidae, Physalopteridae, Steinernematidae, Heterorhabdi- tidae, Rhabditidae, Diplogastridae, and Panagrolaimidae. Fossil records of mermithids, tetradonematids, allantonema- tids, and diplogastrids associated with ants reveal the evolu- tionary history of these associations, some of which date back 40 million years. 2. Mermithidae The family Mermithidae includes parasites of invertebrates, especially insects. Because of their large size, mermithids are easily detected in ants upon dissection (Figure 1) or as they leave their hosts (Figure 2). Most mermithid species, includ- ing those that attack ants, parasitize only a specific host spe- cies, genus, or family while others can infect representatives of several insect orders. Mermithids that attack aquatic in- sects, such as midges (Chironomidae, Ceratopogonidae) and mosquitoes (Culicidae), have a direct life cycle. Direct life cycles occur when, after growth and development is complet- ed in the host, the mermithid emerges, molts to the adult stage, mates, and oviposits in the host’s environment. The in- fective stage mermithid emerges from the egg, actively locates and enters a host, and initiates development in the hemocoel. Some mermithids have an indirect life cycle, which is more complicated but allows hosts to be parasitized in envi- ronments hostile to nematodes. In an indirect cycle, the mer- mithid emerges from the host, molts, mates, and oviposits in the environment. But instead of emerging from the egg to search for a developmental host, the infective stage remains in the egg, waiting to be ingested by an invertebrate that serves as a paratenic host. When mermithid eggs are ingest- ed by a paratenic host, the hatching infective stage penetrates the gut wall and enters the body cavity. But instead of devel- oping, the mermithid encysts and enters a diapause. The en- cysted nematode can be carried through the different stages of host metamorphosis, but for its cycle to be completed, the paratenic host must be captured and fed to the brood of the developmental host. At the completion of the mermithids growth phase in the development host (like an ant), the latter is attracted to an aquatic or semiaquatic habitat favorable 2 Psyche Figure 1; Mermithid exposed in the gaster of Camponotus sp. from the Sierra Nevada, California. Figure 2: Parasitic juveniles of Allomermis solenopsi removed from the gaster of a fire ant worker. Photo courtesy of S. D. Porter, USDA- ARS. to the nematode. This is when the mermithid exits, leaving the dying host behind. The developmental host is usually not only larger, but usually in a completely different taxonomic category and environment from the paratenic host. While the developmental host can live in a relatively dry habitat, the paratenic host usually inhabits an aquatic, semiaquatic, or damp biome. Also, both hosts can be widely separated taxo- nomically and may not even belong to the same phylum. The first written account of a nematode parasite of ants was made by the Reverend William Gould in his 1747 book An account of English Ants (Table 1) [2]. The “white and long kind of worm, which is often met within their bodies” certainly refers to mermithid nemato- des. For a number of years, mermithids were listed under “Filariaf “Gordiusf or “Mermisf and that is why mermithid systematics can be confusing and why early names for Gould’s ant mermithid included Gordius formicarum Diesing [3] and Filaria formicarum von Siebold [4]. The first described ant mermithid was Pheromermis myrmecophila from Fasius spp. [5] . However it was originally described in the genus “Merrnis"] then assigned to the genus Table 1: Section from Gould [2] referring to the first reported instance of mermithid parasitism of ants. Amongft other Incidents that tend to Icflen and deftroy Ant-Flies, it is obfcrvable that abun- dance of them are demoliOied by a white and long Kind of Worm, which is often met with in their Bodies. You mav frequently take three from the Infides of the large, but feldom more than one from a fmall Ant-Fly. 7'hefe Worms lie in a fpiral Form, and fome of them may be extended Half an Inch. Table 2: Mermithid nematodes described from ants. Mermithid Host Reference Agamomermis cephaloti Cephalotes minutus [11] Agamomermis costaricensis Odontomachus hastatus [11] Agamomermis ecitoni Eciton burchellii [11] Allomermis solenopsi Solenopsis invicta [12] Camponotimermis bifidus Camponotus aethiops [13] Comanimermis clujensis Formica fusca Camponotus aethiops [14] * Heydenius formicinus Prenolepis henschei |15] * Heydenius myrmecophila Finepithema sp. [11] Meximermis ectatommi Fetatomma ruidum [11] Pheromermis lasiusi Fasius niger [16] Pheromermis myrmecophila Fasius spp. [5] Pheromermis villosa Fasius flavus, F. niger |17] fossil. Pheromermis [6], then moved to the genus Allomermis [7] and lastly to the genus Gamp onotimer mis [8]. Its position in the genus Pheromermis was recently confirmed by Kaiser, who showed its similarity with the European ant mermithid, Pheromermis villosa [9]. Over the years, a large number of ant species have been reported parasitized by mermithids. A list of Holarctic parasitized ants was presented by Passera [10] and Neotropical parasitized ants by Poinar et al. [ 1 1 ] . A compilation of all described mermithids from ants is present- ed in Table 2. Fossils, such as the postparasitic juvenile of Heyde- nius formicinus emerging from a male Prenolepis henschei (Figure 3) [ 15] , as well as from a worker ant (Figure 4) in Bal- tic amber [ 1 ] show that ants have been parasitized by mer- mithids for at least 40 million years and probably much longer. The fossil record of Neotropical mermithid parasites of ants is represented by a parasitic juvenile of Heydenius myrmecophila adjacent to its ant host, Finepithema sp. in 20- 30 -million-year-old Dominican amber (Figure 5) [11]. It is assumed that the traumatic events of the ant host entering the resin caused the mermithid to emerge prematurely from an opening in the gaster of the ant. Psyche 3 Figure 3: The fossil nematode, Heydenius formicinus, emerging from a male Prenolepis henschei in Baltic amber. Figure 4: Heydenius formicinus adjacent to its worker ant host in Baltic amber. Depending on the caste and length of time the mermithid is associated with its host, various degrees of host intercastes and abnormalities appear. Wheeler [18] was the first to pro- vide an explanation for these phenomena by correlating the unusual morphological conditions with mermithid infec- tions (Figured). Parasitized queen ants (mermithogynes) are shorter, have a smaller thorax (stenothoracy), reduced wings (brachyptery), enlarged abdomen (physogastry), and smaller head (microcephaly) than their uninfected counter- parts. Parasitized worker ants (mermithergates or macroer- gates) often develop morphological features characteristic of queens and soldiers. Attacked male ants (mermithaners) have shorter wings but enlarged heads, eyes, and gasters. Infected soldiers (mermithostratiotes) have reduced heads, an ocellus, and changes in pilosity (Figure 7) [19-24]. The life cycle of most ant mermithids remains a mystery. Crawley and Baylis [5] assumed that P. myrmecophila has a direct cycle, where infection is brought about by the eclosing V Figure 5: Heydenius myrmecophila adjacent to its Linepithema ant host in Dominican amber. (c) Figure 6: Plate (modified) of Pheidole dentata (referred to as P. commutata) from [18] showing the first evidence that mermithid nematodes could cause intercastes of ants, (a) Normal soldier; (b) normal worker; (c) parasitized worker (mermithergate). preparasitic mermithid entering the ant host. When develop- ment is completed, the postparasitic juvenile emerges, molts to the adult stage in the ant’s habitat, mates, oviposits and the cycle continues. However, no one has demonstrated a direct cycle for any mermithid parasite of ants. In 1934, Vandel [25] studied a mermithid parasite of Pheidole pallidula and real- ized that the infection must be initiated in the ant larva. He assumed that the nematodes were in the soil surrounding the ant colony so the infective stages could penetrate directly into 4 Psyche Figure 7; Two mermithid-infected soldiers (mermithostratiotes) (arrows) of Pheidole pallidula adjacent to smaller workers and an uninfected soldier (with large head). Note smaller heads on infected soldiers. Photo courtesy of Luc Passera. Figure 8; Pheromermis villosa in a carpenter ant from Holland. the ant larva; however he was unable to confirm the infec- tion process. The first life cycle of an ant mermithid was achieved by Kaiser with the European Pheromermis villosa [17, 26] (Figure 8). Kaiser showed that P. villosa had an indirect cycle involving oligochaetes as paratenic hosts. Workers of Lasius spp. collecting protein for the brood capture oligochaetes containing the infective stages of P villosa and, unknowingly, feed them to the developing larvae. At this point, the nema- tode becomes active, penetrates into the ant larva’s hemo- coel, and initiates development. It was a significant discov- ery and raises the question whether all mermithid infections of ants have indirect life cycles. Other possible paratenic hosts for Pheromermis could be small aquatic insects that ingest mermithid eggs from the bottom debris of seepage areas or the edges of other water sources. The wasp parasite, Pherome- rmis p achy soma [6], also has an indirect cycle and uses caddis flies as paratenic hosts, which the eusocial wasps (Vespidae) feed to their brood [27] . Thus far, seven genera of mermithids are known to infect ants, namely, Agamomermis, Allomermis, Camponotimermis, Comanimermis, Heydenius, Meximermis, and Pheromermis (Table 2). All of the ant hosts of these mermithids feed their brood animal protein (in contrast to other genera, such as the leaf cutting ants), and this behavior suggests they have an indirect life cycle involving a paratenic host. The two genera, Agamomermis and Heydenius, are collective group genera for immature extant and fossil mermithids, respectively [ 1 ] . There are some morphological and behavioral patterns that characterize mermithids with indirect cycles. They normally have smaller eggs with thicker shells than the eggs of direct development soil or freshwater mermithids. Also their eggs are completely embryonated when laid. Finally, the deposited eggs will not hatch in the environment even though the enclosed parasitic juvenile is fully developed. Hatching only occurs when a potential invertebrate paratenic host ingests the eggs. The eggs of Pheromermis spp. are small, numerous, fully embryonated when laid and do not hatch in the environment. Fully embryonated eggs ensure that the in- fective stages are ready to enter paratenic hosts as soon as they are ingested [6, 17]. The ant mermithid, Allomermis solenopsi [12], possess an unusual morphological feature on the mature eggs that could play a crucial role in its life cycle. The surface of the eggs is covered with elongate, erect, spiny adhesive processes. How these function in the life cycle is unknown, but the related species, A. trichotopson, possesses similar structures [28]. Since A. solenopsi parasitizes the fire ant, Solenopsis invicta in Brazil (Figure 2), the related A. trichotopson, whose host is unknown, may infect Solenopsis geminata in Jamaica. Could these egg processes somehow be connected with parasitism of Solenopsis spp.? Can mermithids be manipulated to control ants? Aside from killing the ant host upon emergence, mermithids drain the host of food, reduce the flight muscles and fat body, and cause morphological modifications as mentioned above [9, 17, 24, 26]. Since mermithid-infected Solenopsis has reduced reproductive organs and die shortly after the nematodes emerge [11, 12, 29], it has potential as a biological control agent. However, if the cycle is always indirect as shown for P villosa, it would be very difficult to artificially infect the ant brood. It would be necessary to first infect the paratenic host and then supply large numbers of these infected invertebrates to worker ants for transport back to the nest. Working with a mermithid that has a direct cycle would be easier; however there is still the problem of raising and disseminating the nematodes. 3. Tetradonematidae The tetradonematids are a diverse group of nematodes that have traditionally been aligned with the Mermithidae. How- ever, aside from some distinctive morphological characters, female tetradonematids normally mature, mate, and produce eggs within the host, which does not occur with mermithids. Two tetradonematids have been described from extant ants. Tetradonema solenopsis is a parasite of the red imported fire ant, Solenopsis invicta, in Brazil [30, 31]. Very little is known about this nematode aside from the scant description showing that females contained eggs and worker infection levels reached 12.5%. Parasitized ants that succumbed to the infections could be recognized by their slightly enlarged gaster with scallop-appearing dorsal sclerites. Psyche 5 Figure 9: Mature female of Myrmeconema neotropicum in the early stages of egg production removed from a pupa of Cephalotes atratus. Arrow shows position of vulva. Figure 10; Mature females of Myrmeconema neotropicum packed with eggs in the gaster of a Cephalotes atratus worker in Peru. The second tetradonematid from extant ants is Myrme- conema neotropicum from Cephalotes atratus in Peru and Panama [32] . Myrmeconema is the only nematode that causes its ant host to radically change color (from black to red), which is crucial for completion of its life cycle [33] . This color change was a mystery for early taxonomists and the variety Cephalotes atratus var. rufiventris was erected solely on the basis of its red abdomen, which was later shown to be the result of Myrmeconema infections [32]. Developing females of M. neotropicum occur in ant pupae (Figure 9) but do not produce masses of eggs until they are carried into the adult ant (Figure 10). As the females deterio- rate, eggs are released into the ant’s hemocoel (Figure 1 1). At this stage of development, the gasters of the infected worker ants turn from black to red and are held high in the air (Figure 12) [33]. Birds mistake the red gasters for fruits and the nematode eggs are passed through the birds’ digestive Figure 11: Eggs of Myrmeconema neotropicum released from the gaster of an infected Cephalotes atratus worker in Peru. Figure 12: Worker of Cephalotes atratus infected with Myrmecone- ma neotropicum. The raised, red abdomen occurs when the nema- tode eggs are infective and ready for transport by birds. Photo cour- tesy of Stephen P. Yanoviak. system and end up in the droppings, which are deposited on leaves and branches. Cephalotes workers collect and feed the infested excreta to their brood, which is how the larvae be- come infected [33]. Aside from their red gasters, parasitized ants are smaller with reduced head widths. They are sluggish, clumsy, gener- ally less aggressive, and about 40% heavier than nonparasit- ized workers. They do not bite when handled, and their alarm/defense pheromone supply is significantly reduced or absent. Myrmeconema is probably widely distributed throughout the Neotropics since this association has been in existence for some 20-30 million years. The fossil worker ant, Cephalotes serratus in Dominican amber, is surrounded by the eggs of Myrmeconema antiqua (Figure 13) [1]. The ant has a hole in its abdomen that quite possibly was made by a bird. Many of the eggs, which closely resemble those of M. neotropicum in size and shape, contain fully developed juveniles (Figure 14). All indications suggest that M. antiqua had a similar life his- tory to the extant M. neotropicum and involved bird carriers. 6 Psyche Figure 13: Worker of Cephalotes serratus infected with Myrme- conema antiqua in Dominican amber. Note microscopic eggs widely distributed in the amber that were released from a hole in the ant’s gaster. Figure 15; Female of the allantonematid Formicitylenchus orego- nensis removed from the body cavity of Camponotus vicinus in Oregon, USA. Figure 14: Detail of eggs of Myrmeconema antiqua in various stages of development in Dominican amber. 4. Allantonematidae It is curious why so few cases of allantonematid infections have been reported in ants. Since ants are probably one of the most investigated insect groups, is the absence of tylenchid parasitism due to a lack of observations or its rarity? The first and only described allantonematid parasite of extant ants is Formicitylenchus oregonensis that was parasitizing a queen Camponotus ant in Western Oregon, USA [34]. The queen had already chewed off her wings and appeared to be searching for a nesting site. There was a single large parasitic female (Figure 15) and 120 third-stage juvenile nematodes in the ant’s gaster. The third-stage juveniles exited through the ants reproductive and digestive tracts and molted twice to reach the adult stage. The enlarged pharyngeal glands in the free-living females suggest that they penetrate the cuticle to enter the body cavity of the host, probably ant larvae. Although the complete life cycle is unknown, the nematodes are clearly distributed by infected queen ants. The gonads of the infected ant were greatly reduced, and her eggs were abnormal. Since carpenter ants can be damaging to struc- tures, F. oregonensis can be considered as a potential biologi- cal control agent. Since the original report of this parasite, the present author recovered a worker carpenter ant also infected with F. oregonensis, thus indicating that Formicitylenchus is probably restricted to ant hosts, especially members of the genus Cam- ponotus. Formicitylenchus shows a close relationship with the allantonematid beetle parasite, Metaparasitylenchus [34]. It is possible that their last common ancestor parasitized beetles and the host shift from arboreal beetles to arboreal ants occurred during the anagenesis of Formicitylenchus. The close physical association between wood-boring beetles and Camponotus ants maybe significant. Rogers [35] commented that “. . .the potential parasite would be expected to find its hosts in organisms which occupy the same niche largely in- dependent of their phylogenetic position. In fact the speci- ficity of many parasites is based on the ecological relationship of the hosts, especially in groups which have only recently become parasitic.” Another reason that allantonematid parasitism of ants may be more widespread than presumed is the discovery of juveniles of a fossil allantonematid, Palaeoallantonema ceph- alotae, in the ant, Cephalotes serratus, in Dominican amber [1] (Figure 16). Just before this fossil was discovered, Stev- en Yanoviak submitted an extant worker of Cephalotes Chri- stopher seni from Peru that was also infected with an alla- tonematid. The parasitic female (Figure 17) of this still unde- scribed species and the developing juveniles inside her body (Figure 18) show features typical of the family. 5. Seuratidae The discovery of adults of Rabbium paradoxus [36] inside the gaster of worker Camponotus castaneus in Florida (Figure 19) was a surprise since all known nematodes of the Seuratidae are heteroxenous and develop to the adult stage in the diges- tive tract of vertebrates [37]. Flowever, in R. paradoxus, the Psyche 7 Figure 16: Three juveniles of the allantonematid, Palaeoallan- tonema cephalotae, that emerged from a worker Cephalotes ant in Dominican amber. Figure 17: Parasitic female of an undescribed allantonematid from workers of Cephalotes christopherseni in Peru. 4 Figure 19: Adults of Rabbium paradoxus adjacent to their ant host, Camponotus castaneus, in Florida. Figure 20: Flead of a female of Rabbium paradoxus. Arrow shows anteriorly located vulva. Figure 18: Juveniles developing inside the body of the female allantonematid shown in Figure 17. vertebrate host is obviously not required for adult develop- ment. The females of R. paradoxus have an anteriorly placed vulva (Figure 20), and the eggs embryonate inside the uterus (Figure 21). Since the other member of the genus, R. caballeroi, occurs in the gut of lizards in the Bahamas [38], it is likely that R. paradoxus originally had (or still has) a liz- ard definitive host. If the complete life cycle occurs just in ants, then C. castaneus would serve as both intermediate and definitive hosts. C. castaneus is a generalist feeder and will in- gest vertebrate feces so it could acquire nematode eggs from lizard droppings. Parasitized worker ants had swollen gasters and showed unusual behavior by foraging during the day instead at night. This would make them easily captured by vertebrate predators. 8 Psyche Figure 21: Embryonated eggs in the uterus of Rabbium paradoxus. The original life cycle of R. paradoxus may have been sim- ilar to that of the seuratoid Skrjabinelazia galliardi, a parasite of sphaerodactyline lizards in Brazil [37]. The female nema- todes living in the gut of the lizard produce eggs that are passed out and ingested by insects. These eggs hatch in the insect gut and the juveniles enter the body cavity without fur- ther development. Growth is resumed when the insect inter- mediate hosts are eaten by lizards [38]. Unfortunately, the complete life cycle of R. paradoxus remains a mystery, but its precocious development is quite interesting. 6. Physalopteridae There are few reports of heteroxenous nematodes utilizing ants as intermediate hosts, that is, hosts where the nematodes develop only to the third-stage infective juveniles. Maturity to the adult stages occurs when the intermediate host is eaten by a vertebrate definitive host. One such nematode is the physalopterid, Skrjabinoptera phrynosoma that lives in the stomach of the Texas horned lizard, Phrynosoma cornutum, and uses the harvester ant, Pogonomyrmex barbatus, as an in- termediate hosts [39] . However instead of depositing isolated eggs that would pass from the lizard, the gravid nematodes die with the retained eggs enclosed in thick walled capsules. The females with their enclosed eggs pass out of the lizard and are collected by worker ants that feed them to their brood. The nematode eggs hatch in the gut of the ant larvae and the juveniles enter the fat body, where they develop only to the third stage. These juveniles are carried through the pupal and into the adult stage of the ant, where they eventual- ly reside in membranous capsules. The nematodes complete their development to the adult stage when infected ants are eaten by the lizards. Worker ants with more than 10 nemato- des were still active but had enlarged, lighter colored gasters. The interesting, pivotal stage in this life cycle is the attract- iveness of the dead, egg-laden female nematodes to worker ants. 7. Rhabditidae, Diplogastridae, and Panagrolaimidae This category includes juvenile nematodes living in the post- pharyngeal glands of ants (internal phoresis) or being carried on the outside surface of ants (external phoresis) (Table 3). While these might not be considered parasites, in some instances where the association has been examined critically [40] damage has been inflicted on the ant’s postpharynge- al glands and some of the nematodes increased in size during their stay in this location. Thus at most, they could be consid- ered weak parasites. If they break through the glands and introduce microbes into the body cavity of the ant, they could even be regarded as pathogenic. However the latter scenario has not been documented. Most of the nematodes in the postpharyngeal glands are dauer juveniles of free-living microbotrophs living in the ant’s environment. The dauers enter the glands when exter- nal conditions become unsuitable (low humidity or dimin- ished food supply). These resistant dauer juveniles can sur- vive for relatively long periods. The nematodes may leave the glands when the environment is more suitable (moist with associated microbes), if the ant dies and the dauer initiates development within the decomposing ant, or when the nem- atodes are transferred from ant to ant during trophallaxis. Janet [43] was the first to discover postpharyngeal rhab- ditids {Oscheius dolichurus) in Lasius flavus and Formica ru- fa in France. Wahab [42] was the first to systematically study these associations in the ant genera Lasius, Formica, Tetramo- rium, and Myrmica in Germany (Table 3). More recently Kohler [41] examined nematodes in the heads of ants collected from sap fluxes and rotten wood on trees in Germa- ny. The most common ant that visited these fluxes was Lasi- us brunneus and, from a total of 262 workers collected, 43.5% carried nematodes, with Koerneria histophora being the most common associate. While most ants carried a single nemato- de, numbers occasionally reached up to 85 dauers per ant. Kohler [41] also found diplogastrid dauers in 4 males and a queen of L. brunneus. The infection rate of ants associated with L. brunneus workers varied depending on the weather cycle. There were more nematodes in ants during the dry per- iod in August than during the rainy months of April and May. Also important in determining the rate of nematodes being carried by the ants was the location of the nests. Rates of in- festation by nematodes in L. brunneus were much higher when the ants were collected from sap fluxes and rotten wood [41] , than were collected from under stones and leaf litter [42] . Kohler [41] was able to infest ants by placing them in a Petri dish with rotten wood containing waving dauer stages. Both Wahab [42] and Kohler [41] provided evidence that the dauers can be transmitted from ant to ant via trophallax- is, which was supported in part by the experiments of Naar- mann [53] showing that Formica ants mix food with secre- tions from the postpharyngeal gland before regurgitating it to nest mates. These ant-dauer associations probably occur worldwide since Markin and McCoy [40] reported Diplo- scapter lycostoma in the postpharyngeal glands of the Argen- tine Ant, Linepithema humile in California and Nickle & Ayre Psyche 9 Table 3: Juvenile nematodes of Rhabditida and Tylenchina associated with ants. Nematode Family Host Reference Diplogasteroides spengelii Diplogastridae Lasius brunneus [41] Diploscapter lycostoma Rhabditidae Formica spp., Lasius spp. [42] u cc Myrmica rugulosa [42] cc cc Linepithema humile [40] * Formicodiplogaster myrmenema Diplogastridae Azteca alpha [1] Halicephalobus similigaster Panagrolaimidae Lasius brunneus [41] Koerneria histophora Diplogastridae Lasius spp. [42] cc cc Lasius brunneus [41] Oscheius dolichurus Rhabditidae Formica rufa, [42, 43] u cc Lasius flavus [42, 43] u cc Tetramorium caespitum [42] u cc Camponotus herculeanus [44] u cc Lasius claviger [44] u cc Lasius brunneus [41] Pristionchus Iheritieri Diplogastridae Formica rufa, Lasius spp. [42] * Unknown cc Azteca spp. [1] Unknown cc Formica obscuriventris Present work (Figure 22) * Fossil. Figure 22: Dauer juveniles of a diplogastrid in the postpharyngeal glands of Formica obscuriventris clivia from Oregon. [44] found Oscheius dolichurus in the head glands of Catn- ponotus herculeanus and Lasius claviger in Ontario, Canada. The author has also found dauer diplogastrids in the post- pharyngeal glands of workers of Formica obscuriventris in Oregon (Figure 22). The association between dauer nematodes and ants is at least 20-30 million years old. Evidence for this is the discov- ery of dauer juveniles of the fossil diplogastrid, Formicodiplo- gaster myrmenema, carried by Azteca alpha workers in Do- minican amber [1] (Figures 23 and 24). The dauer stages appear to be associated with the abdomen of the ants, sug- gesting that they were being carried in the segmental mem- branes of the gaster (external phoresis). None of the fossil stages occurred around the mouthparts of the ants. Also, de- veloping stages of F. myrmenema were associated with nest material adjacent to worker Azteca ants in Dominican amber Figure 23: Three dauer juveniles of Formicodiplogaster myrmenema adjacent to a worker of Azteca alpha in Dominican amber. [ 1 ] . This indicates that F. myrmenema was developing in the nests of A. alpha, which is probably the case with extant nematodes in the head glands of ants. Whether the dauers of F. myrmenema were also in the postpharyngeal glands of the fossil ants is unknown. 10 Psyche Table 4: Ants infected by entomopathogenic nematodes {Steinernema carpocapsae and Heterorhabditis bacteriophora) under laboratory and/or field conditions. Ant Nematode System Reference Acromyrmex octospinosus S. carpocapsae Aqueous [45] Camponotus sp. S. carpocapsae Sucrose [46] Camponotus sp. S. carpocapsae Aqueous [47] Myrmica sp. S. carpocapsae Aqueous [47] Pogonomyrmex sp. S. carpocapsae Sucrose [48] Solenopsis spp. S. carpocapsae Alginate capsules [46] Solenopsis geminata S. carpocapsae Aqueous [49] Solenopsis invicta S. carpocapsae Aqueous [50, 51] Solenopsis invicta H. bacteriophora Aqueous [52] Solenopsis richteri H. bacteriophora Aqueous [52] Solenopsis richteri S. carpocapsae Aqueous [50] Figure 24; Detail of a dauer juvenile of Formicodiplogaster myrmen- ema adjacent to a worker of Azteca alpha in Dominican amber. Figure 25: Developing stages of Steinernema carpocapsae removed from the body of an infected queen of Solenopsis invicta. 8. Steinernematidae and Heterorhabditidae Included in this section are the so-called entomopathogenic nematodes belonging to the genera Steinernema and Het- erorhabditis. It is quite likely that entomopathogenic nema- todes infect ants under natural conditions, but no reports are known. Infection is initiated by a third-stage infective juvenile that enters the host’s body cavity, apparently per os [50]. After reaching the hemocoel, the infective stage ini- tiates development and, in so doing, releases a symbiotic bac- terium {Xenorhabdus spp. in Steinernema nematodes and Photorhabdus spp. in Heterorhabditis nematodes) that is car- ried in the infective stage’s gut lumen. The bacterium kills the insect soon after it is released in the body cavity. The nematodes feed on the mixture of bacteria and insect hemo- lymph and develop to the adult stage in the body cavity. With adequate nourishment, the nematodes undergo a sec- ond generation but when nourishment is limited, the juve- niles form third-stage infective juveniles. By introducing the bacteria that quickly kill the hosts, these nematodes avoid specific defense responses and have a wide host range, attack- ing representatives of many insect orders and even other arthropods [54]. Laboratory experiments have shown that these nemato- des can infect a number of ant species (Table 4) and they also have been used in the field against pest ants [50, 52, 54- 56] . Poole [50] attempted to control field populations of ants {Solenopsis richteri and S. invicta) with Steinernema carpocap- sae. Using a dose of I million infective stages per mound for S. invicta, the nematodes caused 35% mortality in the fall and 80% mortality in the spring. With S. richteri, the death rate was 80% in the spring and 36% in the fall. Poole [50] noted that workers were infected less than other stages, possibly be- cause of their greater activity and grooming behavior. How- ever, workers regurgitated infective stages to the alates and larvae. Queen ants were more susceptible and up to 3,000 in- fective stage juveniles could be produced in some infections (Figure 25). Further field trials of S. carpocapsae and Heterorhabditis bacteriophora against the red imported fire ant, S. invicta. Psyche 11 Table 5 (1) Nematodes represented as dauer or postdauer juveniles in the pharyngeal glands of ants Rhabditidae, Diplogastridae and Panagrolaimidae Nematodes developing in the body cavity of ants (2) (2) Only juvenile nematodes present (3) Adult nematodes with or without juveniles (4) (3) Elongate nematodes normally over 15 mm in length at completion of development; not enclosed in membranous capsules Mermithidae Nematodes under 10 mm in length; enclosed in membranous capsules Physalopteridae (4) Nematodes reproducing in dead ants; infective juveniles produced (5) Nematode adults, eggs and/or juveniles in living ants; infective juveniles absent (6) (5) Males with a bursa; females with a pointed tail Heterorhabditidae Males without a bursa; females with a bluntly rounded tail, often bearing a small point at tip Steinernematidae (6) Eggs and juveniles present Allantonematidae Eggs, but no juveniles present (7) (7) Vulva positioned at middle or lower half of body Tetradonematidae Vulva positioned in upper fourth of body Seuratidae gave control rates of 37.5% with S. carpocapsae but less with H. hacteriophora [52]. In field trials comparing applications of Steinernema carpocapsae and amidinohydrazone against S. invicta, Morris et al. [55] estimated that nematode applica- tions at a rate of 2 million per gallon per mound resulted in 47% mortality. Controlling fire ants in the field is difficult because of the small mound opening through which the nematodes are introduced. Also, it is desirable to have recycling of the nema- todes in the nests, but healthy ants appear to remove infected individuals before the cycle is completed. Since the number of nematodes needed to overwhelm a colony of ants is quite high using inundative methods, consideration was given to the development of baits or other more efficient delivery sys- tems [46, 48, 52, 55, 56]. These other methods are still under investigation. 9. Unknown Nematodes Gosswald [57] reported the presence of several encysted nematodes in the flight muscles of a queen Teleutomyrmex schneideri in Germany. The cysts were quite small, being only 25 pm in diameter. Except for their small size, the cysts are similar in appearance to those of the vespid mermith- id, Pheromermis pachysoma, formed in the body wall of Tri- choptera paratenic hosts [27] and the ant parasite, R villosa, in the body of oligochete paratenic hosts [26]. However, the Pheromermis cysts are 60-100 f/m and 80 pm in diame- ter, respectively. It is possible that juvenile nematodes of a mermithid parasite were acquired after the queen was fully formed and the nematodes preferred to encyst rather than initiate development. The other likelihood is that the nema- todes were the infective stages of a heteroxenous nematode parasite and were waiting for transfer to a vertebrate defini- tive host. However, the only cysts of heteroxenous nemato- des known from ants are those of the physalopterid, S. phryn- osoma, the smallest of which measures 633 pm in diameter [39]. In 1907, Janet [58] found nematodes 7-8 mm in length developing in the head cavities and emerging from the labial region of workers of Formica fusca. Just before the nemato- des emerged, the infected ants began trembling and eventual- ly died. The head cavities of infected ants were empty upon nematode exit. This behavior of developing in the head of ants is known for some phorid flies but not for nemato- des. Whether this was a mermithid with an unusual develop- mental location or a heteroxenous nematode using the ant as an intermediate host is unknown. 10. Identification Key to Nematode Families Associated with Ants See Table 5. 11. Conclusions Representatives of most invertebrate parasitic nematode families attack ants, with the exception of sphaerulariids, en- taphelenchids, and oxyurids. While mermithids are the most commonly encountered nematode parasites of ants, the com- plete life cycle of only a single species is known. The life cycle of ant mermithids can be quite complicated when it involves paratenic hosts living in completely different habitats. Even less is known about the life cycles of other ant parasitic nematodes, certainly not enough to consider using them as biological control agents. While the inundative applica- tion of entomopathogenic nematodes {Steinernema and Het- erorhahditis) can control ants in isolated colonies, establish- ing nematodes for the sustained control of ant populations has not been achieved. There are probably many additional nematode parasites of vertebrates utilizing ants as intermediate hosts. Reptiles, 12 Psyche mammals, and amphibians eat ants, and it follows that nem- atodes other than Skrjabinoptera phrynosoma would have devised methods of cycling themselves through ants to reach their definitive hosts. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 497087, 6 pages dohlO.l 155/2012/497087 Research Article Survival of Wild Adults of Ceratitis capitata (Wiedemann) under Natural Winter Conditions in North East Spain E. Penarrubia-Maria,^ J. Avilla,^ and L. A. Escudero-Colomar^ ^ Sustainable Plant Protection, Mas Badia Field Station, Institute for Food and Agriculture Research and Technology (IRTA ), La Tallada d’Empordd 1 71 34, Girona, Spain ^ Centre UdL-IRTA, University ofLleida, Avenue Alcalde Rovira Rome 1 91, 251 98 Lleida, Spain Correspondence should be addressed to L. A. Escudero-Colomar, adriana.escudero@irta.es Received 12 July 2011; Revised 25 September 2011; Accepted 21 October 2011 Academic Editor: Nikos T. Papadopoulos Copyright © 2012 E. Penarrubia-Maria et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The overwintering of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) at the northern limits of its geographic distribution is not yet well known. With the aim of estimating the survival rate of medfly adults in northeast Spain under natural winter conditions, a two-winter-season trial was carried out. A control was carried out in a climatic chamber at 25° C. The results showed that medfly adults were unable to survive the entire winter season in the Girona area. Climatic conditions, including the daily minimum temperature, daily maximum temperature and the high rainfall, appeared to be involved in adult mortality in winter. 1. Introduction Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (the Mediterranean fruit fly or medfly) has a worldwide geo- graphic distribution and it is well adapted to the climatic conditions [ 1 ] , but at the northern border of its distribution the population density is reduced at least once a year by winter temperature [2]. Therefore, in order to design ef- fective control methodologies against this insect, we require knowledge of the strategy that it uses to survive the most hostile winter periods. The northern limits of the medfly distribution include the northeastern part of Spain (the fruit growing area of Girona), where, unlike in more southern latitudes (such as the middle and southern coast of Spain), adults have not been observed during the coldest period of the year [20] . Adults of C. capitata are present throughout the year in several areas, including the southern coast of Spain [3] but have not been recorded during the coldest period of the year in the Girona area, north east (NE) Spain [4]. There are two hypotheses on medfly overwintering to explain the appearance of new populations in spring. The first one assumes that infestations are of a temporary nature, originating from infested fruit imported from warmer areas that contain a large quantity of winter hosts [5]. This theory has been questioned in a number of areas, including the central mountains of Israel [5, 6] and the Balearic Islands [2]. The second theory assumes that C. capitata adapts to temperate climates because of its ability to withstand low temperatures [7], a fact that enhances successful overwinter- ing for part of the population, as seen in central Italy [8]. In northern Greece, medfly has been observed to overwinter as larvae within infested host fruit even if temperatures fall below zero [9] . These findings were also verified in the region of Tarragona, where medfly larvae were found inside late- ripening varieties of orange [ 10] . Survival of a small percentage of individuals each winter and regeneration of the entire population from these indi- viduals in spring and early summer probably represents a strong selection pressure in this insect for the evolution of a mechanism to withstand the cold [9] . Studies on medfly population dynamics have shown that the main factor affecting population buildup in the tropics is the abundance and availability of fruit [II-I3], whereas in temperate areas, such as northern Greece, low winter temperatures and the absence of host fruits are the two main factors that inhibit overwintering [14-16]. Temperature can 2 Psyche also affect the appearance of the population after the winter period, retarding or advancing adult presence, as seen in Girona [4]. Although medfly distribution appears to be ultimately restricted by the severity of the winter, the existence of a vari- ety of microclimates in a particular area implies that other climatic factors may limit or at least regulate the population dynamics of the species [17]. Survival in insects depends on both temperature and duration of exposure [18], and the duration of low temperatures could be used to test the medfly ’s tolerance to cold [19]. Therefore, the combination of factors such as dry and cold stress [17] and duration of low temperatures could explain the low incidence of the pest in a particular location. Recent studies performed in Girona support the hypoth- esis of an overwintering population in this area [4], but the conditions of medfly overwintering are still unknown. Therefore, the aim of this study was to estimate the survival rate of medfly adults in the Girona fruit area, NE Spain under natural winter conditions over a period of two winter seasons and to test the hypothesis that adult medflies survive winter conditions in this area. 2. Material and Methods The trial was performed over two consecutive winters: from mid-December 2008 to mid-January 2009 and from mid- November 2009 to late December 2009. It was performed on a 962 m^ north-facing commercial plot of “Golden Deli- cious” apples, enclosed by a wood structure with walls and a roof made of plastic mesh. The plot was divided into three sealed compartments with the same dimensions, each with its own access door. Glimatic conditions in the three compartments, including temperature and relative humidity, sometimes differed slightly. The control treatment was ar- ranged in a chamber maintained at 25° C ± 1°C, 50-80% RH with a photoperiod of 14 h light and 10 h darkness in order to determine the viability and longevity of the population used in the field. In 2008, 236 five-to-seven-day-old second-generation (F2) adults originally from an autochthonous population reared under controlled conditions in Girona were used, and in the following year 212 six-day-old wild flies were used, with the aim of avoiding any influence from laboratory rear- ing conditions. Individuals were placed in cages (61 by 61 by 61 cm) provided with an ad libitum diet composed of 1:4:5 parts of hydrolysed protein (Biokar Diagnostics, Beauvais, France), sugar, and water. In the second year, a mesh was placed on the base of the cages in order to prevent the flies from sinking into rain water. Each cage was placed on two wooden supports in order to avoid direct contact with the ground. They were also fixed to the soil surface with two 1.5 cm diameter iron pegs to avoid movement caused by the wind. Three cages (replicates) each containing 45-60 individ- uals were established on 16 December 2008 and 16 Novem- ber 2009. The cages were evenly distributed among the com- partments of the plot and were maintained under natural winter conditions. In each year a similar control cage was maintained in an environmental chamber at 25° G ± 1°G. Once the cages had been installed, individual mortality was recorded on a daily basis until the death of the last individual. During both experimental periods, meteorological data were recorded outside the observation orchard at a weather station located 700 m from the survey plot [20]. Moreover, inside each compartment of the observations plot, as well as in the chamber, temperature and relative humidity were recorded hourly using data loggers hung 1.60 m above ground level (Hobo Pro V2-ext. Temp/RH, Onset Gom- pany). The survival analysis was carried out using the Kaplan- Meier estimates of survival and standard error followed by the log-rank test for pairwise comparisons (P = 0.05). Hence, the three replicates combined and compared against control. This statistical analysis was carried out using the SPSS V.15 software. The relationship among the following factors was stud- ied: year of the study, age of adults, daily minimum tem- perature, daily maximum temperature, daily average tem- perature, and daily rainfall, using the General Einear Model (Proc GEM) procedure of the Statistical Analysis System (SAS Institute Inc., Gary, NG, USA) to determine significant differences at a level of P < 0.05. 3. Results The results of these observations suggest that adults of C. capitata are unable to survive throughout the winter season in the Girona fruit-growing area. Adults subjected to external conditions remained immobile inside field cages, resting on the mesh walls or in the iron-clad corners, but adults inside cages in the environmental chamber were more active. Under natural winter conditions in Girona, adult flies survived an average of 8.43 to 9.88 days in the first study period (starting in mid-December) and 28.45 to 30.24 days in the second (starting in mid-November), but under cham- ber conditions they survived an average of 15.5 days and 12.87 days, respectively (Table 1). The maximum survival period under natural conditions was 11 days in the first winter and 35 days in the second; under chamber conditions, the figures were 29 and 38 days, respectively. Graphs of cumulative survival rates over the study period are given in Figure 1. The log- rank test indicated differences in survival rates between field exposed and laboratory-held control individ- uals {)^ = 41.02, P < 0.5). Similar results obtained when control was compared against each replicate within the same year. The analysis comparing the survival in the control versus the survival in each replicate provided information related to the possible sources of variability between repli- cates that are difficult to measure separately in each replicate, such as the wind, normally strong in the study area and usually flowing in NE-SE direction. Due to the orientation of the orchard involved in the study (N-S), each of the replicates could have been differently exposed to it. The temperature and relative humidity recorded in each cage using data loggers were very similar to those recorded Psyche 3 Table 1: Number of individuals, average survival rates, and maximum survival for Mediterranean fruit fly adults exposed in field conditions in December and November 2008 and 2009, respectively. Year Replicate No. of adults Survival average ± SE (days) Maximum life span for the longest lived individual 1 60 8.62 ± 0.47 18 2008-2009 2 60 9.88 ± 0.33 16 3 60 8.43 ± 0.44 18 Control 56 15.5 ± 1.02 34 1 54 30.24 ± 0.85 41 2009 2 45 29.04 ± 0.78 38 3 55 28.45 ± 1.11 41 Control 58 12.87 ± 1.30 43 15 / 12/08 22 / 12/08 29 / 12/08 5 / 01/09 12 / 01/09 Dates (a) Dates (b) Figure 1: Cumulative survival rates of adult medflies exposed to field conditions on (a) December 16, 2008, and (b) November 16, 2009, and respective controls maintained at 25° C in laboratory conditions (dotted line, control; solid line, average for three replicates). at the nearby meteorological station, with maximum dif- ferences of ±1°C. The absolute maximum temperature was 18.8°C in the first year and 22.3°C in the second (Figure 2). The absolute minimum temperature was -2°C in the first year and -8.1°C in the second. The average temperature recorded inside the plot was 6.8°C in the first year and 8.3°C in the second. Relative humidity in the field cage was 47.54% to 100% in 2008 and 40.38% to 62.49% in 2009. Accumu- lated rainfall in the first year was 67.2 mm, with a maximum daily rainfall of 54.6 mm recorded on 26 December 2008. In the second year, these figures were 7.6 and 2.6 mm, respec- tively. In the second year of the study, one day after the absolute minimum temperature (-8.1°C) was recorded, 20.4% and 21.8% of the remaining living individuals from replicates 1 and 3 died, respectively. In the first year, between 16 November and the death of the last individual, the maximum number of cold hours below 9°C tolerated by adults was 173 hours, while in the second year, between 16 October and the end of the trial, this value was 464 hours. The GLM analysis showed a good fit of the model {R^ = 0.6983) and evidenced significant differences in the following factors: rainfall (F = 287.26, df = I, P < 0.0001), daily minimum temperature (F = 9.27, df = I, P = 0.0026), and daily maximum temperature (F = 11.20, df = 1,F = 0.001). The factors year of the study (F = 0.3, df = I, P = 0.5870), age of adults (F = 0.14, df = I, P = 0.7117), and daily average temperature (F = 2.63, df = I, P = 0.1067) were not significant. 4. Discussion The results showed that medfly adults were unable to survive the entire coldest period of the Girona fruit-growing area. Although adults withstand a high number of cold hours by reducing their movement and keeping still, all of them died 4 Psyche Figure 2: Temperature and rainfall recorded within the observation plot and temperature in the control under chamber conditions (line with squares for temperature in the control; line with triangles for daily maximum temperature; line with circles for daily average temperature; line with rhombus for daily minimum temperature; dotted line for rainfall). after a strong freeze temperature (-8.1°C). It was observed that in semifield conditions the age reached by adults was higher than that of the control. The main factors affecting survival of adult medfly in natural winter conditions of Girona were minimum temperature, maximum tempera- ture, and rainfall. Taking into account these results, the hypothesis that adult medfly may survive the natural winter conditions in the Girona fruit-growing area must be rejected. Medfly adults are unable to survive low winter temper- atures in some Mediterranean areas, including Greece [9]. This was corroborated in the present overwintering study with adults from a population native to Girona. Adults sur- vived winter conditions in this area from mid-November to late December. The insect’s resistance to cold is affected by its micro - habitat, which determines the availability of moisture, the developmental temperature, and parameters such as humid- ity and desiccation tolerance [21]. Other biological factors involved are age, body size during adult development, and feeding. In our study, cold temperature led to a reduction in medfly movements until they kept completely still, at which point they surely did not feed; because they were provided with food ad libitum, we may assume that when they kept still they had enough energy reserves to withstand extreme temperatures. However, it was demonstrated that this process consumed a great amount of energy and that survival is sub- sequently compromised. On the other hand, Nyamukondiwa and Terblanche [22] showed that the minimum critical ther- mal limit (Gtmin) decreases with age up to 14 days old, after which older flies have less tolerance to low temperatures; these authors also found no interaction between age and feeding in C. capitata. During cold and temperate winters, most species are inactive, leading to a seasonal state of quiescence, dormancy, or even diapause that varies with species and circumstances [21, 23]. It has been shown that, at lower latitudes in temperate regions, populations of certain tephritid species (e.g., Eurosta solidaginis (Fitch)) are less cold tolerant than those from higher latitudes [18]. Nevertheless, in some of the southern Mediterranean areas, a small number of medfly adults might be active during winter [15, 24] . In Grete adults survived the whole winter with minimum temperatures between 1°G and 4.5° G [25] . In the present study carried out in Girona, all individuals died during the winter. The tem- perature threshold for population growth is 12° G to 35° G, [17] and maximum temperatures in both years were always below the upper limit. However, minimum temperatures in the study periods sometimes fell below the lower threshold. There is high variability in the severity of the minimum temperature and the duration of exposure to low temper- atures in the temperate zone [19]. Similar results to those found in the present study were recorded using the fruit fly Dacus tryoni at an overwintering site, where mortality was related to the minimum temperatures and mortality rate increased when subzero temperatures occurred [26]. The Gtmin for medfly is the temperature at which each individual insect loses coordinated muscle function and consequently the ability to respond to mild stimuli [22]. Adults exposed to this threshold recovered, so it was not immediately lethal. Depending on the age of the flies, Gtmin was 5.4° G to 6.6° G [22] . Taking into account these thresholds and the minimum temperatures recorded in Girona during the two study periods (-2°G and -8.2°G), it is possible that the study population lost coordinated muscle function, in which case they would have suffered a rapid demise. The effect of rainfall on the medfly population has been related to a decrease in adult captures on rainy days and an increase a few days later, because flies are generally inactive during periods of moderate to heavy rainfall [27, 28]. In the current trial, the mortality of adults was observed to be affected by rainfall. In the first year, rain fell for only a few hours, but this had a negative effect on adult survival. The relationship between the accumulated number of cold hours below 9°C and the survival of adults in the first period was also influenced by the high rainfall of 26 December 2008. Therefore, flies in the second year endured more than twice as many cold hours as those in the first year. Despite the results achieved in the two years of observa- tion, there is a stable population in NE Spain. In the entire Girona fruit-growing area, the first adult medfly captured in Psyche 5 the fruit season, using a wide monitoring network (one per orchard) installed from April to January, coincides year by year (mid- June to early July), as does the only population peak (late September to early October) [4] . In some microcli- mates of the region, medfly larvae may survive inside apples, the only fruit species available for overwintering in the area, as suggested by other authors [9]. It is clear from studies carried out in the area that the previous winter determines the level of the population in the following season. In years with a mild winter, after the capture of the first medfly in the season, the population developed fast and could reach high levels in the peak captures. On the other hand, after cold winters, even if the first medfly is captured on the same date, the population increases slowly, peaking as usual but never reaching the same level as in years following a mild winter [4]. An interesting fact is that, though temperature limits development, it does not necessarily limit the geographic distribution of this species [22]. This could explain why the medfly occurs in southern France, where it is frequently detected [29]. Some studies have developed models for the potential distribution of medfly, and in all of them the Girona area was included [17, 30]. One of the key points to elucidate is the threshold for classifying a winter as mild or cold, from the point of view of the medfly biology of the Girona population. How many cold hours below its Gtmin can the Girona medfly population withstand in order to have enough surviving individuals to quickly develop a new population in the following fruit season? At present we have no answers to this question, and a greater effort must be made to elucidate it. The implications of this knowledge are great, because it would allow us to forecast the development of the population, offering a great advantage for protecting fruit against this pest. 5. Conclusions Medfly adults were unable to survive the entire winter season in the Girona fruit-growing area in both years studied. Glimatic conditions, including low minimum temperatures, maximum temperature, and high rainfall, were responsible for adult mortality in winter. 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H Foote, “Biology of fruit flies,” Annual Revieve of Entomology, vol. 5, pp. 171-192, 1960. [29] J. P. Cayol and R. Causse, “Mediterranean fruit-fly Ceratitis capitata Wiedemann (Dipt, Tripetydae) back in Southern France,” Journal of Applied Entomology, vol. 116, pp. 94-100, 1993. [30] M. De Meyer, M. P. Robertson, A. T. Peterson, and M. W. Mansell, “Ecological niches and potential geographical dis- tributions of Mediterranean fruit fly (Ceratitis capitata) and Natal fruit fly (Ceratitis rosa),” Journal of Biogeography, vol. 35, no. 2, pp. 270-281, 2008. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 820245, 9 pages dohlO.l 155/2012/820245 Review Article Antitermite Activities of C. decidua Extracts and Pure Compounds against Indian White Termite Odontotermes obesus (Isoptera: Odontotermitidae) Ravi Kant Upadhyay,^ Gayatri Jaiswal,^ Shoeb Ahmad, ^ Leena Khanna,^ and Subhash Chand Jain^ ^ Department of Zoology, D. D. U. Gorakhpur University, Gorakhpur 2730 09, India ^ University School of Basic and Applied Sciences, Guru Gobind Singh Indraprastha University, Sector 1 6G Dwarka, New Delhi 110075, India ^ Department ofGhemistry, University of Delhi, Delhi 110 007, India Correspondence should be addressed to Ravi Kant Upadhyay, rkupadhya@yahoo.com Received 18 June 2011; Revised 25 September 2011; Accepted 15 October 2011 Academic Editor: Abraham Hefetz Copyright © 2012 Ravi Kant Upadhyay et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In the present investigation, we have tested antitermite responses of Gapparis decidua stem, root, flower, and fruit extracts and pure compounds to Odontotermes obesus in various bioassays. Crude stem extract has shown very high susceptibility and very low LD50 values, that is, 14.171 jWg/mg in worker termites. From stem extract, three pure compounds were isolated in pure form namely, heneicosylhexadecanoate (CDS2), triacontanol (CDS3), and 2-carboxy-l, 1-dimethylpyrrolidine (CDS8) which have shown very low LD50 value in a range of 5.537-10.083 /rg/mg. Similarly, one novel compound 6-(l-hydroxy-non-3-enyl)-tetrahydropyran-2- one (CDFl) was isolated from flower extract that has shown an LD50 8.08 pgigm. Repellent action of compounds was tested in a Y-shaped glass olfactometer in which CDFl compounds have significantly repelled termites to the opposite arm. Besides this, C. decidua extracts have shown significant reduction (P < 0.05 and 0.01) in termite infestation in garden saplings when it was coated on cotton tags and employed over tree trunks. Further, C. deciduas stem extract was used for wood seasoning, which gave very good results as test wood sticks have shown significantly (P < 0.05 and 0.01) very low termite infestation. 1. Introduction The Indian white termite, Odontotermes obesus Rambur (Isoptera: Odontotermitidae), is highly destructive polypha- gous insect pest, lives in huge mounds, and feeds on cellulose material and almost anything which contains carbohydrate. It causes economic damage to commercial wood, fibers, cel- lulose, sheets, papers, clothes, woolens and mats, and woody building material and infests green standing foliages, cereals stored in godowns. Both worker and soldier termites harm nonseasoned commercial wood and its formed materials. Whether it is a rural area or an urban domestic site, termite menace is everywhere. However, for controlling termite population in the field, various synthetic pesticides such as chlorodane [1], cypermethrin [2], hydroquinone, and indox- acarb [3] have been used. But all such synthetic pesticides are highly poisonous and kill nontarget organisms. Due to their longer residual persistence in the environment, these have been banned and new alternatives are discovered in form of natural pesticides. These plant-origin natural pesticides provide wide range of control and efficiently cut down the population of all kinds of pests even applied in very low quantity. These plant-origin pesticides are much safer and easily biodegradable in the medium and show no residual effect. So far numbers of plant species have been screened to explore potential antitermite agents by the researchers to control termite menace. Few natural products such as flavonoids [4], sesquiterpenes [5], 2 Psyche and thiophenes [6] isolated from different plants species were found effective against termites [7]. In addition, for enhancing the insecticidal potential of crude plant extracts and its target specificity, few synergists were applied in form of poison baits which successfully exploit feeding, tunneling [8], and reproductive behavior in termites [9]. Similarly, application of Summon disks and filter paper disks coated with few chitin synthesis inhibitors, that is, diflubenzuron, hexaflumuron, and chlorfluazuron [10] controlled the aggre- gation, feeding, and recruitment behavior in Coptotermes formosanus termites. We, in continuation to our phytochemical studies on various Indian medicinal plants, have already evaluated Cap- paris decidua for insecticidal and ovipositional inhibitory activity against Bruchus chinensis [11]. We observed that this plant is not attacked by termites at all, and this inspires us to study its antitermite activity, in order to find out some potent antitermite components of this plant. In the present study, different extracts of Capparis decidua and compounds iso- lated from its various parts have been evaluated for antiter- mite activity. C. decidua belongs to the family Capparidaceae [12] and is an indigenous medicinal plant, commonly known as “Kureel” in Hindi. It is a densely branching shrub with scanty, small, caduceus leaves. Barks, leaves, and roots of C. decidua have been claimed to relieve variety of ailments such as toothache, cough, asthma, intermittent fever, and rheumatism [ 13] . The powdered fruit of C. decidua is used in antidiabetic formulations [14], while the bark of its leafless shrub is used for the treatment of asthma, cough, inflam- mation, and acute pain [15]. Seeds of C. decidua showed antibacterial activity against Vibrio cholerae ogava, inaba, and ettor [16]. In this study, we conducted preliminary investigation on the antitermite activity of C. decidua extracts and pure com- pounds. For this purpose, active components were isolated and fractionated to obtain and pure compounds employed to test the toxicity and repellent action in workers of O. obesus both in laboratory and field conditions. The overall aim of this study was to identify potential anti-termite compound that could be used to develop an effective repellent and toxic formulation to kill field termites. Further, we have also used active ingredients from C. decidua in spray, repellent tags, and wood seasoning to protect the wood from termite infestation on garden trees and wood sticks. 2. Materials and Methods 2.1. Insects and Plant Material. Termite O. obesus were col- lected from infested logs found at the University of Gorakh- pur, India, and nearby forest area of eastern Uttar Pradesh, India. Termites removed from plant biomass and logs were maintained in glass jars (height 24", diameter 10") in complete dark conditions at 28 ± 2°C, 75 ± 5 RH. Termites were fed on green leaves. Stems, flowers, and fruits of Capparis decidua were col- lected from different places of Rajasthan and western Uttar Pradesh, India. These were separately shaved, dried, and sub- sequently pulverized to obtain fine powder. O (a) CH 2 OH CDS3 CDS8 (b) (c) OH CDFl (d) Figure 1; Pure compounds obtained from C. decidua after solvent fractions being eluted and purified by column chromatography. Compounds CDS3 (25 mg) and CDS8 (50 mg) were identified as triacontanol and 2-carboxy-l,l-dimethylpyrrolidine. While com- pound CDS2 is characterized as heneicosylhexadecanoate. The column was prepared in petroleum ether using silica gel (60-80) as an adsorbent and eluted with petroleum ether/ chloroform, chloro- form, chloroform/methanol mixtures of increasing polarity. CDFl was isolated (15 mg) from flowers from the chloroform fraction of the extract. It was identified as 6- (1 -hydroxy- non-3-enyl) tetrahydropyran-2-one and is colorless. 2.2. Extraction of Plant Foliage Chemicals. The powdered stems (200 g) were extracted using CHCb/MeOH (1:1), cold MeOH, and hot MeOH sequentially to obtain dry extracts CDSl (14 g), CDS2 (5 g), and CDS3 (2 g), respectively, while its flowers (40 g) and fruits (50 g) were only extracted with CHCI3 MeOH (1:1) to obtain dry extracts CDS7 (3 g) and CDFl (0.2 g), respectively. 2.3. Isolation and Characterization of Active Components from 50% Methanol/Chloroform Extract (CDSl ) of Capparis Stems. The solvent free extract CDSl from C. decidua stem was found to be a mixture of several components of varying polarity on TLC and further fractioned by column chro- matography to isolate the minor active components. The column was prepared in petroleum ether using silica gel (60- 80) as an adsorbent and eluted with petroleum ether/chloro- form, and chloroform, chloroform/methanol mixtures of in- creasing polarity. Twelve fractions were obtained, and three compounds, namely, CDS2, CDS3, and CDS8 have been isolated and characterized. Compounds CDS3 (25 mg) and CDS8 (50 mg) were identified (4) as triacontanol and 2-car- boxy- 1,1- dimethylpyrrolidine (Figure 1). While compound CDS2 is characterized as heneicosylhexadecanoate, and this is the first report of the isolation from the genus Capparis and species decidua. 2.4. Characterization of Compound CDS2. Compound CDS2 (20 mg) was obtained as colorless oil. Its ^H NMR spec- trum revealed its aliphatic nature. Its IR spectrum showed absorptions at 1735 and 1175 cm~^ which were characteristic Psyche 3 for C=0 and C-O stretching of an ester linkage. Its NMR spectrum exhibited a triplet, at S 2.29 integrating for two protons of a-methylene linked to carbonyl group. Also, downfield at 8 4.05, another triplet was observed which was assigned to the methylene directly attached to oxygen atom. Apart from this, NMR spectrum exhibited a triplet at 8 0.88 corresponding to two terminal methyl groups indicating it to be a middle ester. Compound CDS2 displayed a molecular ion peak at m/z 550 and hence analyzed for C37H74O2. Its mass spectrum showed prominent peaks for McLafferty rearrangement at m/z 355 and 257 thereby revealing the presence of hexadecanoate as the acid moiety and heneicosane as the alcohol moiety in the molecule. On the basis of above-mentioned spectral data, com- pound CDS2 was identified as heneicosylhexadecanoate. This is the first report of its isolation from the genus Cap- pans. 2.5. Heneicosylhexadecanoate (CDS2). Compound CDS2 was obtained as colorless oil (12 mg). It showed a single spot on TLC using petroleum ether as the developing solvent, Rf = 0.3. IR Vmax (KBr): 2918, 2850, 1735, 1463, 1175, 758, 719cm-C NMR (5, CDCI3, 300 MHz): 0.88 (t, 6H, 2x- CH3), 1.25 (m, 60H, 3OX-CH2), 1.58 (m, 4H, -CH2CH2- C=0 & -CH2CH2-O), 2.29 (t, 2H, -CH2-C=0-), 4.05 (t, 2H, -O-CH2-). Mass Spectral data, EIMS m/z (%): 550 (10, M+), 453 (5), 355 (24), 257 (31), 240 (8, M+-0(CH2)2oCH3), 202 (15), 174 (10), 111 (20), 97 (35), 71 (48), 57 (100), 41 (80). Extract from Capparis decidua flowers when checked on TEC gave one major spot. It was subjected to column chro- matography using silica gel (60-80) as an adsorbent and eluted with petroleum ether/chloroform, chloroform, and chloroform/methanol mixtures of increasing polarity. One major compound CDEl (15 mg) was isolated from the chlo- roform fraction of the extract. It was assigned constitution as 6-(l-hydroxy-non-3-enyl)-tetrahydropyran-2-one. This is the first report of isolation of this compound from any natural and synthetic source. Extract CD 10 (0.2 g), when checked on TLC, gave one major spot, similar to that of compound CDEl. Some more amount of CDEl was isolated from the extract by column chromatography for bioassays. 2.6. Triacontanol (CDS3). White solid, mp 82-85° C IR Vmax (KBr): 3398, 2918, 2849, 1463, 1360, 1061, 720cm-E ^NMR (6, CDCI3, 300 MHz): 0.88 (t, 3H, -CH3), 1.25 (brs, 54H), 1.54 (m, 2H, -CH2CH2OH), 3.63 (t, 2H, -CH2OH). NMR (6, CDCI3, 75.47MHz): 14.01 (-CH3), 25.67-32.76 (-CH2 ), 63.04 (-CH2OH). EIMS m/z (%): 420 (M+-18, 36), 392 (8), 364 (5), 167 a8), 153 (20), 125 (950), 57 (100). 2.7. 2-Carboxy-l,l-Dimethylpyrrolidine (CDS8). White sol- id, mp 268° C. It gave a single spot on TLC, Rf = 0.5 using chloroform/methanol (60 : 40) as the developing solvent. IR ymax (KBr): 3401, 1621, 1531, 1479, 1400, 1368, 1326, 1002, 961 cm-\ ^NMR (6, CD3OD 300 MHz): 2.13 (m, 2H, H-4), 2.32 (m IH, H-3'), 2.49 (m, IH, H-3), 3.14 (s, 3H, N-CH3), 3.31 (s, 3H, N-CH3), 3.67 (m, IH, H-5), 3.71 (m, IH, H- 5'), 4.01 (t, IH, H-2). NMR (6, CD3OD, 75.47 MHz): 20.22 (C-4), 27.23 (C-3), 46.80 (N-CH3), 53.17 (N-CH3), 68.43 (C-5), 78.17 (C-2). NMR DEPT 135 (6, CD3OD, 75.47MHz): 20.21 (C-CH2), 27.02 (-CH2), 46.73 (-CH3), 53.09 (-CH3), 68.42 (CH2), 78.14 (-CH). EIMS m/z (%): 144 (M+, 2), 117 (7), 115 (6), 101 (5), 99 (3), 85 (7), 59 (100), 55 (13), 45 (42), 43 (70). 2.8. Toxicity Bioassay. Eor evaluation of observation of toxic responses in termites, serial concentrations, that is, 1.0, 2.0, 4.0, 8.0, 16, and 32 pg of different extracts were loaded on separate Whatman paper strips (1x1 cm^) and air dried to remove the solvent. These precoated solvent free strips were placed in the center separate Petri dishes (42 mm diameter) as tests and uncoated as control. Twenty- five worker termites were released in the Petri dish to observe the mortality. After setting the experiment, green leaves were provided as food for both tests and control insects and containers were covered with black paper sheets. Mortality was recorded on the basis of dead and living termites, and observations were made in triplicate for each extract and pure compounds up to 24 hrs. Insects were treated as dead when they become immobile and have shown no further activity to the external stimuli. The LD50 after 24 hrs of exposure to each was calculated by using Probit analysis tested using the method of Einney [17]. 2.9. Repellency Bioassay. Repellent responses were observed in a glass Y-tube olfactometer by using serial concentrations 0.001, 0.002, 0.004, 0.008, 0.016, and 0.032 mg of different extracts loaded on separate Whatman paper strips (1 x 1 cm^) and air-dried to remove the solvent. These precoated solvent free strips were placed in right arm of Y-tube olfactometer ( 16 mm diameter X 90 cm length) as tests, while similar strips uncoated were placed in left arm as control. Twenty- five worker termites were released inside the opposite triarm to observe the repellent activity. After introduction of termites tube, openings were closed by Teflon tape and number of termites oriented towards uncoated strips or nonscented area were counted as repelled. Individuals that did not enter at least one of the arms were scored as unre- sponsive. Tests were conducted for 18 hrs at 27° C tempera- ture. Same tests were conducted after reversing the arms to test directional bias. A Chi^ test was used to compare the number of termites responding to the olfaction generated by C. decidua active ingredients. 3. Field Experiments 3.1. Thread-Binding Assay. Eor control of termite infestation in garden plants, presoaked cotton threads were tagged around the tree trunks at a hight of 5-6 feet above the ground. Eor this purpose, threads were soaked in Capparis decidua aqueous extract for 24 hr and dried in shade. Early age saplings of Tectona grandis (4 year old) trees in 8 different rows each having 24 plants were selected and tagged with the cotton threads and sprayed regularly at 15 days interval with same extract. In controls, the uncoated threads were tagged at 4 Psyche Table 1 : LD50 values obtained in solvent extracts and pure compounds isolated from Capparis decidua against Indian white termite Odontotermes obesus. Extracts Single hr FD50 values ipg/mg) (P < 0.05)" FCF'^ UCF'^ t-ratio Slope value Heterogeneity Chi test Root 24 14.515 11.421 17.459 11.215 0.081 2.070 6.210 Stem 24 14.171 10.48 17.566 12.422 0.093 3.3741 0.124 Fruit 24 14.781 11.106 18.350 11.933 0.088 3.204 9.612 Flower 24 15.274 11.878 18.778 11.982 0.088 2.995 8.986 Mixture CSTMl (stem) 24 13.246 5.663 21.147 6.159 1.62 0.796 3.982 CSTM2 (stem) 24 6.616 0.563 0.825 4.796 1.058 0.470 2.348 CSMM3 (stem) 24 4.973 0.057 16.028 4.088 0.769 0.736 3.679 Pure compounds CDSl* 24 7.514 2.054 10.995 7.123 0.093 1.614 4.833 CDS2 24 7.290 3.178 10.295 8.799 0.135 2.154 6.462 CDS3 24 7.434 5.277 9.179 6.864 0.087 0.533 1.60 CDS4 24 6.531 2.920 9.106 8.655 0.141 1.7475 5.24 CDS5 24 5.537 1.192 8.157 4.853 0.059 0.782 2.345 CDS6 24 8.655 5.711 11.405 9.578 0.149 1.9415 5.824 CDS8 24 10.083 8.39 11.759 7.353 0.093 0.906 2.717 CDFl* 24 8.086 5.40 10.47 9.366 0.147 1.545 4.636 LD 50 values represent lethal dose that causes 50% mortality in the test insects. ’^LCL and UCL mean lower confidence limit, and upper confidence limit respectively, “^t-ratio, slope value, and heterogeneity were significant at all probability levels (90, 95, and 99%). t- ratio: difference in degree of freedom at 0.5, 0.05, and 0.005 levels; slope value shows the average between LD50 and LDso, from which LD50 value is calculated; heterogeneity value, shows the effect of active fraction on both susceptible and tolerant insects among all of the treated insects. CSTM indicates combined tincture of Capparis deciduas, coconut oil, terpene oil, glycerol, elemental sulphur, and liter water. CDS* represents pure compound isolated from C. decidua stem fraction, while CDFl* represents compound isolated from flower. similar height without coating any active fraction on threads. Separate rows were chosen for spray, thread binding, and both. 3.2. Wood Seasoning. For evaluation of termiticidal action of C. decidua stem aqueous extract dried solid wood sticks of Tectona grandis each having 3 feet length were used to dug and treated wood sticks were planted into the pits. For this purpose, set of six wood sticks were seasoned with three different concentrations of C. decidua named as CSTMl, CSTM2, and CSTM3. Antitermite mixture or tincture was prepared by mixing different ingredients (90 gm Capparis decidua, 50 mL coconut oil, 50 mL terpene oil, 50 mL glycerol, and 11 gm elemental sulphur in 15 liter water). In CSTM2 and CSTM3, mixtures C. decidua powder was mixed 132 gm and 180 gm while rest of the ingredients were the same. Seasoning of the wood sticks was done by dipping them in the above mixtures separately for 24 hours. Then, seasoned wood sticks were dried for 12 h and planted inside soil by making separate pit of 2.75 feet depth at a distance of 3 feet. Similarly, six control wood sticks were also used which were unseasoned. After 30-day interval, each one of control and test wood stick was dug out for evaluation of antitermite activity. % weight loss and % infestation, exposure period, and concentration of ingredients were considered for determination of antitermite activity in wood sticks in garden soil. Experiments were run up to 180 days. and wood sticks were marked with colored marker for corresponding control. 4. Statistical Analysis Standard deviations chi-square, t-significance, correlation, and AN OVA were calculated from the means of two replicate, using three equal subsamples from each replicate by using method of Sokal and Rohlf [ 18] . In the experiments, analysis of variance (ANOVA) was done whenever two means were obtained at a multiple test range and P < 0.05 probability level. The LD50 after 24 hrs of exposure to each was calculated by using Probit analysis tested using the method of Finney [17]. 5. Results Toxic and repellent responses of various extracts and pure compounds isolated from C. decidua were evaluated against Indian white termite O. obesus. For this purpose, insects were treated with increasing dose of both extracts and compounds separately. The mortality rate was found dose and time dependent as it was found to be increase with an increase in dose and exposure period. The LD50 values for different extracts of 24 h are given in Table 1. Solvent extracts have shown LD50 in a range of 14.171-15.274 f/g/mg, while com- bined mixtures of C. decidua have shown synergistic activity Psyche 5 Table 2: Percent repellency obtained in solvent extracts and pure compounds isolated from Capparis decidua against Indian white termite Odontotermes obesus. Compounds Concentration in mg Mean number, of Insects repelled Expected number, of insect repelled Value ED 50 Single fractions (acetone) Root 0.001-0.016 14.0 10 NS" 0.009 Stem 0.001-0.016 12.4 10 NS" 0.011 Fruit 0.001-0.016 14.4 10 NS" 0.006 Flower 0.001-0.016 13.2 10 NS" 0.008 Mixture CSTMl extract 0.005-0.08 11.44 10 NS" 0.015 CSTM2 extract 0.005-0.08 10.96 10 NS" 0.042 CSTM3 extract 0.005-0.08 11.24 10 NS" 0.038 Pure compounds CDSl 0.001-0.016 10 10 0.50'’ 0.012 CDS2 0.001-0.016 8.6 10 0.80'’ 0.017 CDS3 0.001-0.016 12.2 10 NS" 0.008 CDS4 0.001-0.016 10 10 0.50'’ 0.013 CDS5 0.001-0.016 10 10 0 . 20 ^ 0.012 CDS 6 0.001-0.016 12 10 NS" 0.010 CDS 8 0.001-0.016 12.2 10 NS" 0.009 CDFl 0.001-0.016 10.4 10 0 . 20 '’ 0.011 "'Not significant as the calculated values were less than the table values at all prohahility levels (90%, 95%, and 99%). '^Significant at all probability levels (90%, 95%, and 99%). The data responses lines would fall within 95% confidence limits, and thus the model fits the data adequately. UCL-LCL* : upper confidence limit and lower confidence limit. CSTM indicates combined tincture of Capparis decidua, coconut oil, terpene oil, glycerol, elemental sulphur, and liter water. against termites and caused comparably high mortality with LD50 4.973-13.246 |Wg/mg (Table 1). From fractionation of stem extract, three compounds were isolated in pure form, which were identified as heni- cicosyl-hexa decanoate (CDS2), tricantanol (CDS3), and 2 carboxy-l-l-dimethylpyrrolidine (CDS8) (Figure 1). All these three compounds were evaluated for their antitermite activity which have shown very low LD50 values, that is, 7.290, 7.434, and 10.083 f/g/mg body weight of termite (Table 1). Similarly, flower extract was fractionated and a single major compound CDFl was identified as 6-(l- hydroxy-non-3-enyl)-tetrahydropyran-2-one. It has shown very high antitermite potential against O. obesus with an LD50 value of 8.086 |Wg/mg (Table 1). It is highly noticeable that C. decidua fractions in termites remain active for longer duration and cause high lethality. The index of toxicity estimation indicates that the mean value was within the limit at all probabilities (90, 95, and 99%) as it is less than 0.05 values of t-ratio. Besides this, regression was also found significant. The steep slope values indicate that even small increase in the dose causes high mortality. Values of the heterogeneity less than 1.0 denote that, in the replicate test of random sample, the dose response time would fall within 95% confidence limit, and, thus, the model fits the data adequately. In olfactometry tests, solvent extracts prepared from stem, flower, fruits, and pure compounds have shown sig- nificant repellency at a very low dose. Interestingly, solvent extract has repelled mean number of insects 12.4, while 8.6 mean numbers of insects were repelled by pure compound in olfactometer. ED50 values obtained in pure compounds (CDSl-CDFl) range 0.008-0.017 f/g/mg body weights, and solvent extracts have shown ED50 in between 0.006- 0.042 /.ig/mg (Table 2). Besides this, for control of termites in the garden, presoaked cotton threads impregnated with Capparis decidua stem extract were tagged around tree trunks of Tectona grandis. By employing these precoated threads, termite infestation and tunneling activity were significantly decreased (P < 0.05 and 0.01) (Table 3). Flowever, P-values obtained in experiments have shown successful random control of termites in the groups. (P0.05 = 2.895, Po.oi = 4.455); F is significant for X value, while, for Y values, it is nonsignificant and Fxy = 5.38. It was also tried to adjust the values by computation for adjustment of SS for Y that shows the termite killing was significant {df = 31, fo.os = 2.04, fo.oi = 2.75) (Table 3). There was observed a significant decrease in mud plastering after regular spray on the infested trees as it was found, and no further termite infestation was observed even after 6 months of experiment. Besides this, C. decidua extracts were also used in wood seasoning for the protection of wood from termite infes- tation. C. decidua fractions have shown good termiticidal action as almost no infestation was observed in test wood sticks up to 6 months. The percent weight loss obtained was also minimized up to 3.25%, while, in untreated sticks 52.82% weight was lost (Tabled). Infestation was found to 6 Psyche Table 3: Termite management after employment of tag binding and spray on infested garden plants. Number of termites % infestation % inhibition in tunneling activity Treatment Mean + SE Mean + SE Mean -I- SE Before treatment After treatment Before treatment After treatment Before treatment After treatment Spray 21.50 ± 0.021 15.5*± 0.017 76.75 ± 0.14 19.0* ± 0.03 55.4 ± 0.01 21.73* ± 0.04 (100) (41.89) (100) (19.8) (100) (28.17) Tag binding 19.4 ± 0.019 10.75* ± 0.09 63.2 ± 0.07 11.25* ± 0.02 38.6 ± 0.05 13.75* ± 0.01 (100) (35.65) (100) (15.11) (100) (26.26) Spray and tag 20.6 ± 0.03 3.375* ± 0.004 67.8* ± 0.07 8.97* ± 0.01 37.2 ± 0.06 9.2* ± 0.08 binding (100) (14.07) (100) (11.68) (100) (19.82) Observations were made at every 15-day time interval. * Significant at P < 0.01 levels. Table 4: Effect of C. decidua stem extract on weight loss and infestation in seasoned wood sticks planted in garden soil. Eractions 0 month 1 month Time duration 2 month 3 month 4 month 5 month 6 month Control (-ve) 543 ± 2.09 500.1 ± 2.74 (7.90)* 469.3 ± 1.60 (13.5)* 449 ± 2.50 (17.31)* 368.5 ± 2.66 (32.13)* 286.5 ± 1.72 (47.23)* 256.16 ± 1.75 (52.8)* 0.00 (0.00) 79.66 ± 1.37 (100)+ 113.83 ± 1.33 (142.89)+ 122.6 ± 1.55 (153.93)+ 135.33 ± 1.43 (169.88)+ 152.66 ± 1.22 (191.63)+ 192.16 ± 1.03 (241.22)+ Control (-l-ve) 532 ± 2.01 524.8 ± 1.81 (1.35)* 516.8 ± 1.63 (2.85)* 497.3 ± 1.74 (6.52)* 493.5 ± 1.12 (7.2)* 469 ± 1.74 (11.84)* 424.16 ± 2.68 (20.27)* 0.00 (0.00) 9.3 ± 0.97 (100)+ 13.8 ± 1.03 (148.38)+ 21.83 ± 1.25 (134.73)+ 24 ± 1.05 (158.06)+ 25.66 ± 0.97 (175.91)+ 31.16 ± 1.20 (235.05)+ Capparis T1 533 ± 1.51 527.5 ± 0.97 (1.03)* 485.3 ± 1.43 (8.9)* 472.6 ± 1.40 (11.33)* 467 ± 1.49 (12.38)* 461.1 ± 1.05 (13.48)* 447.5 ± 1.65 (16.04)* 0.00 (0.00) 0.00 (0.00)+ 0.00 (0.00)+ 0.00 (0.00)+ 9.3 ± 0.86 (7.2)+ 11.33 ± 0.86 (8.01)+ 16.3 ± 0.86 (9.2)+ Capparis T2 532.6 ± 1.46 523.3 ± 1.26 (1.74)* 519.8 ± 1.70 (2.29)* 513 ± 1.41 (3.68)* 509.5 ± 1.81 (4.33)* 513.8 ± 1.70 (3.52)* 510 ± 1.13 (4.24)* 0.00 (0.00) 0.00 (0.00)+ 0.00 (0.00)+ 0.00 (0.00)+ 5.8 ± 0.82 (4.47)+ 8.0 ± 0.903 (5.53)+ 8.66 ± 0.97 (4.71)+ Capparis T3 553.5 ± 1.48 551.6 ± 1.05 (0.003)* 545.6 ± 1.43 (1.42)* 542.8 ± 1.82 (1.93)* 539.5 ± 3.28 (2.52)* 537.1 ± 2.62 (2.96)* 535.5 ± 0.97 (3.25)* 0.00 (0.00) 0.00 (0.00)+ 0.00 (0.00)+ 0.00 (0.00)+ 0.00 (0.00)+ 0.00 (0.00)+ 0.00 (0.00)+ * Values in bracket depict percent weight loss represented in grams. ■•■Values in brackets depict percent weight loss and percent termite infestation. % Wt loss is mean of weight loss obtained in six wood sticks planted in soil after seasoning. It is represented in grams. % infestation represents damage caused by the termites in six wood sticks. It is also mean of number of termites available on six wood sticks. be decreased with increasing concentration of C decidua. Statistical analysis of infested and uninfested data have shown significant correlation between tests and control, as the values of correlation were found positive (0.5416) in the weight loss and infestation in comparison to tests. Test wood sticks have shown significantly very low termite infestation after wood seasoning (P < 0.05 and 0.01). 6. Discussion In present time, termite menace is a serious problem in tropical and subtropical regions. Indian white termite is a dreadful insect pest which causes economic damage to commercial wood, fibers, paper sheet, clothes, woolens, and mats and seriously infests agricultural crops and forest products. In the present study, we have tried to control termite infestation in garden soil by applying nonchemical plant-based extracts and pure compounds isolated from C. decidua. Our results show that C. decidua solvent extracts and pure compounds possess enough antitermite potential. By applying very small dose of these natural products orientation, movement, feeding, and tunneling behavior in termites were found to be significantly suppressed. Further, infestation was to be significantly decreased in seasoned wood sticks even after six months of treatments. In toxicity bioassays, C. decidua solvent extracts have shown very high lethality which is proved by very low LD 50 value, that is. Psyche 7 14.171-15.274 /Ug/mg obtained. Further, addition of coconut and terpene oil, glycerol, and sulphur in C decidua have shown synergistic activity. In such combinations, LD50 was found in a range of 4.973-13.246 /ig/mg (Table 1). On the other hand, LD50 in pure compounds isolated from C. decidua obtained was in a range of 5.537-10.083 |Wg/mg (Table 1). Among pure compounds, CDFl identified as 6- (1 -hydroxy- non-3-enyl)-tetrahydropyran-2-one has shown very high antitermite potential against O. obesus with an LD50 value of 8.086 fig/mg (Table 1). In addition to it, pure compounds isolated from C. decidua (CDSl, CDS2, CDS3, CDS4, CDS5, CDS6, CDS8, and CDFl) have shown significant {P < 0.05) repellent activity at a very low dose with an EC50 ranging between 0.008 and 0.017 /Wg/gm (Table 2). Repellency provided extra advantage to wood seasoning which significantly decreases the infestation and damage done by O. obesus in the field. Similar toxic and repellent activity of plant products have been reported by Blaske and Hertel [19]. Besides this, Chamaecyparis nootkatensis. Sequoia sempervirens, and Pseu- dotsuga menziesii show high antifeedant and toxic activities against termites due to presence of potential antitermitic ingredients [20]. Further, treatment of infested saplings by both spray and tag binding has significantly reduced the number of termites (14.0%), % infestation (11.68), and tun- neling activity (19.825) in garden saplings (Table 3). Further, active ingredients of C. decidua repelled large number of termites in seasoned wood sticks that were planted in three- feet deep small pits made in garden soil. It has protected the wood weight loss up to 3.25%, and no infestation was observed even after 6 months of digging (Table 4). Similarly, Aleurites fordii have shown anti-termite potential (Tung tree) extracts against Reticulitermes flavipes at 0.1 to 5.0% w/w [21]. 2'acetonaphthone also obstructed tunneling and feed- ing behavior in Formosan subterranean termite Coptotermes formosanus Shiraki at 8.33 mg/kg concentration [22] . Besides this, natural amides such as nootkatone [23], valencenoid derivatives [24], and imidacloprid [25] also deter feeding in termites and suppress adult survival [26]. Similarly, larch wood flavonoids [27] and stilbene-rich compounds isolated from bark of Picea glehnii such as piceid (3,4,5 tri- hydroxystilbene glucoside), isorhapontin (3-methoxy-3,4,5 trihydroxystilbene-3-d-glucoside), and astringin (3, 3,4,5- tetrahydroxystilbene-3-d-glucoside) also deter termites at a very low concentration 0.63 to 2.5 /^mol/disc [28]. Similarly, limonoids from meliaceae and rutaceae family showed strong antifeedant activity in Reticulitermes speratus Kolbe in no- choice bioassay. Both obacunone and nomilin showed a drastic antifeedant effect at 510 and 1360 ppm concentra- tion [29]. Isorhapontin exhibited better antioxidant poten- tial than toxifolin (3, 3,4,5, 7 pentahydroxyflavone) in no- choice tests [28]. Amides isolated from piperaceae family {Piper nigrum) have shown high-insecticidal activity against Coptotermes formosanus Shiraki [30]. It is a biodegrad- able environmental friendly natural product with minimal mammalian toxicity [30]. Similarly, in a filter paper-based bioassay for termiticide, guineesine, a minor constituent isolated from Piper nigrum, has shown >90% mortality in termites at 1% wt/wt application. Moreover, root extracts of Diospyros sylvatica impose significant repellent activity and cause high mortality in subterranean termite, Odontotermes obesus in filter paper disc bioassay due to presence of plumbagin, isodiospyrin, and microphyllone or quinnones [31]. Similarly, diterpene acids are screened as good antifeedants [32], while pine resin and its derivatives, cis/trans-deisopropyl dehydroabi- etanol, showed promising performance against termite [32]. Similarly leaf extracts of Polygonum hydropiper (L) and Pogostemon paviflorus (Benth) have shown high toxicity and mortality in tea termite Odontotermes assamensis (Holm) [33]. Similarly, both heartwood and sapwood of Taiwania cryptomerioides were found effective against C. formosanus at lOmg/g concentration [34], while solvent and aqueous extracts of Gloriosa superba [35], Paeonia emodi [36], Cory- dalis incise [37], Cassia obtusifolia [38], Artemisia annua [39], Teucrium royleanum [40], Andrachne cordifolia [41], Angelica archangelica and Geranium sylvatica significantly killed cer- tain harmful insects by inhibiting enzyme activity [42]. Besides plant extracts, essential oils have also shown very strong repellent and toxic activity against Formosan subter- ranean termite due to presence of volatile compounds [43]. Moreover, plant origin monoterpenes were proved highly toxic to Coptotermes formosanus [44]. Similarly, essential oils such Calocedrus formosana (Cupressaceae) effectively work against Coptotermes formosanus at very low dose 27.6 mg/g [45] , while maca {Lepidium meyenii) essential oil effectively kills Coptotermes formosanus at 1% (w/w) concentration [46] . Similarly, clove bud oils kill Japanese termite R. speratus at 7.6mL/liter air by fumigation [47]. Vulgarone B isolated from Artemisia douglasiana Besser, apiol isolated from Li- gusticum hultenii (Fern), and cincin isolated from Centau- rea maculosa have shown higher mortalities in Formosan subterranean termites Coptotermes formosanus [30]. Sim- ilarly, patchouli oil and patchouli alcohol have shown high toxicity and repellency against same species [48]. Both have caused tissue destruction inside exoskeleton of the termites due to contact activity [48]. Similarly, vetiver oil, nootkatone, and disodium octaborate tetrahydrate affect termite tunneling, feeding, and wood digestion by symbiont protozoa resides inside the termite gut [30]. Vetiver oil is a confined novel termiticide with reduced environmental impact for use against subterranean termites [23]. Similarly is a similar bioassay 40% CSNL + 1% CuCb and 40% CNSL + 2% CuCb which have shown least damage done by the termites [49] after 10-day exposure. Similarly, boron was established as a termiticide [50] . Sulfonated wattle tannins alone combined with copper chloride at different concentrations and cashew nut shell liquid without or with copper chloride have successfully prevented termite attack [49]. Similarly, wood treated with copper II compounds tri- and dialkylamine-boric acid complex showed lesser termite damage and acted as good preservatives [27]. Nootkatone affect wood consumption termite survival and affects growth of flagellate symbionts [23]. Similarly, in the present study, sulfur, added in C. decidua combinatorial mixture signifi- cantly decreased the termite attack and damage. It may be due to effect of combinatorial mixture on exoskeleton of termites and antimicrobial effect of sulfur on termite gut 8 Psyche microfauna. It is also possible that sulfur inhibits growth of microbes in the soil if used in seasoned woods, or sprayed over tree trunks, it destructs microbial population that may work as an attractant for termites. Because in rainy season after gaining extra humidity due to pouring rain water, tree bark provides substratum for growth of fungi and bacteria. It becomes extra soft due to rain water, fungal and bacterial activity that might be patable for termites and induce mud plastering in termites. As presence of sulfur in natural products such as amide caused higher mortality in termites [51], many commercial termiticides are available in the market to combat the destructive termites but none are entirely natural. The main purpose of present work was to contribute to the development of new termiticide from plant natural resource that may have better activity than synthetic termiticides and might be environmentally more acceptable than any other synthetic pesticide. No doubt, C. decidua possesses enough antitermite po- tential to control Indian white termite, O. obesus population. However, it can be concluded that C decidua active compo- nents can be used for controlling the damage and termite infestation if used as spray, fumigant or in form of poison baits. Hence, strong recommendations are being made to develop ecofriendly antitermite formulation from C. decidua plant for effective control of field termites. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 591570, 7 pages doi:10.1155/2012/591570 Review Article Evolutionary Perspectives on Myrmecophily in Ladybirds Amelie Vantaux,' Olivier Roux,^ Alexandra Magro,^’^ and Jerome OriveF ^ Entomology Laboratory, Zoological Institute, Catholic University of Leuven, Naamsestraat 59, Box 2466, 3000 Leuven, Belgium ^Institut de Recherche pour le Developpement, UMR MiVEGEC-Maladies Infectieuses et Vecteurs Ecologie, Genetique, Evolution et Contrdle, Antenne de Bobo-Dioulasso, 01 BP 171, Bobo Dioulasso 01, Burkina Laso ^CNRS, UMR EDB-Evolution et Diversite Biologique, 118 Route de Narbonne, 31062 Toulouse, Lrance ^ENLA, UMR EDB-Evolution et Diversite Biologique, Universite de Toulouse, 2 Route de Narbonne, 31320 Castanet Tolosan, Trance ^ CNRS, UMR EcoEoG-Ecologie des Eorets de Guyane, Campus Agronomique, BP 316, 97379 Kourou Cedex, Prance Correspondence should be addressed to Amelie Vantaux, amelie.vantaux@gmail.com Received 4 October 2011; Accepted 4 December 2011 Academic Editor: Volker Witte Copyright © 2012 Amelie Vantaux et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Myrmecophiles are species that usually have developed specialized traits to cope with the aggressiveness of ants enabling them to live in their vicinity. Many coccinellid species are predators of Hemiptera; the latter is also often protected by ants. Therefore these ladybirds frequently interact with ants, and some species have become myrmecophilous. In this paper, we aim to provide an overview of the evolution of myrmecophilous traits in ladybirds. We then discuss the costs and benefits of myrmecophily and the dietary shift to myrmecophagy observed in a few species. 1. Introduction Ants represent a highly ecologically successful and most often dominant group of insects. Their predominance in almost all terrestrial ecosystems leads them to interact with many other organisms. One of the best-known examples of such inter- action is their mutualism with Hemiptera. Ants protect the sap-feeding insects, and in return they benefit from the honeydew provided by the Hemiptera [1-3]. Honeydew is rich in carbohydrates and in some amino acids, which are attractive and nutritionally valuable for ants [4-7 ] . Addition- ally, the ants sometimes use aphids as a source of protein by consuming them [8-11]. Aside from protecting the Hemi- ptera, ants may also reduce their risk of getting fungal infec- tions via hygienic behaviors [12-14], reduce indirect com- petition with untended Hemiptera [15], and they can even transport the Hemiptera to suitable feeding sites when a host plant’s quality deteriorates [16]. Nevertheless, the main ben- efit for the Hemiptera when tended by ants is the protection the ants provide from natural enemies [1, 3, 13, 17-21]. Untended colonies experience higher predation and para- sitism rates. The colonies of the aphid Tuberculatus quercicola (Matsumura), for example, had lower survival rates when ants were excluded [17], and the black cherry aphid Myzus cerasi (Fabricius) reached higher densities of individuals on trees with ants than on those without ants [ 18] . As a result of the protection they provide to Hemiptera, ants are in com- petition with predators such as ladybirds and syrphid larvae as well as parasitoids. Ladybird species are well known for their aphido- and coccidophagy, which have popularized them as biocontrol agents in agricultural systems and private gardens. They ex- hibit, however, a large trophic diversity from mycophagous and phytophagous to predatory species. The latter species mainly eat coccids or aphids and, also to some extent, aley- rodes, psyllids, chrysomelids, and mites, although cocci- dophagy is considered more primitive than aphidophagy [22, 23]. The fact that many ladybird species prey on Hemiptera brings them into frequent contact with ants. Some of them use behavioral, physical, and chemical characteristics to cope with these aggressive competitors. Species found living regu- larly or only with ants are called “myrmecophiles” (from the Greek words for ants, “myrmex”, and loving, “philos”). Their interactions span from facultative and diffuse relationships. 2 Psyche which in ladybirds stems from their general defensive traits, to more obligate and integrated interactions which rely on specific adaptations. In this paper, we aim to provide an over- view of the evolution of myrmecophilous traits in ladybirds [22-24]. We then discuss the costs and benefits of such spe- cialization on ant-tended Hemiptera and the dietary shift to myrmecophagy demonstrated in a few species. 2. Diversity and Biology of Myrmecophilous Species The Coccinellidae family consists of seven subfamilies [25] among which five include myrmecophilous species: Scymni- nae, Ortaliinae, Chilocorinae, Coccinellinae, and Cocciduli- nae [26-31] (Table 1). Interestingly, only one myrmecophi- lous species has been recorded to date in each of the three subfamilies Ortaliinae, Chilocorinae, and Coccinellinae and two in the subfamily Coccidulinae whereas there are eight species from the Scymninae subfamily belonging to four dif- ferent tribes: one species from the Diomini tribe, two species from the Hyperaspidini and Brachiacanthadini tribes, and three species from the Scymnini tribe. Therefore, myrme- cophilous species seem to be more strongly represented at the base of the phylogenetic tree [22-24] . Some myrmecophilous species have evolved more obligate relationships with ants and use chemical mimicry (i.e., the passive or active acqui- sition of a chemical signature by the myrmecophile allowing acceptance by the host) to evade ant aggressiveness. This adaptive trait has appeared in two different subfamilies with mimicry of the ant brood in the Scymninae [28, 32] and mimicry of the aphid prey in the Chilocorinae [33]. All of these characteristics and their distribution in the phylogeny show that myrmecophily appeared independently several times during the evolution of ladybirds [22-24], even in the case of the dietary shift to myrmecophagy which appeared both in the Scymninae [27, 28] and the Ortaliinae [34]. 2.1. General Defensive Traits 2.1.1. Physical Traits. Many ladybird species, though not strictly myrmecophilous, encounter ants and show variation in their sensitivity to ant aggressiveness or to their venom [35]. Adults can, for example, hold the body tightly pressed against the plant surface when attacked by ants. Some species can completely conceal their legs under the body when cow- ering, such as individuals from the Chilocorinae subfamily, so that ants cannot seize any appendages which usually caus- es ant aggression to quickly cease [33, 35]. Moreover, lady- birds use reflex bleeding as a general defense mechanism against their natural enemies. It has a mechanical protective effect since, as the haemolymph coagulates, it becomes more viscous and sticky, impeding the ants’ movements [36]. Adult ladybirds are rather well protected by their sclero- tized elytrae, while their larvae and pupae have soft bodies that are more sensitive to ant bites. The pupae are often pro- tected by the larval skin shielding them and sometimes by their ability to use reflex bleeding [36] or by a dense covering of hair [33]. In some species, the pupae are also able to move up and down in response to a tactile stimulus [37], which could deter ant attacks. Many ladybird larvae have a waxy covering protecting them from their natural enemies, includ- ing ants [38]. When ants try to attack the larvae, their man- dibles become covered by the covering’s sticky filaments, and this usually causes them to stop and start grooming them- selves. The two myrmecophilous species, Scymnus nigrinus (Kugelann) and S. interruptus (Goeze), are able to prey on ant-tended aphid colonies and to better survive predator attacks thanks to their waxy covering [30]. Myrmecophilous Brachiacantha quadripunctata (Melsheimer) and B. ursina (Fabricius) even get inside the ants’ nest where they feed on ant-tended coccids and adelgids (Flemiptera, Aldegidae) [39, 40] . The waxy filaments also allow the myrmecophilous lady- bird Azya orbigera (Mulsant) to feed on coccids tended by Azyeca instabilis (E Smith) [29], and the larvae of the lady- bird Ortalia pollens (Mulsant) to feed on Pheidole punctulata (Mayr) workers [34]. However, this waxy coating does not always provide an efficient protection as Pheidole megacepha- la (Fabricius) ants prey on Cryptolaemus montrouzieri (Mul- sant) and A. orbigera ladybird larvae since they are able to remove their protective coating [19]. Some ladybird larvae that are devoid of a waxy covering, such as Diomus thoracicus (Fabricius) [28], Platynaspis lute- orubra (Goeze) [33], and Scymnodcs FH/ws (Blackburn) [27], show a convergent adaptation to ants in their general mor- phology: they are all ovate and flat with expanded marginal setae and short, stout legs. This body shape, with few exposed extremities, could be considered a protective type as has been found in other myrmecophilous Coleoptera [41]. 2.1.2. Behavioural Traits. In myrmecophilous species, the larvae tend to move slowly and inconspicuously, as has been observed for Coccinella magnifica (Redtenbacher), P. lute- orubra, and B. quadripunctata [33, 40, 42]. The comparison between the myrmecophilous species C. magnifica and its close nonmyrmecophilous relative C. septempunctata (Lin- naeus) has shown that C. magnifica uses physical, behavioral, and chemical defenses adapted from the general defenses observed in Coccinellidae [42, 43]. No novel behavior or de- velopment of specific traits have been observed in C. mag- nifica [43]. One way for C. magnifica larvae to limit ant ag- gressiveness is to minimize the time spent on an aphid colony and thus the chance of encountering ants; for example, the larvae frequently pick up and carry their prey away from the colony before consuming it [42]. The fact that the nonmyrmecophilous C. septempunctata is also sometimes observed near ant-tended aphid colonies, especially at the end of the colonies’ cycle when aphid colonies become scarce, and that reciprocally C. magnifica has been observed preying on untended colonies, suggests that the scarcity of prey may have been a selective pressure in the evolution of myrme- cophily [43, 44]. Indeed, during prey shortages, the limited availability of untended colonies might have forced ladybirds to prey on ant-tended colonies, opening the path to devel- oping a tolerance towards ant aggressiveness. However, the myrmecophily of C. magnifica ladybirds has not been observed throughout its European habitat, suggesting that Psyche 3 Table 1: Taxonomy and some biological characteristics of myrmecophilous ladybirds. Facultatively myrmecophilous species and species for which no reliable information is available are not included. Taxon Ant associate Larval diet Myrmecophilous traits References Chilocorinae Platynaspis luteorubra Lasius niger, Myrmica rugulosa, Tetramorium caepsicum Ant-tended aphids Behavior, prey odor mimicry, flat body, marginal setae, short and stout legs 133] Coccinellinae Coccinella magnifica Formica rufa Ant-tended aphids Behavior, chemical deterrent [42, 43] Coccidulinae Azya orbigera Azteca instabilis Ant-tended coccids Waxy covering |29] Bucolus fourneti Unknown Ant workers Waxy covering [31] Ortalinae Ortalia pallens Scymninae Pheidole punctulata Ant workers Waxy covering [34] Brachiacantha quadripunctata Lasius umbratus, Formica subpolita Ant-tended aphids and adelgids Behavior, and Waxy covering [40] Brachiacantha ursina Lasius sp. Ant-tended aphids and adelgids Waxy covering 139] Hyperaspis reppensis Tapinoma nigerrimum Apparently ant-tended fulgorids Body oval, and waxy covering [60] Scymnodes bellus Iridomyrmex sp. Ant workers Flat body, marginal setae, short and stout legs |27] Scymnus interruptus Lasius niger Ant-tended aphids Waxy covering 130] Scymnus nigrinus Formica polyctena Ant-tended aphids Waxy covering 130] Prey odor mimicry, flat body. Diomus thoracicus Wasmannia auropunctata Ant brood marginal setae, short and stout |28] legs Thalassa saginata Dolichoderus bidens Unknown Ant mimicry 132] myrmecophily might be facultative or limited to some popu- lations [45]. Concerning oviposition, two strategies have been observed. The eggs can be laid close to untended aphid colonies on which the emerging larvae can feed such as in C. magnifica [42], or females may try to oviposit directly in the Hemiptera colony despite possible ant aggressiveness. In the case of A. orhigera, females oviposit in the coccid colonies and lay the eggs under scale exuvia or carcasses to protect them from predation [46]. 2.1.3. Chemical Traits. In addition to its mechanical imped- iment of ant movement, the haemolymph released during a reflex bleeding event often has a repellent effect due to the presence of alkaloids [47]. The alkaloids are synthesized by the ladybirds and seem to originate from fatty acids, as has been shown for the biosynthesis of coccinelline in C. septempunctata fat bodies [48]. Furthermore, their presence in eggs also provides them with a chemical protection that deters predators [47]. They could also act as an ant repellent but this remains to be demonstrated. The extremely repellent effect of the myrmecophilous ladybird C. magnifica has been suggested, but it has not been demonstrated yet [42, 43]. Finally the waxy coating might also possess chemical prop- erties helping to attenuate ant aggressiveness as it does for Scymnus louisianae (Chapin) [49]. 2.2. Chemical Adaptation. Some myrmecophilous species employ a chemical strategy using a specific chemical signa- ture on the cuticle [28, 32, 33]. They rely on a specific cutic- ular profile which can be obtained through passive or active acquisition [50] and results in the chemical mimicry of its prey odour, such as in P. luteorubra [33], or of the ants brood in Thalassa saginata (Mulsant) [32] and D. thoracicus [28]. Thus, chemical mimicry helps some ladybirds to decrease ant aggressiveness, as in the case of P. luteorubra larvae, and even to disguise themselves as nestmates as in the case of T. saginata and D. thoracicus. Interestingly, chemical mimicry probably results from an adaptation as opposed to a preex- isting trait, and it has only been observed in larvae thus far. Indeed, we would expect a preexisting trait to be observed in adults too, as it would help them to get the same benefits as their larvae, in particular avoiding ant aggressiveness at emergence. This adaptation might not be necessary in adults since they are protected by their hard elytrae and can readily fly away from the aphid colony in the case of P. luteorubra or even occupy a different niche as in the two nest- integrated ladybird larvae. 3. Why Specialize on Ant-Tended Hemipteran Colonies? Since myrmecophilous interactions vary from facultative to obligate, the extent of the associated costs and benefits varies 4 Psyche accordingly with the most integrated species having the high- est costs but also the highest benefits. 3. 1 . Benefits. The first and most obvious benefit of being able to prey on ant-tended Hemiptera is gaining access to better food sources [19, 20, 26, 44]. Indeed, ant-tended hemipteran colonies are usually larger, have a longer lifespan, and thus persist longer in late summer in temperate regions than untended colonies. For R luteorubra, better foraging success was measured in ant-tended colonies and resulted in a high- er adult weight, which is likely to positively influence adult fitness and survival [33]. This better foraging success has been explained by a decrease in the defensive behavior of aphids and a shorter searching distance in ant-tended colo- nies [33]. A second and important benefit of myrmecophily may arise from the access it provides to an enemy- free space. Ants limit interspecific and intraguild competition as well as access to the parasitoids and predators of ladybirds [29, 30, 33, 51, 52]. The competitors of S. interruptus and S. nigrinus, for example, have been noted as being less present on ant- tended colonies [30]. Moreover, the presence of ants reduced larval parasitism by Homalotylus platynaspidis (Hoffer) (Hymenoptera, Chalcidoidea) in P. luteoruhra [33], and the aggressive behaviour of A. instabilis disturbed the oviposition behavior of H. shuvakhinae (Trjapitzin), the most common parasitoid of A. orbigera [29]. Nonetheless, the observed decreases in parasitism rates do not always directly result from ant protection. C. magnifica larvae are less parasitized by the parasitoid Dinocampus coccinellae (Schrank) than the larvae of its close nonmyrmecophilous relative C. septem- punctata found in the same area. Laboratory studies have shown that this is linked to the unsuccessful parasitism of C. magnifica and not to the presence of ants [53] . Ant parasitoids might also influence the interaction between ants and ladybirds. Phorid flies affect ant worker behavior by decreasing their activity [54-57] . During periods of low ant activity induced by the disturbance generated by phorid flies, A. orbigera adults can prey on the coccids at the same rate as in untended colonies and oviposit in the colony [46]. 3.2. Costs. As discussed above, the specialization in preying on ant-tended Hemiptera depends mostly on the ability of ladybirds at all stages to be protected from ants. Ants aggres- sively protect the Hemiptera colonies, which disturbs lady- bird foraging and can cause them to leave and stop exploiting a patch, and most importantly they can be injured or killed. Another possible cost is that adaptation to ants would render the respective coccinellid species difficult to live with- out them. Myrmecophilous ladybirds could be poor com- petitors or poorly defended against predators and parasites as suggested by Majerus et al. [26] . The production of chem- ical defenses might be at the cost of other traits such as immunity or strong defenses against predators or parasites [26]. Furthermore, the association with ants is likely to de- crease the habitat range available for the ladybird, especially for the most specific parasites specialized on one ant species. Consequently, any reduction in the host habitat or abun- dance would directly affect the ladybirds’ fitness and survival. 4. Dietary Shift to Myrmecophagy Only four ladybird species larvae are currently known to feed on ants: Bucolus fourneti (Mulsant) [31], O. pallens that eats P. punctulata ants [34], S. bellus feeding on Iridomyrmex sp. [27], and D. thoracicus feeding on Wasmannia auropunctata (Roger) [28] . The first three species feed on ant workers out- side the ant nest, relying on ant’s foraging habits to get close to them. Thus, these species can stay relatively immobile and wait for prey to approach. In the case of D. thoracicus^ the larvae are parasites that live inside the ant nest. The larvae are usually found in or near the brood pile where they have access to a constant food source. The integration of this species into the ant colonies relies on the chemical mimicry of its cutic- ular profile with the one of the ants’ brood [28]. Only the adults leave the colonies early after emergence to avoid being attacked, as they do not share the same cuticular profile as the ants. Such a dietary shift to myrmecophagy provides several important benefits. First, the ladybirds gain access to a food source available all year round and for many years since ant colonies are usually long-lived. Second, in the case of D. tho- racicus, the larvae might be better protected from predators, parasitoids, and competitors lacking the adaptations needed to enter the ant nest. They may also benefit from a rather homeostatic environment in which temperature and humid- ity are rather constant and individuals are protected from climatic events. The shift by ladybirds to myrmecophagy most probably followed the development of myrmecophily and as such bears the same costs, such as a more restricted niche due to specialization on ants. Nevertheless, these costs are largely balanced out by the access to a constant food resource both in time and quantity. This removes the constraints of re- source limitation which are important in the evolution of habitat preferences and diet in predatory ladybirds [58]. Hemipteran colonies, and especially aphids, are a transient resource, and even if adult ladybirds can track them down by moving between patches, ladybird larvae are less mobile and limited to the colonies surrounding them. Therefore, the females of aphidophagous species tend to lay eggs early in the development of an aphid colony, known as the “egg window” [59] , to ensure that their larvae have sufficient food. By feeding on ants, such limitation does not occur, which probably compensates for the costs of a myrmecophilous life style. 5. Perspectives Despite being a highly species-rich group with around 6000 species described, the biology of most ladybird species, espe- cially those found in the tropics, remains largely or even completely unknown. Only a few myrmecophilous species have been identified to date, but because most coccinellids encounter ants very frequently and often supplement their Psyche 5 essential food sources with other food items (which might help in their being able to shift to new diet and habitat), it would not be surprising that many more myrmecophilous species still remain to be discovered. Among the seven sub- families of Coccinellidae, myrmecophilous species have been identified in only five of them with, moreover, most of them concentrated in the Scymninae subfamily [26-30]. The predominance of myrmecophily in the Scymninae raises the question of the evolution of traits that promoted such inter- actions with ants. Gaining more knowledge on the biology of myrmecophilous ladybirds, especially the ones having shifted to myrmecophagy, would provide insights on the ev- olution of myrmecophily and myrmecophagy in this family. The ladybird diet is usually similar in larvae and adults; nonetheless, ants attack adult D. thoracicus suggesting that they rely on a different food source than do the larvae. The biologies of adult O. pollens and S. hellos ladybirds are un- known as well. Unraveling the diet of all stages in these three species would shed light on the origin of the dietary shift to myrmecophagy. The adults might be adapted to preying on ant-tended Hemiptera colonies which would have favored a dietary shift in the larvae. It has also been hypothesized that the limited availability of prey at some point in time might have been a selective pressure in the evolution of myrme- cophily in the case of C. magnifica [43, 44]. A similar con- straint could apply to the tropical and subtropical myrme- cophagous species with the advantage of a dietary shift to ants associated with a food source available all year round since there is no dormancy period. In temperate areas, lady- birds overwinter at the adult stage. Thus, one can hypothesize that the lifecycles of ladybirds and ants might not be synchro- nized enough to have permitted myrmecophagy to appear in these regions. Finally, another hypothesis might be that since both ladybird species from subfamilies rooted at the base of the phylogenetic tree and coccids are more abundant and diversified in the Southern hemisphere, there might be more opportunities for myrmecophily to arise in these areas, both from a larger number of possible interactions and a longer common evolutionary history. Financial Support Financial support was provided by the French Ministere de VEcologie et do Developpement Durable — Programme Eco- systemes Tropicaux and the Programme Convergence 2007- 2013, Region Guyane from the European Community. Acknowledgments The authors would like to thank Andrea Yockey-Dejean for proofreading the paper. They are grateful to J. P. Lachaud for his invitation to contribute to this paper, to two reviewers for their useful comments on the paper, and to the Lahoratoire Environnement de Petit Saut (HYDRECO) for logistical help during their fieldwork on Diomus thoracicus. References [1] M. J. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 170483, 7 pages doi:10.1155/2012/170483 Research Article Effect of Habitat Type on Parasitism of Ectatomma ruidum by Eucharitid Wasps Aymer Andres Vasquez-Ordonez,'’^ Inge Armbrecht,^ and Gabriela Perez-Lachaud^ ^ Departamento deBiologia, Universidad del Valle, Calle 13 No. 100-00 Cali, Valle, Colombia ^Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Apartado Aereo 7495, Bogota, Colombia ^El Colegio de la Frontera Sur, Entomologia Tropical, Avenida Centenario km 5.5, 77014 Chetumal, QROO, Mexico Correspondence should be addressed to Gabriela Perez- Lachaud, igperez@ecosur.mx Received 26 August 2011; Accepted 23 October 2011 Academic Editor: Volker Witte Copyright © 2012 Aymer Andres Vasquez-Ordonez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Eucharitidae are parasitoids that use immature stages of ants for their development. Kapala Cameron is the genus most frequently collected in the Neotropics, but little is known about the biology and behavior of any of the species of this genus. We aimed to evaluate the effect of habitat type on eucharitid parasitism and to contribute to the knowledge of the host-parasite relationship between Kapala sp. and the poneromorph ant Ectatomma ruidum (Roger) in Colombia. Twenty £. ruidum colonies were extracted from two different habitat types (woodland and grassland), and larvae and cocoons (pupae) were examined in search for parasitoids in different stages of development. Globally, 60% of the colonies were parasitized, with 1.3% of larvae and 4% of pupae parasitized. Planidia (first-instar larvae), pupae, and adults of the parasitoid were observed. All of the pupae and adult parasitoids belonged to Kapala iridicolor Cameron. All the colonies collected in the woodlands were parasitized and contained more parasitized larvae (2%) and parasitized cocoons (8%) than those collected in grasslands (4/12 parasitized colonies, 0.5% parasitized larvae, 0.8% parasitized cocoons). The relationship observed between habitat type and parasitism prevalence is a novel aspect of the study of eucharitid impact on ant host populations. 1. Introduction Several dipteran, strepsipteran, and hymenopteran para- sitoids are natural enemies of ants [1-9]. Among the hymenopterans, the Eucharitidae sensu stricto is the only monophyletic group, at the family level, where all of its members are parasitoids of ants. They are also one of the largest and most diverse groups attacking social insects [8]. Eucharitidae have a specialized life cycle that includes oviposition away from the host, on or into a host-plant [2]. Although there are more than 400 species of Eucharitidae already described [8], the hosts and host-plants of only a few species are known [10], and knowledge on the life history and ecology of these wasps is even scarcer. In the New World, detailed studies on selected species have only been carried out in a few localities in Mexico, Argentina and North America (e.g., [1, 11-17]). Eor Colombia, there is no detailed report on the biology of any species of this family. The impact of eucharitids on their host populations has recently been explored in detail for some Mexican and South American ant populations [12, 17-19]. These, and earlier reports (e.g,, [2, 11, 20]), signaled the aggregated nature of eucharitid populations. In fact, prevalence of parasitism by eucharitids varies greatly in time and space [2], with 100% of colonies parasitized at some sites, and other colonies escaping from parasitism (e.g,, [18, 19]). Differences in local parasitism, in general, can be attributable to several different factors such as the presence of resources, other than hosts, necessary for maintaining high parasitoid populations locally (e.g., floral and extrafloral nectar, and refuge sites for adults), suitable host-plants, microclimatic differences, and/or dispersal capacity of adult parasitoids [21, 22]. In some cases, for example, parasitoids may be less effective at parasitizing hosts in sites with simpler vegetation [23] . In the case of eucharitids, an aspect not yet studied in detail is the effect of the habitat on the impact of these parasitoids on 2 Psyche their ant-host populations, though preliminary results of a recent study suggest that differences in management in coffee agroecosystems (i.e., shade, pruning, weed management) might affect parasitism by eucharitids [24] . Ectatomma ruidum (Roger) (Hymenoptera: Formicidae: Ectatomminae) is a diurnal, earth-dwelling. Neotropical ant that nests in the soil. This ant is found from southern Mexico to Brazil, from sea level to an altitude of 1500-1600 m [25- 27], and is dominant in several ecosystems such as forests [28], or economically important cultivated areas [29, 30]. Two species of Kapala (Eucharitidae) have been reported to parasitize this ant in Mexico [14, 31], and parasitism of E. ruidum by Kapala sp. is also known from Colombia (C. Santamaria and J. Herrera, unpub. data). The purpose of this study is to report observations of the host-parasite relationship between Kapala sp. and E. ruidum in Colombia and to compare the impact of this eucharitid on its ant host population in two different habitat types. 2. Materials and Methods This study was carried out on the grounds of the Melendez Campus at the Universidad del Valle (3° 22' N, 76° 32' W), located at the south of the city of Cali, Department of Valle del Cauca, Colombia. The Campus has an area of approximately 100 ha, 8 ha of which are occupied by buildings, 44 ha by woodlands, 46 ha by grasslands, and 1 ha by two ponds. The average elevation is 970 m; mean annual temperature is 24.1°C and average relative humidity 73% [32]. Average annual rainfall is around 1500 mm, with two rainfall peaks, from March to May and from September to November (Instituto de Hidrobiologia, Meteorologia y Estudios Ambientales-IDEAM, unpublished data, cited by [32] ). According to the Holdridge system, the study site is located in an area classified as Tropical Dry Forest (bs-T) [33] . Five sites on the campus were examined: 2 sites in grasslands dominated by Poaceae and other creeping plants and with no trees, and 3 sites located in woodlands. The sites in the latter habitat had a lesser amount of Poaceae among the creeping vegetation and had, in some cases, abundant litter. Common tree species in these sites were Pithecellobium duke (Roxb.) Benth., Samanea saman (Jacq.) Merr., and Calliandra pittieri Standi. (Fabaceae); Mangifera indica L. (Anacardiaceae); Ceiba pentandra (L.) Gaertn. (Bombacaceae); Eicus elastica Roxb. (Moraceae); and Tabebuia chrysantha G. Nicholson, T. rosea (Bertol.) A. DC., and Spathodea campanulata P. Beauv. (Bignoniaceae) [32]. In each of the 5 sites chosen, we determined the number of E. ruidum nests in a plot of 8 X 8 m. One additional plot, placed 50 m from the closest grassland plot and comparable to the others, was censused for nest density evaluation in the grassland area, to get an even sample size. During April, May, and November 2009, 20 nests chosen at random were excavated (8 in woodlands and 12 in grasslands) and transported to the laboratory for examination. Ant larvae were inspected for planidia (eucharitid first- instar larvae) attached to their cuticle by means of a stereoscopic-microscope (Nikon SZ645). Cocoons were kept in petri dishes at room conditions for 5 days or more and were examined once daily to record emergence of adult eucharitids. At the end of the observation period, all of the cocoons were dissected to look for adults and pupae of dead, or not yet emerged, parasitoids, and to register the caste and sex of ants attacked by the parasitoids. Adult wasps were individually placed in vials covered with cloth mesh, and their survival time was evaluated. No food or water was provided. Pupae and adult eucharitids were identified with available keys [8, 34, 35], and their sex was determined, when possible, based on the dimorphism present in the antennae [8]. The material collected was measured using a stereomicroscope equipped with an ocular micrometer and preserved in 96% alcohol. Voucher specimens of both the ants and the parasitoids have been deposited in the Grupo de Investigacidn en Ecologia de Agroecosistemas y Habitats Naturales (GEAHNA) collection, at the Museo de Entomologia of the Universidad del Valle, Colombia (MEUV), and at the Arthropod Collection of El Colegio de la Erontera Sur, Unidad Chetumal, Mexico (ECO-CH-AR). A Eisher’s exact test was carried out to establish whether there were significant differences between the proportions of parasitized colonies found in woodlands and in grasslands, and Z tests were used to search for differences in the number of parasitized larvae and parasitized pupae according to habitat. Nest density and colony size according to habitat (woodlands or grasslands), and colony size according to the presence or absence of parasitoids (both habitats), were com- pared using a Mann-Whitney test. Spearman nonparametric correlation was used to explore the relationship between the size of the colony (adults + brood) and total parasitized brood, between the number of larvae per colony and total parasitized larvae, and between the number of cocoons per colony and total parasitized cocoons. All statistics were cal- culated using STATISTICA 8.0 (StatSoft, Inc.) and R 2.13.1 (The Eoundation for Statistical Computing) programs. 3. Results Of the 20 E. ruidum colonies examined, 12 (60%) were parasitized (Table 1). The global rate of parasitism in the study area was 2.3% (parasitized brood per total ant brood, 27/1162), with 1.3% (9/714) of the larvae and 4.0% (18/448) of the pupae parasitized. In total, 29 eucharitid individuals or their remains were observed, with 2.4 ± 2.6 (mean ± standard deviation; n = 12 colonies; range: 1-10) parasitoids per parasitized colony. Parasitoids in 3 different stages of development were found: planidia in 7 colonies (1.6 ± 0.8 parasitized larvae per parasitized colony; range: 1-3), pupae in 3 colonies (3.3 ± 3.2 individuals; range: 1-7), and adults in 5 colonies (1.6 ± 0.9 individuals; range: 1-3). Pupae and adults were identified as belonging to Kapala iridicolor (Cameron). All of the colonies collected in the woodlands were parasitized {n = 8), while in the grasslands only 33.3% (4/12) contained eucharitids (Table 1). Prevalence of parasitism and type of habitat were not independent (Eisher’s two- tailed exact test: P = 0.0047), and there was a greater frequency of parasitized nests in the woodlands than in Psyche 3 Table 1: Composition of Ectatomma ruidum colonies in two different habitat types, percent parasitized brood, and number and stage of development of Kapala iridicolor individuals. Ectatomma ruidum Kapala iridicolor Nest Habitat Number Queen Number Gynes Males Workers Larvae Pupae Total Parasitized larvae (%) Parasitized Planidia/ pupae (%) scar Pupae Adults 1 Woodland 0 0 5 34 17 23 79 2 (11.8) 1 (4.4) 3 0 l( c/, o c o Oh O Oh Number of days Control Aspergillus ochraceus (10^ conidia/mL) A. ochraceus (10^ conidia/mL) A. ochraceus (10^ conidia/mL) A. ochraceus (10® conidia/mL) A. ochraceus (10^ conidia/mL) Figure 2: Cumulative time-related survival proportion (Kaplan- Meier curves) of the Atta bisphaerica worker ants after being treated with different suspension concentrations of the Aspergillus ochraceus fungus. performed with fungi widely used within the microbial control of insects, such as Metarhizium anisopliae (Metsch.) and Beauveria bassiana (Bals.). They have proved to be highly virulent in the laboratory, although field tests do not reproduce the same results [20-22]. In our study, M. anisopliae was not found and B. bassiana was obtained only once. The Aspergillus ochraceus was the most frequent and, for the first time, reported to infect ants frequently. The mortality test results of ants infected with A. ochraceus and B. bassiana are similar to those obtained in other pathogenicity tests of B. bassiana or M. anisopliae against leaf-cutting ants [22-24]. Although the high control mortality indicates that the ants were stressed in some way (e.g., social isola- tion), and thus probably more susceptible to diseases, the parasite treatments caused significantly increased mortality. This allows us to say that A. ochraceus could be also a promising biological control agent of ants. Surely, more research should be undertaken before significant field use of the pathogen. Even though that Aspergillus are unusual pathogens of most insects, these fungi produce a diverse range of compounds that can be potent insect toxins and potentially useful as pesticidal [25]. However, the safety of these compounds is a major concern and more studies are 4 Psyche Table 2: P values of Log- rank test to compare two Kaplan-Meier survival curves for Atta bisphaerica workers treated with different conidial concentrations of Aspergillus ochraceus. P values less than 0.05 were considered significant. Conidial concentrations 0 10^ 10*5 10^ 10^ 10^ 0 — 10^ P = 0.90 — 10^ P = 0.06 P = 0.07 — 10^ P = 0.02 P = 0.04 P = 0.93 — 10^ P < 0.01 P < 0.01 P = 0.15 P = 0.10 — 10^ P < 0.0001 P < 0.0001 P < 0.001 P < 0.0001 P < 0.01 — Table 3: P values of Log- rank test to compare two Kaplan-Meier survival curves for Atta bisphaerica workers treated with different conidial concentrations of Beauveria bassiana. P values less than 0.05 were considered significant. Conidial concentrations 0 105 10*5 10^ 10^ 10^ 0 — 10^ P = 0.80 — 10*5 P = 0.03 P = 0.02 — 10^ P < 0.0001 P < 0.0001 P < 0.01 — 10^ P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 — 10^ P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.77 — Number of days Control Beauveria bassiana (10^ conidia/mL) B. bassiana (10^ conidia/mL) B. bassiana (10^ conidia/mL) B. bassiana (10^ conidia/mL) B. bassiana (10^ conidia/mL) Figure 3; Cumulative time-related survival proportion (Kaplan- Meier curves) of the Atta bisphaerica worker ants after being treated with different suspension concentrations of the Beauveria bassiana fungus. needed to evaluate potential risks to humans and nontarget species. Cladosporium fungi are phytopathogens [26] and may be transported by various insects causing their death. In a study of leaf-cutting queen ants, the Cladosporium fungus was prevalent in Atta laevigata and in A. capiguara [27]. It is believed that these fungi are frequently found due to their cosmopolitan distribution, being acquired from the environment and dispersed during the founding of a colony by queens or forage worker ants carrying leaves. The role of the Cladosporium species inside the ant colonies is not known for sure, but they have been considered potential antagonists of the fungus garden [28]. The same kind of reasoning can be adopted for the Mucor fungi species, which have also been found in the nests of leaf-cutting ants, however, without knowing exactly how they interact with these ants or the fungus garden. The fungi of this genus are considered invertebrate pathogens. For example, Mucor hiemalis can cause the mortality of Cupiennius and Ischnothele spiders [29]. Toxic metabolites of these organ- isms present an insecticidal activity against the adults of Bactrocera oleae and Ceratitis capitata (Diptera: Tephritidae) [30]. Considering the diversity of fungi found in this study, the importance of new surveys and tests of entomopathogenic fungi isolates with the potential of being used in micro- bial control of these ants becomes evident. Beauveria and Metarhizium are the species which have been most studied and which have been evaluated on tests on their pathogenic potential. It is evident now that A. ochraceus should be further investigated for its pathogenic potential for leaf- cutting ants as well as other fungi genera found here. Psyche 5 Acknowledgment The authors would like to thank Dr. Harry Evans of CAB International for fungi identification. References [1] B. Holldobler and E. O. Wilson, The Ants, Harvard University Press, Cambridge, Mass, USA, 1990. [2] F. A. M. Mariconi, As Sauvas, Agro nomica Ceres, Sao Paulo, Brazil, 1970. [3] C. A. Lima, T. M. C. della Lucia, R. N. C. Guedes, and C. E. da Veiga, “Development of granulated baits with alternative attractants to Atta bisphaerica forel (Hymenoptera: Formicidae) and their acceptance by workers,” Neotropical Entomology, vol. 32, no. 3, pp. 497-501, 2003. [4] M. C. Oliveira, T. M. C. Della Lucia, D. Nascimento Junior, and C. A. Lima, “Especies forrageiras preferidas para o corte porAtta bisphaerica Forel, 1908 (Hymenoptera: Formicidae),” Revista Ceres, vol. 49, pp. 321-328, 2002. [5] E. Diehl-Fleig, M. E. Silva, A. Specht, and M. E. Valim- Labres, “Efficiency of Beauveriana bassiana for Acromyrmex spp. control (Hymenoptera: Formicidae),” Anais da Sociedade Entomologica do Brasil, vol. 22, pp. 281-286, 1993. [6] M. A. L. Bragan 9 a, A. Tonhasca, and T. M. C. Delia Lucia, “Reduction in the foraging activity of the leaf-cutting ant Atta sexdens caused by the phorid Neodohrniphora sp,” Entomologia Experimentalis etApplicata, vol. 89, no. 3, pp. 305-311, 1998. [7] A. Tonhasca Jr., M. A. Braganca, and T. M. C. Della Lucia, “Delayed suppression of the foraging activity of the leaf- cutting ant Atta sexdens rubropilosa by a phorid parasitoid,” Bulletin of the Ecological Society of America, vol. 78, p. 322, 1997. [8] R. Isenring and L. Neumeister, “Recommendations regarding derogations to use alphacypermethrin, deltamethrin, feni- trothion, fipronil and sulfluramid in ESC certified forests in Brazil,” ESC Editor, p. 99, 2010. [9] W. O. H. Hughes, L. Thomsen, J. Eilenberg, and J. J. Boomsma, “Diversity of entomopathogenic fungi near leaf- cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliaef Journal of Invertebrate Pathology, vol. 85, no. 1, pp. 46-53, 2004. [10] E. Lopez and S. Orduz, '"Metarhizium anisopliae and Tricho- derma viride for control of nests of the fungus-growing ant, Atta cephalotesf Biological Control, vol. 27, no. 2, pp. 194-200, 2003. [11] A. Silva, A. Rodrigues, M. Bacci, F. C. Pagnocca, and O. C. Bueno, “Susceptibility of the ant- cultivated fungus Leu- coagaricus gongylophorus (Agaricales: Basidiomycota) towards microfungi,” Mycopathologia, vol. 162, no. 2, pp. 115-119, 2006. [12] E. H. Varon Devia, Distribution and Eoraging by the Leaf- Cutting ant, Atta cephalotes, in Coffee Plantations with Different Types of Management and Landscape Contexts, and Alternatives to Insecticides for Its Control, University of Idaho, Moscow, Idhao, USA, 2007. [13] E. Diehl and L. K. Junqueira, “Seasonal variations of meta- pleural secretion in the leaf-cutting ant Atta sexdens piriventris santschi (Myrmicinae: Attini), and lack of fungicide effect on Beauveria bassiana (Bals.) vuillemin,” Neotropical Entomology, vol. 30, no. 4, pp. 517-522, 2001. [14] A. Kermarrec and M. Decharme, “Ecopathological aspects in the control of Acromyrmex octospinosus Reich (Form., Attini) by entomophagous fungi,” in The Biology of Social Insects, M. D. Breed, C. D. Michener, and H. E. Evans, Eds., p. 148, Westview Press, Boulder, Colo, USA, 1982. [15] A. Kermarrec, G. Febvay, and M. Decharme, “Protection of leaf-cutting ants from biohazards: is there a future for microbiological control?” in Eire Ants and Leaf Cutting Ants : Biology and Management, C. S. Lofgren and R. K. Vander Meer, Eds., pp. 339-356, Westview Press, Boulder, Colo, USA, 1986. [16] S. B. Alves, “Fungos entomopatogenicos,” in Controle Micro- biano de Insetos, S. B. Alves, Ed., pp. 289-381, FEALQ, Piracicaba, Sao Paulo, Brazil, 1998. [17] G. L. Barron, The Genera ofHyphomycetesfrom Soil, Robert E. Krieger Publishing, New York, NY, USA, 1972. [18] G. Barson, N. Renn, and A. F. Bywater, “Laboratory evaluation of six species of entomopathogenic fungi for the control of the house fly {Musca domestica L.), a pest of intensive animal units,” Journal of Invertebrate Pathology, vol. 64, no. 2, pp. 107- 113, 1994. [19] T. M. C. Della Lucia, As Eormigas Cortadeiras, Folha de Vi 9 osa, Vi^osa, MG, Brazil, 1993. [20] E. Diehl-Fleig and M. E. Silva, “Patogenicidade de Beauveria bassiana e Metarhizium anisopliae a formiga sauva Atta sexdens piriventrisf Boletim do Grupo de Pesquisadores de Controle Bioldgico, vol. 6, p. 15, 1986. [21] E. Diehl-Fleig, M. E. Silva, and M. M. Pacheco, “Testes de pato- genicidade dos fungos entomopatogenicos Beauveria bassiana e Metarhizium anisopliae em Atta sexdens piriventris (Santschi, 1919) em diferentes temperaturas,” Ciencia e Cultura, vol. 40, pp. 1103-1105, 1988. [22] A. M. C. Castilho, M. E. Fraga, E. L. Aguiar- Meneze, and C. A. R. Rosa, “Selection of Metarhizium anisopliae and Beauveria bassiana isolates pathogenic to Atta bisphaerica and Atta sexdens rubropilosa soldiers under laboratory conditions,” Ciencia Rural, vol. 40, no. 6, pp. 1243-1249, 2010. [23] E. S. Loureiro and A. C. Monteiro, “Patogenicidade de isolados de tres fungos entomopatogenicos a soldados de Atta sex- dens sexdens (Linnaeus, 1758) (Hymenoptera: Formicidae),” Revista Arvore, vol. 29, pp. 553-561, 2005. [24] A. V. Santos, B. L. De Oliveira, and R. I. Samuels, “Selection of entomopathogenic fungi for use in combination with sub- lethal doses of imidacloprid: perspectives for the control of the leaf-cutting ant Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae),” Mycopathologia, vol. 163, no. 4, pp. 233-240, 2007. [25] R. A. Humber, “Fungal pathogens and parasites of insects,” in Applied Microbial Systematics, F. G. Priest and M. Goodfellow, Eds., pp. 203-230, Kluwer Academic Publishers, Dordrecht, Netherlands, 2000. [26] G. M. Richards and L. R. Beuchat, “Infection of cantaloupe rind with Cladosporium cladosporioides and Penicillium expan- sum, and associated migration of Salmonella poona into edible tissues,” International Journal of Pood Microbiology, vol. 103, no. 1, pp. 1-10, 2005. [27] F. C. Pagnocca, A. Rodrigues, N. S. Nagamoto, and M. Bacci, “Yeasts and filamentous fungi carried by the gynes of leaf- cutting ants,” Antonie van Leeuwenhoek, vol. 94, no. 4, pp. 517- 526, 2008. [28] A. Rodrigues, M. Bacci, U. G. Mueller, A. Ortiz, and F. G. Pagnocca, “Microfungal “weeds” in the leafcutter ant symbiosis,” Microbial Ecology, vol. 56, no. 4, pp. 604-614, 2008. [29] W. Nentwig, “A zygomycetous fungus as a mortality factor in a laboratory stock of spiders,” The Journal of Arachnology, vol. 18, pp. 118-121, 1990. 6 Psyche [30] M. A. Konstantopoulou, P. Milonas, and B. E. Mazomenos, “Partial purification and insecticidal activity of toxic metabo- lites secreted by a Mucor hiemalis strain (SMU-21) against adults of Bactrocera oleae and Ceratitis capitata (Diptera: Tephritidae) Journal of Economic Entomology, vol. 99, no. 5, pp. 1657-1664, 2006. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 198084, 19 pages doi:10.1155/2012/198084 Review Article Fire Ants {Solenopsis spp.) and Their Natural Enemies in Southern South America Juan Briano, Luis Calcaterra, and Laura Varone USDA-ARS- South American Biological Control Laboratory, Bolivar 1559, B1686EFA Hurlingham, Argentina Correspondence should be addressed to Juan Briano, jabriano@speedy.com.ar Received 8 August 2011; Revised 4 October 2011; Accepted 14 October 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 Juan Briano et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We review the fire ant research conducted by the ARS-South American Biological Control Laboratory (SABCL) since 1987 to find a complex of natural enemies in southern South America and evaluate their specificity and suitability for field release as self- sustaining biological control agents. We also include those studies conducted by the ARS-Center for Medical, Agriculture, and Veterinary Entomology in the United States with the SABCL collaboration. Ecological and biological information is reported on local fire ants and their microsporidia, nematodes, viruses, phorid flies, eucharitid wasps, strepsiptera, and parasitic ants. Their biology, abundance, distribution, detrimental effect, field persistence, specificity, and phenology are discussed. We conclude that the objectives of the ARS program in South America are being achieved and that the pioneering studies have served to encourage further investigations in the United States and other countries and advanced the implementation of biological control programs to decrease imported fire ant densities and damage. Still, several promising organisms should be further investigated for eventual field release in the near future. 1. Introduction The ant genus Solenopsis Westwood (Hymenoptera: Formi- cidae: Myrmicinae) is represented in South America by 16 native species known as “fire ants” [1]. While, in general, these ants cause occasional local problems in their homeland, two species accidentally introduced into the southern United States in the early 1900s are considered pests with a high neg- ative impact in rural and urban areas [2]. These pest species are the red imported fire ant, Solenopsis invicta Buren, and the black imported fire ant, S. richteri Forel, both included in a revision of the S. saevissima complex [3]. During the last decade, S. invicta has been considered one of the 100 worst invasive exotic species [4]; this fire ant became a more global problem when it invaded ecosystems in the Caribbean Islands [5], Australia [6], New Zealand [7], Hong Kong, Taiwan [8], and mainland China [9]. Its eradication has been accomplished only in New Zealand [ 10 ]. In the United States, the imported fire ants cause many problems in the southeast and in some patches in California. They are a major public health concern because of their aggressive stinging behavior [11], Although, for most indi- viduals, this is just an irritating nuisance, for several hundred thousand people in the United States, sensitive to fire ants or highly allergic, the sting might cause severe reactions and eventually death [12], Fire ants also injure domestic animals, livestock, affect wildlife [13, 14], native ants, and other arthropods [15, 16]. Structures, electrical devices, and agri- cultural crops can also be damaged [17, 18]. In disturbed North American environments, imported fire ants are dominant terrestrial arthropods [19]. Solenopsis invicta has displaced Neartic species of fire ants in the United States and adversely affected the diversity of the ant assemblages [15, 16]; however, the nature of the impact on native ant species has been controversial [20]. Some benefi- cial effects of fire ants such as predation on several agricul- tural and livestock pests have been also reported [20, 21]. In the United States, chlorine insecticides were used to control fire ants in the 1950s and 1960s, but they brought negative consequences to the environment [21]. A new bait with the insecticide mirex was believed to make fire ant eradication possible. However, in 1971, its use was highly restricted because of many environmental concerns and 2 Psyche mirex registration was cancelled in 1977 [11, 21]. Since the 1980s, more environmentally friendly products have been used in the United States [11] and in other invaded countries. Still, the chemical approach is expensive, only provides temporary control, is detrimental to several nontarget organ- isms, and is not appropriate for large and/or sensitive envi- ronments. Consequently, the need of implementing control methods with less negative environmental impacts became a priority. The first serious interest in biological control of fire ants was shown by the United States in the late 1960s. Scientists from the University of Florida and the ARS-Insects Affecting Man and Animals Research Laboratory (lAMARL, now the Center for Medical, Agricultural, and Veterinary Entomology, CMAVE), both in Gainesville, EL, conducted the first surveys for natural enemies in Brazil, Uruguay, and Argentina and provided information on several potential candidates [22-24]. In late 1987, after three years of cooperative work with Brazilian researchers in Mato Grosso and Mato Grosso do Sul, Brazil, scientists from the lAMARL formally estab- lished the fire ant biological control program at the ARS- South American Biological Control Laboratory (SABCL) in Hurlingham, Buenos Aires province, Argentina [25]. Since then, the main objective of the program has been to find a complex of natural enemies of fire ants in their homeland, evaluate their specificity, and determine their suitability for eventual use in the United States against the red and black imported fire ants. In this paper, we review the fire ant research conducted by SABCL researchers in southern South America since 1987. Several studies carried on in the United States by CMAVE scientists in collaboration with SABCL researchers are also included. We cover not only the occurrence of fire ant natural enemies and aspects of their biology and ecology, but also ecological studies on other South American fire ants. 2. Fire Ants in Southern South America South American fire ants occur in almost all habitats from the Amazon Basin of Brazil to 42° S in Rio Negro province, Argentina [1, 26-28], and up to more than 3,200m of altitude in the Puna region in the Andes [29]. Solenopsis invicta occurs along most of the Rio de la Plata basin from the vicinity of Rosario, Santa Fe province, Argentina, to Paraguay, southern Brazil and eastern Bolivia [1, 28] (Figure 1). Recent surveys revealed that mitochondrial DNA haplotypes of S. invicta are distributed in Argentina up to 33°41' S in Mercedes, San Luis province, and 64°52' W and 1,100 m of altitude in the Calilegua National Park, lujuy province [29-3 1 ] . The occurrence of S. invicta in the Amazon basin has been controversial since it has been previously recorded in Porto Velho, Rondonia state, Brazil [28], but it was virtually absent in more recent studies [30, 31]. Solenopsis richteri is native to central Argentina, southern Uruguay, and Brazil. In Argentina, it occurs mainly in the pampas surrounding Buenos Aires and along the lower reaches of the Rio de la Plata basin, up north to Rosario area. Other common fire ant species in southern South America are S. quinquecuspis Forel, mostly cooccurring with S. richteri; S. magdonaghi Santschi, mostly cooccurring with S. invicta; S. interrupta Santschi, mostly cooccurring with S. electra Forel in northwestern Argentina and Bolivia; S. weyrauchi Trager, mostly occurring presumably alone above 2,000 m of altitude throughout the Andes from Peru to Argentina [1, 30]. Hundreds of studies on introduced populations of the red and black imported fire ants have been published since the 1970s, several of which have attributed their invasion success to the adaptation to disturbed habitats, the escape from natural enemies, or the competitive superiority [20, 32]. Despite their widespread impact in invaded habitats, little was known about these species in their homeland. The first studies in their native range focused on the occurrence and detrimental effects of natural enemies such as pathogens, social parasites, and parasitoids [20, 24, 33-36]. Several ecological studies on ant assemblages were con- ducted during the last decade in Argentina and Brazil [30, 37- 42]. These studies were oriented (1) to know the position of S. invicta in the hierarchy of dominance of the ant assem- blages, cooccurring not only with many competitor ants but also with their natural enemies, and (2) to investigate if its success in the introduced range is the consequence of a low- competitive environment more than the relative absence of their natural enemies [38-40]. These works revealed that, in several ant assemblages in Argentina and Brazil, overall, 5. invicta occupied the top in the ecological dominance hierarchy, being the ant most frequently captured (64-82% of the samples) and numerically abundant (23-27% of total individuals captured) without showing the highest biomass. Most assemblages included at least 8-10 ant species that were also very common [38-42]. The ecological studies also showed that S. invicta was frequently a slow discoverer but almost always a good dominator of the food resources, allowing other cooccurring species of ants to be abundant [38-40]. This would indicate that its success was not necessarily based on the break of the discovery- dominance tradeoff, as it has been found in other invasive species, such as the Argentine ant, Linepithema humile (Mayr). Despite not being a good discoverer, S. invicta won, on average, 75% of the interactions in five ant assemblages in northern Argentina [38, 39] and Brazil [40]. In Argentina, its main competitor was Pheidole obscurithorax Naves (also exotic in the United States). Its ecological dom- inance was based on (1) the large numbers of individuals, (2) the well-developed recruitment system, (3) the aggressive behavior, and (4) the uninterrupted-foraging activity [30, 38- 40]. The situation in southern South America strongly con- trasts with that observed in North America, where S. invicta is the unique dominant ant representing most ant biomass [17, 20, 32]. At least in Argentina, the strong competitive environment and the indirect effect of natural enemies were suggested to be the most important factors limiting the success of S. invicta. Competitors and natural enemies would likely be locally adapted to the genetically divergent S. invicta populations inhabiting different parts of South America. An assessment of its genetic variation using 2,144 colonies from Psyche 3 Figure 1: Red dots showing localities in Argentina, Brazil, Uruguay, and Chile mentioned in the paper. 75 sites worldwide revealed that around 97% of all known mt DNA haplotypes of S. invicta only occur in the native range [31]. The dominant haplotypes in the United States and other newly invaded areas occur only at low frequencies (<5%) in eight populations in Formosa province (Figure 1) in northeastern Argentina [31], indicating that this area is more likely to be the source of S. invicta in the United States [43]. 3. Natural Enemies 3.1. Pathogens. Preliminary explorations for fire ant diseases in Argentina were conducted by researchers from lAMARL and SABCL in 1987 in the provinces of Buenos Aires, Entre Rios, and Santa Fe [25]. The vial sampling of 425 fire ant colonies in 47 sites and the subsequent microscopic exam- ination revealed the presence of the following pathogens: (1) Kneallhazia {=Thelohania) solenopsae Knell, Allen, and Hazard (Microsporidia: Thelohaniidae) at 41% of the sites and 11% of the colonies; (2) Vairimorpha invicta Jouvenaz and Ellis (Microsporidia: Burenellidae) at 11% of the sites and 2% of the colonies; (3) Myrmecomyces annellisae Jouve- naz and Kimbrough (Deuteromycotina: Hyphomycetes) at 15% of the sites and 2% of the colonies; (4) Mattesia sp. (Neogregarinida) at 7% of the sites and 1% of the colonies; (5) a mermithid nematode at 7% of the sites and 0.5% of the colonies. This preliminary overall occurrence of K. solenopsae and U invictae in 13% of the colonies of S. richteri and S. quinquecuspis almost doubled the prevalence (7.6%) of the same infections on S. invicta in the area previously surveyed of southwestern Brazil [25]. 3.1.1. Microsporidia. Kneallhazia solenopsae and U invictae are obligate intracellular pathogens first discovered infecting mainly the fat body of fire ants collected in the area of Cuiaba, Mato Grosso, Brazil [22, 44-46] (Figure 1). Both microsporidia show immature vegetative stages and reproductive stages represented by spore dimorphism with basically eight meiospores (octospores) bound by a mem- brane and nonbounded, or free, binucleate spores. More recent ultrastructural studies on K. solenopsae showed the presence of several other spore morphotypes [47-50]. 4 Psyche Field Surveys. Subsequent surveys conducted in 1988 were mostly concentrated on microsporidia of S. richteri and S. quinquecuspis in Buenos Aires province to select a field site for long-term ecological studies [51, 52], The microscopic (phase-contrast) examination of 1,836 samples of fire ant colonies from 185 roadside sites revealed the presence of K. solenopsae (Figure 2) at 25% of the sites and 8% of the colonies and invicta (Figure 3) at 5% of the sites and 1% of the colonies. In some sites, K. solenopsae showed epizootic levels infecting 40-80% and Vi invictae infecting 60% of the colonies. This prevalence was the highest reported for South America. Simultaneous, or dual, infections of K. solenopsae and Vi invictae in the same colony were not detected. The area of Saladillo, 180 km SW of Buenos Aires (Figure 1), was selected for long-term studies on S. richteri populations infected with K. solenopsae (see Detrimental Effect), At this stage, the fungus M, annellisae was found in 6% of the sites and 1% of the colonies; Mattesia sp. and the mermithid nematode were not found. From 1991 to 1999, explorations were extended to northern Argentina in the search for K. solenopsae and, mainly, Vi invictae infecting S. invicta [53]. The sampling of 2,528 fire ant colonies in 154 sites revealed the presence of K. solenopsae at almost 43% of the sites and in 12% of the colonies and Vi invictae at 13% of the sites and in 2,3% of the colonies. Again, some sites in northcentral Santa Fe province showed more epizootic levels of Vi invictae with up to 50% of the colonies infected; some of these sites were selected for long-term studies on S. invicta populations infected with 14 invictae (see Detrimental Effect). Both microsporidia were sympatric in 12 sites, in three of which 7 dual infected colonies (S. richteri and S. macdonaghi in Entre Rios and S. invicta in Santa Ee) were found. This very low overall prevalence of dual infections (7/2,528 = 0.0028 = 0.3%) was identical to the combined probability of finding at random K. solenopsae (12%) and 14 invictae (2.3%) simultaneously in the same colony (0,12 X 0.023 = 0.0028 = 0.3%). In 1993, a brief and opportunistic sampling of 61 S. invicta colonies at 18 sites in the area of Cuiaba (type locality for K. solenopsae and 14 invictae) revealed the presence of 21% of the colonies infected with K. solenopsae and 6.6% with 14 invictae (Briano and Patterson, unpublished data). At least in Argentina and Paraguay, K. solenopsae and 14 invictae showed the ability to infect both monogyne and polygyne colonies of S. invicta and S. richteri. In a sampling of 20 S. invicta colonies infected with K. solenopsae, 45% were polygyne and 55% were monogyne colonies; from 15 S. invicta colonies infected with 14 invictae, 46% were polygyne and 54% were monogyne colonies [54]. Similarly, it was found that in a population of 41 colonies of S. richteri infected with K. solenopsae, 42% of the colonies were polygyne and 58% were monogyne [55]. It is important to remark that, during the course of the investigations in Argentina on K. solenopsae, this microsporidium was suddenly discovered in the United States [56] and subsequently found in most southern states. This fact redirected some of the studies on this candidate. Figure 2; Phase contrast view (400x) of meiospores of Kneallhazia solenopsae in workers of Solenopsis richteri. Because of the grinding process, the octect membrane usually brakes and most spores are released in the aqueous extract. A few intact octects (arrows) are shown with meiospores inside. Figure 3; Phase contrast view (400x) of ovoid meiospores (octospores) and bacilliform binucleate free spores of Vairimorpha invictae in workers of Solenopsis invicta. Again, most octects with meiospores brake during the grinding process. since its presence in the US represented a change in the biological control approach for S. invicta. New surveys for 14 invictae and K. solenopsae were conducted from 2001 to 2005 in several central and Northern provinces (temperate and subtropical) of Argentina and in limited areas of Paraguay, Brazil, Chile, and Bolivia [57], including large western regions previously unexplored. A total of 2,064 colonies were sampled in 262 sites in roadsides, pastures, and recreational areas. Vairimorpha invictae was found at 12% of the sites and 10% of the colonies in Argentina, Brazil, Paraguay, and Bolivia. The provinces of Santa Fe and Entre Rios showed the highest prevalence of infected colonies (20 and 7%, resp.); in certain S. invicta sites in Santa Fe (San Javier and vicinities; Figure 1), the prevalence was 50-54%, and, in S. richteri sites in Entre Rios (Medanos), the prevalence was 60%. The prevalence of 14 invictae in Paraguay and Bolivia was very low. Kneallhazia solenopsae showed a wider distribution occurring at 25% of the sites and 13% of the colonies and was reported for the first time in western and northwestern Argentina and Bolivia, at altitudes of almost 2,300 m and colder weather. It was also first reported infecting S. interrupta [57]. The province of Buenos Aires showed the highest prevalence with 68% of Psyche 5 infected sites and 34% of infected colonies. This time, both microsporidia cooccurred in 1 1 sites, 10 of which showed 46 dual infected colonies in several provinces. This prevalence of dual infections (46/2,064 = 0.0223 = 2.2%) was higher than the combined probability of finding K. solenopsae (13%) and invictae (10%) simultaneously in the same colony (0.13 X 0.10 = 0.013 = 1.3%) and was the consequence of repeated and planned samplings in sites with high prevalence of dual infections. The highest prevalence of dual infections was found in Santa Fe with 3.9% of S. invicta infected colonies and in Entre Rios with 2.7% of S. richteri infected colonies. Intracolonial Prevalence. The intracolonial prevalence of K. solenopsae in fire ant colonies (mainly S. richteri) was very high. Vegetative stages (Figure 4) infected 28% (range 20- 45%) of the immature fire ants including eggs and only 1.2% of the queens, while mature stages (spores; Figure 2) infected 42.3% (range 34-95%) of the workers, sexual adults, and queens and 0.8% of the pupae [58]. The presence of infected eggs revealed transovarial (vertical) transmission of K. solenopsae. The mean number of meiospores per worker ranged from 9 X 10^ to 6.7 X 10^. Free spores were extremely rare. The intracolonial prevalence of V invictae in fire ant colonies (mainly 5. invicta) was also very high in most ant castes and stages [53]. Vegetative stages infected 30% (range 17-52%) of the fire ant larvae and 4.8% of the queens. Low prevalence of vegetative stages was also detected in a few eggs, providing evidence for transovarial transmission. However, the importance of the vertical transmission in the life cycle of V invictae remained uncertain. Meiospores and binucleate spores of V. invictae (Figure 3) were found in all fire ant castes except eggs. Meiospores infected 33% (range 5-56%) of mature ants, and binucleate spores infected 39% (range 9.5-63%) of immature and mature ants. The occurrence of V invictae was much more common in sexual males than in females. The mean number of meiospores per worker ranged from 1.2 X 10"^ to 6.4 X 10"^. Free binucleate spores of V invicta in S. invicta were much more common than those of K. solenopsae in S. richteri, ranging from 3.2 X 10^ to 1.6 X lOl Dual infections showed lower intracolonial prevalence. In S. invicta, it ranged from 4.5 to 22% of the individual pupae, workers, and sexual females. In S. richteri and S. macdonaghi, dual infections were found only in 2.7% of the workers [53]. Dual infections were suggested to represent an important mortality factor for fire and colonies, but it was never confirmed with appropriate laboratory tests. Detrimental Effect. The long-term field effect of K. solenopsae on S. richteri was studied in 6 plots established in natural pastures in the area of Saladillo, Buenos Aires province, and monitored 4-10 times per year from October 1988 to January 1993 for the density of colonies and the infection rates [51, 59, 60]. The study included the identification, sampling, and mapping of 1,348 active colonies. Although the fire ant densities showed cyclic variations unrelated to seasons, the overall density decreased from 162 to 28 colonies per hectare by the end of the study. The proportion of infected Figure 4: Cell of a Solenopsis richteri larva infected with a binucleate vegetative stage of Kneallhazia solenopsae. Giemsa’s stain, lOOOx. colonies was very variable during the study and was positively related to rainfall. A weak negative association was found between the density of colonies and the rate of infection. The reduction of fire ant densities was attributed to the presence of K. solenopsae, although the loss of control plots because of natural dissemination of the infection obscured conclusive results. During 9 months in 1992, the mound volumes of 84 K. solenopsae-infected colonies were compared with 88 healthy colonies from two different areas [55]. The presence of polygyny, number of queens per colony, and presence and abundance of brood and myrmecophiles were also compared between infected and healthy colonies. There was a strong negative association between mound size and infection with K. solenopsae. The mounds of infected colonies (mean ± SD: 4.9 ± 1.0 liters) were substantially smaller than those of healthy ones of the two different areas (14.7 ± 1.8 and 18.7 ± 1.7 liters). No difference was found in the volumes of infected monogyne and polygyne colonies. The presence of multiple queens was common in both infected and healthy colonies, and the number of queens per colony did not differ significantly. The infection of K. solenopsae had no effect on worker brood presence, but there was less sexual brood in infected colonies. All the myrmecophiles found, Neoblissus parasigaster Bergroth (Hemiptera: Lygaeidae), Martinezia sp. {Myrmecaphodius) (Coleoptera: Scarabeidae), Myrmecosaurus sp. (Coleoptera: Staphylinidae), and the social parasite Solenopsis daguerrei (Santschi) were as common in infected as well as in healthy colonies, but N. parasigaster was more numerous in infected colonies. Within the Saladillo plots, no effect of K. solenopsae was reported on the fire ant colony movement [61] . The lethal effect of K. solenopsae was suggested for polygyne colonies of S. richteri originally collected in the field and later fragmented in the laboratory to one queen and 100 workers. The fragmented colonies were reared in small plastic containers for their residual longevity [62]. After 3 months of rearing, the mortality was 92% for infected colonies {n = 14) and only 49% for healthy colonies {n = 22). In 1995 and 1996, the survival of 224 starved individual minor and major workers and 13 sexual females selected at random from infected and healthy colonies of S. richteri was compared [62]. At 27° C, the mortality rate of infected 6 Psyche workers was higher than that of healthy workers. Although the final mortality rate of infected and healthy sexual females was similar, the mortality rates occurred much sooner for infected sexuals. No differences were detected in the live weight of infected and healthy workers (minor or major). In a similar survival test with workers of S. invicta infected with Vi invictae, mortality rates of infected workers were much higher than those of healthy ones [53]. The long-term field effect of K. solenopsae and Vi invictae on S. invicta was studied in 8 roadside plots established in Santa Fe province and monitored 3-5 times per year from May 2000 to March 2004 for the density of colonies, the USDA population index (PI), and the infection rates [63]. As in the study of S. richteri, control plots were lost since they suffered the natural spread of the infections, making difficult the analysis of the results. Although the mean PI per plot showed abrupt reductions followed by reinfestations, important reductions of 53-100% were observed at the end of the test in 7 of the 8 plots, resulting in an overall PI reduc- tion of 69%. From the total 394 colonies sampled, 82.5% were healthy and 17.5% were infected. The percentage of infection with both microsporidia also showed fluctuations and an overall reduction from 26 to 5% of infection rates. Only 3 colonies were found with dual infections in 2 plots. The proportion of infected and healthy colonies in the PI categories was significantly different for medium and large colonies with worker brood. More than 97% of the large colonies were healthy, suggesting that infected colonies did not produce large colonies. Field Persistence. Several observational studies on field per- sistence were conducted to check the occurrence of the microsporidia over time. These repeated samplings were important to recognize field sites with high prevalence of infections to be used as source sites for eventual shipping of infected colonies to the US. One S. quinquecuspis site in the area of Pergamino, 240 km N of Buenos Aires (Figure 1), infected with K. solenopsae was monitored every 1-2 months from October 1988 to luly 1990 (Briano, unpublished data). The mean number of fire ant colonies sampled per monitoring date was 23 (range: 12-36), andiC. solenopsae was always found infecting the colonies with ranges of 17-64%. Another site in Saladillo with S. richteri infected with K. solenopsae was monitored 9 times from Oct 1992 to lun 1998 (Briano, unpublished data). The mean number of fire ant colonies sampled per monitoring date was 14.2 (range: 6- 50). In seven (78%) of the monitoring dates, K. solenopsae was found infecting from 22 to 67% of the fire ant colonies examined. In the other two sampling dates, no infected colonies were found. Two S. richteri sites in Entre Rios and one S. invicta site in Santa Fe infected with K. solenopsae and U invictae were sampled 5-10 times from luly 2001 to March 2005 [57]. On average, 18 colonies (range 7-70) were sampled per monitoring date and a high prevalence of both infections was detected, reaching epizootic levels in most occasions. The total prevalence of both microsporidia ranged from 46 to 78% of the colonies; in two occasions, 100% of the colonies were infected. Each microsporidium exhibited a characteristic enzootic/epizootic wave; U invictae occurred more sporadically, with sudden fluctuations in prevalence, while K. solenopsae showed a more sustained prevalence with fewer fluctuations. High peaks in prevalence of K. solenopsae coincided with low peaks of prevalence of U invictae and vice versa. The mutual interference of both microsporidia was never confirmed with laboratory tests; but it was suggested that the successive high levels of both infections, one at a time, might represent a more constant pressure against fire ant host populations. However, this assumption was never checked with appropriate filed tests. Transmission. Many tests for the artificial horizontal trans- mission of K. solenopsae to individuals or colonies of S. invicta and S. richteri were conducted in the laboratory from 1992 to 2000 (Briano, unpublished data). Several approaches were used, such as (1) inoculation of healthy laboratory colonies with spore suspensions obtained from infected workers; (2) transference of infected fire ant larvae to healthy receptor colonies; (3) transference of infected fire ant adult workers to healthy receptor colonies; (4) transference of the myrmecophile N. parasigaster to healthy receptor colonies; (5) inoculation of N. parasigaster with spore suspensions obtained as above; (6) mixing of queenless infected colonies with healthy polygyne colonies; (7) inoculation of healthy field colonies with spore suspensions using various methods. After several days or weeks (depending on the approach used), the microscopic examination of the inoculated indi- viduals, colonies, or myrmecophiles did not reveal infections. However, horizontal transmission of K. solenopsae was achieved by scientists at CMAVE by transferring S. invicta infected brood to healthy colonies (approach number 2 above) and by mixing colonies (approach number 6 above) [64—66]. Tests were conducted to obtain artificial dual infections in S. richteri with K. solenopsae and U invictae (Bri- ano, unpublished data). Colonies infected with U invictae were used as inocula with the following approaches: (1) transference of Vairimorpha-infected larvae to Kneallhazia- infected colonies; (2) transference of Vairimorpha-mfected workers to Kneallhazia-infected colonies; (3) inoculation of Kneallhazia-infected colonies with Vairimorpha spore suspensions obtained from infected workers. Again, infec- tions were not detected in the subsequent microscopic examination of the inoculated colonies. In 2003, colonies of S. invicta infected with U invictae were collected in Santa Fe, Argentina, and transported to quarantine at CMAVE for additional transmission tests. The following approaches were considered [67]: (1) inoculations of incipient S. invicta colonies reared from newly mated queens with larvae from the infected field colonies; (2) inoculations of incipient S. invicta colonies reared from newly mated queens with nonmelanized pupae from the infected field colonies; (3) inoculations of incipient S. invicta colonies reared from newly mated queens with larvae or melanized pupae from infected laboratory colonies; (4) inoculation of incipient S. invicta colonies with dead adults from the infected field colonies. The subsequent microscopic examination revealed, for the first time, positive transmission Psyche 7 in 40% (2/5) of the inoculated colonies in approaches number 1 and number 2, 100% (3/3) in approach number 3, and 33% (2/6) in approach number 4. Due to the limited number of colonies inoculated in each approach, the statistical analysis was not conducted. Also, the colony growths and brood volumes were significantly lower in infected than in healthy colonies. Specificity. The field host range of K. solenopsae and 1C invictae was first studied from 1993 to 2000 in eastcentral Argentina and southern Brazil by sampling terrestrial ants cooccurring with infected fire ants. Ants were sampled using 520 bait traps (glass vial with pieces of canned “Vienna sausage”) in 52 preselected infected roadside sites and by hand sampling of 585 colonies (S. invicta, S. richteri, and other ants species) in 90 sites [68] . Kneallhazia solenopsae and V invictae were found infecting only S. invicta, S. richteri, S. macdonaghi, and Solenopsis sp. (unidentified fire ant species), while the other ants baited/sampled in the genera Pheidole, Camponotus, Crematogaster, Linepithema, Brachymyrmex, Nylanderia {=Paratrechina), Acromyrmex, and Wasmannia were not infected. A preference of V invictae for S. invicta was suggested [68]. The infection in S. macdonaghi was a new host record. A few meiospores of K. solenopsae also had been found in some individuals of the myrmecophile N. parasigaster and the parasitic ant S. daguerrei [51, 59], but infections in host tissue were not confirmed. More recently, empirical evidence of K. solenopsae infections on S. geminata and S. geminata x S. xyloni hybrid was reported from Texas and Mexico [69]. In 2004, additional studies were conducted in 5 polygyne sites in Corrientes and Santa Fe to confirm the specificity of V invictae for Solenopsis ants [70]. All sites had high levels of V invictae infections in fire ant colonies. As above, baits and hand samplings were used to collect ants and other arthropods in the immediate areas of infected fire ants. Vairimorpha invictae infections were detected only in fire ants by microscopy and PCR. The other ants tested were in the genera Ectatomma, Pachycondyla, Acromyrmex, Cremato- gaster, Pheidole, Wasmannia, Cephalotes {=Zacryptocerus), Dorymyrmex, Linepithema, Camponotus, Brachymyrmex, and Nylanderia. The other tested arthropods were in the orders Aranae, Odonata, Orthoptera, Homoptera, Hem- iptera, Psocoptera, Coleoptera, Diptera, Lepidoptera, and Hymenoptera. The host specificity of V invictae was studied in the laboratory at CMAVE evaluating the tropical fire ant, S. geminata, the southern fire ant, S. xyloni, and the Argentine ant, Linepithema humile [71]. Inoculations of S. invicta brood infected with V invictae into lab colonies of the three recipient ant species resulted in infections only in the control S. invicta in 60% of the colonies. However, the adoption of congeneric brood was not consistent, and, within the first two days, all the S. geminata and most of the S. xyloni colonies had moved the inoculated brood in the trash pile. In the case of the Argentine ant, the inoculated S. invicta brood was initially tended in 2 of the 6 nests. However, inocula appeared to be finally discarded from all nests. Since alien brood survival seemed to have been temporary, whether the lack (a) (b) Figure 5: (a) Parasitized (left) and nonparasitized Solenopsis work- er. (b) Juvenile Allomermis solenopsi emerging from worker. of infection in the test ants was due to limited interspecific cross -fostering of brood or lack of physiological susceptibility was not determined [71]. 3.1.2. Nematodes. Few records of parasitic nematodes exist for fire ants [72] . The diagnostic character for the occurrence of mermithid nematodes in ants is the enlarged gasters of the workers (Figure 5(a)). In the late 1980s, three species were reported for southern South America: (1) Tetradonema solenopsis Nickle and Jouvenaz from central-western Brazil in 2.9% of the 2,250 fire ant colonies examined; (2) a mermithid nematode from central- eastern Argentina in 0.5% of the 425 colonies examined; (3) an unidentified nematode from central- eastern Argentina, Uruguay, and southern Brazil in 4.3% of the 600 colonies sampled [73, 74]. In 2006, while researching for decapitating phorid flies in Corrientes province, Argentina, CMAVE scientist Sanford Porter redetected the presence of mermithids in S. invicta. The nematode was recently described and named as Allomer- mis solenopsi [72]. Surveys and Parasitism Rates. From 2006 to 2008, examina- tion of 489 fire ant colonies in Buenos Aires and northeastern Argentina revealed infections in 17.3% of the 29 sites, where an average of 52.3% of S. invicta or S. richteri colonies was infected. The mean number of parasitized workers per colony was 52 (range 1-500). Also pupae were found with enlarged gasters and juvenile nematodes inside. Several positive sites were further revisited for the persistence of the infections; although the infection persisted over more than one year, the parasitism rates showed great variations (Varone, unpublished data). Life Cycle. The life cycles of terrestrial or semiterrestrial mermithid nematodes are completely known only in a few cases [75-77]. In general, adults are free-living organisms, and developing stages are parasitic. Females usually lay eggs during periods of high moisture. Juveniles undergo one molt in the egg and emerge as a second-stage juvenile. The emerged juvenile enters the insect host through the anus, spiracles, or direct penetration through the cuticle to reach the body cavity [78]. Some species of mermithids develop little until the host reaches the adult stage and then migrate to the abdomen, mature, and kill the host on emergence [79] . 8 Psyche (a) (b) Figure 6: (a) Adults of Allomermis solenopsi forming a mating cluster and laying eggs (lOx). (b) Eggs (arrow) and juvenile A. solenopsi (400x). The A. solenopsi parasitism mechanism on fire ants remains unknown. However, in the laboratory, it seems that water is needed for reproduction and oviposition. It was observed that juvenile nematodes came out from dead parasitized workers only when placed in water (Porter, unpublished data). The emergence took several minutes to three days (Figure 5(b)) but failed in 65.8% of the cases. Once in the water, juvenile nematodes molted to adults and formed “mating clusters” (Figure 6(a)) with the subse- quent egg laying. Juveniles were observed developing inside the eggs, and some emerged when the eggs were crushed (Figure 6(b)). Rearing mermithid parasites of insects have been espe- cially difficult. Since mermithids are relatively common par- asites of aquatic insects, laboratory cultures have been estab- lished for a few mosquito parasites [77, 80, 81]. Creighton and Fassuliotis [82] cultured a mermithid parasite for the control of a terrestrial insect, Diahrotica halteata (Coleoptera: Chrysomelidae). Several tests were conducted to spread the infection artificially under laboratory conditions by transferring eggs or newly emerged juveniles of A. solenopsi to uninfected host colonies. The juveniles were introduced to receptor colonies using hard-boiled egg, tenebrionid larvae, and crickets, or located by hand in the ant brood pile using a humid and folded wiper (Kimwipes), Parasitism was obtained only using crickets and the wiper but at very low rates. Detrimental Effect. An important change in the behavior of infected workers was detected in the laboratory by voluntar- ily exposing for 10-minute 5 SABCL staff members to the fire ant sting (Varone, unpublished data). All infected workers tested {n = 10) lost their stinging behavior. Infected ants did not show the typical stinging position of a curved gaster, probably due to the enlarged gaster. Linear relationships were found between the sizes of the ant heads and the venom glands in 20 healthy and 20 infected workers, indicating no atrophy of the venom gland in infected workers (Varone, unpublished data). An effect was also observed with aggression tests among workers from different colonies. Contests involving two ants, one being infected and the other not, usually only resulted in antennation, while interactions between two healthy workers mostly ended in aggression and/or fighting. The infection with A. solenopsi also affected the residual survivorship of the worker ants. On average, parasitized workers {n = 872) survived 17.5 ± 12.6 days and nonpar- asitized ones {n = 1,078) survived 35.8 ± 26.1 days after the colony was removed from the field. Allomermis solenopsi adult survivorship as a free form in water was 10.7 ± 9.2 days, showing great variability among individuals with a range of 2-72 days (Varone, unpublished data). 3.1.3. Viruses. Until recently, little effort has been invested on virus infections in fire ants [36], Virus-like particles were reported from Solenopsis ants in Brazil [83]. More recently, three positive-strand RNA viruses were discovered infecting S. invicta in the United States: SINV-1, SINV-2, and SINV- 3 [84-90]. These viruses were successfully transmitted to uninfected workers by feeding food. The viruses replicated within S. invicta [88] and were associated with significant mortality among workers and larvae once infected [89]. Surveys for these viruses in Argentina were conducted from 2005 to 2008 mainly on S. invicta and some S. richteri and S. quinquecuspis. Almost 400 colonies were sampled at 32 sites in the provinces of Corrientes, Chaco, Formosa Santa Fe, and Entre Rios. In addition, 6 colonies of S. weyrauchi and S. interrupta were sampled in two sites in Bolivia along with 17 S. macdonaghi at one site in Uruguay. In all cases, workers were preserved in ethanol 96%. Reverse transcription polymerase chain reactions (RT-PCR) and multiplex PCR were used [91 ] . In Argentina, the viruses occurred in 22 (73.3%) sites with a mean colony infection of 26%, SINV-1 was present in 12 (40%) sites and in 24 (12.8%) colonies; SINV-2 in 8 (26.7%) sites and 11 (5.8%) colonies and SINV-3 in 2 (6,7%) sites and 5 (3.2%) colonies. Although SINV-1 was the most common, it frequently occurred in combination with the other two; only 29.2% of the infected colonies were exclu- sively infected with SINV-1, 37.5% had double infections (SINV-1 plus SINV-2 or SINV-3), and the remaining 33.3% were positive for all three viruses [91]. Only one S. invicta colony was exclusively infected with SINV-2, In Bolivia and Uruguay, infected colonies were not detected (Varone and Calcaterra, unpublished data). 3.2. Parasitoids 3.2.1. Phorid Flies. At least 30 Pseudacteon species (Diptera: Phoridae) are parasitoids of Solenopsis fire ants in the New World and 23 attack South American fire ants in the Solenop- sis saevissima species group [92, 93], Fire ant decapitating flies are parasitoids of individual workers [94, 95]. Female flies chase live worker ants (Figure 7) and, in a rapid aerial attack, insert their eggs into the thorax. After hatching, the larva migrates into the ant’s head, consumes all the tissue, and ultimately decapitates the host ant. A single adult fly emerges from the oral cavity 2-6 wk after the egg was laid [96]. Once the adult fly emerges, it has only a few days to look for a mate to repeat the cycle. Psyche 9 Figure 7; Pseudacteon fly chasing a Solenopsis invicta worker for oviposition (photo by S. D. Porter). Field Surveys. Before the mid 1990s, the biology, geograph- ical distribution, and abundance of most of these South American flies on fire ants were scarcely known. Borgmeier described most of the species [97], and extensive collections were conducted in the early 1970’s [98, 99]. Large-scale surveys were conducted by SAB CL and CMAVE researchers from 1995 to 2002 documenting the occurrence of P. tricuspis Borgmeier, P. curvatus Borgmeier, P. borgmeieri Schmitz, P. litoralis Borgmeier, P. obtusus, P. nocens Borgmeier, and P. affinis Borgmeier at several sites in the provinces of Buenos Aires, Santa Fe, Chaco, Formosa, Corrientes, and Fntre Rios (Briano, unpublished data). Several sites with high densities of flies were used later to run field host preference tests. Five species of flies (P. tricuspis, P. curvatus, P. litoralis, P. obtusus, and P. nocens) were shipped to the quarantine facilities at CMAVF for mass rearing, host- specificity tests, and eventual field releases. Fater, surveys were extended to western Argentina, southern Bolivia, southern Paraguay, Uruguay, and central Chile [29, 30, 100]. Fourteen Pseudacteon species were col- lected from 52% of the 720 fire ants mounds examined at 146 sites: P. curvatus, P. litoralis, P. tricuspis, P. nocens, P. obtusus, P. cultellatus Borgmeier, P. nudicornis Borgmeier, P. borgmeieri, P. solenopsidis Schmitz, and Pseudacteon near obtusus (small biotype) were associated with S. invicta. Nine species were sympatric at one site in Corrientes. Pseudacteon obtusus showed the southernmost geo- graphical distribution in Corralito, Rio Negro, Argentina (Figure 1; 40°44' S), and the westernmost in Bulnes, Bio Bio, Chile (Figure 1; 72°20' W), where it was recorded attacking Solenopsis gayi (Spinola) and Solenopsis weyrauchi in Santa Cruz (Figure 1; 2,280 m), Tucuman province. Prepuna biore- gion. Pseudacteon curvatus was one of the most abundant and widely distributed species. Its density was negatively correlated with the densities of P. obtusus and P. tricuspis [30, 101], suggesting differential habitat/host preferences and/or competitive replacement. Pseudacteon cultellatus was found attacking S. invicta in a gallery forest gap next to the Rio Parana, in Corrientes province [100]. A total of 356 P. tricuspis and 204 P. obtusus males were collected from disturbed fire ant mounds while chasing females for mating, showing sex ratios female : male of 2 : 1 and 1:1, respectively [30]. These ratios were similar to those observed in the lab- oratory at CMAVE (S. Porter, pers. comm.). However, the primary field sex ratio remained unknown. A new species, P. calderensis Calcaterra, was discovered attacking the fire ant S. interrupta in Salta and Jujuy provin- ces [ 100, 102] , a region scarcely surveyed after the last fire ant decapitating fly was discovered in South America [ 103] . Phenology and Phylogeny. Seasonal activity of phorid flies was studied at two S. invicta sites in Corrientes [ 104] . Species showed different annual and/or daily activity patterns. The highest abundance was recorded in spring and the lowest in summer. Abundance was higher close to dusk, and species diversity was highest at midday. Weather conditions affected the presence and abundance of most species except P. litoralis and P. nocens, which represented 71-79% of all female flies captured at these two sites in Corrientes. These flies were genetically very similar and showed similar patterns, suggesting a shared derived trait from a recent common ancestor. In contrast, P. cultellatus and P. nudicornis were genetically quite similar but showed different activity patterns [104]. Natural Parasitism and Detrimental Effect. Studies on nat- ural parasitism conducted at multiple sites in northeastern Argentina revealed a very low overall rate of 0.2-0. 5% worker parasitism. The highest rates per site (1.2%) and per colony (2.8%) were reported for a gallery forest in spring [104]. The presence of phorids affected the foraging capacity of S. invicta. A 50% decrease in the numbers of workers baited after the arrival of the flies suggests a potential shift in the use of food resources in favor of other ant species. However, an effect on the hierarchy of dominance of the ant assemblage was not observed [39]. Vectors. Using PCR techniques, Pseudacteon flies from Argentina were tested in the United States for their potential as vectors of the bacterium Wolbachia [105]. Seven of ten species tested were positive for four Wolbachia strains. Multiple infections were detected only in P curvatus. Strains infecting the flies were not closely related to the sequences obtained from strains infecting S. invicta and S. richteri, indicating that these flies were not vectoring Wolbachia into these fire ant species. Pseudacteon decapitating flies do not appear to vector fire ant viruses [ 106] . More recently, Pseudacteon flies were tested as potential vectors of the microsporidia K. solenopsae and V invictae [ 107] . Several species of flies that were reared from S. invicta- infected workers were confirmed as carriers of K. solenopsae. Detrimental effects on the development of fly pupae and on emergence of adult flies were not observed. These results indicated that Pseudacteon flies might vector K. solenopsae but actual vectoring remains to be confirmed. In contrast, U invictae did not infect phorids reared from infected fire ants [107]. Field Releases in the United States. In 1995, the first releases of P. tricuspis from South America (without the participa- tion/collaboration of SABCF) were conducted in Texas by 10 Psyche scientists of the University of Texas at Austin. Later, other releases of R curvatus and P. obtusus were conducted by the Texas Cooperative Extension Program in collaboration with APHIS and ARS [108]. The establishment and expansion of the flies have been systematically monitored [109-1 11]. Since 1997, five Pseudacteon species have been released by CMAVE researchers. These species and biotypes are those originally found in Brazil and Argentina and later tested in the US and/or in South America by CMAVE scientists with close collaboration of Brazilian and SABCE researchers. The species released are (1) P. tricuspis, biotype Sao Paulo, Brazil, released from 1997 to 2000 [112]; (2) P. curvatus, biotype Eas Elores, Argentina, released in 2000 [113]; (3) P. curvatus, biotype Eormosa, Argentina, released in 2003 [114]; (4) P. Utoralis, biotype Eormosa, released in 2005 [115]; (5) P. obtusus, biotype Eormosa, released in 2008 (Porter, unpublished data); (6) P. cultellatus, biotype Corrientes, released in 2010 (Porter, in progress). Several postrelease studies were conducted in the United States mainly on P. tricuspis and P. curvatus. These studies (1) confirmed predictions of the high host specificity of the pre-release studies [116, 117], (2) documented their estab- lishment and spread [108, 109, 118, 119], (3) documented seasonal abundance and rates of parasitism [120], and (4) indicated little detrimental effect of P. tricuspis on S. invicta population densities [ 121] . Other studies on distribution and efficacy are in progress. 3.2.2. Eucharitid Wasps Biology. Almost all species of the small wasps, Orasema spp. (Hymenoptera: Eucharitidae), are brood parasites of myrmicine ants in the genera Pheidole, Solenopsis, Tetramor- ium, and Wasmannia [24, 122-124]. Adult females lay their eggs into host plants, and the emerging larvae (planidia) attach themselves to foraging ant hosts and are carried into the nest [123, 125]. Adult wasps of O. simplex Heraty showed a short life span in the laboratory, estimated in 3.6 ± 1.5 days, and a single female had more than 600 mature oocytes in the ovarioles indicating a high fertility at emergence [126]. The short female survivorship and high fertility strongly suggested that O. simplex is a proovigenic species, representing a way to counteract the low probability of the phoretic transport provided by foraging worker ants to reach the nest. Occasional observations reported several types of plant tissues as oviposition sites [122, 127-130]. Recent laboratory nonchoice oviposition tests with plants of economic and ornamental importance such as corn, soybeans, lemon, red pepper, and Vinca rosea confirmed that all plants tested resulted appropriate substrates for oviposition [126]. Similarly, field observations in the surroundings of para- sitized fire ant colonies located at three sites in the provinces of Corrientes and Entre Rios revealed that 87% of the shrubs and grasses present in the genera Smilax, Paspalum, Grindelia, Eupatorium, Sesbania, Asclepias, Verbena, Sida, and Stemodia showed oviposition marks [126]. While associated with the brood, immature Orasema species produce or assimilate compounds that mimic the cuticular hydrocarbon profile of the ant host, thus avoiding detection [131]. Pupation occurs in the brood, followed by adult emergence within the ant nest. The adults exit the nest for mating and oviposition [125, 132] (Eigure8). Surveys. Eifty-five species of Orasema have been described worldwide [124, 133], and more than 200 species have been estimated for the Neotropic (Heraty, pers. comm.). Orasema parasitoids were first reported on fire ants of the S. saevissima complex in Uruguay [134]; O. xanthopus (Cameron) was later found parasitizing up to 40% of the colonies of S. invicta and other fire ant species of the same complex in Brazil [24, 25, 99, 135, 136]. In Argentina, 11 species were reported, three of which were parasitoids of fire ants in Buenos Aires, Ea Pampa, and some of the northwestern provinces [123, 137]. Between 2005 and 2007, the distribution of Orasema spe- cies and their ant hosts were intensively studied in Argentina and neighboring countries by excavating Solenopsis colonies in 73 sites in roadsides, pastures, and recreational areas [ 138] . A total of 731 colonies with brood were transported to the laboratory, separated from the soil by flotation [139] and the brood isolated [140] for Orasema individuals. Orasema was found in 29 sites parasitizing 13.5% of the 443 colonies in Argentina and 4.2% of the 288 colonies in Paraguay, Uruguay, and Bolivia. Eive species were identified: (1) O. simplex was the most abundant, occurring at 17 sites and in 63.7% of the 72 parasitized colonies; (2) O. xanthopus and (3) O. salebrosa Heraty were found only at two sites; (4) O. aenea Gahan was found parasitizing fire ants for the first time at one site in Argentina; (5) O. pireta Heraty was found at one site parasitizing an unidentified Solenopsis species in Bolivia. In Paraguay and Uruguay, only O. simplex was present [ 138] . Two new host species of Orasema within the S. saevissima complex were discovered: S. quinquecuspis in Argentina and S. macdonaghi in Uruguay. The wide variety of habitats and geographic distribution suggested that Orasema is a common parasitoid of fire ants in their native land. However, a second sampling of the Argentine sites conducted 6 to 18 months later revealed a field persistence in only 36.4% of the sites [ 138] . Eaboratory Rearing. After several attempts and different approaches, the laboratory rearing and artificial transfer of this parasitoid to nonparasitized fire ants was achieved by placing planidia together with plant tissue in fragmented receptor colonies with abundant healthy brood [126]. How- ever, this method had a very low success rate of 3.1% (only 12 adult wasps obtained from 385 planidia transferred). As previously observed by Vander Meer et al. [131], immature Orasema individuals were tended by ant workers as their own brood, with no aggression. However, several Orasema adults were found partially preyed, suggesting the loss of host-specific compounds soon after emergence. 3.2.3. Myrmecolacid Strepsipteran Eife Cycle. The ant parasitoid, Caenocholax fenyesi Pierce (Strepsiptera: Myrmecolacidae), has an unusual life cycle in Psyche 11 Figure 8: Life cycle of Orasema. (a) Planidia (arrows) attached to ant larvae, (b) First- instar larva (arrow), (c) Developed larva (ant larva behind), (d) Pupa, (e) Adult female, (f) Adult male. which males parasitize ants while females parasitize crickets [141, 142]; it is currently the only extant species in its genus. The male of this species has the smallest genome (108 Mb) studied so far [143]. The lack of information on its host associations has led to several speculations. Distribution and Hosts. Caenocholax fenyesi has a wide geographical distribution occurring from southern United States to Chile and Argentina and infects seven ant species from three subfamilies with discontinuous distributions [144-146]. In Mexico, Central America, or Ecuador where S. invicta did not occur, C. fenyesi parasitized other ant species [147]. Males of C. fenyesi had been previously collected in Salta province in northwestern Argentina where mtDNA haplotypes of S. invicta occur [31, 145]. Parasitoid-Host Association. In 2003-2004, 15 C. fenyesi males were isolated in the laboratory from four S. invicta colonies originally collected in Corrientes and Formosa provinces. This was the first report of C. fenyesi parasitizing S. invicta in South America [144]. However, the parasitism rate was less than 0.2%. In 2003 and 2005, additional surveys for C. fenyesi females using light traps, pitfall traps, and sweep nets were conducted in areas of Corrientes and Formosa where parasitized S. invicta colonies have been found previously. None of the 456 orthopterans, 9 dyctiopterans (Mantodea), and 6 phasmodeans (Proscopidae) collected were parasitized by C. fenyesi [ 144] . Cryptic Diversity. Recent molecular analysis revealed that C. fenyesi contained at least 10 cryptic lineages consistent with separate species and that the genetic diversity was strongly structured by geography and host association of the female [147]. Further studies revealed slight variation in key morphological characters, so several species might not be strictly cryptic (J. Kathiritamby, pers. comm.). 3.3. Parasites 3.3.1. Social Parasitic Ant Life History. Only one social parasite, the parasitic ant Solenopsis {=Labauchena) daguerrei, has been effectively reported for fire ants [ 148] . Like most other social inquilines, S. daguerrei has lost the worker caste and produces only reproductive queens and males [149] with a numerical sex ratio female : male of 3 : 1 [149]. The parasite commandeers the host’s workers to care for its own brood and provide them with food [150]. Mature parasite queens that have shed their wings (dealates) are only one-tenth the weight of fire ant host queens [149]; they attach or “yoke” themselves to the neck of the host queen with their mandibles and ride around on her back or sides (Figure 9). A host queen may have two or three parasite queens attached to her neck and another half a dozen to other parts of her body, apparently intercepting food intended for the host queens and inhibiting host queen egg production [ 149, 150] . Parasitic queens attached to hosts survived longer than those not attached [151]. Since the parasites are treated like nestmates by host workers, S. daguerrei apparently avoids the chemical recog- nition system of its hosts by mimicking or assimilating the cuticular hydrocarbons responsible for the host colony odor [150-152]. The ability of S. daguerrei to match host colony odor is likely to be sensitive to the strong patterns of genetic differentiation of the hosts, being consistent with the hypothesis that these parasites are locally adapted to their hosts, and thus specific to their associated host ecotype. 12 Psyche Figure 9: Two queens of Solenopsis daguerrei yoking a queen of Solenopsis richteri. However, many laboratory and field attempts to artifi- cially propagate this parasitic ant using sympatric colonies have failed [153]. Parasitized and nonparasitized colonies were used as source and receptor colonies, respectively. Sev- eral approaches were used, such as transference of parasitic queens, sexuals and/or pupae, contact of entire colonies, transplanting of entire field colonies, and the transfer of newly mated parasitic queens. Field Surveys. Surveys for S. daguerrei in fire ant populations were conducted in Argentina, Brazil, Bolivia, Paraguay, and Uruguay by ARS scientists from 1974 to 1996 [154]. The examination of 12,180 fire ant colonies revealed occurrence of this parasitic ant in a variety of habitats in northeastern Argentina, Uruguay, and southern Brazil. Parasitization occurred in S. richteri, S. quinquecuspis, S. invicta, S. mac- donaghi, and S. saevissima, all members of the S. saevissima species group. The overall parasitism rates ranged from 1 to 7% of the colonies. The sites with the highest parasitism rates were San Eladio, Buenos Aires, Argentina, with 7% and Dourados, Mato Grosso do Sul, Brazil with 6,2% (Figure 1). However, surveys in the 1970s revealed that in some localities S. daguerrei was found in 24-70% of the colonies [59, 150]. Phenology and Mating. Most of the available information on S. daguerrei phenology and breeding biology was reported from S. richteri host populations in Buenos Aires province, Argentina. Adults of S. daguerrei were more common in fall- early winter, contrasting with the low seasonality showed by parasitic populations in northern Argentina and southern Brazil [148]. Mating flights were not observed. In a labo- ratory study, a total of 756 individuals of S. daguerrei were captured flying out from S. richteri host colonies; of those, 738 (98%) were females (87% were inseminated) and only 18 (2%) were males [151]. A later examination of the host colonies showed that no parasitic males were found inside and that 40% of the parasitic females were inseminated. As previously suggested [150], these observations confirmed that copulation occurred mainly inside the nests with nest- mates, resulting in a high level of inbreeding [ 155] . However, cryptic dispersal of males and mating with noninseminated females might occur, thereby reducing inbreeding. Also, the apparent poor dispersal ability of this parasite suggested a strong genetic differentiation on both a micro- and macro- geographic scale (Bouwma, unpublished data). Detrimental Effect. Field studies conducted in the area of San Eladio in Argentina on S. richteri colonies [149, 156] showed that, compared to nonparasitized colonies, parasitized colonies (1) had less worker brood, (2) produced the sexual caste later in the season, and (3) had fewer queens (2.9 versus 5.5). This suggested that the parasite might drive the host toward monogyny. Also, in field populations of S. richteri, lower mound densities were found in areas with presence of the parasite compared to parasite-free sites, suggesting some potential for the biological control of fire ants. In a few laboratory studies, S. daguerrei was reported to kill the host queens by decapitation [148] and to cause the colony to collapse [ 150] . Evolutionary Traits, hike other social parasites, it is believed that S. daguerrei must be highly specialized and has evolved the ability to exploit the social system of their hosts [155]. Recent studies on the evolutionary history of members of the S. saevissima species group were based on morphological characters [1] and mtDNA sequences [157]. These studies showed that S. daguerrei occupied a basal position in the group and that it was a close relative of its several hosts. It belonged to a larger clade, sister of the host clade, following the loose version of Emery’s rule [158] and indicating that S. daguerrei would not have evolved directly from their hosts within the S. saevissima group (strict version of Emery’s rule) [159-162]. This is supported by the single origin of social parasitism suggested for S. daguerrei collected from S. invicta and S. richteri host colonies over a vast geographic area [ 157] . In 2007, molecular studies to determine the genetic structure of S. daguerrei in Argentina, southern Brazil, and Paraguay revealed a high genetic variability and the probable presence of a complex with new species (Bouwma, unpublished data). Vectors. To test if S. daguerrei was a vector of the bacterium Wolbachia, three individuals of the parasitic ant were found to be infected representing a new host record for Wolbachia [105]. Sequence analyses revealed that each individual con- tained the unusual number of eight Wolbachia variants. In total, nine unique sequences or strains were found, two of which were identical to the sequences obtained from their fire ant hosts S. invicta and S. richteri. This suggested horizontal transmission of Wolbachia between S. daguerrei and its hosts. 4. Conclusions Native fire ants in southern South America, mainly S. invicta, were dominant ants in several regions, but these ant communities usually included several abundant competitors. This strong competitive environment in their homeland contrasts with the situation in invaded communities in North America. The release from interspecific competition in the new habitats and the escape from coevolved natural enemies seem to strongly contribute to S. invicta s successful invasions in North America. Psyche 13 Since 1987, the field surveys and the examination of ap- proximately 14,000 fire ant colonies in almost 1,000 col- lecting sites in Argentina and neighboring countries have documented the presence of the microsporidia Kneallhazia solenopsae and Vairimorpha invictae, the nematode Allom- ermis solenopsi, three S. invicta viruses, 14 species of Pseu- dacteon decapitating flies, 5 species of the parasitoid wasp Orasema, the strepsipteran Caenocholax fenyesi, and the par- asitic ant Solenopsis daguerrei. Kneallhazia solenopsae was the most common pathogen of native fire ants, showing a wide distribution and high field persistence, mainly infecting S. richteri. On the other hand, invictae showed a narrower distribution, a lower and disjunct overall occurrence, and higher prevalence in S. invicta. Both diseases showed the ability to infect monogyne and polygyne populations, and, at times and in certain areas, they reached epizootic levels, representing the highest infection rates ever reported for South America. Despite this, the natural occurrence of dual infections in the field was very low and similar to probability predictions (combined probability of finding at random K. solenopsae and invictae simultaneously in the same colony). Their high intracolonial prevalence indicated that these microsporidia were important chronic diseases of fire ants. Both diseases showed several deleterious effects on individual colonies and field populations, and their ecological and phys- iological host ranges were restricted to closely related ants in the genus Solenopsis. These facts suggested that they might be good self-sustaining organisms for the classical biological control of the imported fire ants in the United States, with little or no risk to native ants and other arthropods. The horizontal transmission of both pathogens accom- plished by CMAVE scientists in the United States has allowed specificity and efficacy trials under laboratory and field conditions. Once field release of U invictae is approved for the United States, the ability to transmit this disease into S. invicta colonies will accelerate the artificial field infection, its dispersal, and the eventual faster decline in imported fire ant population densities. The finding and identification of the nematode A. solenopsi represented a new species discovery. The overall occurrence was low, and the parasitism rates in the field were highly variable. Laboratory rearing was difficult, and many aspects of its life cycle remain unknown. However, infected fire ants showed shorter longevity and interesting changes in their behavior. Further efforts with this organism are recom- mended. The use of molecular techniques facilitated the screening for fire ant viruses. Of the three viruses found, SINV-1 was the most common and abundant and was frequently found in combination with the other two. Although no detrimental effect was observed in field infected colonies in the United States, under certain stress conditions, colonies might col- lapse. Consequently, a combination of these viruses and their genetic manipulation represent a potential alternative to traditional insecticides for controlling imported fire ants. Among the parasitoids, by far, Pseudacteon flies were the most frequent and abundant, with 14 species collected, many of which were sympatric. Pseudacteon curvatus was the most abundant in many areas followed by P. obtusus, one of the most widely distributed. However, the overall natural parasitism rate was very low, indicating low direct effect on worker mortality. As expected, the presence of phorids decreased the foraging capacity of the workers. A new species was discovered, and several new fire ant hosts were docu- mented. Many of the species exhibited different annual and daily activity patterns, and some of them showed the possibility of vectoring K. solenopsae. Five Pseudacteon species were released by ARS in the United States. Several postrelease studies documented their establishment, spread, seasonal abundance, and parasitism rates and confirmed their high specificity for the imported fire ants. Unfortunately, significant reductions of S. invicta population densities have not been observed yet. A considerable amount of new information on the biolo- gy of Orasema wasps was obtained. Their overall occurrence was fairly high, and they were found in many habitats over a wide geographic range. In addition, five species were identified and two new Solenopsis hosts were discovered. The laboratory rearing and artificial transmission was obtained for the first time but at very low rates. A wide variety of economically important plants were confirmed as oviposi- tion substrates. Cosmetic damage to many plants during the oviposition process probably precludes this organism from further testing for fire ant biological control. Similarly, useful biological information was gathered for Caenocholax fenyesi mainly on its host associations. The finding of parasitism on S. invicta represented the first record in South America. However, parasitism rates were extremely low. In addition, preliminary attempts to elucidate its complicated life cycle by finding the females in the field have failed. At this point, the use of this organism as a biological control agent of fire ants seems very unlikely. The parasitic ant S. daguerrei parasitizes several fire ant species within the S. saevissima group. The overall occur- rence was low, but, in certain areas, it reached high parasitism rates, mainly on S. invicta in Brazil and S. richteri in Argentina. New biological observations were reported on phenology and mating, and several detrimental effects were documented for field populations of S. richteri. In addition, molecular studies on its evolutionary history revealed close host relatedness and wide genetic variability, suggesting the potential presence of a complex of species. Future studies on parasite-host matching are needed to identify the most suitable species or biotype for biological control of S. invicta in the US. Unfortunately, unsolved rearing problems and the inability to transfer this parasite to nonparasitized fire ant colonies have discouraged further testing. In summary, after 23 years of intensive field work and laboratory research, the main objective of the program to find a complex of fire ant natural enemies and to evaluate their specificity and suitability for field release has, indeed, been accomplished. Many natural enemies were found, investigated, and developed in close collaboration with ARS scientists in the United States. Several of these organisms were field released, and their ecology and efficacy in the new habitats continue to be evaluated. Many of these biological 14 Psyche control agents could be available for use in other countries or regions invaded by fire ants. The pioneering studies conducted in South America on native fire ants and their natural enemies have served to greatly encourage further investigations by many scientists and institutions in the United States and other countries. These efforts have advanced the implementation of area- wide biological control programs. Still, promising organisms such as U invictae, S. daguerrei, A. solenopsi, viruses, and, maybe, more species or biotypes of Pseudacteon flies should be further investigated for eventual field release in the near future. It is expected that the final outcome of current and future programs will be the decrease of the imported fire ant population densities and their damage. Acknowledgments The authors thank all past and current ARS research leaders, scientists, and technicians at the Imported Fire Ants and Household Insects Unit in Gainesville, FL, for their continued support to initiate and codevelop the Imported Fire Ant Project in Argentina. Several scientists, D. P. Wojcik, D. P. Jouvenaz (deceased), R. S. Patterson, D. F. Williams, D. H. Oi, and S. D. Porter, have periodically visited SABCL and intensively collaborated on the progress of the project. Recent visits by S. M. Valles, A. Bowma, D. D. Shoemaker, and M. S. Ascunce have resulted in considerable molecular work. A number of local technicians and students, M. Carranza, J. Jara (deceased), A. Delgado, J. Livore, S. Cabrera, L. Ramirez, L. Nunez, D. lele, J. Sacco, and M. Manteca Acosta, assisted with field and lab work. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 345932, 7 pages dohlO.l 155/2012/345932 Research Article Herbivore Larval Development at Low Springtime Temperatures: The Importance of Short Periods of Heating in the Field Esther Miiller^ and Elisabeth Obermaier^ ^ Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland ^ Department of Animal Ecology and Tropical Biology, University of Wurzburg, Am Huhland, 97074 Wurzburg, Germany Correspondence should be addressed to Elisabeth Obermaier, o.maier@biozentrum.uni-wuerzburg.de Received 4 July 2011; Revised 14 October 2011; Accepted 29 October 2011 Academic Editor: Panagiotis Milonas Copyright © 2012 E. Muller and E. Obermaier. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Temperature has been shown to play an important role in the life cycles of insects. Early season feeders in Palaearctic regions profit by the high nutritional quality of their host plants early in the year, but face the problem of having to develop at low average springtime temperatures. This study examines the influence of short periods of heating in the field on larval development and on mortality with the model system Galeruca tanaceti L. (Coleoptera: Chrysomelidae), an early season feeder, that hatches at low springtime temperatures. Eield and laboratory experiments under different constant and variable temperature regimes were performed. While in the field, the average daily temperature was close to the lower developmental threshold of the species of 10.9°C; maximum temperatures of above 30° C were sometimes reached. Larvae developed significantly faster, and pupae were heavier, in the field and in an assay with short periods of heating than at the same average temperature under constant conditions in the laboratory. We conclude that larvae profit substantially from short periods of heating and temperature variation in the field and that intervals of high temperature enable insect survival and exploitation of nutrient- rich food resources at early times in the season. 1. Introduction With their early arrival in spring, “early season” or “flush” feeders take advantage of the high nutritional quality of their potential food source, itself the result of a high concentration of nitrogen in growing leaves [ 1 ] . Many insects prefer young plants or tissues to old ones and are therefore restricted to feeding at certain times of the season [2]. These herbivores may profit by a fast development and high pupal weight due to the readily assimilated nitrogen at this time of the year [3] . On the other hand, at least in Palaearctic regions, herbivores which appear early in the season are vulnerable to low mean and minimum daily temperatures, which might often drop below the lower developmental threshold of the species in question and severely reduce growth and development. Insects that specialize in using ephemeral resources (e.g., young leaves) should be especially sensitive when the timing of the availability of those resources is unpredictable. Asyn- chrony with plant phenology and factors that promote it, such as climate change, have a considerable impact on the dynamics of spring-feeding herbivores [4]. Synchronization between bud burst and egg hatch in a Lepidopteran species varies widely with spring temperatures, while an artificial ele- vation of temperature prolongs the total period of budburst but shortens the period of egg hatching [5]. Climatic parameters in general have been shown to play an important role in insect life. The most important micro- climatic parameters are humidity, solar radiation, and wind, as insects essentially heat up by radiation and lose this heat through convection. Insects, and especially their larvae, are highly sensitive to these variables because of their small size and their relatively large surface area [6]. Temperature plays a major role within the abiotic factors, representing one of the most important environmental factors in the life cycle of insects. In particular, it has been shown to have a con- siderable influence on their development [7-9]. In general, there is an optimal temperature for the development of a species within a favoured range, where mortality is especially low and development time short. A number of adverse phys- iological reactions can occur when development takes place 2 Psyche at temperatures below this optimum. The chemical reactions of the endocrine system slow down with the cold [10], and growth rate is reduced. Some insects step into a diapause to escape low temperatures [11]. Cold temperatures are also able to change the correlation between body size and the beginning of metamorphosis. Larvae that mature at lower temperatures often produce under- or oversized adults [10]. Below a certain threshold, many insects come to a de- velopmental arrest, but can survive. The temperature at which growth stops is referred to as the “lower developmental threshold.” It is specific to each species and is known pre- cisely for only a few. For the wax moth Galleria mellonella Linnaeus, for example, the lower developmental threshold is 19° C, while for the Lepidopteran Xestia C-nigrum Linnaeus, it is only 5° C [10] — despite their distribution area being very similar. As with growth rate, development time is also related to temperature. Typically, development time decreases expo- nentially with increasing temperature [12-14] . The influence of temperature upon insect development is related not only to the daily mean average, but also to the rate of temperature change. Likewise, growth is expected to be related to both duration and quantum of temperature above thresholds. Insects have frequently been shown to de- velop more rapidly, lay more eggs, suffer a lower mortality, or complete their life cycle within a wider temperature range when temperatures are fluctuating, like they predominate in the field, than at constant temperatures, as long as the max- imum and minimum of the fluctuating temperature are within the optimal range of development for the organism [ 15-17] . While the effects of fluctuating versus constant tem- peratures have already been well studied, little is currently known about the effects of short periods of heating for larval development and for the growth of Palaearctic insect species in the field. This might be achieved for larvae and adults through, for example, their basking behaviour [18], but Richards and Suanraksa [19] have also shown that energy re- serves for embryonic development were sufficient for consid- erable periods spent below the constant temperature thresh- old, provided that enough time was spent at much higher temperatures beforehand. The polyphagous leaf beetle Galeruca tanaceti Linnaeus was used as the model organism for studying the influence of short periods of heating and temperature variations in the field on the larval development of an early season feeder. The adult females deposit their egg clutches with the beginning of the fall in herbaceous vegetation, preferentially on high and dry blades of grass. While the adult beetles die, the eggs are the overwintering form. Between March and April, at cold springtime temperatures, the larvae emerge and develop while feeding on the first young leaves of their host plants [3] . After approximately four weeks of feeding, the larvae pupate after the fourth larval stage, in the soil. In this study, we examine the influence of short periods of heating and of temperature variation in the field on herbivore larval development. An adaptation to low temperatures close to the lower developmental threshold is discussed as a pre- requisite for early season feeders in Palaearctic regions, so that they are able to exploit the high nutritional quality of their food resource at this time of the year. Larval development was studied under field conditions, and under different temperature regimes in the laboratory, to calculate the lower developmental threshold of the species and to help evaluate the field data. 2. Material and Methods 2.1. Study System. The field experiment was performed on dry grassland in the Flohe Wann Nature Reserve in Lower Frankonia (Northern Bavaria, Germany, 50°03' N, 10°35' E). The study site was grazed by sheep until a few years ago. In the two years prior to the study, the site was no longer man- aged. Randomly picked plots were chosen with the help of GIS and GPS. The tansy leaf beetle, Galeruca tanaceti, is polyphagous and feeds on species of the families Asteraceae, Brassicaceae, Garyophyllaceae, Dipsacaceae, Liliaceae, Lamiaceae, Polyg- onaceae, and Solanaceae [3]. In the study area, one of the main host plants of G. tanaceti is yarrow, Achillea millefolium L. (Asterales: Asteraceae) [20] , but larvae can also be found feeding on Gentaurea jacea L. (Asterales: Asteraceae) and Salvia pratensis L. (Lamiales: Lamiaceae). In fall, females of the tansy leaf beetle deposit their egg clutches on vertical structures within the herbaceous vegeta- tion layer, where the egg clutches then hibernate [21]. After hatching in March-April, the larvae seek suitable host plants close to the oviposition site, on which they feed for about four weeks until pupation [3]. After pupation, the adults can be found from early lune onwards before they enter re- productive diapause in midsummer. 2.2. Larval Development and Mortality. Prior to the experi- ments, in fall, egg clutches of G. tanaceti were collected from different sites of the reserve and stored over the winter in a closed cage under natural climatic conditions. In spring, egg clutches were transferred to the laboratory and kept at room temperature until hatching of the larvae. Egg clutches were checked daily for hatching larvae. In the field as well as in the climate chambers, the development time of larvae from eclosion to pupation, pupal weight, and mortality rate were all registered. Only larvae that hatched within 24 h of the start of the experiment were used. The larvae of the different egg clutches were mixed prior to use, to ensure a random assignment to treatments. 2.3. Experimental Setup 2.3.1. Field Experiments. After transfer to the field, larvae developed on a dry grassland site in the Hohe Wann Nature Reserve in 40 completely closed gauze cages with a size of 40 X 40 cm. The cages consisted of a wooden frame covered with gauze mesh on all sides, including the top. The mesh width of the gauze was 1.2 mm because of the very small size of the newly hatched larvae. It is possible the gauze may have shaded the larvae and reduced T max; however, the negative effects of closed cages on larval development in comparison to treatments with open cages could not be observed [Muller, unpublished data]. To avoid the escape of the larvae, the cages were placed flush with the soil. Psyche 3 Additionally, the bottom rim was sealed with soil. All cages included the same number of plants of the main host plant of the beetle, Achillea millefolium L.. 10 larvae, hatched within 24 hours of the start of the experiment, were positioned on the same host plant in the centre of each cage. The larvae were placed together in groups of 10 larvae per cage to simulate natural conditions as closely as possible, for usually multiple larvae hatch out of the egg clutch at the same time. After 28 days, at the end of the feeding phase, the remaining larvae were counted, collected, kept singly in boxes under natural conditions, and provided with food until pupation. All pupae were weighed immediately after pupation. The experiment was repeated once (1st cycle: 4/22-5/19 and 2nd cycle: 5/19-6/15). Additionally the air temperature was recorded during both cycles of the experiment. For this, a thermobutton (Dallas Semiconductor “DS 1921L-F5XTher- mochron iButton”) was installed in each cage at a 30 cm height (mean height of egg clutches) and shaded. The tem- perature was recorded once every hour during both experi- mental periods. 2.3.2. Laboratory Experiments. Laboratory treatment groups were installed as follows: constant temperatures at 15°C (1) and 23° C (2), variable temperature with short periods of heating at a temperature of 18°C for 22.5 hours, and at 28° C for 1.5 hours (daily mean: 18.6°C) (3). All three climatic chambers received the same daylight conditions, according to the conditions in the field (L/D: 14/10 hours). The experi- ment began when the larvae, which had hatched within less than 24 hours, were exposed to a certain temperature according to their treatment group. The larvae were kept singly in plastic boxes to exclude interaction influences and were fed with their main host plant, Achillea millefolium ad libitum. 17 larvae were kept in each climatic chamber. along with the duration in calendar days for all larvae investigated (field and laboratory). Degree days are a measuring unit for the amount of heat that acts on animals or plants above a specific developmental threshold temperature. This amount is counted over a period of 24 hours. One degree day is counted for every degree over the specific developmental threshold (lower developmental threshold). Thus, multiple degree days can be accumulated over a period of 24 hours [26] . Different kinds of calculations are possible. If the minimal temperature does not drop below the lower developmental threshold, the so-called “average method” is used (1). This method was used for the climate chamber data. DD (maximal temperature + minimal temperature) 2 -base temperature. ( 1 ) If the minimal temperature drops below the lower de- velopmental threshold, the “modified sine wave method” is used. This method takes advantage of the fact that daily temperatures behave similarly to sine functions. The sum of degree days is calculated via the areas under the sine waves. A reference table is available for ease of use, where the degree days can be read off for precise minimal temperatures [26]. The “sine wave method” was used for calculation of the degree days of the field experiment, because the temperatures fluctuated and dropped below the base temperature. For calculation purposes, the maximum and minimum temper- ature of each cage on each day was noted. Afterwards, the average maximum and the average minimum temperature across all cages were calculated, separately for each run. With these averages, the number of degree days was taken from the reference table for each day. 2.3.3. Calculation of Lower Developmental Threshold and Degree Days. In this study, for the calculation and graphical definition of the lower developmental threshold, develop- mental data from two constant temperatures were used (15°C and 23°C), after the line-fitting method of Ikemoto and Takai [22]. A linear regression was calculated of the rate of development (1/D) for the larvae of both chambers and related to the linear degree-day model (e.g., [23, 24]). This is based on the assumption that the rate of development (1/D) increases linearly with incubation temperature T in the range of temperatures usually experienced. The lower de- velopmental threshold results in the intersection of the regression line with the x-axes. In poikilothermic organisms, it is assumed that the de- velopmental rate depends on temperature in such a way that the product of the duration of development D (days) and the incubation temperature T (degrees) above the species- specific lower developmental threshold to is represented by a constant k (degree days). Thus, a specific number of de- gree days, the so-called “thermal constant k” (measured in degree days [DD]), are required for an individual to com- plete development (e.g., [25, 26]). For identification of the development time, the duration in degree days was calculated 2.3.4. Statistical Analysis. The calculation of the degree days was performed after Herms [26]. Treatment groups of larval weight, development time, and mortality were compared by a GLM after testing for normal distribution. All statistical anal- yses were performed with Excel 2003 or SPSS 14 for Micro- soft Windows. 3. Results 3.1. Laboratory Experiment. The climate chamber experi- ment showed a significant difference in the development time of the larvae over all treatment groups (F = 1911.806; P < 0.001; ni = 5; n 2 = 13; ^3 = 14) (Table 1). Mean de- velopment time of larvae differed between 24 days (constant temperature group: 23° C) and more than 52 days (constant temperature group: 15° C). In the treatment with variable temperatures and a short period of heating (18°C/28°C; mean daily temperature: 18.6° C) and a mean development time of 32 days until pupation, development was already strongly accelerated when compared to the 15°C group with constant temperatures. Likewise, the weights of the pupae differed significantly between the treatment groups (F = 52.483; P < 0.001; ni = 5; n 2 = 13; n^ = 14). The pupae of the 15° C treatment group 4 Psyche Table 1: Comparison of larval development of the different treatment groups in the climate chambers and in the field with a statistics column, showing the differences between all groups. Different letters indicate significant differences between treatment groups, shown with development time, weight, and mortality. Treatment group 18°C/28°C Climate chamber 23°C 15°C (1) run (15.0°C ± Field 0.3) (2) run(13.0°C±0.2) Statistics Development time [days] (x ± SD) 32.00 ± 1.03^ 24.00 ± 0.75'' 52.00 ± 0.89" 31.00 ± 0.0" 31.00 ± 0.00" F = 1063.43; P < 0.001 Physiological development time [degree days] 265.64 290.40 221.40 293.00 200.00 Pupal weight [mg] (x ± SD) 42.00 ± 5.5D 49.00 ± 3.76" 18.00 ± 1.95" 41.00 ± 14.76" 39.00 ± 16.55" F = 17.985; P < 0.001 Mortality [%] 18^ 23" 70'' 90'' 90'' F = 36.442; P < 0.001 were significantly lighter than those of the other treatment groups in spite of their long development time (Table 1). Regarding pupal weight, the pupae of the treatment group with variable temperatures and a short period of heating in the climatic chamber were heavier than those in the constant 15°C group. Among the laboratory treatment groups, mortality of the 15°C treatment was highest (>70%) and differed sig- nificantly from the mortality of the larvae of the two other groups (F = 6.204; P = 0.001; n = 17). At 15°C, 12 larvae died, at 23°C, 4 larvae died, and at 18°C/28°C, 3 larvae did (Table 1). Based on the developmental data of the two chambers with constant temperatures (15°C and 23°C), and an extrapolation after the line-fitting method [22], a “lower developmental threshold” of To = 10.9° C was calculated for the development of Galeruca tanaceti. 3.2. Field Experiment. In the field experiment, the mean daily temperature differed between the two cycles of the experiment where larvae were exposed. The mean daily temperature of the first cycle (15°C ± 0,33) (4/22-5/19) was higher than that of the second cycle (13°C ± 0.2) (5/19- 6/15) (Figure 1). Maximum daily temperatures varied from 15°C to 33°C in the first cycle and from 13°C to 32°C in the second cycle, reaching 30° C and above during several days in each experimental cycle. Regarding the degree days, the first cycle contained more degree days and got a higher physiological development time of /ci = 293 °d compared to fewer degree days and lower physiological development time {k 2 = 200° d) in the second cycle, caused by lower min- imum daily temperatures and more days with a lower mean temperature (Figure 1, Table 1). Nevertheless, all larvae of both groups took 3 1 days for development. The mean pupal weight was 41 mg ±14.76 {n = 400) in the first cycle and 39 mg ±16.55 {n = 400) in the second one. The rate of mortality was very high, and 90% of the larvae in both cycles died or disappeared. 4. Discussion This study investigates how larvae of an early season feeder, the leaf beetle G. tanaceti, manage to develop at relatively low Figure 1: Temperature gradation for both cycles of the field experiment with mean (♦), maximum (■), and minimum (A) mean daily temperatures measured at 0.3 m height (n = 40). springtime temperatures and profit at the same time by the high nutritional quality of their host plants at that time of the year. Field and laboratory experiments under different tem- perature regimes were performed. Mean daily temperatures in the field turned out to exceed the lower developmental threshold of the species (10.9°C) by only a few degrees Cel- sius. The lower developmental threshold can vary between different Coleopteran species. The Curculionidae species Cionus latefasciatus Voss, for example, has a threshold tem- perature of 7.7° C [27], whereas for the lower developmental threshold of G. tanaceti-larvae, a temperature of 10.9° C was determined according to the developmental data of the two chambers with constant temperatures (15°C and 23° C) and an extrapolation after the line-fitting method [22] . Mean daily temperatures during both cycles of the field experiment ((1) cycle: 15°C ± 0.33; (2) cycle: 13°C ± 0.20) therefore exceeded, by two to four degrees on average, the lower developmental threshold of the species, at which no growth or development occurs. Minimum daily temperatures were almost always below the lower developmental threshold, sometimes even dropping to zero degrees Celsius. In general. Psyche 5 low temperatures can affect development negatively by caus- ing low pupal weight and prolonged development time and thereby reducing fitness via predation pressure [28], dis- advantages in mating [29-31] or fewer and smaller offspring [32]. Mean daily temperatures in the field were therefore either lower than or equal to the constant 15°C climatic chamber. In spite of this, larval development in the field, provided with short periods of heating, differed significantly from that of the larvae in the constant 15°C climatic chamber. Larvae in the field showed an almost twice as fast development and were more than twice as heavy as in the constant 15°C cli- matic chamber. We assume that larval development at very low temperatures, even if partly below the lower develop- mental threshold, is possible if there are heating periods with higher temperatures in between and which can be taken advantage of by the larvae. Maximum daily temperatures in the field varied between 15°C and 33° C, reaching values of 30° C and above during several days in each experimental cycle. The use of short periods of high temperature and the regulation of body temperature to maximise radiative gain can be achieved, for example, by basking behaviour [6, 19, 33]. Insects are often found basking on leaves where temperatures are reached that are several degrees higher than the surrounding air, caused by reflected radiation, long- wave radiation reradiated from the warm leaf, and possibly convection and heat conducted from the warm leaf [19]. Obviously, basking is especially important during colder weather periods. For adult G. tanaceti-heetles, surface tem- perature was on average 4°C higher while basking in the sun as compared to that of the plant surface on which they were resting (Tearasa, pers. communication). Beside exposure to short periods of heating, temperature variation in comparison with constant temperatures can also help explain the more rapid development of insects at the same mean daily temperatures [17]. Blanckenhorn [11] has described, using the yellow dung fly, how development time is shorter at variable temperatures in the field than with the same mean constant temperature. The same was found by Sehnal [10] in the context of development at low temperatures. This phenomenon, therefore, seems to be fairly widespread among insects; its underlying mechanisms, however, remain poorly understood. With the field and laboratory data available in this study, it is difficult to dis- criminate between the two mechanisms. T max, however, seems to be a very important factor for larval development at relatively cold average springtime temperatures, as indicated by the climatic data from the field and larval development in the 18/28° C chamber. The 18/28°C chamber, with a relatively low mean daily temperature (18.6°C), shows that a short time spent heating per day (in the case of the 18/28°C chamber, 1.5 h per day) seems to be sufficient to accelerate larval development and change developmental parameters, such as development time and pupal weight, to values comparable to those in the field. Mortality is much lower than in the field, probably because of the constant conditions in the climatic chamber and the absence of natural enemies or adverse abiotic factors such as rain and wind. The short period of heating per day in the cli- matic chamber might be equivalent to heating by sunshine or the higher temperatures over midday in the field (Figure 1). In the field, however, larvae may also regulate body temper- ature independently from ambient temperatures by basking, and this hinders the comparison of field with lab work. The larvae of the constantly heated 23° C chamber showed, in spite of this, the shortest development time, with 24 days on average and the highest pupal weight compared to the 15° C chamber. This temperature might resemble one close to the temperature optimum of the species. Further- more, this optimal development demonstrates that the pro- longed larval development and high mortality in the 15°C chamber were not due to insufficient conditions in the laboratory, but rather that the chosen temperature regime and progression was responsible for the values obtained. A comparison of the two constant chambers of 15°C and 23° C shows, additionally, that with the chosen temperature regimes, there was neither a positive correlation between body size and development time (calendar days) — as is com- monly described in life history theory — nor was there a negative correlation of body size and development time (ex- pressed as degree days), as found by Blanckenhorn [11]. This might be due to extremely unfavourable conditions at constant 15°C which could not be completely compensated for by a longer time of development. In any case, surviving pupae stayed rather small at this low temperature. The findings of Ratte [12] suggest yet another explana- tion for the better results of the field study in comparison to the results of the constant 15°C treatment group in the laboratory. He concluded that some insects grow faster if they are also exposed to temperatures below their lower de- velopmental threshold. The larvae of G. tanaceti were ex- posed to temperatures below their lower developmental threshold Tq in the field, but not in the constantly heated 15°C chamber. The differing development times of 52 days in the climate chamber and only 31 days in the field might also be partly explained by this observation. The identical development time of all larvae of both field runs is of considerable interest. One explanation could be that the transport from the field to the laboratory after the larvae stopped feeding represented some kind of signal for pupation. Pupation may have been induced by a temperature change, the handling itself, or some alteration of other mi- croclimatic factors. G. tanaceti is well adapted to its early appearance in March/April at low springtime temperatures. Larvae are black in colour and so absorb the sunlight and use it for movement, feeding, and metabolism. It has been shown that, at least in some instances, specimens from the warmer parts of the range are generally brighter and paler than those from the same taxon collected in cooler areas [6]. Furthermore, overwintering in the egg stage enables larval development at precisely the time of the first bud burst, when the quality of the food resource is especially high. Additionally, these “early season feeders” have only a few feeding competitors at the time of their major growth and development. Finally, the reduced development larvae, suffering from low temperatures close to or below the lower 6 Psyche developmental threshold, can be compensated for by short periods of heating or temperature variation. They enable the larvae to develop at almost normal speed even at early springtime conditions in the field. Disadvantages, such as a possible (and possibly worsening) lack of synchronization of hatching with the availability of the host plants due to climate change [4, 5], and slow development at temperatures close to the lower developmental threshold, are at least partly compensated for in the aforementioned ways. As long as the extreme values of thermal conditions are not too high to induce stress in the organisms [34], short periods of heating in the first place enable the exploitation of nutrient-rich food resources at this time of the year. Acknowledgments The authors thank H.-J. 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Naturalists’ Handbook, Richmond Publishing, Slough, UK, 1991. [19] A. G. Richards and S. Suanraksa, “Energy expenditure during embryonic development under constant versus variable tem- peratures Oncopeltus fasciatus (Dallas),” Entomologia Experi- mentalis et Applicata, vol. 5, no. 3, pp. 167-178, 1962. [20] T. Meiners and E. Obermaier, “Hide and seek on two spatial scales: plant structure differentially influences herbivore ovi- position and host-finding of egg parasitoids,” Basic and Ap- plied Ecology, vol. 5, pp. 87-94, 2004. [21] T. Meiners, B. Randlkofer, and E. Obermaier, “Oviposition at low temperatures — late season negatively affects the leaf beetle Galeruca tanaceti (Goleoptera: Galerucinae) but not its spe- cialised egg parasitoid Oomyzus galerucivorus (Hymenoptera: Eulophidae),” European Journal of Entomology, vol. 103, no. 4, pp. 765-770, 2006. [22] T. Ikemoto and K. 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Ward, “The effects of size on the mating behaviour of the dung fly Sepsis cynipseaf Behavioral Ecology and Sociobiology, vol. 13, no. 1, pp. 75-80, 1983. [32] R. Thornhill and J. Alcock, Evolution of Insect Mating Systems, Harvard University Press, Cambridge, UK, 1983. [33] T. D. Schultz, “The utilization of patchy thermal microhabitats by the ecto thermic insect predator, Cicindela sexguttataf Eco- logical Entomology, vol. 23, no. 4, pp. 444-450, 1998. [34] S. A. Estay, S. Clavijo-Baquet, M. Lima, and F. Bozinovic, “Be- yond average: an experimental test of temperature variability on the population dynamics of Tribolium confusumf Popula- tion Ecology, vol. 53, no. 1, pp. 53-58, 2011. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 635761, 7 pages doi:10.1155/2012/635761 Review Article On the Use of Adaptive Resemblance Terms in Chemical Ecology Christoph von Beeren, Sebastian Pohl, and Volker Witte Department of Biology II, Ludwig Maximilians University Munich, Grofhaderner Strafe 2, 82152 Planegg-Martinsried, Germany Correspondence should be addressed to Volker Witte, witte@biologie.uni-muenchen.de Received 12 September 2011; Revised 7 November 2011; Accepted 8 November 2011 Academic Editor: Alain Lenoir Copyright © 2012 Christoph von Beeren et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Many organisms (mimics) show adaptive resemblance to an element of their environment (model) in order to dupe another organism (operator) for their own benefit. We noted that the terms for adaptive resemblance are used inconsistently within chemical ecology and with respect to the usage in general biology. Here we first describe how resemblance terms are used in general biology and then comparatively examine the use in chemical ecology. As a result we suggest the following consistent terminology: “chemical crypsis” occurs when the operator does not detect the mimic as a discrete entity (background matching). “Chemical masquerade” occurs when the operator detects the mimic but misidentifies it as an uninteresting entity, as opposed to “chemical mimicry” in which an organism is detected as an interesting entity by the operator. The additional terms “acquired” and “innate” may be used to specify the origins of mimetic cues. 1. Introduction Social insects, especially ants and termites, dominate many terrestrial habitats in terms of abundance, biomass, and en- ergy turnover [1,2]. They accumulate considerable amounts of resources that can be of potential use for other organisms, in the form of living biomass, infrastructures (e.g., nest sites), or stored products [3] . The ecological success of social insects comes with the cost that predators and parasites may exploit their societies [4-6]. Since Wasmann’s [7] extensive study on organisms that developed close relationships with ants, a multitude of so-called myrmecophiles has been found to exploit ant colonies and their social resources in a variety of ways [5, 8]. Parasitic relationships may escalate in an evolu- tionary arms race where the hosts adapt towards protecting themselves from exploitation, while parasites adapt towards avoiding expulsion from the host [9]. In this context it is crucial that members of a society can be recognized reliably and distinguished from aliens, which can thus be aggressively expelled [10]. An efficient social re- cognition system is essential for a colony to function as a closed unit. The better such recognition works, the more ef- fectively social exploitation can be prevented. Complex pro- files of cuticular hydrocarbons (CHCs) are known to carry information necessary for recognition of colony members in ants, bees, and wasps [10]. Macroparasites of ants have evolved a variety of strategies to cope with their hosts’ elaborate recognition system [5]. Potential strategies for avoiding or resisting the hosts’ defense behavior include the use of morphological, acoustical, and behavioral adaptations or the use of chemical repellents or attractants [1, 5, 11-13]. Particularly widespread and important are chemical strategies for avoiding recognition, either by not expressing relevant recognition cues or by matching host recognition cues [11, 14, 15]. For simplicity, we use the term “cue” referring to any chemical information that is potentially perceivable, irrespective of whether the information transfer is “intentional” or “unintentional” sen- su Steiger et al. [16]. Chemical resemblances work analogously to other bio- logical resemblances, such as acoustic or visual mimicry [ 17] . Unfortunately, different definitions exist in chemical ecology (see below), and thus different authors may describe different forms of chemical resemblances with identical terms or the same type of resemblance with different terms. The aim of this paper is threefold. First, we identify how definitions of resemblances are generally used in biology. Second, we analyze the terminology that is used in chemical 2 Psyche Table 1: Summarized table of adaptive resemblance terms in general biology as used in important reviews. Systems can either be considered according to what a mimic pretends to be or according to what an operator perceives. We adopted the latter view. not detected as a discrete entity (causing no reaction) By an operator, the mimic is. . . detected as an uninteresting entity (causing no reaction) detected as an interesting entity (causing a reaction beneficial to the mimic) Reference(s) Crypsis Masquerade Mimicry Endler 1981 [21], 1988 [22] Eucrypsis Mimesis Homotypy Pasteur 1982^ [23] Eucrypsis Plant-part mimicry Mimicry Robinson 1981 [24] Crypsis Masquerade Mimicry Ruxton et al. 2004 [25], Ruxton 2009 [17] Cryptic resemblance Cryptic resemblance Sematic resemblance Starrett 1993 [18] Crypsis Masquerade — Stevens and Merilaita 2009*^ [26] Crypsis Crypsis Mimicry^ Vane-Wright 1976 [27], 1980 [20] Camouflage or mimesis Camouflage or mimesis Mimicry Wickler 1968 [19] — : not considered. ^Pasteur [23] uses the term “camouflage” as generic term for both eucrypsis and mimesis. '^The term “camouflage” is used by Stevens and Merilaita [26] to describe all forms of concealment, including crypsis and masquerade. “^For the imitation of inanimate objects, Vane-Wright [27] uses the expressions “decoys” or “deflective marks”. ecology. Finally, we attempt a synthesis and suggest a ter- minology that agrees best with the general biological defi- nitions and with the chemical strategies observed in nature. 2. General Definitions of Biological Resemblances Since the resemblance of organisms to elements of their environment (e.g., other organisms or background) is often not coincidental, but rather evolved for the benefit of the mimic, the term adaptive resemblance was coined [18]. In adaptive resemblance one organism (the mimic) modifies its appearance, pretending to be something different (the model), in order to dupe another organism (the operator) [19, 20]. Many different terms have been used to describe adaptive resemblance, including mimicry, camouflage, cryp- sis, masquerade, and mimesis. These terms have been debated intensively and defined repeatedly according to different criteria (see Table 1). For the purpose of this paper, we adopted an operator’s view to narrow down the existing definitions of adaptive resemblance into a unified system. This means that we distinguish the cues of a mimic with respect to whether and how they are perceived by the operator. The resulting categories are only valid within a given perceptive channel between mimic and operator, and they can differ in other channels or if other organisms are considered. The first column of Table 1 defines resemblances in which a mimic is not perceived as a discrete entity by the operator and consequently causes no reaction in the operator. In such cases the mimic frequently blends with the background. We adopt the term “crypsis'" for this phenomenon according to Endler [21], who first distinguished this type of resemblance from “masquerade". In the latter a mimic is perceived by an operator as a discrete entity, which is however misidentified as uninteresting so that the operator also shows no reaction to the mimic. Accordingly, crypsis relies on the relationship between the organism and the background, whereas the benefit of masquerade is thought to be independent of the background [28]. A stick insect, for example, is likely to be recognized as a stick by a potential predator independent of its surroundings (e.g., when lying on grass). A cryptic organism, however, depends strongly on the background. This fact allows testable predictions to be made. For example, a mimic performing masquerade should be treated similarly by the operator independent of its background. On the other hand a mimic that performs crypsis should be treated differently (e.g., recognized and attacked) by the operator when the background changes. The third column of Table 1 defines adaptive resem- blances in which a mimic is perceived by the operator as an entity of interest. This category was first described in a biological context by Bates [29] as “mimicry"", and this term is currently most frequently used, hence we adopt it here. Finally, another mechanism exists to avoid detection by an operator, which is however not based on resemblance. The term “hiding"" has been applied to cases in which the absence of informative cues is achieved by behavioral adaptations, making detection by an operator impossible [17]. In visual systems, for example, a rabbit is hiding if it stays in its burrow in the presence of a predator (operator), thereby avoiding detection [17]. If a hiding organism would be removed from the environment, the perceptive input of the operator will not change in the concerning channel. Hiding is not included in Table 1 because it does not fall into categories of resemblance; nevertheless this term will be of importance in our discussion on chemical interactions below. 3. The Use of Adaptive Resemblance Terms in Chemical Ecology Compared to visual adaptive resemblances, chemical adap- tive resemblances had initially been paid less attention to in scientific literature, despite the fact that chemical com- munication is the most widespread form of communication Psyche 3 among organisms [ 16, 30, 3 1 ] . However, more recent reviews on this topic show that understanding of chemical adaptive resemblance has increased markedly [11, 15, 32, 33]. According to this special issue on ants and their parasites, we focus here particularly on important reviews about para- sites of social insects and on reviews about adaptive chemical resemblance. Reviews are suitable for analyzing how the terminology is used, since they provide overviews about specific fields, summarize the literature, and therefore mirror common practices. We used the same categorization as in Table 1, adopting an operator’s point of view. Note that two resemblance types were combined, that is, resemblances in which a mimic is not detected as discrete entity and resemblances in which a mimic is detected as an uninteresting entity (Table 2). We combined these two types of resemblances because none of the reviews distinguished them. Additionally, we included the origins of mimetic compounds in the table, since this is an interesting point regarding chemical resemblances and several authors based their terminology upon it. Table 2 shows that the terms chemical mimicry and chemical camouflage are not used consistently. Some authors used the terms according to criteria similar to those used in general biology (see Table 1). They distinguished between chemical mimicry as the imitation of an interesting entity and chemical camouflage either as the imitation of an uninteresting entity or as the resemblance of background cues (sensu Dettner and Liepert [15]). This use of terms did not include the origins of mimetic compounds. In contrast, other authors focused primarily on the origin of mimetic cues. According to their terminology, chemical mimicry implies that mimetic cues are biosynthesized by the mimic, while chemical camouflage implies that the mimic acquires mimetic cues from the model (first defined by Howard et al. [38]). Additional definitions specifically focused on a mimic’s avoidance of being detected as a discrete entity (Table 2). Chemical resemblances that allow mimics to avoid detection by background matching were defined as chemical mimesis by Akino [14] or as chemical crypsis by Stowe [31]. In addition to adaptive resemblances, another mech- anism exists among parasites to prevent detection by an operator. This mechanism was called “chemical insignifi- cance” [39]. However, chemical insignificance was originally brought up to describe the status of freshly hatched ant workers (callows), which typically carry very low quantities of cuticular hydrocarbons [39]. The term insignificance referred to these weak chemical cues, which are frequently not colony or even species specific, allowing the transfer and acceptance of callows into alien colonies [11]. The term chemical insignificance was also adopted to describe a status of ant parasites, which may benefit from displaying no or only small quantities of recognition cues to sneak unnoticed into host colonies [3, 11, 39, 40]. We discuss this point in more detail at the end of the following chapter. Furthermore, chemical transparency was recently de- scribed as a chemical strategy in a wasp social parasite [41]. This strategy is somewhat similar to chemical insignificance, except that it refers particularly to a subset of cuticular com- pounds that are presumably responsible for recognition. We discuss both strategies, chemical insignificance and transpar- ency, in more detail at the end of the following section. 4. Suggestions for a Consistent Terminology As described above, adaptive resemblance terminology is used inconsistently in important reviews of chemical ecology, likely mirroring inconsistent use in this field generally. Most importantly, the terms chemical camouflage and chemical mimicry are inconsistently used by different approaches. While some authors distinguish them according to different models that are mimicked, others distinguish them according to the origin of mimetic cues (Table 2). To avoid confusion, we suggest a consistent terminology that is in line with the definitions used in general biology (Table 1). Consequently, adaptive resemblance of an entity interesting for the operator should be referred to as “chemical mimicry \ irrespective of the origin of mimetic cues. Nevertheless, an additional distinction between biosynthesis and acquisition of mimetic cues might often be useful. Hence, we suggest using addi- tional terms to distinguish the origins of mimetic cues; “ac- quired chemical mimicry” indicates that mimetic cues are acquired from the model, while “innate chemical mimicry” (as first mentioned by Lenoir et al. [11]) indicates that a mimic has an inherited ability to biosynthesize mimetic compounds. The two different mechanisms may affect coevolutionary dynamics in different ways. For example, a consequence of the acquisition of recognition cues by a parasite from its host is that the mimetic cues of model and mimic are of identical origin [3]. Coevolutionary arms races select in such cases for effective ways of acquiring chemical host cues by the mimic, for example, through specific behaviors such as intensive physical contact to the host. In the host, selection favors counterdefenses which prevent the acquisition of chemical cues. Selection pressures are somewhat different when a parasite biosynthesizes the mimetic cues [3]. In this case, the origins of the chemical cues of mimic and model are different, which allows coevolutionary arms races to shape on the one hand the accuracy of chemical mimicry of the mimic and on the other hand the discrimination abilities of the operator. Mimics that are not detected as discrete entities or that are detected but misidentified as uninteresting entities by an operator have rarely been addressed in chemical ecological reviews, although they are common in general biology (first two columns of Table 1). Since the term camouflage is not used in general biology to distinguish these two forms of resemblances (Table 1) and since the term chemical cam- ouflage is used inconsistently in chemical ecology (Table 2), we suggest abandoning this term so as to avoid confusion. Instead, we suggest using terms consistent to general biology. Accordingly, “chemical crypsis” describes cases in which an operator is not able to detect a mimic as a discrete entity, while “chemical masquerade” describes cases in which an operator detects a mimic as an uninteresting entity. In both cases, the operator shows no reaction. The terms “acquired” and “innate” can be applied to these categories as well to add further information on the origin of the disguising cues. Note that it is challenging but logically possible to 4 Psyche qJ Ch U O ^ TO fi Xi ^ KA qj 0/j Sh ^ ^ 2 O Ch e e qj ;-( 4 ^ ^ o .£( ’■M Cl, o 'T3 ^ rt B O ”5 !-, ct 3 ;3 t/) Ph ti u c/5 5 -h TO 0^ Dh ^ o d o X5 ct 3 c/) d (U ^ s ^ •5 o C/) (U tiD d cd d ^ x> o e 8 a> 03 a> ^ !-, O -in ^ d 'd TO ^ U ( 1 > g K a> u u ;-H ^ s ^ f c/> ^ fi 2 . w bX) ^ •S ^ ^ Cb d c /5 £ a ■” 1^1 '+H B o S qj e a d o Ph o o o c/5 d o -d a> a> N rP !-h d TO ^ 5 S d '6 g -G d d o CO 03 i2 . . o g ^ ’Bb w d hj a> -d H CO nd i-t o C/> O 6 6 (U 4 d ■M Ch ;-, !-h (U Ph c/) O CJ O Ch §.& Bh g ^ CU d bO 8 -S u ^ • .— ( ^ •§ .a a ^ o ^ d 2 .bp g O ns C/> » ^ U a> M-» O TO Uh a> CP o c TO DC (i> u d (i> p— ( (i> D< c/5 O 4 C O f ^ P-H d o c/> ( 1 > 4 C -M d >-> c/5 O cu -M TO d d >-> d (i> DO d c/5 ( 1 > ;-H TO ( 1 > -M U ( 1 > -M ( 1 > XJ XJ ( 1 > -M U ( 1 > -M a> O p 3 J— ( ° -P •d s d ^ TO u a> -M a> nd -M o d a> DD TO qd d o TO U TO U - o3 O Uh O Ph qu ,—1 H- 1 ' — ' aj u CO G O TO G O c/5 TO CnI a> Uh Uh <1> o g G bD 'M TO a> PD Q rO O o CnI ■M 4 G 4 G u U CnI rO LO o o CnI d d^ pq Td d o3 Td d o X LO rO o o CnI ’d H-H o o CnI ■M - 4 d >- a a rO bO o o CnI 03 O O Pd Td d o3 4 d CO o3 QJ bJD o3 qd d o o3 O o3 O CO rO CnI o o CnI ■M 4d rO bO bO On 1— H I o ■M CO p p p p p TO 1 4 d u p .o .o .o .o .o U : u .o ’■M ’■M ’■M ’■M ’■M *a 1 ’■M CU CU CU CU CU 1 CU .g .g .g .g .g 4d U o3 O - !-, o ’d o OX) d d .a 3 a o cu cu < 03 o *c o U X u TO Cu 0/ G a G •M 0/ Q 0/ u a cu UD cu a o 03 & o S ® d cu o 2 3 d eg O 0/ G ^ .a M 4-t -M ~ o o bd ^ 2 Lh 0/ ■HH cu 4d H d C4d ^ b> in "d (U 32 Sh _ ■J ^ hX (JJ X3 "S G u 0/ G cu ‘•D cu O UD 4-* U bb G cu Q ^ G ^ O 2 ^ ^ U r! TO 41 4-1 O) G UD c g OJ 3 ^ * 4—1 ■SP-d O 0/ ■d d cu CO '4b 4 h +H O d lh o o Psyche 5 Table 3; Proposed terminology for chemical adaptive resemblances. Chemical cues of a mimic can either be “acquired” from the environment (including the host), or they can be “innate”, that is, biosynthesized. In all cases of chemical adaptive resemblance, the operator is deceived by the mimic so that the mimic benefits. Suggested term By an operator, the mimic is. . . . . . not detected as a discrete entity due to Chemical crypsis the expression of cues that blend with the environment (causing no reaction in the operator). . . . detected but misidentified as an Chemical masquerade uninteresting entity (causing no reaction in the operator). Chemical mimicry . . . detected as an entity of interest (causing a reaction in the operator). empirically separate cases of masquerade and crypsis [28], but this has yet to be done in a nonvisual context. Table 3 gives an overview on our proposed terminology for chemical adaptive resemblances. Please note that in our terminology it is only important whether and how mimics are perceived by an operator. Similarities in the chemical profiles of parasites and hosts maybe important diagnostic tools, but they are not part of the definitions. Finally, we want to stress the special case of organisms that suppress the expression of chemical cues which can potentially be detected by the operator. Following our aim of applying a consistent biological terminology, “chemical hiding” is the most appropriate definition. This definition includes two slightly different scenarios, the total absence of relevant cues and the presence of cues below the operator’s perceptive threshold. In both cases chemical perception of the organism is impossible. A host’s inability to detect any chemical cues of a parasite was also referred to as “chemical insignificance” [3]. However, the term chemical insignifi- cance is unfortunately used ambiguously regarding the im- portant point whether there are no detectable cues [3] or small yet detectable amounts of cues are present [39] . Clearly, it should be distinguished whether an operator is able to detect an organism or not. If resemblance cues are present and perceived (irrespective of the quantitative level), the phenomenon will fall per definition into one of the cate- gories chemical crypsis, chemical masquerade, or chemical mimicry (Table 3). For example, if a callows’ weak chemical signature was expressed by a parasite and adult host ants misidentified this parasite as a callow, we would follow Rux- ton [17] by assigning this to chemical mimicry (since callows are certainly interesting entities). Empirical evidence for a chemical mimicry of callows could result in practice from a combination of chemical data (callow resemblance) and behavioral data (hosts treat parasite as callows). However, an exhaustive discussion about methods is beyond the scope of this conceptual paper. Consequently, the original definition of chemical insignificance as a “weak signal” [39] appears not applicable to parasites without the risk of confusing it with chemical mimicry. If chemical cues are below an operator’s perceptive threshold, the definition of chemical hiding will apply. However, the term chemical insignificance may be used as a functional term describing the lack of chemical information in a certain context. For example, callows are chemically insignificant in terms of nestmate recognition due to a lack of chemical information in that context. Nevertheless, callows carry apparently sufficient in- formation in the context of caste identity since workers show characteristic behaviors towards them; for example, they receive assistance during hatching and are transported to new nest sites in migratory ants. The above discussion on chemical insignificance applies also to the phenomenon of chemical transparency. If no cues are expressed that are perceivable by the operator, the focal organism would show chemical hiding, regardless of the presence of any other compounds. In contrast, if perceivable cues are present, chemical crypsis, chemical masquerade, or chemical mimicry applies. In the described case of chemical transparency [41], the parasite is most likely recognized and misidentified as an interesting entity (e.g., as brood), since social parasites usually exploit the brood care behavior of their hosts. Notably, a parasite may alternatively avoid chemical de- tection through behavioral mechanisms by “hiding” accord- ing to the definition in general biology (see above) rather than “chemical hiding.” For example, if it avoids detection by staying in a cavity so that its chemical cues do not reach the operator, it is hiding. A parasite that performs “hiding” could potentially be detected if it was somehow confronted with the operator. In contrast, a parasite that shows “chemical hiding” cannot be detected by chemical senses of the operator at all. 5. Examples for the Use of Adaptive Resemblance Terms In this section we want to discuss examples to clarify the use of terms regarding adaptive resemblances. The mimicking of CHC profiles of the host is widespread among ant parasites, and this is generally assumed to facilitate integration into the host colonies. Parasites are indeed frequently not recognized as alien species [11,33]. This strategy of avoiding recognition as an alien species by expression of host CHCs could poten- tially be referred to as chemical crypsis (if the colony odor is regarded as the background) or as chemical masquerade (if a nestmate worker is regarded as an uninteresting entity). However, we argue that the strategy is best described by chemical mimicry for the following reasons. First, workers are certainly able to detect other workers, and hence parasites that mimic them are discrete entities, excluding the term chemical crypsis. Second, workers are certainly interesting entities to other workers because social actions are shared, such as grooming or trophallaxis. Consequently, a mimic that uses a worker as model resembles an entity of potential interest to ant workers, so that chemical mimicry rather than chemical masquerade applies. It becomes more complicated when a parasite mimics the nest odor of its host. Lenoir et al. [42] demonstrated that the inner nest walls of the ant species Lasius niger are coated with the same CHCs as those that occur on the cuticle of workers. However, the CHCs on the walls occurred in different 6 Psyche proportions and showed no colony specificity. If a mimic resembles such a chemical profile, chemical crypsis will be the most appropriate term, because the mimic represents no discrete entity and rather blends with the uniform nest odor. To our knowledge, no clear evidence exists for this case. Another example is worth highlighting in this context which was already pointed out by Ruxton [17]. The CHCs of Biston robustum caterpillars resemble the surface chemicals of twigs fr om its host plant [43] . Formica jap onica and Lasius japonicus workers do not recognize the caterpillars on their native host plant, but when caterpillars were transferred to a different plant, the ants noticed and attacked them. In this case it depends on the operator’s perception whether the example should be considered as chemical crypsis or chemical masquerade. If the ants did not detect a twig (and hence a caterpillar) as a discrete entity, but as background, chemical crypsis would apply. If the ants detected the caterpillar as a discrete but uninteresting entity, for example, as a twig, then chemical masquerade would apply. As Ruxton [17] emphasized, twigs are of huge dimension compared to the size of ants. Hence, it is more likely that ants do not detect caterpillars as discrete (uninteresting) entities, but rather perceive them as (uninteresting) background. Accordingly, chemical crypsis appears to be the most appropriate term for this example. These examples may demonstrate that it can be rather difficult to assign appropriate terms to particular adaptive resemblance systems. Nevertheless, the definitions we pro- posed are generally straightforward, and they can be applied unambiguously if the necessary information about a system is available. We hope that this paper contributes to a careful and consistent use of adaptive resemblance terminology in chemical ecology. Acknowledgments The authors thank the behavioral ecology group at the LMU Munich and Graeme D. 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Wakamura, “Diet-induced chemical phytomimesis by twig-like caterpillars of Biston ro- bustum Butler (Lepidoptera; Geometridae),” Chemoecology, vol. 14, no. 3-4, pp. 165-174, 2004. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 238959, 8 pages dohlO.l 155/2012/238959 Research Article Exploitative Competition and Risk of Parasitism in Two Host Ant Species: The Roles of Habitat Complexity, Body Size, and Behavioral Dominance Elliot B. Wilkinson and Donald H. Feener Jr. Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA Correspondence should be addressed to Elliot B. Wilkinson, ebwilkinson@yahoo.com Received 24 August 2011; Revised 31 October 2011; Accepted 4 November 2011 Academic Editor: Volker Witte Copyright © 2012 E. B. Wilkinson and D. H. Eeener Jr. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Habitat structural complexity can slow resource discovery by ants but can also lower the risk of parasitism during foraging. The relative importance of these two ecological facets of habitat complexity may differ in a species- specific manner and thus may be important in the outcome of exploitative competition over food resources. Eor the host ant species Pheidole diversipilosa and P. bicarinata, we used in situ experimental manipulations to explore whether the effects of habitat complexity on exploitative competition depended on host body size and behavioral dominance, two characteristics likely to affect mobility and utilization of refuge from specialist Dipteran parasitoids {Apocephalus orthocladius and A. pugilist, resp.). We found that habitat complexity affected the resource discovery and harvest components of exploitative competition in an opposing fashion for each species and discuss these results in light of the differences in body size and behavioral dominance between the two hosts. 1. Introduction Characteristics of habitats in which animals forage influence their mobility, ability to compete, and the likelihood of encountering predators, among other things. The structural complexity of a habitat can act on several aspects of animal foraging simultaneously. In particular, exploitative compe- tition or the consumption of a common resource without direct competitive interaction may be directly affected by habitat complexity because it constrains animal movement in a species-specific manner [1]. Architecturally complex substrates often take more energy and time to traverse [2- 5], which can reduce a species’ ability to find and efficiently harvest a resource. A given degree of habitat complexity will be more difficult for relatively small species to traverse because they must move around or through the substrate, instead of over it [1]. As a result, habitat complexity can mediate exploitative competition for a common resource because species of different sizes are differentially affected. Although habitat complexity may have negative effects on exploitative competitive ability by constraining animal movement, it may also have positive indirect effects on competitive ability by providing refuge from predators or parasitoids during resource acquisition. Numerous studies on a wide range of taxa have noted the importance of habitat complexity in providing refuge from predators ([6- 13], but see [14, 15], e.g., of habitat complexity increasing predation). By impeding movement and providing refuge, habitat complexity may have opposing effects on resource discovery and acquisition, but the degree to which this is true may depend on species-specific characteristics such as body size or use of refuge from predators. Ant communities are well suited for studying the role of habitat complexity in exploitative resource competition and escape from predators. Evidence suggests that exploitative competition between species has fitness consequences and is important in determining community composition [16-19]. Ants exhibit a wide range of body sizes [ 1 ] , with larger ants being able to navigate complexity in the microhabitat more easily than small ants [20-23]. Increased habitat complexity does not appear to have an energetic cost to foragers [24], but does increase the time required to harvest resources and necessarily decreases harvest rate [25]. 2 Psyche Ant communities are not traditionally considered to be structured by top-down forces from predators. However, community composition can be influenced by specialist Dipteran parasitoids (Apocephalus: Phoridae) that attack host ant species, induce behavioral responses in their hosts, and alter the outcome of interspecific competition in the community [26-32]. Habitat complexity has been shown to benefit the host ant species Pheidole diversipilosa and P. hicarinata during interference competition with nonhost ant species by providing refuge from parasitoids. Refuge allows hosts to maintain similar numbers of soldiers during head-to-head competition as in competitive bouts without parasitoids [13]. These two host ants cooccur in the same habitat and are dominant to most other ants in the com- munity, but P. hicarinata is behaviorally subordinate to P. diversipilosa [30]. This difference in dominance has the potential to impact benefits derived from habitat complexity during exploitative competition. Previous research indicates that P. diversipilosa wins a majority of contests, has access to the majority of resources, and experiences a resource environment that is not restricted by competition [29] . If such a host is attacked by its specialist parasitoid while exploiting an uncontested resource, it can simply abandon the resource, wait for parasitoids to leave, and return to the resource at a later time [33] . As a result, any refuge provided by habitat complexity would have marginal benefit to the colony during exploitative competition. Predictions are different for the more subordinate species, P hicarinata. Subordinate species only have access to a limited proportion of total available resources because they often lose resources to dominants [29]. Previous work has shown that subordinate hosts simply cannot afford to leave a resource when parasitoids arrive because successful foraging bouts are too rare [33]. For subordinates, a higher mortality risk must be accepted in order to satisfy energy requirements. Work on damselfly, passerine bird and ant communities has demonstrated that solutions to this ecological trade-off have evolutionary repercussions: subordinate competitors or species with higher resource requirements display little predator avoidance regardless of any pressure from dominant competitors [33-35]. Such observations have led to the hypothesis that subordinate species, who typically experience a more limited resource environment than dominants, will sacrifice predator avoidance to a greater extent than dominants in order to meet energy requirements [33, 36, 37]. While harvesting uncontested resources, subordinate hosts are likely to benefit from refuge provided by habitat complexity to a greater extent than dominant hosts because subordinates under attack by parasitoids must continue to forage even when resources are not contested by competitors, whereas dominants can avoid parasitism by returning to the nest. The ecological and evolutionary consequences of host dominance discussed above suggest that benefits derived from habitat complexity may depend on whether foraging is occurring in an interference or exploitative competitive context. The benefits derived from habitat complexity during interference competition (head-to-head competition for resources) are investigated in a previous study [13]. In con- trast, this study focuses on whether habitat complexity affects the exploitative component of competition (depression of the resource base in the absence of competitors). We explored the benefits derived from habitat complexity separately in interference and exploitative contexts because parasitoids have a greater impact on hosts engaged in interference competition (versus exploitative harvest of uncontested resources), due to a positive feedback between recruitment pheromones used during defense of resources and parasitoid behavior [29]. In addition, this study expands upon a previous study [13] by exploring whether habitat complexity affects the “discovery” and “harvest” components of exploitative com- petition separately. The effects of habitat complexity on each component of exploitative competition are interpreted in light of the body size and behavioral dominance of two host ant species. Of the two focal species, P. diversipilosa is approximately twice as large as P. hicarinata (workers: 0.12 versus 0.05 mg, resp.; soldiers: 0.44 versus 0.26 mg, resp.), and wins 15% more of its interactions with all other species in the local assemblage [30]. First, we determine whether habitat complexity influences the time it takes each host species to find resources (the “discovery” component of exploitative competition, [38]). Second, we ask whether the benefits hosts receive from refuge during harvest of uncontested resources (the “harvest” component of exploita- tive competition) depends on their dominance within the community. Benefits provided by habitat complexity during harvest of uncontested resources are measured in terms of the number of soldier ants because (1) soldier ants are crucial for the defense and harvest of large resources and (2) only soldiers are attacked by parasitoids. We then interpret our findings in the context of variation in habitat complexity. 2. Materials and Methods 2.1. Study Site and System. This study was conducted in oak, pine, and juniper woodlands in the Chiricahua Mountains of Southeast Arizona. The two focal ant species P. diversipilosa and P. hicarinata coexist in this habitat and are hosts to species-specific parasitoids {Apocephalus orthocladius and A. pugilist, resp., [39]). In July- August of 2003, P. diversipilosa was studied on National Forest land surrounding the South- western Research Station (31°52'N 109° 14' W). In August- September of 2004, P. hicarinata was studied nearby on land owned by the Southwestern Research Station (31° 53' N 109° 12' W). Colonies of P. diversipilosa, P. hicarinata, and their respective parasitoids are found at both of these sites within meters of each other, but their relative abundances at each site differ (see Section 4). 2.2. Experimental Design. To investigate how habitat com- plexity affects exploitative competition for resources and host-parasitoid interactions, we forced field colonies to forage up into plastic bins and recorded their behavior under different levels of habitat complexity and parasitism. Cookie baits measuring 2x2 cm were placed 50 cm away from the nest entrance, and the number of soldiers harvesting and Psyche 3 defending these baits was recorded every 10 minutes for 2.5 hours in all treatments. Cookie baits are examples of large resources that require soldiers to break them into small pieces for efficient transport by workers. Placing baits 50 cm away from colony entrances ensured that baits were discovered and that colonies traversed a distance during which they were susceptible to parasitoid attack. Foraging bins were 30 X 60 cm Sterilite storage contain- ers, and had a 6 cm diameter hole at one end that could be placed directly over colony nest entrances. Using foraging bins allowed us to (1) minimize disturbance around nest sites and control exactly which resources hosts were harvesting and (2) introduce or exclude parasitoids from treatments using bridal veil to cover the foraging bin. We used soldier number as a response variable because soldiers (1) are able to carve up large resources for transport to the nest by workers, and thus are critical to harvesting resources, (2) can defend resources against competitors, and (3) are the only caste attacked by parasitoids in this system. We also recorded the time it took colonies to discover cookie baits. We used a multifactor design with two levels of habitat complexity (complex or simple) and parasitoid exposure (parasitoids present or absent). Complex habitat treatments contained 5000 cm^ of leaf litter that had been ovendried for 72 h, while simple habitat treatments occurred in empty foraging bins. The addition of leaf litter closely approximated average leaf litter depth found in habitat where both species coexisted. Parasitoids were captured by aspiration during recruitment events instigated at unused host colonies nearby. In parasitoid-present treatments, two parasitoids were introduced after soldiers had recruited to resources. Foraging bins were covered tightly with bridal veil in all treatments to ensure that parasitoids could not escape from parasitoid-present treatments and that parasitoids could not gain access to parasitoid-absent treatments. Treatments were replicated on seven colonies of P. diver- sipilosa and eight colonies of P. hicarinata. The experiment was performed in areas where P. diversipilosa and its specialist parasitoid A. orthocladius cooccurred with P. hicarinata and its specialist parasitoid A. pugilist in order to control for the surrounding competitive environment. Colonies were randomly assigned the order in which they received treatments such that all colonies on a given trial day received different treatments. This allowed us to control for the effects of environmental variation and cumulative treatment effects. In addition, we rested colonies for two days between treatments to control for energetic state after foraging on cookies. It was not possible to monitor all colonies at once due to time constraints and distance between colonies, so all replicates were divided roughly into two groups, and groups experienced treatments within 24 h of each other to control for environmental conditions. All treatments were shaded to control for temperature and humidity differences between colony locations. 2.3. Analysis. Exploitative competition can generally be di- vided into two components: discovery and harvest of re- sources. To determine the impact of habitat complexity on resource discovery for each host, we conducted paired t-tests on the time it took hosts to discover cookie baits (TTD) in complex and simple habitat treatments. This experiment resembles a randomized block or repeated measures design, in which colonies are blocks and treatments are implemented within blocks. Since parasitoid treatments were not imple- mented until after hosts discovered cookie baits, TTD values were averaged across both levels of parasitism (e.g., for each complexity treatment, TTD values for each colony were averages of TTD in parasitoid absent and parasitoid present levels). Paired t-tests were then performed to compare each colony’s average values for complex and simple habitats. To compare discovery speed between hosts within either simple or complex habitat treatments, we used two-sample t-tests because hosts were not intrinsically paired. To control for the multiple comparisons made within habitat complexity treatments and maintain an experiment-wide a of 0.05, we used Bonferroni adjustments. To determine whether the refuge benefits provided by habitat complexity during harvest of resources depend on host dominance level, we constructed a randomized block/ repeated measures General Linear Model to test for dif- ferences among treatments. For each host, post hoc com- parisons among means were conducted using Tukey’s HSD method with degrees of freedom appropriate for randomized block/repeated measures designs and 0.05 experiment-wide a levels. Replicate means were calculated by averaging re- corded values of soldiers at cookie baits over the 2.5 h for- aging period. Recorded values were averaged from the time colonies discovered the cookie bait for treatments without parasitoids, and from the point of parasitoid introduction for treatments with parasitoids. Means were transformed [log (mean + 1)] to meet homogeneity of variance and normality assumptions. 3. Results The time it took P diversipilosa to discover cookie baits did not differ significantly between complex and simple habitat treatments (fi,6 = -0.870, P > 0.05; Figure 1), although R diversipilosa discovered resources slightly faster in complex habitat treatments. In contrast, P. hicarinata discovered resources in simple habitats much more quickly than in complex habitats {tij = 5.276, P < 0.005; Figure 1). Within complex habitats, P diversipilosa discovered resources more quickly than P hicarinata (ti,i3 = 2.538, P < 0.05; Figure 1), butP. hicarinata discovered resources more quickly than P. diversipilosa in simple habitats (fi,i3 = -2.923, P < 0.005). For both P. diversipilosa and P hicarinata, general linear models indicated that significant differences in the number of soldiers harvesting resources existed between at least two treatments (^3,17 = 5.070, P < 0.05; ^3,21 = 4.139, P < 0.05 resp.). P. diversipilosa maintained significantly more soldiers at resources in complex habitats without parasitoids than either complex or simple habitats with parasitoids (closed circle compared to closed and open triangles in Figure 2(a): Qt = 4.244, P < 0.05; Qt = 4.683, P < 0.05). There was a little 4 Psyche 100 80 a ^ 60 a 8 40 c/5 5 20 0 Complex Simple Habitat complexity • P. diversipilosa (0.12 mg) O P. bicarinata (0.05 mg) Figure 1; Differences in resource discovery time between P. diver- sipilosa and P bicarinata in complex and simple habitats. Means and standard errors are presented. difference in soldier number between simple and complex habitats without parasitoids (open and closed circles: Qt = 1.358, P > 0.05), There was also no difference in soldier number between simple habitats without parasitoids and both simple and complex habitats with parasitoids (open circles compared to open and closed triangles: Qf = 3.379, P>0.05;Qf = 2.886, P >0.05). P. bicarinata maintained significantly more soldiers at resources in complex and simple habitats without parasitoids than simple habitats with parasitoids (open and closed circles compared to open triangle in Figure 2(b): Qt = 4.199, P < 0.05; Qt = 4.191, P < 0.05), In contrast, no difference in soldier number existed between complex and simple habitats without parasitoids and complex habitats with parasitoids (open and closed circles compared to closed triangles: Qt = 2.355, P > 0.05; Qt = 2.347, P > 0.05). Soldier number was also statistically indistinguishable between complexity levels in both parasitoid and no parasitoid treatments (open compared to closed triangles and open compared to closed circles: Qf = 1.844, P > 0.05; = 0.008, P > 0.05). 4. Discussion 4.1. Exploitative Competition. For a given habitat complexity level, such as the leaf litter used in this study, smaller species perceive their environment as more rugose than larger species. This theory, known as the size-grain hypothesis, predicts that larger species will traverse a moderately rugose habitat with greater ease than smaller species [1]. Results on resource discovery time show that smaller P. bicarinata take longer to discover resources in complex habitats than do larger P, diversipilosa, which is consistent with the size-grain hypothesis. However, the observation that smaller P, bicar- inata find resources in simple habitats more quickly than larger P. diversipilosa runs somewhat contrary to the predic- tions of the size-grain hypothesis. This observation suggests that, in addition to the limitations on movement predicted by the size-grain hypothesis, these two species either (1) differ in the degree to which they tolerate desiccation, (2) have different exploratory or recruitment strategies, or (3) exhibit differential sensory bias toward habitat complexity. First, differences in the degree to which species tolerate desiccation is not a plausible explanation for P. bicarinata discovering resources more quickly than P. diversipilosa in simple habitats because smaller ants such as P. bicarinata are more sensitive to desiccation stress than larger ants, and soil temperatures are much higher in more open, simplified environments [40-44]. Physiological limitations are also not a plausible explanation in the context of our experimental setup because physiological conditions between treatments were controlled (see Section 2). Second, P. diversipilosa and P. bicarinata may differ in their exploratory [45] or recruitment behaviors [46]. Unfortunately, the small scale of our experimental arena caused a rapid attenuation of recruitment curves, making insight into exploratory and recruitment behavior difficult in this study. Further work should be conducted to determine whether differences in exploratory or recruitment behavior can explain P. bicarinata discovering resources more quickly than P. diversipilosa in simple habitats. Finally, P. diversipilosa and P. bicarinata may exhibit differential sensory bias towards habitat complexity, a possibility that is discussed in detail below. Regardless of the mechanism behind these results, the ultimate consequence is that smaller P. bicarinata can discover resources faster in simple habitats, while larger P. diversipilosa can discover resources faster in complex habitats. Thus, habitat complexity has important but contrasting effects on the resource discovery component of exploitative competition for both species. During initial attempts to find host colonies for this study, 44% of P, bicarinata and 64% of P. diversipilosa for- aging bouts to cookie baits went unchallenged (data not shown). Thus refuge from parasitoids during uncontested harvest of resources may have important fitness conseq- uences. P. diversipilosa and P. bicarinata harvesting resources in the absence of direct competition respond differently to habitat complexity, and this difference is best explained by the parasitoid avoidance behavior and dominance of each host. We predicted that P. diversipilosa, being behav- iorally more dominant and having access to the majority of resources, would abandon uncontested resources when under attack by parasitoids regardless of the presence of refuge in complex habitats. This prediction follows from the resource loss-predation trade-off suggested to exist in a variety of systems [33, 36, 37], We found that P. diver- sipilosa under attack by parasitoids do abandon uncontest- ed resources regardless of whether refuge from habitat com- plexity is present. However, we also found that the number of soldiers P. diversipilosa maintains at resources in simple habi- tats without parasitoids is not statistically distinguishable from the number of soldiers maintained in simple habitats with parasitoids. Psyche 5 A lower physiological threshold for open habitats is one explanation for this pattern but is unlikely for reasons explained above. In addition, if desiccation tolerance were solely responsible for the observed foraging patterns of P. diversipilosa during exploitative competition, significant differences between complex habitat treatments in the presence and absence parasitoids should not exist. However, we cannot rule out the role of desiccation tolerance in R diversipliosa foraging behavior. A more plausible explanation is that P. diversipilosa exhibits a sensory bias towards habitat complexity and is less willing to forage in any habitat that does not offer refuge from parasitoids. Numerous studies on a wide range of taxa suggest that animals make patch choices based on perceived predation risk ([47] and references therein, [48-50]). Work on vole, deer mouse and passerine bird populations suggests that competitive dominants may choose to forage in habitats with less predation risk, thereby forcing subordinates to forage in habitats with greater predation risk [50-53] . These patch choices take place in eco- logical time and are considered solutions to the problem of maximizing energy intake while minimizing mortality risk. As predicted by the resource loss-predation trade-off, subordinates must accept a higher mortality in order to satisfy energy requirements. Therefore, we predicted that P. hicarinata under attack by parasitoids would benefit from refuge even while foraging on uncontested resources. The number of P. hicarinata soldiers at resources in complex habitats was similar regardless of parasitoid presence, but soldier number in simple habitats with parasitoids was much lower than without parasitoids. These observations support the predictions of the resource loss-predation trade off and suggest that refuge benefits associated with habitat complexity depend on host dominance during exploita- tive competition. Subordinate hosts harvesting uncontested resources benefit from habitat complexity because their need for resources does not allow them to avoid parasitism by ceasing foraging. In contrast, dominant hosts harvest- ing uncontested resources receive no benefit from habitat complexity because they can afford to cease foraging in the presence of parasitoids. The potential for resource loss increases when resources are directly contested by competi- tors (interference competition). As the potential for resource loss increases during interference competition, dominant hosts should become more willing to accept the risk of parasitism in order to retain resources, and refuge provided by habitat complexity may allow hosts to strike a balance between retaining resources and risking mortality. Prior work in this system has shown that P. diversipilosa under attack by parasitoids will not abandon resources if they are directly contested by competitors, as long as habitat complexity provides some refuge from attacking parasitoids [13]. This study expands upon previous work [13] by demonstrating that behavioral dominance and refuge pro- vided by habitat complexity interact to influence how species balance the resource loss-predation trade-off in different competitive contexts. The acts of discovering resources and harvesting resources in the absence of competitors are two important components of exploitative competition between the focal species of this study. Habitat complexity provides an advantage to R diversipilosa during the discovery phase of exploitative competition because P. diversipilosa is larger and can traverse complex habitats more easily than P hi- carinata. The opposite is true while harvesting resources: habitat complexity provides an important refuge benefit to P. hicarinata, but no refuge benefit to P diversipilosa. Dur- ing exploitative competition, habitat complexity plays a dual role in impeding movement and providing refuge. These mechanisms work in opposing manners in this system be- cause the focal species differ in body size and behavioral dominance. The degree to which the discovery and harvest components of exploitative competition are opposing will depend on the relative strength with which habitat complex- ity impedes movement and offers refuge for P. diversipilosa and P. hicarinata. 4.2. Impact of Natural Heterogeneity on Movement and Ben- efits from Refuge. For ants, the degree to which movement is impeded by habitat complexity depends largely on the abundance and quality of litter on the ground surface. Nat- ural heterogeneity in habitat complexity could lead to local pockets in which movement was strongly impeded by habitat complexity, favoring P. diversipilosa s resource discovery abil- ities, and other pockets where movement was unimpeded, favoring P. hicarinata s discovery abilities. In extremely het- erogeneous environments, the relative discovery abilities of both species may, therefore, be similar when summed ac- ross the community. Further work is needed to determine whether natural heterogeneity in habitat complexity could facilitate coexistence between these host species. The degree to which habitat complexity provides refuge depends both on variation in litter and on the abundance of parasitoids. While under attack by a constant number of par- asitoids, P. hicarinata benefits more from refuge than P. diver- sipilosa (Figure 2). If there is reliable parasitoid pressure on both hosts, P. hicarinata would experience a greater relative benefit from refuge. Working in the same system, LeBrun and Feener [29] found that parasitoids discovered P. diversipilosa exploiting resources in the absence of competitors ~50% of the time. Parasitoid discovery of P. hicarinata is less pre- dictable, as A. pugilist exhibits wide fluctuations in abun- dance through space and time, but is rarely more than 50% [33]. Based on observed parasitoid abundance for each host, it appears that the potential to benefit from refuge is greater for P. hicarinata. Pockets of low habitat complexity will not counteract benefits that P. hicarinata receives from areas nearby with higher habitat complexity because, unlike P. diversipilosa, P. hicarinata forages willingly in simplified ha- bitats, and also unlike P. diversipilosa, P. hicarinata main- tains some foraging presence at resources regardless of whether refuge from parasitoids is available (see Figure 2). 4.3. Conclusions. While P. diversipilosa should have greater relative discovery abilities in complex habitats, natural het- erogeneity in structural complexity will minimize this advan- tage by favoring P. hicarinata in simpler habitats. P. hicarinata is also likely to benefit from refuge from parasitoids during harvest of uncontested resources to a greater degree than 6 Psyche (a) (b) O No parasite; simple A Parasite; simple # No parasite; complex ^ Parasite; complex Figure 2; Number of (a) P. diversipilosa and (b) P. bicarinata soldiers harvesting resources in the absence of head-to-head competition when parasitoids are absent (circles) or present (triangles) in complex habitat (filled symbols) or simple habitat (empty symbols). Means and Tukey’s minimum significant difference (MSD) comparison intervals are presented. Means whose comparison intervals overlap are not significantly different. Means whose comparison intervals do not overlap are significantly different at an experiment- wide a of 0.05. R diversipilosa, and this advantage will not be affected by natural heterogeneity in structural complexity. These advan- tages in exploitative competitive ability experienced by P. bicarinata may partially explain why it is able to coexist along with P. diversipilosa, who is a superior interference competitor [29], This study demonstrates how the dual roles of habitat complexity in impeding movement and providing refuge from parasitoids impact the exploitative competitive abilities of two host ant species. These two mechanisms by which habitat complexity mediates competition may function in an opposing manner because of differences in host body size and behavioral dominance. However, further work should be conducted to determine whether differences in exploratory or recruitment strategies offer additional insight into the effects of habitat complexity on each host [45, 46]. Natural variation in habitat complexity or variation caused by disturbance such as fire [13] may impact the relative importance of these mechanisms for each host, the degree to which they are opposing, and therefore the potential for coexistence between these species. Knowledge of the prevalence of complex versus simple substrates within and between habitats is important for predicting the degree to which these mechanisms oppose each other, but is currently lacking. Acknowledgments The authors thank Jessica Pearce, Philipp Wiescher, and three reviewers for providing valuable comments on earlier drafts. Stefan Cover and Ed LeBrun helped identify host ants. This study benefited from the facilities of the Southwestern Research Station and the assistance of the Cuenca Los Ojos Foundation. 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[51] J. J. Christian, “Social subordination, population density, and mammalian evolution,” Science, vol. 168, no. 3927, pp. 84-90, 1970. [52] M. A. Bowers and H. D. Smith, “Differential habitat utilization by sexes of the deermouse, Peromyscus maniculatusf Ecology, vol. 60, pp. 869-875, 1979. [53] J. Suhonen, “Predation risk influences the use of foraging sites by tits,” Ecology, vol. 74, no. 4, pp. 1197-1203, 1993. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 869415, 5 pages doi:10.1155/2012/869415 Research Article Termiticidal Activity of Parkia biglobosa ( Jacq) Benth Seed Extracts on the Termite Coptotermes intermedins Silvestri (Isoptera: Rhinotermitidae) Bolarinwa Olugbemi Division of Termite Control and Ecology, Termite Research Laboratory, P.M.B. 656, Akure 340001, Nigeria Correspondence should be addressed to Bolarinwa Olugbemi, drolugbemi@yahoo.com Received 5 October 2011; Revised 14 November 2011; Accepted 28 November 2011 Academic Editor: Arthur G. Appel Copyright © 2012 Bolarinwa Olugbemi. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The chemical and mineral composition of raw and boiled seeds of the African locust bean, Parkia biglobosa (Jacq) Benth, was determined while the termiticidal action of the aqueous, alcoholic, and acetone extracts of the bean seeds were investigated. Varia- tions in the proximate and mineral composition of the raw and boiled seeds were obtained while heavy minerals such as cadmium, cobalt, lead, nickel, and copper had been leached out of the seed during the process of boiling. Extracts from the raw seeds exhibited varying degree of termiticidal activity, while extracts from the boiled seed had no effect on the workers of Coptotermes intermedins Silvestri. Alcoholic extracts were more active than the aqueous and acetone extracts. Termites die within 30 min, 40 min, and 110 min when exposed to concentration of 4 g mL“^ treatments of alcoholic, aqueous, and acetone extracts, respectively. 1. Introduction Termites cause the most serious damage of all wood-feeding insects. In addition to timber and wood products, they attack growing trees, leather, rubber, and wool as well as agricultural crops [ 1 ] . Significant damage is caused by ter- mites to man-made fabrics, polythene, plastics, metal foils, books, furniture, wooden telephone poles, wooden railway sweepers, and insulators of electric cables [2]. Damage caused by termites to wooden structures in the United States of America is estimated to be over 3 billion Dollars annually, with subterranean termites accounting for at least 80% of these damages [3]. Costs attributable to Coptotermes formosanus in the Hawaiian Islands alone are greater than 60 million Dollars per annum [4]. Termites are so destructive in that they derive their nutrition from wood and other cellulotic materials. In Africa and elsewhere in the developing countries, there is hardly any data on either the quantum of damage done by termites to agricultural crops, construction timbers, paper, and paper products, or the cost of control or repairing the damage done by these insect pests. The damage done by various termite species in Nigeria [2] ranged from scavenging on tree barks and dead branches, to eating out grooves in the roots and stems of plants. Past research efforts had focused more on chemical methods of control, with an obvious lack of attention placed on understanding the behavior and history of these termites. In view of mounting concerns over the side effect caused by the use of these toxic and environmentally unfriendly chemicals, direction of research is now focusing on alterna- tive nontoxic, biological, and environmentally friendly meth- ods of control. These methods include baiting systems, use of asphyxiant gases, application of extreme temperatures, barriers of various types, as well as biological control organ- isms [3, 5]. Extractives with insecticidal properties from naturally resistant wood and plant species in form of phenolic, ter- penoid, and flavonoid compounds, show great promise for prevention of termite attack [6-9]. Some of these substances may also act as feeding deterrent [10-12]. The termite Coptotermes formosanus was found to be at- tracted and preferentially feed upon the amino acids, glu- tamic and aspartic acids [ 13] . These could be used to improve 2 Psyche the effectiveness of baiting systems. Many of the chemicals causing attraction and avoidance in several tree species are polar molecules [14]. Investigation has shown that steaming of the heartwood of the Japanese larch, degraded or removed the chemicals responsible for the inhibition of termite attack [15]. A number of tree species such as the Alaska cedar, redwood, and teak [16] are resistant to termite attack. Neem was found to be a strong repellent to Coptotermes formosanus and was suggested as a barrier tree to protect more vulnerable plants [17]. The use of high levels of carbon dioxide, for extended period of time has been successfully used to control termites in contained spaces [4]. The application of heated air to kill termites has shown to be successful in laboratory bioassays [18]. Liquid nitrogen has also been shown to be effective in eliminating termites in the laboratory [3]. These temperature-based control methods are showing great promise, but need more field studies on their effectiveness in natural settings. In other studies [19] Inundation with water was shown to cause a decline in foraging worker population. This could indicate possible applications to control, for example, the controlled flooding of the territories of specific termite colonies to reduce damage by foragers. Barriers to foraging termites that are being tested include sand, crushed granite, glass splinters, and metal shields. These methods have had mixed successes, thereby pointing to the need for more research in this area [3]. The African locust bean, Parkia biglobosa (Jacq) Benth, is a perennial leguminous tree, found growing wildly in forested and savanna belts in Nigeria. Fermented Parkia seeds are locally used in traditional soup seasoning, medicinal preparations and food additives [20]. In addition, boiled water obtained during fermentation process of P biglobosa seeds is used in controlling termite infestation at the local level. In spite of this practice, few reports exist on the termi- ticidal properties of aqueous solution of FI biglobosa seeds. The effect of various concentration levels of aqueous and acetone extracts of P biglobosa was reported to record total mortality within 40-110 minutes after application [21]. This study is therefore aimed at investigating the effect of aqueous, alcoholic, and acetone extracts of P. biglobosa seeds on the rhinotermitid, Coptotermes intermedius Silvestri in order to ascertain its efficacy in the control of this termite pest. 2. Materials and Methods 2.1. Parkia biglobosa . P. biglobosa seeds were obtained from the open market at the King’s market in Akure, Southwest Nigeria. The raw seeds were washed and sun-dried before grind- ing to powdered form. 100 mL of deionized water, 70% alcohol, and acetone were added to each of 20 g of the powdered seeds of P. biglobosa in covered 250 mL glass beakers, respectively, for 12 h. The extract was filtered, and the filtrate was kept for the bioassay. 20 g of the dried boiled seeds were also grounded to powdered form, and 100 mL of de-ionized water, 70% al- cohol and acetone were added to beakers containing the powdered seeds, respectively. The mixtures were left for 12 h, filtered, and the filtrate was kept for subsequent bioassay. 2.2. Termites. Colonies of Coptotermes intermedius were collected from a decaying branch of Cassia sp. at the Federal University of Technology, Akure, and brought to the Termite Research Laboratory, Akure, Southwest Nigeria. The termite colony with its decaying wood was kept and maintained in a plastic container that was kept moist with the aid of a water-filled cotton wool-capped McCarthy bottle affixed to the side of the container. The termites were maintained in the laboratory for at least one month before use, at a temperature of 27° C ± 3°C and relative humidity of 70% ± 5%. 2.3. Determination of Proximate Composition ofP. biglobosa Seeds. The moisture content, ash, crude fiber, and fat con- tents (raw and boiled seeds) were determined by the meth- ods of the Association of Official Analytical Chemists [22] while the nitrogen component was determined by the micro- Kjeldahl methods [23]. The percentage nitrogen value ob- tained was converted to crude protein by multiplying with a factor of 6.25 [21]. The carbohydrate constituent was cal- culated from a difference of all the components from 100. 2.4. Mineral Analysis. The mineral components were deter- mined by dry- ashing 1 g of the seed powder at 550° C in a furnace. The ash obtained was dissolved in 10% hydrochloric acid, filtered, and made up to standard volume with de- ionized water. The Atomic Absorption Spectrophotometer (Buck Model 200-A) was thereafter used to determine the following minerals in the seed preparation: sodium, potas- sium, calcium, magnesium, iron, copper, lead, manganese, cadmium, cobalt, nickel and phosphorus. 2.5. Bioassay to Determine Termiticidal Activity of Extracts of the Seeds ofP. biglobosa (Raw and Boiled). One milliliter aliquot of the different extract concentrations, ranging from 0.1 gmL“^ to 0.4gmL~\ were each applied unto the surface of a Whatman No. 1 filter paper, allowed to air dry, and placed at the bottom of the covered petri dish arenas, respec- tively [21, 24]. 50 workers of Coptotermes intermedius were placed in each of the arenas containing the different extract concentrations, and they were monitored every 10 min for 3 h to determine the conditions of the termites. The experi- ments were replicated 5 times. For the control experiments, one milliliter of deionised water, 70% alcohol, and acetone were each applied on the surface of a Whatman No. 1 filter paper, allowed to dry, and placed at the bottom of the covered petri dish arenas, respectively. A bioassay was carried out as described above, and replicated 5 times. 3. Results The proximate determination of P. biglobosa seeds showed varying values between the raw and boiled seeds. Moisture content, crude fiber, ash, and carbohydrate values are slightly higher in the raw seeds as against the values for boiled seeds (Table 1). Values for crude protein, fat, and nitrogen are higher in the boiled seeds than in the raw seeds. Psyche 3 Table 1: Proximate composition (mean of 5 replicates) of Parkia biglobosa seeds. Chemical components (%) ± SD Raw (%) ± S D Boiled Moisture 13.40 ± 0.01 11.20 ±0.02 Crude protein 25.44 ± 0.01 30.19 ±0.01 Crude fat 6.60 ± 0.01 18.20 ±0.01 Crude fiber 8.81 ±0.02 7.53 ±0.01 Ash 4.81 ±0.01 3.63 ± 0.01 Carbohydrate 40.94 ± 0.01 29.25 ± 0.01 Nitrogen 4.07 ± 0.01 4.83 ±0.01 Table 2: Mineral composition of Parkia biglobosa seeds. Minerals Raw (mg ■ mL ^ ) ± SD * Boiled (mg ■ mL ± SD Sodium 165.09 ± 0.03 299.50 ± 0.02 Potassium 120.54 ± 0.05 451.00 ± 0.03 Calcium 548.40 ± 0.02 271.05 ± 0.01 Magnesium 62.60 ± 0.01 426.90 ± 0.03 Zinc 16.34 ± 0.01 20.02 ± 0.01 Iron 7.85 ± 0.02 9.00 ± 0.01 Lead 2.45 ± 0.01 ND Manganese 0.97 ± 0.01 0.66 ± 0.01 Cadmium 0.18 ±0.10 ND Cobalt 4.21 ±0.01 ND Nickel 4.01 ±0.01 ND Phosphorus 4.39 ± 0.01 65.72 ± 0.02 Copper 6.97 ± 0.02 ND : Mean of 5 replicates; SD: standard deviation; ND: not detected. The mineral composition of boiled seeds of P. biglobosa such as sodium, potassium, magnesium, zinc, iron and phos- phorus, has higher values than in the raw seeds (Table 2). However, calcium and manganese levels were higher in the raw seeds than in the boiled ones. Elements such as lead, cad- mium, cobalt, nickel, and phosphorus present in the raw seeds are absent in the boiled seeds (Table 2). The termiticidal action of the aqueous seed extracts showed that mortality was achieved faster at higher con- centrations than at lower concentration levels. At 0.4 g mL“^ treatment, all the termites died within 40 minutes while it took 60 minutes for all the termite workers to die when exposed to the 0.1 gmL“^ treatment (Figure 1). For the alcoholic extracts, it took just 30 minutes to elim- inate all the termite workers at the 0.4 gmF“^ concentration level compared to 0.1 gmF~\ which took 60 minutes to kill all the termites (Figure 2). The acetone extracts showed termiticidal activity at 0.3 g mF“^ and 0.4 g mF“^ concentration levels while no sig- nificant activity was detected at the 0.1 gmF“^ and 0.2 gF“^ concentration levels (Figure 3). Termiticidal activity was not detected in bioassays involv- ing aqueous, alcoholic, and acetone extracts of boiled seeds of P. biglobosa, as no death was recorded in all the tests. Similarly, no termite death was obtained in the control ex- periments. O.lgmL”^ 0.3gmL“^ 0.2gmL“^ 0.4gmL“^ Figure 1: Antitermitic activity of aqueous extracts of Parkia biglo- bosa. 4. Discussion The variations obtained in the values of proximate and mineral composition of raw and boiled seed powder of P. biglobosa may be due to the effect of boiling, which increases, reduces, or totally eliminates some minerals from the boiled 4 Psyche O.lgmL”^ 0.3gmL“^ 0.2gmL“^ 0.4gmL“^ Figure 2: Antitermitic activity of 70% alcoholic extracts of P. biglo- bosa. Time (min) O.lgmL”^ 0.3gmL“^ 0.2gmL“^ 0.4gmL“^ Figure 3: Antitermitic activity of acetone extracts of P. biglobosa. seeds. An earlier study [25] had reported that boiling, soak- ing in water, and dehulling of African locust bean, led to the reduction in ash, crude fiber, and mineral contents of the seeds. However, the overall increase in crude fat and crude protein contents in the boiled seed extract may be due to reduction in concentration of other chemical components during the boiling process. These differences may account for the termiticidal activity in extracts of the raw seeds when compared with extracts from the boiled seeds. It was reported [26] that a larger percentage of the mineral constituent in P. biglobosa may reside in the hull of the seed, and are therefore leached out during processing. Raw P. biglobosa have been found to contain some heavy metals such as cadmium, cobalt, nickel, lead, and copper [27], which are basic natural components of the earth crust and are toxic at low concentrations. Heavy metals such as Cd, Co, Cu, Ni, and Pb were found in extracts of the raw seeds, but absent in extracts of the boiled seeds. These elements might have been lost during the boiling process [25] . The presence of these heavy metals in the raw seeds, in addition to the factor of polar organic compounds arising from the interactions between the mineral constituents and the extracting medium may therefore explain the termiticidal action of these extract on workers of C. intermedins. Termiticidal activity of aqueous and alcoholic extracts of raw seeds of P. biglobosa increases as concentration levels increases while significant termiticidal activity for acetone extract was obtained only at higher concentration levels (0.3gmL“^ and 4gmL“^). At lower concentration levels (O.lgmL”^ and 0.2gmL“^), acetone extracts of raw P. biglobosa did not confer significant activity on the test termites. This result confirmed an earlier observation made in a study on the termiticidal effect of P biglobosa extracts on the termite, Amitermes evuncifer [21]. Aqueous, alcoholic, and acetone extracts of boiled seeds did not have termiticidal effect on these termite workers. This may explain why the boiled water obtained during the fermentation stage of preparation is effective in the tradi- tional control of termite infestation in the rural areas [1,21]. Extracts from several plants have been reported to have biocidal effects or feeding deterrents to insects such as termites [8, 9, 11, 12, 28]. It has also been well established that certain extractives found in plant materials acted as natural repellent for termites [7, 14, 28, 29]. Extracts from raw seeds of P biglobosa, which appears to be relatively nontoxic to people, have therefore shown to be an effective termiticide candidate for the control of the termite, Coptotermes intermedins. Since there has never been any adverse documented toxicological effect on local pro- cessors of P. biglobosa as well as consumers of the finished product, toxicity of the raw extracts may be unharmful to people. 5. Conclusion This study has shown that extracts of the African locust beans (P. biglobosa) seeds have termiticidal properties, and could be effectively used for the control of termite infestations. The presence of some heavy metals as well as the interacting polar organic compounds in the raw seed extracts could account for this termiticidal property. Further research work is, however; required to determine the contributions of each of these chemical constituents to the overall termiticidal action of raw locust bean seed extracts. References [1] S. L. O. 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Clement, “Terpenes from the maritime pine Finns pinaster: toxins for subterranean termites of the genus Reticulitermes (Isoptera; Rhinotermitidae)?” Biochemi- cal Systematics and Ecology, vol. 18, no. 1, pp. 13-16, 1990. [7] F. S. Nakayama, P. Chow, D. S. Bajwa, J. A. Youngquist, J. H. Muehl, and A. M. Krzysik, “Preliminary investigation on the natural durability of guayule {Parthenium argentatum) based wood products ,” in Proceedings of the 31st International Research Group on Wood Preservation Annual Meeting, Kona, Hawaii, USA, May 2000. [8] J. Morisawa, C. S. Kim, T. Kashiwagi, S. I. Tebayashi, and M. Horiike, “Repellents in the Japanese cedar, Cryptomeria japon- ica, against the pill-bug, Armadillidium vulgare,” Bioscience, Biotechnology and Biochemistry, vol. 66, no. 11, pp. 2424-2428, 2002. [9] A. A. M. Coelho, J. E. Paula, and L. S. Espindola, “Insecticidal activity of cerrado plant extracts on Rhodnjus milesi Car- cavallo, Rocha, Galvao and Juberg (Hemiptera: Reduviidae) under laboratory conditions,” Neotropical Entomology, 2006. [10] R. H. Schefifrahn, “Allelochemical resistance of wood to ter- mites,” Sociobiology, vol. 1, no. 1, pp. 257-281, 1991. [11] K. Chen, W. Ohmura, S. Doi, and M. Aoyama, “Termite feed- ing deterrent from Japanese larch wood,” Bioresource Technol- ogy, vol. 95, no. 2, pp. 129-134, 2004. [12] A. M. Taylor, B. L. Gartner, J. J. Morrell, and K. Tsuoday, “Effects of heartwood extractive fractions of Thuja plicata and Chamaecyparis nootkanensis on wood degradation by termites or fungi,” Journal of Wood Science, vol. 52, pp. 147-153, 2006. [13] J. Chen and G. Henderson, “Determination of feeding pre- ference of Eormosan subterranean termite (Coptotermes for- mosanus Shiraki) for some amino acid additives,” Journal of Chemical Ecology, vol. 22, no. 12, pp. 2359-2369, 1996. [14] J. K. Grace, “Influence of tree extractives on foraging prefer- ences of Reticulitermes flavipes (Isoptera; Rhinotermitidae),” Sociobiology, vol. 30, no. 1, pp. 35-42, 1997. [15] S. Doi, M. Takahashi, T. Yoshimura, M. Kubota, and A. Adachi, “Attraction of steamed Japanese larch (Larix leptolepis (Sieb. et Zucc.) Cord.) heartwood to the Subterranean Termite Cop- totermes formosanus Coptotermes formosanus Shiraki (Iso- ptera: Rhinotermitidae),” Holzforschung, vol. 52, no. 1, pp. 7- 12, 1998. [16] J. K. Grace and R. T. Yamamoto, “Natural resistance of Alaska cedar, redwood, and teak to Eormosan subterranean termites,” Eorest Product Journal, vol. 44, pp. 41-44, 1994. [17] K. M. Delate and J. K. Grace, “Susceptibility of neem to attack by the Eormosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera; Rhinotermitidae),” Journal of Applied Entomology, vol. 119, pp. 93-95, 1995. [18] R. J. Woodrow and J. K. Grace, “Thermal tolerances of four termite species (Isoptera: Rhinotermitidae, Kalotermitidae),” Sociobiology, vol. 32, no. 1, pp. 17-25, 1998. [19] B. T. Eorschler and G. Henderson, “Subterranean termite behavioral reaction to water and survival of inundation: impli- cations for field populations,” Environmental Entomology, vol. 24, no. 6, pp. 1592-1597, 1995. [20] E. O. Ajayeoba, “Phytochemical and antibacterial properties of Parkia biglobosa and Parkia bicolorParkia bicolor leaf extracts,” African Journal of Biomedical Research, vol. 5, pp. 125-129, 2002. [21] T. O. Eemi-Ola, V. A. Ajibade, and A. Afolabi, “Ghemical com- position and termicidal properties of Parkia biglobosa (Jacq) benth,” Journal of Biological Sciences, vol. 8, no. 2, pp. 494-497, 2008. [22] AOAG (Association of Official Analytical Ghemists), Official Methods of Analysis, Washington, DG, USA, 15th edition, 1990. [23] D. Pearson, Chemical Analysis of Poods, Ghurchill Livingstone, London, UK, 7th edition, 1976. [24] E. L. Garter, A. M. Garlo, and J. B. Stanley, “Termiticidal com- ponents of wood extracts: 7-methyljuglone from Diospyros virginianaj Journal of Agricultural and Pood Chemistry, vol. 26, no. 4, pp. 869-873, 1978. [25] B. O. Omafuvbe, O. S. Ealade, B. A. Oshuntogun, and S. R. A. Adewusi, “Ghemical and biochemical changes in African locust bean {Parkia biglobosa) and melon {Citrullus vulgaris) seeds during fermentation to condiments,” Pakistan Journal of Nutrition, vol. 3, no. 3, pp. 140-145, 2004. [26] D. A. Alabi, O. R. Akinsulire, and M. A. Sanyaolu, “Qualitative determination of chemical and nutritional composition of Parkia biglobosa (Jacq.) Benth,” African Journal of Biotechnol- ogy, vol. 4, no. 8, pp. 812-815, 2005. [27] H. H. Gheng, Pesticides in the Soil Environment: Processes, Impacts, and Modeling, W/S Soil Science Society of America, Madison, Wis, USA, 1990. [28] B. Neya, M. Hakkou, M. Petrissans, and P. Gerardin, “On the durability of Burkea Africana heartwood; evidence of biocidal and hydrophobic properties responsible for durability,” Annals of Barest Science, vol. 61, no. 3, pp. 277-2^2, 2004. [29] V. U. Blaske and H. Hertel, “Repellent and toxic effects of plant extracts on subterranean termites (Isoptera; Rhinoter- mitidae),” Journal of Economic Entomology, vol. 94, no. 5, pp. 1200-1208, 2001. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 150418, 6 pages doi:10.1155/2012/150418 Research Article Incorporating a Sorghum Habitat for Enhancing Lady Beetles (Coleoptera: Coccinellidae) in Cotton P. G. Tillman^ and T. E. CottrelP ^ USDA, ARS, Crop Protection and Management Research Laboratory, RO. Box 748, Tifton, GA 31793, USA ^ USDA, ARS, Southeastern Fruit & Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA 31008, USA Correspondence should be addressed to P. G. Tillman, glynn.tillman@ars.usda.gov Received 27 September 2011; Accepted 29 November 2011 Academic Editor: Ai-Ping Liang Copyright © 2012 P. G. Tillman and T. E. Cottrell. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lady beetles (Coleoptera: Coccinellidae) prey on insect pests in cotton. The objective of this 2 yr on-farm study was to document the impact of a grain sorghum trap crop on the density of Coccinellidae on nearby cotton. Scymnus spp., Coccinella septempunctata (L.), Hippodamia convergens Guerin-Meneville, Harmonia axyridis (Pallas), Coleomegilla maculata (De Geer), Cycloneda munda (Say), and Olla v-nigrum (Mulsant) were found in sorghum over both years. Lady beetle compositions in sorghum and cotton and in yellow pyramidal traps were similar. Lor both years, density of lady beetles generally was higher on cotton with sorghum than on control cotton. Our results indicate that sorghum was a source of lady beetles in cotton, and thus incorporation of a sorghum habitat in farmscapes with cotton has great potential to enhance biocontrol of insect pests in cotton. 1. Introduction Lady beetles (Coleoptera: Coccinellidae) have a significant impact on aphids (Hemiptera: Aphididae) [1-4], including the cotton aphid {Aphis gossypii Glover) attacking cotton {Gossypium hirsutum L.) [5] and the corn leaf aphid {Rhopalosiphum maidis (Fitch) and greenbug {Schizaphis graminum (Rondani) attacking grain sorghum {Sorghum bicolor (L.) Moench spp. bicolor) [6-8]. In the southeastern USA, cotton fields commonly are closely associated with other agronomic crops, especially corn {Zea mays L.) and peanut {Arachis hypogea L.), and in these farmscapes poly- sphagous pest species are known to move from corn and peanut into cotton to find newly available, suitable food, or oviposition sites [9]. As part of a larger pest management strategy, strips of grain sorghum planted between a source crop and cotton have proved useful as a trap crop to reduce pest movement, especially stink bugs (Hemiptera: Pentat- omidae), into cotton [10, 11]. Additionally, a grain sorghum trap crop is beneficial to natural enemies by hosting the corn leaf aphid and greenbug [12]. Thus, grain sorghum, when planted adjacent to cotton, can perform as a trap crop for stink bugs and possibly as a source of natural enemies mov- ing into cotton. In fact, many species of Coccinellidae are commonly found inhabiting grain sorghum: Harmonia axyridis (Pallas), Hippodamia convergens Guerin-Meneville, H. sinuata Mulsant, H. parenthesis (Say), Coccinella septem- punctata L., Coleomegilla maculata (De Geer), Cycloneda munda (Say), Scymnus spp., Olla v-nigrum (Mulsant), Exo- chomus sp., and Psyllobora vigintimaculata (Say) [13-16]. These same species colonize cotton [4, 16, 17], and their presence within a grain sorghum trap crop may lead to these predators moving into cotton and facilitating insect pest management. The objective of this study was to document the impact of a grain sorghum trap crop on the density of Coccinellidae on nearby cotton. Two treatments were used: (1) cotton fields without sorghum and (2) cotton fields bordered on one side by a strip of grain sorghum. Within the grain sorghum, Coc- cinellidae were not only sampled on plants but also from yel- low pyramid traps that predominantly served in the larger pest management scheme to kill stink bugs in sorghum. 2. Materials and Methods 2.1. Study Sites. Six cotton fields, ranging from 5 to 18 ha in size, were sampled each year, 2006 and 2007, in Irwin County GA (Table 1). Recommended agricultural practices 2 Psyche Table 1: Planting date (PD) and variety for cotton (Ct) with sor- ghum trap crops, control cotton, and sorghum (So) in 2006 and 2007. Year Treatment Rep Crop Variety^ PD Cotton w/trap crop 1 Ct DP 555 4/28 Cotton w/trap crop 2 Ct DP 555 5/4 Cotton w/trap crop 3 Ct DP 555 5/10 2006 Sorghum trap crop 1-3 So DK 54 4/14 Control cotton 1 Ct DP 555 5/1 Control cotton 2 Ct DP 555 5/4 Control cotton 3 Ct DP 555 5/26 Cotton w/trap crop 1 Ct DP 555 5/9 Cotton w/trap crop 2 Ct DP 555 6/7 Cotton w/trap crop 3 Ct DP 555 6/11 2007 Sorghum trap crop 1-3 So DK 54 6/13 Control cotton 1 Ct DP 555 5/11 Control cotton 2 Ct DP 555 5/11 Control cotton 3 Ct DP 555 6/11 ''Seed companies; DK: DeKalb; DP: Deltapine. for production of sorghum [18] and cotton [19] were fol- lowed. Row width was 0.91 m for each crop, and rows for each crop were parallel to each other. 2.2. Yellow Pyramidal Traps. These traps consisted of a 2.84- liter clear plastic polyethylene terephthalate jar (United States Plastic Corp., Lima, OH, USA) on top of a 1.22 m-tall yellow pyramidal base [20, 21] . An insecticidal ear tag (Saber Extra, Coppers Animal Health Inc., Kansas City, KS, USA) was placed in the plastic jar at the beginning of a test to prevent escape of captured specimens. Active ingredients in the ear tag were lambda- cyhalothr in (10%) and piperonyl butoxide (13%). As part of the larger strategy to reduce pest movement into cotton, stink bug attraction to the traps was enhanced by placing Euschistus spp. stink bug lures {40 pL of the Euschistus spp. pheromone, methyl {E, Z)-2,4-decadienoate (CAS registry no. 4493-42-9) (Degussa AG Fine Chemicals, Marl, Germany, loaded onto rubber septa) in traps and replacing lures weekly. Insects from weekly collections were taken to the laboratory for identification. 2.3. Experimental Design. Two treatments were used each year: control cotton (without a sorghum trap crop) and cot- ton bordered by a sorghum trap crop and yellow pyramidal traps within the trap crop. At the beginning of the study, six commercial cotton fields were selected in Irwin County, Georgia, and each treatment was assigned randomly to three cotton fields similar to a completely randomized design. For the sorghum trap crop treatment, sorghum was planted in a strip (4 rows) along one edge of the cotton field; row 1 of sorghum was adjacent to a peanut field and row 4 was ad- jacent to the cotton field. Then 25-28 yellow pyramidal traps (depending on field width) were placed 12 m apart in row 1 of sorghum. 2.4. Insect Sampling. Each year of the study, crops and yellow pyramidal traps were examined weekly for the presence of lady beetles; from the week of 5 July to the week of 16 August in 2006 and from the week of 19 July to the week of 23 August in 2007. Due to time constraints of sampling these large fields, not all farmscapes were sampled on the same day of the week, but crops and/or yellow pyramidal traps within a field were sampled on the same day. For each sorghum sample, all plant parts within a 1.83 m length of row were visually checked for all lady beetles. For each cot- ton sample, all plants within a 1.83 m length of row were shaken over a drop cloth, and the aerial parts of all plants were visually checked thoroughly for all lady beetles. Voucher specimens are stored in the USDA-ARS, Crop Protection and Management Research Laboratory in Tifton, GA, USA. For sampling purposes, the edge of a cotton field adjacent to a peanut field was labeled as side A, and in a clockwise direction the other 3 sides of a field were labeled as sides B, C, and D. Each year, samples were obtained from within the cotton field at 3 distances from the edge of side A (i.e., at rows 1,2, and 5), and at 6 interior locations along the length of the field (i.e., rows 16, 33, 100, 167, 233, and 300 from the edge of the field on side A). In both years, the 300-row samples were not close to the edge of side C; 24-31 m from side C in 2006 and 61m from side C in 2007. For sides B-D, samples were taken from 2 edge locations, rows 1 and 5 from the edge of the field. The number of samples from each field on each date was as follows: 9 from each row on side A, 3 from each row on sides B-D, and 6 from each interior location. For both years, the 4-row strip of sorghum was sampled by taking 9 samples from each of the 4 rows. 2.5. Statistical Analysis. Lady beetle species compositions in sorghum strips, cotton fields, and yellow pyramidal traps were similar for both years, and then data for the two years were combined. Means were obtained for number of lady beetle adults per sample for sorghum and yellow pyramidal traps using PROC MEANS [22]. The number of lady beetle adults per sample in cotton with sorghum trap crops and control cotton was compared using f-tests. In 2007, one cot- ton field with a sorghum trap crop was excluded from data analysis on week 6 due to an insecticide application after sampling on week 5. One control cotton field was excluded from data analysis on weeks 5 and 6 due to an insecticide ap- plication after sampling on week 4. 3. Results and Discussion Scymnus spp., C. septempunctata, H. convergens, H. axyridis, C. maculata, C. munda, and O. v-nigrum were found in crops and yellow pyramidal traps over both years in Georgia. The corn leaf aphid was observed feeding on sorghum mainly during the vegetative stage, and the greenbug was observed mainly feeding in sorghum grain heads. Cotton aphids were present on cotton for much of the growing season, but they were mainly observed on this crop early in the season (late June-early July). Scymnus spp. and C. septempunctata were the predom- inant species in sorghum; however, C. septempunctata and Psyche 3 Table 2: Percentage composition (within columns) of lady beetle species in sorghum trap crops, yellow pyramidal traps, cotton with sorghum trap crops, and control cotton. Species Percentage in sorghum trap crops (n = 1789) Percentage in yellow pyramidal traps (n = 20,313) Percentage in cotton w/sorghum trap crops (n = 4804) Percentage in control cotton {n = 2879) Scymnus spp. 51.9 16.5 28.6 43.0 C. septempunctata 33.9 38.1 13.7 14.8 H. convergens 6.3 6.3 31.7 22.6 H. axyridis 5.4 32.1 24.6 18.3 C. maculata 2.0 5.7 1.1 0.9 0. v-nigrum 0.4 0.1 0.2 0.2 C. munda 0.1 1.2 0.1 0.2 H. axyridis were the most abundant coccinellids captured in the yellow pyramidal traps (Table 2). Species composition was similar for cotton with or without (i.e., control) a trap crop with the predominant species being Scymnus spp., C. septempunctata, H. convergens, and H. axyridis. The lady beetle species in sorghum and cotton have been previously reported to colonize these crops [4, 13-17]. It was not surprising that yellow pyramidal traps (baited with an aggregation pheromone for Euschistus spp. stink bugs) captured adult lady beetles. Captures of lady beetles in this yellow trap, with or without the stink bug pheromone, are common (T.E.C., personal observation), and yellow sticky cards have been used in previous studies to sample adult Coccinellidae [23-27] . The similarity in lady beetle captures in traps and those sampled on sorghum may indicate that the yellow trap itself does not attract lady beetles from significant distances; lady beetle capture in the trap was likely facilitated by the attractiveness of the surrounding sor- ghum. Nevertheless, modifying the yellow pyramidal traps (intended to attract and kill stink bugs) to reduce lady beetle capture could conserve these predators in sorghum. In 2006, lady beetle density remained relatively low in flowering and milking sorghum and then peaked during the soft dough stage of seed development (Figure 1). Generally, corn leaf aphids are first observed on sorghum when plants have three to five leaves, and then their numbers increase on vegetative sorghum until declining around the boot or early bloom stages [7]. Greenbugs also are present on sorghum during the three to five leaf stage, but their numbers do not increase until after plants have about 10 leaves, with peak abundance at the half bloom or soft dough stage and then de- clining as sorghum matures [7]. After week 4, density of adult lady beetles began an overall decline in sorghum, a like- ly result of prey depletion on sorghum. Apparently, as prey were depleted on sorghum, beetles moved from sorghum to yellow pyramidal traps during week 5 but their capture in these traps dropped precipitously thereafter (Figure 1). Density of lady beetles increased slightly on sorghum during the hard dough stage (i.e., when 75% of the grain dry weight has accumulated) and then declined as sorghum heads matured. In cotton, lady beetles first appeared in relatively low numbers in early July and peaked on cotton in late July- early August. Fady beetle density was significantly Cotton w/sorghum trap crop Sorghum Control cotton Yellow pyramidal traps Figure 1; Mean number of lady beetle adults per sample in cotton with a sorghum trap crop, control cotton, sorghum, and yellow pyramidal traps in 2006. FT; flowers; FR: fruit. Number of lady beetles in yellow pyramidal traps divided by 10. Date refers to middle of sampling week. higher on cotton with sorghum trap crops than on control cotton during weeks 2 through 6 (Table 3). Altogether, these results indicate that sorghum was a source of adult lady beetles moving into cotton fields. Because cotton aphids were observed on cotton early in the season, lady beetles were likely responding to populations of cotton aphids in cotton. Aphidophagous lady beetles, though, can be generalist predators; therefore, when they moved into fruiting cotton, they were likely also preying on other pest insects that feed on cotton fruit such as lepidopteran pests and stink bug eggs [28, 29]. In 2007, lady beetle abundance on sorghum followed a similar pattern as seen during 2006. Beetles first moved to flowering sorghum, and density peaked when sorghum heads reached the soft dough stage (Figure 2). Fady beetle density was significantly higher on cotton with sorghum trap crops than on control cotton during weeks 1 through 5 (Table 3). As above, these results suggest that sorghum can serve as a source of lady beetles dispersing to cotton. 4 Psyche Table 3: Number (mean ± SE) of lady beetle adults per 1.83 m of row in cotton with sorghum trap crops and control cotton in 2006 and 2007. Year Week Cotton w/sorghum trap crop Control cotton \t\ df P 1 0.15 ±0.02 0.106 ±0.022 1.45 937 0.1465 2 0.431 ± 0.039 0.246 ± 0.04 3.2 937 0.0014 3 0.982 ± 0.076 0.349 ± 0.038 lAl 1132 0.0001 2006 4 0.952 ± 0.075 0.545 ± 0.052 4.51 1132 0.0001 5 1.153 ±0.094 0.52 ± 0.057 5.79 1132 0.0001 6 0.918 ± 0.072 0.66 ± 0.053 2.91 1132 0.0037 7 0.65 ± 0.059 0.648 ± 0.065 0.02 1327 0.9819 1 0.611 ±0.051 0.302 ± 0.039 4.6 503 0.0001 2 0.447 ± 0.045 0.309 ± 0.036 2.4 536 0.0168 2007 3 0.732 ± 0.058 0.411 ±0.045 4.39 563 0.0001 4 0.637 ± 0.059 0.487 ± 0.049 1.97 413 0.049 5 0.696 ± 0.075 0.479 ± 0.066 2.03 476 0.0432 6 0.412 ± 0.047 0.338 ± 0.05 1.07 341 0.2845 Cotton w/sorghum trap crop Sorghum Cotton control Yellow pyramidal traps Figure 2: Mean number of lady beetle adults per sample in cotton with a sorghum trap crop, control cotton, sorghum, and yellow pyramidal traps in 2007. BD: buds; EL: flowers; FR: fruit. Number of lady beetles in yellow pyramidal traps divided by 10. Date refers to middle of sampling week. In the current study, results suggest that adult lady beetles dispersed from sorghum into cotton. Previous studies also have demonstrated sorghum as a source of lady beetles mov- ing into cotton. For example, populations of insect predators, including H. convergens, increased when feeding on green- bug in grain sorghum fields adjacent to cotton in Arizona [30, 31]. These predators dispersed into cotton as sorghum matured and the greenbug population declined. Cage studies also indicate that adult lady beetles disperse from sorghum into cotton in response to crop phenology and prey abun- dance [32] . In the current study, lady beetle density similarly declined as sorghum matured. In a study in Texas, as greenbug and corn leaf aphid numbers increased in sorghum, predators, including the predominant Hippodamia spp. predators, also increased [33]. They reported that predator levels in cotton began to increase at about the same time that predator density began to decrease in sorghum indicating that predators dispersed from sorghum into cotton. In fact, fluorescent dust marking demonstrated predator dispersal from sorghum into cotton. In another study using rubidium to mark predators in sorghum and cotton, H. convergens and Scymnus loewii Mulsant were documented to move from sorghum into cotton [32]. In these previous studies, adult lady beetles dispersed from sorghum into cotton in response to sorghum senes- cence and prey decline, but in our study, lady beetles con- tinuously moved from sorghum to cotton throughout devel- opment of fruit in cotton. Although the reason for lady beetles continuously moving from sorghum into cotton was not determined, it was likely due to resource availability (e.g,, prey, pollen, and extrafloral nectaries) in cotton. Perhaps, planting sorghum earlier would result in relaying lady beetles from senescing sorghum into cotton as documented in a 3 yr relay intercropping study in Texas [34] . There, the intercrops acted as a reservoir for predators, including lady beetles, dur- ing the noncotton season. These intercrops “relayed” the aphid predators from canola and wheat in the winter to sorghum in the spring and finally to cotton in the summer. Of the intercrop species tested, predator numbers were high- est in sorghum. Average aphid abundance was lower in relay intercropped cotton than in isolated cotton, and average predator numbers were higher in relay intercropped cotton than in isolated cotton. Predators appeared in higher num- bers earlier in the summer in relay intercropped cotton than in isolated cotton suggesting that this management strategy aids early colonization of predators in cotton, thereby in- hibiting increase of the cotton aphid. In a 2 yr study in Texas, a sorghum relay strip-crop system enhanced numbers of pre- dators, including lady beetles, and suppressed cotton aphid abundance in cotton [35]. It can be concluded from these two studies and the current study that incorporating a source crop for lady beetles in a cotton field can be a successful management tactic for control of cotton aphids. Also, Psyche 5 a multifunctional habitat of sorghum to detract stink bugs from feeding and ovipositing on cash crops, and using pher- omone traps to capture and kill stink bugs has great potential for suppressing stink bugs in cotton while preserving lady beetles. Lady beetles in this study were present in cotton fields with or without sorghum indicating that these natural enemies disperse into cotton from other plants. Peanut fields were adjacent to all the cotton fields, but early-season host plants such as corn and rye were also prevalent in these agricultural landscapes. 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Phatak et al., “Influence of cover crops on insect pests and predators in conservation tillage cot- ton,” /owrna/ of Economic Entomology, vol. 97, no. 4, pp. 1217- 1232, 2004. [37] G. Tillman, “Populations of stink bugs (Heteroptera: Pen- tatomidae) and their natural enemies in peanuts,” Journal of Entomological Science, vol. 43, no. 2, pp. 191-207, 2008. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 532314, 5 pages doi:10.1155/2012/532314 Research Article Eggs of the Blind Snake, Liotypblops albirostris. Are Incubated in a Nest of the Lower Fungus-Growing Ant, Apterostigma cf. goniodes Gaspar Bruner, Hermogenes Fernandez-Marin, Justin C. Touchon, and William T. Wcislo Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Panama, Panama Correspondence should be addressed to Hermogenes Fernandez-Marin, fernandezh@si.edu and William T. Wcislo, wcislow@si.edu Received 15 September 2011; Accepted 25 November 2011 Academic Editor: Diana E. Wheeler Copyright © 2012 Gaspar Bruner et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Parental care is rare in most lower vertebrates. By selecting optimal oviposition sites, however, mothers can realize some benefits often associated with parental care. We found three ovoid reptilian eggs within a mature nest of a relatively basal fungus-growing ant, Apterostigma cf. goniodes (Attini), in central Panama. In laboratory colonies, A. cf. goniodes workers attended and cared for the eggs. Two blind snakes, Liotyphlops albirostris (Anomalepididae), successfully hatched, which is the first rearing record for this species. The ants did not disturb the snakes, and the snakes did not eat the ants; we found no ants in the dissected stomachs of the snakes. We review other associations between nesting fungus-growing ants and egg-laying vertebrates, which together suggest that attine nests may provide a safe, environmentally buffered location for oviposition, even in basal attine taxa with relatively small colony sizes. 1. Introduction The degree to which organisms are buffered from environ- mental fluctuations is often reflected in basic life history strategies; at the extremes, some produce large numbers of offspring that suffer extremely high mortality rates, whereas others produce relatively few offspring but are cared for by their parents, thereby decreasing juvenile mortality [1]. For oviparous animals, a mother can realize some benefits normally associated with parental care (e.g., decreased rates of egg mortality), yet not bear the associated costs, by placing her eggs in a nest where heterospecifics tend the eggs, such as brood parasitic bees [2] or birds [3] that lay eggs in nests of other bees or birds, respectively. In some cases, the associations involve distantly related taxa. Among oviparous snakes, parental care is rare, beyond providing the eggs a relatively secure nesting site [4]. There are scattered reports of nesting associations between both amphibians and reptiles with a variety of ant species [5-9], including fungus- growing ants (Attini, Formicidae) [5, 10, 11]. All attines maintain a homeostatic environment (including humidity and temperature) in order to cultivate their fungal symbiont [12, 13], which is a stable environment that snakes exploit [5, 6]. A stable incubation temperature is important for eggs of many species of reptiles, including snakes, and can determine sex and behavior after hatching [14, 15]. Little is known about the nature of potential snake-ant associations, and whether the snakes provide any benefit to the ants or are harmless commensals. Reports thus far indicate that the ants, including soldiers with greatly enlarged mandibles, do not harm the snake eggs [5]. To date all the reported associations between fungus-growing ants and snake eggs are limited to Atta and Acromyrmex colonies, which contain thousands or millions of workers with large numbers of nest chambers [16], raising the question of whether only large colonies provide suitable abiotic conditions that are sufficiently stable for the development of snake eggs (e.g., [17, 18]). Here, we report finding three snake embryos in a nest of a phylogenetically basal attine ant with relatively small nests, Apterostigma cf. goniodes. We also provide a review of the associations among attines with both squamates and 2 Psyche anurans and briefly sketch possible hypotheses for the origins of these associations. 2. Materials and Methods A nest of a fungus -growing ant, A. cf. goniodes, was excavated in central Panama (Plantation Road, Soberania National Park, Colon Province, Panama) in August 2009, A, cf gon- iodes is a small ant (0.4 mm head width, 0.6 mm head length) with mature colonies having more than 1,000 workers (HFM, personal observation). They cultivate a G4 fungus (Family: Pterulaceae), which is phylogenetically distant from lepiotaceous fungus cultivated by Atta and Acromyrmex (G1 fungus) [17]. The nest contained four chambers with fungus gardens. In one of the four chambers, we found three ovoid snake eggs (3.0 x 0.75 cm, measured along the widest and longest axes) embedded in the fungal garden. The nest con- tents, including the fungus garden, workers, ant brood, and the snake eggs were collected and placed in a small plastic container with wet cotton inside to maintain humidity. In the laboratory, we maintained the embryos, along with the ant colony, in a Petri dish (6 cm X 2.5 cm), which was placed inside a larger plastic container (19 cm X 16 cm X 7 cm) at a temperature of 25° G, on a light: dark cycle of 12 : 12 hr. The ant nest was fed with corn meal and cleaned twice per week. To determine if the ants could distinguish snake eggs from a snake -like egg made of a different material, we ob- served the behavioral responses of ants to natural and artificial snake eggs. We transferred the eggs from the nest to a sterile Petri dish and gently removed the fungal mycelium around the eggs using sterile forceps. We shaped plasticine into a form that mimics a snake egg and sterilized it (115°G for 20 min). Then, we placed one egg, either plasticine or natural, on the top of the fungal garden. Using a stereomicroscope (0.7x), we observed the ants’ behavior toward the egg in 10-minute periods for a total of 120-minute observation for each egg. We recorded the frequency at which ants placed a piece of fungal mycelium on the egg (a known hygienic behavior [ 16] ); made antennal contact with the egg; groomed the surface of the egg using the mouth. We monitored the nest until the snakes hatched. Once hatched, the snakes were maintained for 2 weeks in the ant nest to observe snake-ant interactions with special emphasis on possible agonistic interactions (e.g., did the newly hatched snakes eat the ant workers?). After 18 days, two snakes were found dead in the nest and we stored them in alcohol (95%). These snakes were dissected using a surgical scalpel, and the stomach contents were analyzed for any evidence that they had fed on A. cf. goniodes workers, brood, or eggs. Voucher specimens of the ant species is deposited in the Dry Reference Gollection of the Smithsonian Tropical Research Institute and the Museo de los Invertebrados, Uni- versidad de Panama. 3. Results and Discussion Two of the three embryos hatched nine and 12 days after the nest was collected, and both were identified as Liotyphlops Figure 1: 1, albirostris egg on a colony of A. cf. goniodes. Note that the workers have planted pieces of fungal garden on the egg’s shell. Table 1: Behavioral responses (behavior/ 10 min ±; means ± SD) by Apterostigma cf. goniodes workers to natural and artificial eggs. P* refers to the P value from Wilcoxon signed-rank tests; n = 12 observation periods. Natural egg Artificial egg p* Planting cover 2.41 ± 1.37 0±0 P = 0.019 Antennal contact 1.66 ± 1.55 0.5 ± 1.16 P = 0.003 Grooming 0.66 ± 0.65 0±0 P = 0.011 albirostris (Figure 1), the white-nosed blind snake (Anoma- lepididae), which is a relatively basal, Gentral American endemic snake [22] . These small snakes (adult total length, ~ 223 mm) are fossorial, and little is known about their biology. Furthermore, to our knowledge, there are no documented accounts of their oviposition behavior. Approximately three days prior to hatching, the first embryo had longitudinal grooves on the egg and amniotic fluid was leaking from the egg. This embryo was removed from the nest and placed in a sterile Petri dish. We cut open the egg shell, and a live, well-developed neonate emerged with the yolk sac still connected. After 48 hours, the connection to the yolk sac was lost. The snake appeared healthy and was placed in the ant nest with the remaining embryos. At a similar age prior to hatching, a second egg appeared to have the same grooves, and, two days, later the snake naturally hatched and was unconnected with its yolk sac. With respect to the interactions between ants and snake eggs, A. cf. goniodes workers repeatedly antennated and groomed snake eggs, but were never observed to bite them (Figure 1, Table 1). Moreover, ant workers took fungus garden pieces, with and without substrate, and planted them on the eggs. This behavior is very similar to what the workers do with ant eggs, larvae, and pupae within their fungus garden, as a method for putative control of point Psyche 3 Table 2: General summary of Squamata associated with fungus-growing ants. Ant species Vertebrates Nature of association^ Region Apterostigma Cf. goniodes Liotyphlops alhirostris (A) This study Panama Acromyrmex ambiguous Philodryas patagoniensis (C), Liophis obtusus (C) Oviposition Uruguay [5] Acromyrmex echinatior Unidentified Oviposition Panama (H.F.M. pers. comm) Acromyrmex heyeri Philodryas patagoniensis (C), Liophis obtusus (C) Oviposition Uruguay [5] Acromyrmex heyeri Pseudoblabes agassizii (C), Liophis obtusus (C), Oviposition Uruguay [6] Acromyrmex hispidus Philodryas patagoniensis (C), Clelia rustica (C) Oviposition Uruguay [5] Acromyrmex hispidus Philodryas aestivus manegarzoni (C) Oviposition Uruguay [6] Acromyrmex lobicornis Philodryas patagoniensis (C), Liophis obtusus (C), Micrurus frontalis altirostris (E), Leptotyphlops munoai (L), Liophis jaeger i (C), Pseudoblabes agassizii (C), Elapomorphus bilineatus (C) Oviposition Uruguay [5] Acromyrmex lobicornis Liophis obtusus (C), Clelia rustica (C), Philodryas patagoniensis (C), Liophis obtusus (C), Pseudoblabes agassizii (C) Oviposition Uruguay [6] Acromyrmex lundi Amphisbaena darwini (Am) Oviposition Uruguay [6] Acromyrmex octospinosus Leptodeira annulata (C) Venezuela [19] Acromyrmex octospinosus Stenorrhina degenhardti (C) Oviposition Colombia [10] Acromyrmex octospinosus Tripanurgos compressus (C) Burrow The island of Trinidad [7] Acromyrmex striatus Philodryas patagoniensis (C), Teius teyou (T) Oviposition Uruguay [5] Atta cephalotes Amphisbaena alba (Am) Burrow and Predator The island of Trinidad [20] Atta colombica Leptodeira annulata (C) Oviposition Panama [5] Atta mexicana Sympholis sp. (C) Burrow Southern Mexico [20] Atta sexdens Leptodeira sp. (C) Oviposition The island of Trinidad [7] Atta sexdens Amphisbaena alba (Am) and Amphisbaena mitchelli (Am) Burrow Brazil [21] Atta sp. and Acromyrmex sp. Psuedoboa neuwiedii (C) Oviposition South America [7] Family (A: Anomalepididae, Am: Amphisbaenidae, C: Golub ridae, E: Elapidae, E: Eeptodactylidae, T: Teiidae). ^Tbe associations are debned as follows: oviposition: an egg was found inside a colony; burrow: an adult or a young snake was found inside tbe colony; predator: analyses of intestinal or fecal contents show evidence of prey. sources of infection [16]. When the mycelial cover of an egg was removed, the ants completely recovered the eggs with fungal garden material (as the snake embryos originally were found) but did not do so with the artificial egg. Ants spent substantially more time physically examining the snake egg than the artificial egg (Table 1), suggesting that the ants were not simply responding to natural eggs as a foreign object. We have observed adults of L alhirostris in nests of other attines, including Trachymyrmex cornetzi (one observation in 188 nest excavations), Trachymyrmex sp. 10 (two obser- vations in 35 nest excavations), and Atta cephalotes (one observation in 12 nests excavations), but no embryos have been observed (H.F.M. and G.B. personal observations). The reproductive biology of L. alhirostris is unknown, and few comparative data are available for blind snakes in general, so we do not know if this species regularly oviposits in nests of ants or other social insects. Some blind snakes have a specialized diet of ants and termites and have an olfactory system that allows them to detect the pheromone trails of their prey and conspecifics [23, 24], raising the possibility that L. alhirostris might follow the A. cf. goniodes workers into the nest, but again we have no data to indicate the snakes are feeding on the ants. Alternatively, the adult snakes might use the nest as a temporary refuge [25] and occasionally oviposit there. Large colonies of leafcutter ants appear to provide a suitable environment for oviposition by small vertebrates, such as reptiles and amphibians (Table 2). The most com- prehensive reports are from the subtropical temperate zone in Uruguay, where 82 of 577 nests of Acromyrmex spp. contained squamate eggs [5, 6] (Table 2). Squamate egg development can take up to 90 days [26], and thus a thermally stable refuge for incubation may be more valuable in temperate regions than in the tropics. Some small subterranean reptiles, including blind snakes, are known to prey on ants [23, 24, 27]. We did not observe, however, any disturbance of the fungus garden or any antagonistic interactions between worker ants and the snakes throughout the posthatching period, despite the fact that L. alhirostris are presumably large enough to feed on A. cf. goniodes workers. Thus, the relationship between L. alhirostris and A. cf. goniodes is unclear. To date, the benefits seem more apparent for the snake. Nothing is known about the chemical ecology of snake-ant interactions, nor how the snakes might interact 4 Psyche with possible ant parasites or agropredators, such as Gnamp- togenys and Megalomyrmex [28, 29], and any possible benefit for the ants is unknown. Another possibility is that the snake egg may provide a temporary hygienic platform on which the ants can cultivate incipient gardens, in the same manner that attines use other found objects in their environment [30]. This study also raises the question of whether some chemical components of the snakes or the egg surface have been modified to mimic the ants’ cuticular hydrocarbons used for recognition, and, if so, to what extent does diversification in these chemical signals help explain the diversity of snakes reported in association with fungus -growing ants (Table 2). Our finding is the first report of a squamate-attine in- teraction involving a more basal fungus -growing ant species, and the first report of the oviposition behavior for the Cen- tral America endemic species L. albirostris. Further, we pro- vide the first behavioral observations of squamate-attine interactions, both before and after hatching. Our review demonstrates that at least 20 species of squamates have been reported to oviposit in nests of 13 species of attine ants. These associations are remarkable in part because they occur underground in ant nests, which makes them extremely difficult to locate and observe. Thus, attine nests as oviposition sites for squamate eggs may be substantially more common than previously believed. Acknowledgments The authores thank Francisco Pimentel for help in the laboratory, Ulrich Mueller for help with ant identifications, and the Autoridad Nacional del Ambiente (ANAM) for permits. G. Bruner and FI. Fernandez-Marin were supported by Secretaria Nacional de Ciencia, Tecnologia e Innovacion (SENACYT) grant, H. Fernandez-Marin by a postdoctoral fellowship from STRI, and general research funds from STRI to W. T. Wcislo. J. C. Touchon was supported by the National Science Foundation (DEB-0716923). References [1] R. H. MacArthur and E. O. Wilson, The Theory of Island Bio- geography, Princeton University Press, 1967. [2] W. T. Wcislo, “The roles of seasonality, host synchrony, and behavior in the evolutions and distributions of nest parasites in Hymen optera (Insecta), with special reference to bees (Apoidea),” Biological Reviews of the Cambridge Philosophical Society, vol. 62, no. 4, pp. 415-443, 1987. [3] M. Andersson and M. Ahlund, “Host-parasite relatedness shown by protein fingerprinting in a brood parasitic bird,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 24, pp. 13188-13193, 2000. [4] J. D. Reynolds, N. B. Goodwin, and R. P. Freckleton, “Evolutionary transitions in parental care and live bearing in vertebrates,” Philosophical Transactions of the Royal Society B, vol. 357, no. 1419, pp. 269-281, 2002. [5] R. Vaz-Ferreira, L.C. de Zolessi, and F. Achaval, “Oviposicion y desarrollo de ofidios y lacertilios en hormigueros de Acromyr- mexf Physis, vol. 29, pp. 431-459, 1970. [6] R. Vaz-Ferreira, F. C. de Zolessi, and F. Achaval, “Oviposicion y desarrollo de ofidios y lacertilios en hormigueros de Acro- myrmexf Trabajos del V Congreso Latinoamericano de Zoolo- gia, vol. 1, pp. 232-244, 1973. [7] J. Riley, A. F. Stimson, and J. M. Winch, “A review of Squamata oviposition in ant and termite nests,” Herpetological Review, vol. 16, pp. 38-43, 1985. [8] A. Schliiter, P. Fottker, and K. Mebert, “Use of an active nest of the leaf cutter ant Atta cephalotes (Hymenoptera: Formicidae) as a breeding site of Lithodytes lineatus (Anura: Feptodactylidae),” Herpetology Notes, vol. 2, no. 1, pp. 101- 105, 2009. [9] A. Schliiter and J. Regos, “Fithodytes lineatus (Schneider, 1799) (Amphibia: Feptodactylidae) as a dweller in nests of the leaf cutting ant Atta cephalotes (Finnaeus, 1758) (Hymenop- tera; Attinff Amphibia- Reptilia, vol. 2, pp. 117-121, 1981. [10] E. Velasquez-Munera, A. Ortiz- Reyes, and V. P. Paez, “Ovipo- sition of Stenorrhina degenhardti (Serpentes: Colubridae) in nest of Acromyrmex octospinosus (Hymenoptera; Formici- dae),” Actualidades Bioldgicas, vol. 30, no. 88, pp. 199-201, 2008. [11] B. Baer, S. P. A. den Boer, D. J. C. Kronauer, D. R. Nash, and J. J. Boomsma, “Fungus gardens of the leafcutter ant Atta colombica function as egg nurseries for the snake Leptodeira annulataf Insectes Sociaux, vol. 56, no. 3, pp. 289-291, 2009. [12] M. Bollazzi and F. Roces, “To build or not to build: circulating dry air organizes collective building for climate control in the leaf-cutting ant Acromyrmex ambiguusf Animal Behaviour, vol. 74, no. 5, pp. 1349-1355, 2007. [13] U. G. MueUer, A. S. Mikheyev, E. Hong et al., “Evolution of cold-tolerant fungal symbionts permits winter fungiculture by leafcutter ants at the northern frontier of a tropical ant-fungus symbiosis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 10, pp. 4053-4056, 2011. [14] J. Xiang and W.-G. Du, “The effects of thermal and hydric environments on hatching success, embryonic use of energy and hatchling traits in a colubrid snake, Elaphe carinataf Journal of Herpetology, vol. 35, no. 2, pp. 186-194, 2001. [15] R. Shine, “Fife-history evolution in reptiles,” Annual Review of Ecology, Evolution, and Systematics, vol. 36, pp. 23-46, 2005. [16] N. A Weber, Gardening Ants, the Attines, American Philosoph- ical Society, Philadelphia, Pa, USA, 1972. [17] J. K. Webb, G. P. Brown, and R. Shine, “Body size, loco- motor speed and antipredator behaviour in a tropical snake (Tropidonophis mairii, colubridae): the influence of incubation environments and genetic factors,” Eunctional Ecology, vol. 15, no. 5, pp. 561-568, 2001. [18] J. J. Kolbe and F. J. Janzen, “Impact of nest-site selection on nest success and nest temperature in natural and disturbed habitats,” Ecology, vol. 83, no. 1, pp. 269-281, 2002. [19] G. R. F. Brandao and P. E. Vanzolini, “Notes on incubatory inquilinism between Squamata (Reptilia) and the neotropical fungus-growing ant genus Acromyrmexf Papas Avulsos de Zoologia, vol. 36, no. 3, pp. 31-36, 1985. [20] J. Riley, J. M. Winch, A. F. Stimson, and R. D. Pope, “The association of Amphisbaena alba ( Reptilia: Amphisbaenia) with the leaf-cutting ant Atta cephalotes in Trinidad,” Journal of Natural History, vol. 20, no. 2, pp. 459-470, 1986. [21] G. Azevedo-Ramos and P. R. S. Moutinho, “Amphisbaeni- ans (Reptilia; Amphisbaenidae) in nests of Atta sexdens (Hy- menoptera: Formicidae) in eastern Amazonia, Brazil,” Ento- mological News, vol. 105, no. 3, pp. 183-184, 1994. Psyche 5 [22] O. Rieppel, N. J. Kley, and J. A. Maisano, “Morphology of the skull of the white-nosed blindsnake, Liotyphlops albirostris (Scolecophidia; Anomalepididae),”/oM^«fl/ of Morphology, vol. 270, no. 5, pp. 536-557, 2009. [23] F. R. Gehlbach, J. F. Watkins, and J. C. Kroll, “Pheromone trail- following studies of typhlopid, leptotyphlopid, and colubrid snakes,” Behaviour, vol. 40, no. 3, pp. 282-294, 1971. [24] J. F. Watkins, F. R. Gehlbach, and R. S. Baldridge, “Ability of the blind snake, Leptotyphlops dulcis, to follow pheromone trails of army ants, Neivamyrmex nigrescens and N. opacithoraxf The Southwestern Naturalist, vol. 12, no. 4, pp. 455-462, 1967. [25] F. R. Gehlbach and R. S. Baldridge, “Live blind snakes {Lepto- typhlops dulcis) in eastern screech owl {Otus asio) nests: a novel commensalism,” Oecologia, vol. 71, no. 4, pp. 560-563, 1987. [26] R. Shine, “Egg-laying reptiles in cold climates: determinants and consequences of nest temperatures in montane lizards,” Journal of Evolutionary Biology, vol. 12, no. 5, pp. 918-926, 1999. [27] J. F. Watkins, F. R. Gehlbach, and J. G. Kroll, “Attractant- repellent secretions of blind snakes {Leptotyphlops dulcis) and their army ant prey {Neivamyrmex nigrescens) f Ecology, vol. 50, pp. 1098-1102, 1969. [28] M. B. Dijkstra and J. J. Boomsma, '"Gnamptogenys hartmani Wheeler (Ponerinae: Ectatommini): an agro-predator of Tra- chymyrmex and Sericomyrmex fungus-growing ants,” Natur- wissenschaften, vol. 90, no. 12, pp. 568-571, 2003. [29] R. M. M. Adams, U. G. Mueller, T. R. Schultz, and B. Norden, “Agro-predation: uisurpation of attine fungus gardens by Meg- alomyrmex ants,” Naturwissenschaften, vol. 87, no. 12, pp. 549- 554, 2000. [30] H. Eernandez-Marin, J. K. Zimmerman, and W. T. Wcislo, “Eungus garden platforms improve hygiene during nest estab- lishment in Acromyrmex ants (Hymenoptera, Eormicidae, At- tini),” Insectes Sociaux, vol. 54, no. 1, pp. 64-69, 2007. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 139714, 3 pages doi:10.1155/2012/139714 Research Article Feeding Preferences of the Endangered Diving Beetle Cybister tripunctatus orientalis Gschwendtner (Coleoptera: Dytiscidae) Shin-ya Ohba' and Yoshinori Inatani^ ^ Center for Ecological Research, Kyoto University, Otsu 520-2113, Japan ^Higashi Matsuyama Nature Club, Izumi, 594-0082, Japan Correspondence should be addressed to Shin-ya Ohba, oobug@hotmail.com Received 14 September 2011; Revised 24 November 2011; Accepted 7 December 2011 Academic Editor: Martin H. Villet Copyright © 2012 S. Y. Ohba and Y. Inatani. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The numbers of Cybister tripunctatus orientalis Gschwendtner diving beetles are declining in most regions of Japan, and it is included in the Red Data List of species in 34 of 47 prefectures of Japan. However, basic ecological information about C. tripunctatus orientalis, such as its feeding habits, remains unknown. In order to elucidate the feeding habits of C. tripunctatus orientalis larvae, feeding preference experiments were carried out in 2nd and 3rd instar larvae. The number of Odonata nymphs consumed was significantly higher than the number of tadpoles consumed, indicating that C. tripunctatus orientalis larvae prefer Odonata nymphs to tadpoles. In addition, all the first instar larvae of C. tripunctatus orientalis developed into second instars when they were supplied with motionless Odonata nymphs, but their survival rate was lower when they were supplied with motionless tadpoles. These results suggest that C. tripunctatus orientalis larvae prefer insects to vertebrates. 1. Introduction Cybister tripunctatus orientalis Gschwendtner (adult body length: 24-29 mm) is found in China, the Korean Peninsula, Taiwan, and Japan excluding Hokkaido [1]. The numbers of C. tripunctatus orientalis are declining in most regions of Japan, and it is included in the Red Data List of species in 34 of 47 prefectures of Japan [2, 3]. Cybister tripunctatus orien- talis has become extinct in Tokyo, Kanagawa, Aichi, Kyoto, Osaka, Wakayama, and Hyogo (it was rediscovered in Hyogo in 2010: [4]), Contributing factors, such as a decreasing number of suitable aquatic habitats due to the abandonment of rice paddies, water pollution, pesticide application, and invasion by alien species, are of great concern [2, 5]. In addition, the sizes of predatory invertebrate populations are limited by their food resources, as is true for any predatory insect [6-8]. Thus, understanding their trophic ecology is necessary to support an insect conservation program. However, basic ecological information about C. tripunctatus orientalis, such as their feeding habits, remains unknown. A number of descriptive reports [9-12] have suggested that Cybister larvae feed on tadpoles, fish, and aquatic insects. In studies of C. tripunctatus orientalis, Kunimoto [13] saw 3rd instar larvae capturing tadpoles and Odonata larvae {Pantala flavescens) in rice fields in Tottori, western Japan. Ohba [14, 15] revealed the larval feeding habits of two congeneric Cybister species based on a field census and rearing experiment: C. brevis Aube and C. chinensis Motschulsky (formerly Cybister jap onicus Sharp, see Nilsson and Petrov [16]) larvae preyed mainly on aquatic insects and did not eat vertebrate animals such as tadpoles, except for the 3rd instar of C. chinensis. Moreover, these studies showed that the results of feeding preference experiments performed under laboratory conditions were in accordance with field observations of the Cybister species and their growth performance [14, 15]. Therefore, feeding preference experiments performed under laboratory conditions can contribute to deducing the natural feeding habits of Cybister species. The objective of this study was to reveal whether the larvae of C. tripunctatus orientalis prefer invertebrate prey (Odonata nymph) over vertebrate prey (tadpole). For that purpose, two laboratory experiments were carried out in order to determine the feeding preferences of C. tripunctatus orientalis larvae. 2 Psyche 2. Materials and Methods 2.1. Study Animals. Three male and two female C. tripunc- tatus orientalis adults were collected as breeding stock from an irrigation pond in southern Kochi, Shikoku, Japan, in September 2010 and kept in an aquarium (45 cm X 34 cm in dimension, 20 cm in height) maintained under natural water temperature and day length conditions for overwintering. From April 2011 onwards, they were maintained at a water temperature of 25° C under a 16L : 8D light cycle to stimulate reproduction. River gravel was laid onto the bottom of the aquarium in a 2 cm thick layer, and dechlorinated tap water was added to a depth of 15 cm over the gravel surface. Three water hyacinths {Eichhornia crassipes; ca. 5 cm in stock diameter) were planted in the aquarium as oviposition sites. Flatched larvae were reared individually in small plastic containers (10 cm diameter X 10 cm in height) filled to a depth of 2 cm with dechlorinated tap water and kept under natural temperature and day length conditions from June to July 2011. Larvae of Culex spp., chironomids, and notonectid nymphs were supplied to the C. tripunctatus orientalis larvae as food for the first experiment. The prey animals used in this study were collected from rice fields and irrigation ponds, 2.2. First Experiment. To investigate the feeding preferences of C. tripunctatus orientalis, a feeding preference experiment was conducted (see Ohba [14, 15]) using 2nd and 3rd instar larvae. As the first instar larvae did not capture live tadpoles in the preliminary experiment, we did not use first instar larvae in the feeding preference experiment. Small damselfly nymphs (Platycnemididae: Copera spp. or Lestidae: Eestes spp., 15-20 mm) and large dragonfly nymphs {Sympetrum spp. 20-30 mm) were provided as food to the 2nd and 3rd instar larvae, respectively. In addition, small (snout to vent length: 10-20 mm) and large (20-30 mm) tadpoles of the tree frog Hyla japonica were provided as food to these larval instars, respectively. The prey density in each plastic container was kept constant (3 tadpoles and 3 Odonata nymphs). Each beetle larva was fasted for a day before the experiment. Before the start of the experiment, the Odonata nymphs were fed larval Aedes spp. on a daily ad libitum basis in order to prevent intra- and interspecific predation (attacking the tadpoles) during the experiment. The number of carcasses (consumed by the C. tripunctatus orientalis larva) of each prey animal was counted at 24 hours after the beginning of the experiment. The experiments using the 2nd and 3rd instar nymphs were replicated 10 and 14 times, respectively. To determine the diet choices of C. tripunctatus orientalis larvae, the Wilcoxon signed-rank test was used to compare the number of prey consumed between the tadpoles and Odonata nymphs for each larval instar. In this case, paired nonparametric test should be applied to this data because of discrete-valued data. 2.3. Second Experiment. In the preliminary experiment, C, tripunctatus orientalis larvae hardly ever consumed tad- poles but they did routinely eat Odonata nymphs. This might have been caused by different escape capabilities of tadpoles and Odonata nymphs. To examine only the influence each I I Tadpole □ Odonata Figure 1: The number of tadpoles and Odonata nymphs consumed by Cybister tripunctatus orientalis larvae. *P < 0.05, the Wilcoxon signed-rank test. Data are shown as the mean + S.D. prey item on the development of C. tripunctatus orientalis larvae when the prey were prevented from escaping, motion- less tadpoles and motionless Odonata nymphs were used. The bases of the tadpole tails {H. japonica, ca. 10 mm) or the thoraces of the Odonata nymphs {Eestes spp. ca. 15 mm) were squeezed using forceps for 5 seconds to immobilize them. A first instar C. tripunctatus orientalis larva and one motionless preys specimen were put into a small plastic container. The individual prey were exchanged for new prey every day, and this process was continued for 15 days or until the C. tripunctatus orientalis larva died. The motion- less tadpole and motionless Odonata nymph treatments were replicated 6 times each. Survival analysis was used to test for survival curve differences between the motionless tadpoles and motionless Odonata nymphs. The Kaplan- Meier method for estimating survival and the nonparametric Mantel-Cox log-rank test were used. Survival analysis has been regularity employed in medical science to analyze “incomplete” data recorded before the termination of the event of interest. Because many insects died before the present experiments were terminated, the incomplete data thus resulting is not suitable for traditional nonparametric techniques (e.g., Moore and Townsend [17]). A statistically different significance was assumed to be at P < 0.05. All statistical tests were conducted using the Statcel [18]. 3. Results and Discussion In the first experiment, the number of Odonata nymphs con- sumed was significantly higher than the number of tadpoles consumed in 2nd and 3rd instars ( the Wilcoxon signed-rank test, 2nd: Z = 2.55; 3rd: Z = 2.98, P < 0.02 for both; Figure 1). Psyche 3 Although we did not carry out any field observations, it is assumed that C. tripunctatus orientalis larvae consume mostly aquatic invertebrates in their natural habitats. How- ever, Kunimoto [13] recorded 3rd instar larvae of C. tripunc- tatus orientalis capturing tadpoles in rice fields. This discrep- ancy would most likely have been caused by differences in prey density; that is, their study was carried out in June, when there is a high density of tadpoles in rice fields [13]. Accord- ing to Ohba [ 15] , the emergence of first and second instar lar- vae of closely related species, Cyhister chinensis Motschulsky, coincided with the appearance period of Odonata nymphs and tadpoles but these larvae fed on Odonata nymphs and did not eat tadpoles. Therefore, larvae of C. tripunctatus orientalis may not eat tadpoles well in their fields. In fact, C. tripunctatus orientalis larvae consumed more Odonata nymphs than tadpoles when supplied with the same number of Odonata nymphs and tadpoles (Figure 1). Thus, the 2nd and 3rd instar larvae of C. tripunctatus orientalis preferred Odonata nymphs over tadpoles, as is also observed for closely related species, Cyhister brevis Aube, larvae [14]. In the second experiment, the survival rates of C. tripunctatus orientalis larvae differed significantly between those feeding on motionless tadpole and Odonata nymph treatments (survival analysis, Mantel-Cox = 5.87, P = 0.015). In larvae fed motionless tadpoles, four died after 12.3 ± 4.27 (mean ± S.D.) days and two developed into 2nd instar larvae after 14.5 ± 0.71 days. On the contrary, all six larvae fed motionless Odonata nymphs developed into 2nd instar larvae after 6.2 ± 0.41 days. The mean larval duration of the first instar was longer in the motionless tadpole treatment than in the motionless Odonata nymph treatment irrespective of survival. The results show that C. tripunctatus orientalis larvae can consume tadpoles but obtain insufficient levels of nutrients from them for optimal development. Interestingly, C. brevis larvae died from starvation after being supplied with only motionless tadpoles, indicating that C. brevis larvae do not consume tadpoles; not because they can- not capture tadpoles but because they dislike and/or do not recognize tadpoles as prey animals [ 14] . These different feed- ing habits might be attributable to differences in digestive enzymes between the two species. This should be examined in future studies. These results strongly suggest that environ- ments with abundant aquatic invertebrates are favorable for maintaining C. tripunctatus orientalis populations. Rice fields are an important breeding habitat for aquatic insects [19] including C. tripunctatus orientalis in Japan. However, the species diversity in rice fields has been declining due to recent land consolidation, the modification of tradi- tional earth ditches to (7-shaped concrete ditches in Japan [5, 20]. Therefore, poorly drained paddies, which are wet in winter and kept flooded throughout summer, are suitable to conserve the aquatic invertebrates as well as C. tripunctatus orientalis. Acknowledgments The authors thank N. Katayama and Y. Sakai for their help with the beetle keeping and N. Sonoda and Y. Ohba for their kind assistance during this study. References [1] M. Mori and A. Kitayama, Dytiscoidea of Japan, Bun-ichi Sogo Shuppan, Tokyo, Japan, 2002. [2] Japan Environment Agency, Threatened Wildlife of Japan. Red Data Book, Environment Agency of Japan, Tokyo, Japan, 2nd edition, 2000. [3] Association of Wildlife Research 8c EnVision, “Search system of Japanese red data,” 2007, http://www.jpnrdb.com/index .html. [4] S. Ohba and Y. Inatani, “Record of Cybister tripunctatus orientalis from western Hyogo and eastern Shimane, Japan,” Kiberihamushi, vol. 33, pp. 15-16, 2010 (Japanese). [5] S. Nishihara, H. Karube, and I. Washitani, “Status and con- servation of diving beetles inhabiting rice paddies,” Japanese Journal of Conservation Ecology, vol. 11, no. 2, pp. 143-157, 2006. [6] R. E. Lenski, “Eood limitation and competition: a field exper- iment with two Carabus species,” Journal of Animal Ecology, vol. 53, no. 1, pp. 203-216, 1984. [7] D. L. Pearson and C. B. Knisley, “Evidence for food as a lim- iting resource in the life cycle of tiger beetles ( Coleoptera: Cicindelidae),” Oikos, vol. 45, no. 2, pp. 161-168, 1985. [8] S. A. Juliano, “Eood limitation of reproduction and survival for populations of Brachinus ( Coleoptera: Carabidae),” Ecology, vol. 67, no. 4, pp. 1036-1045, 1986. [9] N. Ichikawa, “Notes on breeding water beetles,” Insectarium, vol. 21, pp. 60-62, 1984 (Japanese). [10] Y. Tsuzuki, A. Taniwaki, and T. Inoda, The Perfect Mannuals for Breeding of Aquatic Insects, Data House, Tokyo, Japan, 1999. [11] R. Uchiyama, An Illustrated Reference Book of Life in Rice Paddy Eield, YAMA-KEI Publishers, Tokyo, Japan, 2005. [12] N. Ichikawa, “Cybister japonicus,” in Endangered Species of the Waterside, pp. 51-68, YAMA-KEI Publishers, Tokyo, Japan, 2007. [13] H. Kunimoto, “Ecology of Cybister tripunctatus orientalis (Part 1): overwintering and breeding site,” Yuragia, vol. 23, pp. 1-7, 2005 (Japanese). [ 14] S. Y. Ohba, “feeding habits of the diving beetle larvae, Cybister brevis Aube (Coleoptera: Dytiscidae) in Japanese wetlands,” Applied Entomology and Zoology, vol. 44, no. 3, pp. 447-453, 2009. [15] S. Y. Ohba, “Ontogenetic dietary shift in the larvae of cybister japonicus (Coleoptera: Dytiscidae) in Japanese rice fields,” Environmental Entomology, vol. 38, no. 3, pp. 856-860, 2009. [16] A. N. Nilsson and P. N. Petrov, “On the identity of Cybister chinensis Motschulsky, 1854 (Coleoptera: Dytiscidae),” Kole- opterologische Rundschau, vol. 77, pp. 43-48, 2007. [17] M. K. Moore and V. R. Townsend, “The interaction of temper- ature, dissolved oxygen and predation pressure in an aquatic predator-prey system,” Oikos, vol. 81, no. 2, pp. 329-336, 1998. [18] H. Yanai, 4 Steps Excel Statistic, OMS Publishers, Saitama, Ja- pan, 1998. [19] H. Saijo, “Seasonal prevalence and migration of aquatic insects in paddies and an irrigation pond in Shimane Prefecture,” Japanese Journal of Ecology, vol. 51, no. 1, pp. 1-11, 2001. [20] N. Ichikawa, “The present condition and the preservation of pond insects lived in village,” Japanese Journal of Environmen- tal Entomology and Zoology, vol. 19, pp. 47-50, 2008. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 924256, 7 pages dohlO.l 155/2012/924256 Review Article Structure Determination of a Natural Juvenile Hormone Isolated from a Heteropteran Insect Toyomi Kotaki,^ Tetsuro Shinada,^ and Hideharu Numata^ ^ Division of Insect Sciences, National Institute of Agrobiological Sciences, Ohwashi, Tsukuba, Ibaraki 305-8634, Japan ^ Graduate School of Science, Osaka City University, Osaka 558-8585, Japan ^ Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan Correspondence should be addressed to Toyomi Kotaki, kotaki@alfrc.go.jp Received 15 September 2011; Accepted 7 November 2011 Academic Editor: Mark M. Feldlaufer Copyright © 2012 Toyomi Kotaki et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Juvenile hormone (JH), which occurs in several forms in different insects, is one of the most important insect hormones. The structure of JH in Heteroptera has not been elucidated until recently, although insects in this suborder have long been used as experimental animals for JH research. Here we review the structure determination of a novel JH in a stink bug, Plautia stali, which was named juvenile hormone III skipped bisepoxide [JHSB 3 : methyl (2R,3S,10R)-2,3;10,ll-bisepoxyfarnesoate], based on the arrangement of two epoxides at C2,3 and C10,ll with a skipped double bond at C6,7. 1. Introduction Juvenile hormone (JH) is an important regulator of many functions in all insects (Figure 1) [1, 2]. It controls various aspects of development including metamorphosis, reproduc- tion, and polyphenism. This hormone was discovered in a blood-sucking bug, Rhodnius prolixus, by Wigglesworth in 1934 [3]. He demonstrated a hormone, produced by a gland behind the brain, the corpus allatum (CA), that was responsible for the maintenance of juvenile characters as the insect grew, hence the name juvenile hormone [3]. He also found that the gland became inactive to allow metamorphosis to the adult and active again in the adult to support reproductive functions such as deposition of yolk in eggs and production of secretions of accessory reproductive glands [4, 5] . Since the chemical structure of JH isolated from a moth, Hyalophora cecropia, was elucidated in 1967 [6], several forms of JH were also determined [1, 2]. JH III is the most common among various insect orders while JH 0, JH I, JH II, and 4-methyl JH I were found in Lepidoptera. JHB3 was identified as a JH specific to higher Diptera (Cyclor- rhapha) [1, 2]. However, the structure of JH in the suborder Heteroptera has remained uncertain in spite of attempts to identify it [7-13]. Recently, we identified the structure of JH in a stink bug, Plautia stali, and named it juvenile hormone III skipped bisepoxide (JHSB3) and demonstrated its biological function as the JH in this stink bug [14, 15]. Here we review the process of structure determination of JHSB3 and its biological function as JH in R stali. 2. Heteropteran Insects in JH Research In his pioneering work, Wigglesworth found a humoral factor controlling metamorphosis and reproduction in R. prolixus, which he first referred to as “inhibitory hormone” [3,5] and later as JH [ 16] . Since then heteropterans have been employed for evaluation of the biological efficacy of synthetic derivatives of natural JHs and anti-JH compounds, which lead to the discovery of a JH analogue specific to Pyrrhocoris apterus, juvabione and anti-JH, precocenes [17-20]. Heteropteran insects are used for studies to elucidate the role of JH in various aspects of insect development. For example, in Oncopeltus fasciatus, JH was demonstrated to control reproduction, adult diapauses, and diapause-as- sociated migration [21, 22]. In diapausing adults of P. apterus, the activity of CA was inhibited by the brain via nervous connections [23, 24]. The production of methyl farnesoate [25] and JH III [26] by the CA in vitro was report- ed in Dysdercus fasciatus, O. fasciatus, and Nezara viridula. 2 Psyche JHI OMe OMe Figure 1: Structure of known JHs. Radiochemical assay for JH biosynthesis MEM containing ^H-methionine (Pratt and Tobe, 1974; Ferenz and Kaufner, 1981) Hex: Et-Ac, 1:1 0 5 XlO^ dpm (Kotaki, 1993) Figure 2: Radiochemical assay for JH biosynthesis. Incubation of CA from Plautia stall adults in a medium containing radiolabeled methionine revealed the presence of an unknown product with an Ry value different from those of JH I, JH III, or JHB 3 in TLC analysis. However, Baker et al. failed to detect any form of known JHs in O.fasciatus [12]. 3. Evidence for a New JH in Stink Bugs In 1989, we reported that the CA product was responsible for control of adult diapause in R stall [27]. This was shown by surgical operations such as CA extirpation and implan- tation. To gain insight into the CA product, the In vitro radio- chemical assay [28, 29] was adopted to this bug (Figure 2). Assuming that the CA product was a methyl ester as with known JHs, the tritium-labeled CA product would be ob- tained by the incubation experiments in the presence of L- [mcf/zy/-^H] methionine in the medium. After the incubation period, the product was extracted with hexane and analyzed by thin-layer chromatography (TLC) using a liquid scintilla- tion counter to detect radioactivity of each part of TLC plate. The CA of P. stall and three other heteropteran species were subjected to this assay. The Rf values for the CA product of four species were almost identical with each other, whereas they were different from those for JH I, JH III, and JHB3 [8, 9]. When precursors of JH III, P,P-farnesol or farnesoic acid were added to the medium, the biosynthesis of CA product was enhanced. This suggested that the product possessed the same sesquiterpenoid skeleton as did JH III [8]. Because radiolabeled JH III added to the incubation medium was not converted to the “CA product,” the possibility that the CA released JH III but the latter was degraded or metabolized to the “CA product” in the incubation medium was ruled out [9]. Thus far, at least 7 species from 4 families of Heteroptera seem to share this product [7, 9, 10, 30]. Although the CA of O. fasclatus, D. fasclatus, and N. vlrldula were reported to produce JH III and/or methyl farnesoate In vitro [25, 26] and JH I was found in the hemolymph of R. pedestrls (formerly R. clavatus) [31], the production of JH III and JH I in N. vlrldula and R. pedestrls, respectively, was not confirmed [9]. To assess the JH activity of the CA product, we developed a bioassay method using P. stall. The CA product, collected as hexane extracts of medium for CA incubation, was topically Psyche 3 (a) (b) (c) Figure 3: Bioassay for juvenilizing activity in Plautia stall. A last instar nymph (a), an adult (b), and a nymph-adult intermediate (c) obtained as a result of application of JH-active sample. Scale bar: 5 mm. Arrows with labels W and S indicate forewing and scutellum lengths and pronotum width, respectively. applied to last instar nymphs, and following the final ecdysis, relative lengths of forewings and scutellum were deter- mined [8]. Adults have fully developed long forewings and scutellum (Figure 3(b)) while in last instar nymphs and metamorphosis-inhibited insects by JH application, forew- ings and scutellum were not developed into the adult form yet but remained in buds or partially developed, short form (Figures 3(a) and 3(c)), The more the CA product was ap- plied to test insects, the shorter their wings and scutellum were. This result indicated that the CA product had the ac- tivity to inhibit metamorphosis of the test insects, hence JH activity. Therefore, JH in R stall was very likely to be a new JH that is different from any known JH, 4. Structure Determination of Stink Bug JH To elucidate the structure of stink bug JH, we first examined the molecular weight of CA product using high resolution fast atom bombardment ionization (FAB) mass spectrometry to estimate its compositional formula. The mass of proto - nated molecule [M-l-H]"'' was estimated to be 283.1885, lead- ing to a compositional formula, C16H26O4, for the CA pro- duct. This formula is identical to that for dipteran JHB3. However, previous studies clearly showed that the R / values of the CA product and JHB3 were not the same despite the supposition that the CA product and JHB3 shared a se- squiterpenoid skeleton in common [8, 9] . Based on these ob- servations, we proposed that the structure of JH in P. stall would be a regio- or geometric isomer of JHB3. To test this hypothesis, we synthesized a compound mix- ture consisting of 32 isomers of bisepoxides of methyl farnesoate with 2E or 2Z double bond, epoxides at C2,3 and at either C6,7 or CIO, 11 by two steps starting from a mixture of E- and Z-geranylacetone. The bioassay for JH activity indicated that the mixture was JH active. In a GC-MS analysis using a DB-35MS column the mixture gave several peaks. Cochromatography of the mixture and the CA pro- duct indicated that one of peaks of the mixture coincided with the CA product in retention time and mass spectrum. Using a normal phase chiral column, Chiralpak lA (DAICEL Co., Ltd) on an HPLC system, the bisepoxide mix- ture was separated into 21 fractions and each of them was subjected to the bioassay for JH activity. Two fractions (reten- tion time: 28 min. and 38 min.) applied topically to last instar nymphs showed a juvenilizing effect at a dose of 0. 1 f/g/insect. These biologically active fractions were subjected to one and two-dimensional ^H NMR analyses. Data obtained indicated that each fraction consisted of a single stereoisomer of a novel JH structure, methyl P,P-2,3;10,ll-bisepoxyfarnesoate (Figure 4). Although stereoisomers of this bisepoxide had dis- tinctly different retention time when separated by the chiral HPLC, their ^H NMR data were not distinguishable from one another. Probably the positions of two epoxides were too distant to give different signal patterns. Therefore, we synthesized four possible stereoisomers 1-4 in an optically pure form (Figure 4), and these were compared with the natural CA product to determine the relative and absolute structure. Each stereoisomer was obtained starting from E,E- farnesol by asymmetric Katsuki-Sharpless epoxidation and Sharpless dihydroxylation reactions in which stereochemical outcomes were reliably controlled by ingeniously designed chiral reagent systems [32]. Bioassay indicated that isomers 1 and 2 showed more JH activity than the other two isomers. The chiral HPLC analysis of these stereoisomers gave four peaks. Elution time for isomers 1 and 2 were almost the same as that for the JH-active fractions obtained by the chiral HPLC separation of the bisepoxide mixture. In consideration of the detection limit of HPLC analysis using a UV detector, the stereoisomers and CA product were subjected to more sensitive GC-MS (chemical ionization. Cl with NH3 as 4 Psyche O' O" O 3 (2S, 3R, lOR) O''' O'" 2 {2R, 3S, lOS) O'" O’ 4 (2S, 3R, lOS) Figure 4: Four stereoisomers of JHSB 3 . y \J \l + 2 and CA product /\ / \CA product \Ai 1 + 2 ___ 30 35 40 45 50 55 Retention time (min) Figure 5; Chiral GC-MS(CI) analysis of synthetic standards of JHSB 3 (1) and isomer 2 and natural product by the CA of Plautia stali using an Rt-j5DEXcst column. Vertical axis indicates signal intensity for m/z 300, [M+NH 4 ] Black, blue, and red lines indicate analysis of JHSB 3 (1) and isomer 2 (10 ng each), CA product, and cochromatography of these two samples, respectively [15]. a reagent gas) using a chiral column, Rt-)SDEXcst. With this column, isomers 2, 3, and 4 showed almost the same reten- tion time of 44.7 min while isomer 1 , with retention time of 45.7 min, was distinctly separated from the others (Figure 5). The CA product produced one main peak at 45.8 min in this system. The mass spectra for isomer 1 and CA product were identical (Figured insets). Cochromatography of isomer 1 and CA product indicated that the peaks for these two over- lapped completely with an increase in peak height in an addi- tive fashion (Figure 5). These results demonstrate that isomer 1 , a novel form of JFI, is the natural JH in P. stali. We named it juvenile hormone III skipped bisepoxide [JHSB 3 : meth- yl (2R,3S,10R)-2,3;10,ll-bisepoxyfarnesoate], based on the arrangement of two epoxides at C2,3 and CIO, 11 with a skipped double bond at C6,7. 5. Biological Function of IHSB 3 Although, in the process of IHSB 3 structure determination, a juvenilizing JH activity was indicated in JHSB 3 and one of its stereoisomers, how active the remaining two stereoisomers at higher doses were was not explored. JH activity of the four isomers were, therefore, compared using last instar nymphs as well as adult females of R stali kept under reproduction- promoting, long-day conditions whose CA were surgically removed. The latter test examined the activity to stimulate reproduction, another function of JH. Topical application of JHSB 3 to last instar nymphs inhibited their metamorphosis in a dose-dependent fashion (Figure 7). Nymphs treated with 0.001 f/g of JHSB 3 molted to normal-looking adults. With an increase in the dose, the rel- ative lengths of forewings and scutellum decreased. At a dose of 0.1 fig or higher, nymphs molted to intermediates with wings and a scutellum reduced to a similar extent to those of normal last instar nymphs. A diastereomer of JHSB 3 , isomer 2, revealed a similar dose-response curve. On the other hand, isomers 3 and 4 were less active than JHSB 3 and iso- mer 2. A dose of 1 fig of isomers 3 and 4 showed little effect on the metamorphosis of bugs. At a dose of 5 fig, bugs molted to an intermediate, but their wings and scutellum were still slightly longer than those of bugs treated with 0.1 fig or 1 fig of JHSB 3 . Application of lOR-JH III showed a similar dose response to those of isomers 3 and 4, but even at the highest dose of 10 fig, its effect was not so evident as that of isomers 3 and 4 at 5 fig. Extirpation of the CA from females reared under long- day conditions inhibited the development of ovaries. More than half (10 of 14) of allatectomized hexane-treated females underwent oosorption when they were dissected 4 days after allatectomy. Topical application of JHSB 3 rescued those adults from the inhibitory effect of CA removal in a dose- dependent fashion (Figure 8 ). Isomer 2 seemed as potent as JHSB 3 whereas isomers 3 and 4 showed virtually no stimulatory effect on oocyte development even at the highest dose of 5 fig. These results indicated that JHSB 3 and isomer 2 were highly JH active while the other two were about 1,000 times less active. The downward epoxide configuration at C2,3, shared by JHSB 3 and isomer 2, seems important for manifestation of JH activity. JHSB 3 structure determination was accomplished by analyzing the product of CA in vitro. The biosynthesis of JHSB 3 by the CA, however, does not automatically imply its presence in the hemolymph in vivo. It must be experiment- ally verified by detecting JHSB 3 in the circulating hemoly- mph. We collected hemolymph samples from reproductively active and diapause females. According to the previous studies [27, 33], in the former the concentration of JH in the hemolymph is expected to be high while in the latter low. These two samples were analyzed using FC-MS. In the hemolymph sample from reproductively active females, a peak was observed at the same retention time accompanied by the same mass spectrum as synthetic JHSB 3 standard. No significant peaks of characteristic fragments corresponding to JH I, II, nor III were observed in the hemolymph samples. This indicated the presence of JHSB 3 alone in the hemo- lymph from reproductively active females, and on the other hand, virtually no peak corresponding to JHSB 3 or any other Psyche 5 : 100,000 5 - 2.5 - 1|53 233 283 300 7 - . 1 ■ ■ . 16.235 I 100 150 200 250 300 350 0 -I -rA "4 / W- 1— I— I— I— p-i— I— I— I— I— I— I— I— I— I— I— I— I— I— I— I— I— r 15 15.25 15.5 r 16 15.75 Retention time (min) (a) I ■ III I ■ III I 16.25 16.5 16.75 17 X 100,000 (b) miz 300 miz 283 (c) Figure 6 : Detection of JHSB 3 (1) from the hemolymph of Plautia stali females. Hemolymph samples collected from reproductively active (a) and diapausing females (b), and 10 ng of synthetic standard of JHSB 3 (c) were analyzed on GC-MS using a DB-35MS column. Vertical axis indicates signal intensity for m/z 283, [M+H]+ (solid line) and m/z 300, [M+NH 4 ]+ (dotted line). Insets in a and c indicate a mass spectrum of the peak at 16.2 min [14]. S 0.001 0.01 0.1 1 10 Dose (/rg) (a) S 0.001 0.01 0.1 1 10 Dose (fig) -o- 1, IHSB 3 4 -■-2 ^5 3 (b) Figure 7: Juvenilizing activity of JHSB 3 (1), its stereoisomers and lOP-JH III on Plautia stali. Last instar nymphs were treated with a test compound. Following the final molt, lengths of forewing (a) and scutellum (b) relative to the width of pronotum were determined. S on the horizontal axis indicates solvent control. Each datum point and error bar represents average value ± SD (n = 8-18). Asterisks indicate that the average value was significantly different from that of the solvent control (Steel’s test, P < 0.05) [14]. 6 Psyche Dose ifig) -o- 1,JHSB3 3 2 4 Figure 8; Reproduction-stimulating effect of JFISB3 (1) and its stereoisomers on allatectomized females of Plautia stall reared under long-day conditions. Females were allatectomized and treated with a test compound on day 4 of adult life, and oocyte diameter was determined on day 8. Solid diamonds labeled with I and S on the horizontal axis indicate results of day 8 untreated and solvent-treated adults, respectively. Each datum point and error bar represents average value ± SD {n = 8-12). Asterisks indicate that the average value was significantly different from that of the solvent control (Steel’s test, P < 0.05) [14]. JHs was detected in the sample from diapause females. JHSB 3 was, therefore, the only molecule found in the hemolymph and its concentration was likely to fluctuate as expected by the previous studies. Dahm et al. [34] pointed out three criteria that have to be fulfilled to chemically identify JH. These points, derived from the classical definition of the hormone, are as follows: (1) production by the CA; (2) titer fluctuation in synchrony with the processes controlled by JH; (3) rescue effect in JH- deprived insects. As indicated above, these three criteria were met for JHSB 3 in R stall. JHSB 3 , therefore, functioned as the JH in this species. It was likely that other heteropteran insects share this new JH in common because the CA of at least seven heteropterans other than P. stall also produced the products In vitro that behaved similarly to that of R stall on the TLC plate [7, 9, 10, 30]. 6. Conclusion JH in Heteroptera has been a long-lasting enigma in spite of that JH research began with morphological studies in R. prollxus [3,4]. We have successfully determined the structure of novel, Heteroptera-specific JH, JHSB 3 . Because JHSB 3 is the only JH with a C2,3 epoxide, heteropterans using this molecule likely possess an enzyme responsible for conversion of the C2,3 double bond to epoxide. The presence of specific JH suggests underlying specific pathways for not only bio- synthesis but also degradation. A JHSB 3 -specific receptor should also play a role in heteropterans. Our discovery has provided a basis for all these suppositions and will enhance further studies on heteropteran JH. An attempt to elucidate the enzymes involved in JHSB 3 biosynthesis is in progress. How far JHSB 3 is shared among the suborder Heteroptera or the order Hemiptera is a question to be answered in the future in the viewpoint of insect endocrinology and practical insect control, as well. Structure-activity relation study is underway to gain insight into specificity of JHSB 3 receptor and development of JHSB 3 -based control agents. Acknowledgments This study was supported in part by Grants-in-Aid for Sci- entific Research (C) (no. 19580059) to T. Kotaki and (B) (no. 20380038) to H. Numata from the Japan Society for the Pro- motion of Science. References [1] E. D. Morgan and I. D. Wilson, “Insect hormones and insect chemical ecology,” in Miscellaneous Natural Products Including Marine Natural Products Pheromones Plant Hormones and Aspects of Ecology, K. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 752815, 11 pages doi:10.1155/2012/752815 Research Article The Biology and Natural History of Apbaenogaster rudis David Lubertazzi^’^ ^ Department of Ecology and Evolutionary Biology, University of Connecticut 75 North Eagleville Road, U-43, Storrs, CT 06269-3043, USA ^Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA Correspondence should be addressed to David Lubertazzi, dlubertazzi@oeb.harvard.edu Received 16 October 2011; Accepted 11 November 2011 Academic Editor: Diana E. Wheeler Copyright © 2012 David Lubertazzi. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Workers from the genus Aphaenogaster are among the most abundant ants in the hardwood forests of eastern North America. The biology of these so-called rwdzs-group ant species, including details about their sociometry, productivity, natural history, and behavior, are synthesized here using published and newly collected data. The latter was collected, in part, using an artificial field nest, and its construction and use are explained. Ants of the rudis group occur in high densities in forest habitats (0.5- 1.3 nests m^), have moderate sized colonies (population means from 266 to 613 workers per nest), and are keystone seed dispersers. Many aspects of their life history and behavior follow an annual cycle that tracks seasonal changes. These include foraging, reproduction, the production of new workers and nest migrations. This synthesis highlights what is known about these ants and reveals gaps in our knowledge that require further study. 1. Introduction Sociometric data and natural history information about ants serve as the raw material for discovering patterns, formu- lating hypotheses, and planning experiments that examine a wide range of ecological, behavioral, and evolutionary processes. Seminal research investigating topics as diverse as population regulation [1], mutualisms [2], kin selection [3, 4], and sex-ratio evolution [5] illustrate how ant studies, using previously published or newly collected sociometry data, have made important contributions to understanding the function and evolution of biological systems. Tschinkel has argued that myrmecologists have been neg- ligent in collecting and reporting basic biological attributes of individual ant species [6, 7]. Also problematic is that the existing data for some of our better studied species are scattered among an eclectic collection of published stud- ies. Collecting, cataloguing, and disseminating information regarding ant species’ biological attributes are important tasks that need greater attention. This paper provides an overview of the biology of Aphaenogaster rudis (s.l). This ant is common in the hard- wood forests of the eastern United States [8-11] and has been the subject of numerous ecological and evolutionary studies (e.g., [12-14]). Newly collected data and previously published information from dozens of studies are synthe- sized here to explain what is known about these ants. A description of an artificial field nest that facilitates collecting and studying whole colonies of this ant and data collected from these nests are also provided. 2. Materials and Methods Data and observations from published studies and from three study populations in the state of Connecticut are used to describe the basic biology of a prototypical rwdis-group ant. This term is used to refer to three different species, as explained in the taxonomy section. Details about the Connecticut study populations, methods used for finding, collecting, and sorting whole nests, and the construction and use of a newly developed artificial wooden field-nest are explained in the following. 2.1. Connecticut A. rudis Populations. The three populations for which new data are presented were located in Connecticut State Forests. These populations are referred to throughout this paper by the names of the forests where they occurred: 2 Psyche Nipmuck (Nipmuck State Forest, 41° 59' 21" N 72° 10' 51" W), Pachaug (Pachaug State Forest, 41° 36' 8" N 71° 53' 22" W), and Mohegan (Mohegan State Forest, 41° 39' 59" N 72° 4' 59" W). Each study site occupied a multi hectare section of mature forest dominated by hardwoods (primarily Acer and Quercus species) and containing a few small groves of conifers {Tsuga canadensis and Pinus strohus). These secondary forests are part of a New England landscape with a well-documented history [ 15] . Colonies were collected by digging up nests (see Section 2.2) in the three Connecticut populations in the spring, summer, and fall of 2001-2003. This field work, and nests collected during these field seasons and in 2004 and 2005 where artificial nests (see Section 2.4) were used for colony collections, are the source of data and observations reported on here as being from Connecticut. 2.2. Collection of Nests. Workers of the rudis group are among the most commonly encountered ants in the hard- wood forests of southern New England. In late spring, summer, and early autumn, nests can be found in the soil, in dead wood on or near the forest floor, under and between rocks, and in the leaf litter. Foragers are readily attracted to many ant baits and nests can be located by following bait- laden foragers as they return to their colonies. Soil nests have a single, small circular nest-entrance that is hidden under the leaf litter. In the summer these nests are compact and shallow (< 15 cm deep). Whole colonies may be excavated from the soil by removing a single inverted cone- shaped plug of earth, centered on the nest entrance, from the ground using a full-sized shovel. Colonies nesting in other locations, for example, in downed wood or in the leaf litter, can be collected in their entirety by placing the ants and their nesting material into large plastic bags. Field-collected nests can easily be separated from their nesting materials in the lab. Material from each field colony collected in Connecticut was spread across the bottom of 55 liter plastic bin that contained two artificial nests, sugar water, and water. Artificial nests consisted of foil covered test tubes with a moistened cotton ball. Small test tubes with water held behind a cotton plug, and a similarly arranged honey-water solution, served as a source of water and carbohydrates. The exposed nesting materials desiccated and within a few days workers would move the queen and brood into the nesting tubes. 2.3. Artificial Wooden Nests. Artificial field nests were con- structed from two rectangular pieces of white-pine lumber measuring 12.7 X 22.8 X 2.54 cm. One piece of wood was partially excavated with a router to hollow out a U-shaped chamber, with an additional shallower cut made from the top edge of the U to the outer edge of the board (Figure 1). The second piece of wood was placed over the top of the hollowed out chamber, forming an enclosed nesting area with a single entrance. The two nest pieces were held together with two screws, secured with wing nuts, placed through predrilled holes in both boards. 22.8 cm Figure 1: Artificial field nest. The U shape of the bottom half of the nest is excavated to a depth of 1.3 cm and the short section that connects the U to the exterior edge, shown in a lighter shade of gray, to a depth of 3 mm. Two holes drilled in each piece allow bolts to be used to hold the two boards together. Twenty-five artificial nests were placed on the forest floor in the Mohegan study site in the spring of 2002. The following spring a majority of the nests were occupied by rudis group ants. These nests were collected in mid-August 2003 (Table 1) by placing each nest in a sealable plastic bag. The contents of each colony were processed (individuals counted and preserved) in the laboratory within a few days of collection. In 2004 additional nests were censused from the same forest, as detailed in Lubertazzi and Adams [14]. 2.4. Worker Head Widths and Caste Dry Weights. Worker head width is commonly used as a reliable measure for assessing variability in worker size [16]. We measured the head widths of 25 randomly chosen workers from each of 39 colonies sampled from the Nipmuck and Pachaug forests. The head of each worker was removed from the body, placed frons side up on a glass slide and measured using a stereomicroscope at 90x magnification. The maximum width of the head was recorded to the nearest hundredth of a mm using an ocular scale. ANOVA was used to test if worker size varied between populations or among colonies within populations. The relationship between colony worker number and worker size was examined by regressing the average colony head width on colony worker number for 27 of the 39 colonies sampled. This reduced data set represented collections deemed to be of whole colonies. A randomly chosen sample of 25 workers from each of eight preserved colonies was used to determine the average dry weight of workers. Individuals were oven dried for three days at 30°C then weighed to the nearest mg using a Mettler balance. The dry weight of 25 males and 25 gynes from the Nipmuck and Pachaug populations was similarly assessed. These individuals were collected and preserved in alcohol in August 2001. Collections were made one week prior to the time sexual adults were no longer found in field nests, which was presumably the time of the mating flight. Psyche 3 Table 1: Worker number and sexual production for colonies from artificial wooden nests collected in the Mohegan State Forest in August 2003. All colonies except G contained a queen. Colony Workers Males Females alates 1 568 68 4 3 660 111 5 4 544 18 17 5 907 75 0 7 322 1 0 8 760 1 0 10 479 0 0 A 827 0 0 C 498 58 0 E 473 140 0 G 183 0 0 H 395 0 0 J 644 112 0 V 563 0 0 X 1033 0 0 Y 846 72 0 Z 724 0 0 2.5. Nest Density Measures. Three Connecticut populations were sampled in July and August of 2003 to determine nesting density. Twelve haphazardly selected 3 m X 3 m plots (plots per forest: Nipmuck 5, Mohegan 3 and Pachaug 4) were intensively searched for nests and tallied. Each plot within a forest was located at least 30 m away from any adjacent plot. 2.6. Foraging Distances. Distances from randomly placed pecan sandies cookie crumb baits to a foragers’ nest entrance were measured in July and August of 2003. From 5-10 baits were placed in a haphazardly fashion over a roughly 10 m square area of the forest floor and rechecked after 30 minutes. If a bait was being foraged upon one laden worker was followed back to its nest entrance. The straight line distance from the bait to the nest entrance was then measured to the nearest cm. The bait was checked for foragers from any other colonies and if present similarly followed and the distance measured. The next bait was then checked and similarly censused. Data was collected from all three Connecticut study populations (colonies sampled: Nipmuck, 22, Mohegan 17, and Pachaug 25). Foraging distances between populations were compared using ANOVA. 3. The rudis-Group Ant North America is home to 26 validly named Aphaenogaster species and 4 undescribed forms [17]. One group of species, the Aphaenogaster rudis-fulva-texanus complex, comprises species largely confined to eastern North America [18]. Cer- tain members of this group are impossible to separate mor- phologically [18-21], Umphrey [18], seeking to define the species and range boundaries of the complex, developed and described complex morphological metrics, electrophoretic markers, and karyotype counts for identifying these ants. He defined and assigned coded names to ten forms with six being matched to named species and the remainder putatively representing undescribed species. Ants from the genus Aphaenogaster are abundant in the wide ranging mesic hardwood forests of eastern North American. These ants have been the subject of dozens of published studies and are often identified and reported as A, rudis. Using range limits and habitat infinities provided by Umphrey [18] we can infer that these studies are likely to involve three species: A. rudis Enzmann or N22a in Umphrey ’s coding system, the undescribed form N17 and A. picea (Wheeler, W.M.) or N18. It is not possible to clearly distinguish these three forms from each other using morphological characters. This paper will describe the biology of these ants as a single prototypical species that will be referred to as a “rwJis- group” ant. This name is used for practical convenience and not in a strict taxonomic sense. While many publications ascribe the name A, rudis to their study form the majority likely involve the two northern forms (N17 and N18). Our biological knowledge is therefore heavily biased by knowing more about populations that experience cooler temperatures and a shorter annual season of productivity than the lower latitude N22a (putatively the true A. rudis). Further work is clearly needed to resolve the taxonomic and nomenclature problems within this complex. In light of these issues, it is imperative that future research involv- ing ants from the rudis-fulva-texanus complex include the deposition of voucher samples in an accessible museum collection. The ranges of the three rwr/is-group species overlap along some of their boundaries but are thought to be predominately allopatric [18]. N22a/A. rudis has the most southern distribution. It is found in the lower elevations of the Appalachians from Virginia to northeastern Alabama and within forests of the piedmont region of the Carolinas. Along the Maryland, Delaware, and New Jersey coastal plain, the range of N22a/A. rudis broadly overlaps with A. carolinensis. To the west N22a/A. rudis is found in the forests of Tennessee, Kentucky, southern Ohio, and southern Indiana and has been found as far west as Missouri. N17 and N18/ picea are, respectively, western and eastern variants that occur to the north or at higher elevations than N22a. N17 is found in and to the north of Ohio, into Ontario, and possibly occurs as far west as Iowa. N18/A, picea is found within, east of, and north of Pennsylvania. It occurs in southeastern Ontario, Quebec and possibly the maritime provinces of Canada. Its preference for cooler mesic forests also allows this form to occur in higher elevation Appalachian mountain forests, potentially extending its range as far south as Georgia and Alabama. Studies cited in this paper that report on A. rudis s.l. are not believed to represent any other forms within the rudis-fulva-texanus complex. This is inferred from what is known about the other forms’ ranges, habitat preferences and/or low nesting densities. A. fulva may be found in low densities in some forests where rwJis-group ants are common. The former can be 4 Psyche reliable identified from the latter by the heavily rugose mesopleural of its queens. Queens of rwdis-group ants have a smooth mesopleura [17]. 4. Colony Characteristics 4.1. Annual Colony Cycle. Mature rudis-group colonies exhibit an annual cycle of activity and productivity that tracks seasonal changes. The description outlined here arbitrarily starts with a renewal of colony activity in the spring and ends with winter diapause. Specific seasonal activities and responses are detailed, along with supporting data and information, in an idealized version of what occurs within the colony over the course of a full year. The seasons are used in a relative sense to indicate times of the year when temperatures are warming (spring), consistently supporting colony growth (summer), cooling (autumn), and cold (winter). The dates associated with these times of the year will vary according to the geographical location of a given population. 4.1.1. Spring. In Connecticut rwdis-group workers are among the first ants to become active on the forest floor during the spring. Once the snow cover has gone and the ground is exposed, foragers can be found above ground on warm and sunny days of early spring (~15°C ambient temperature). Early spring activity explains, in part. Lynch et al.’s [22] finding in a Maryland ant community that a rwdis-group ant was active for a greater portion of the year than other co-occurring ant species. Umphrey [18] suggested N18M. picea was among the most cold tolerant North American Aphaenogaster species. This tolerance helps these ants be poised to begin their annual above-ground activities as early in the spring as possible. As spring progresses entire colonies diurnally move back and forth from underground chambers to protected cavities near the surface, especially favoring locations warmed by sunlight. Daytime spring temperatures at the top of the leaf litter can reach 40° C and may be 20° C warmer than 15 cm below ground [23]. Exposure to these higher temperatures raises metabolic rates, restarting brood development and queen oogenesis. Later in the spring the winter nest is abandoned for a new site on or near the ground surface. This switch in nesting location coincides with the ground surface and subsurface becoming consistently warmer than the more thermally stable, but now regularly cooler, underground nesting chambers. The spring-season nest migration of rudis- group colonies has been observed in Missouri [24] and Connecticut. Maintaining a high degree of flexibility in where and when they move their nests help rwdis- group ants hasten their transition from winter diapause to summer productivity. 4.1.2. Summer. The abandonment of the winter nests puta- tively delimits a time when conditions become consistently conducive to colony growth. In Connecticut this point is reached in late May or early June. Some larval growth and egg production take place in the spring and fall but the bulk of egg laying, larval growth, pupation, and eclosion occurs during the summer. Productivity peaks in late summer and declines sharply in the fall [25, 26]. Maintaining the optimal temperature/humidity microenvironment for the brood and queen is an important focus of the workers. This may involve daily movements within the nest or, if a better nesting location is found, can include a summer-season nest migration [12, 27-29]. Structures such as downed wood that contain hollow chambers or loosely attached bark, areas between exposed rocks and the soil, and other similarly sheltered warm locations can all provide suitable nesting sites during the summer. Food demands are highest during this season. Prior to the summer individuals had to rely on internal reserves, built up during the autumn, to sustain their low metabolic and developmental needs. During the warmer months food is now needed to supply nutrients to the brood, the queen, and the workers. Foraging, worker development, and the production of new sexuals (each explained in greater detail in sections that follow) all assume greater importance during the summer. 4.1.3. Autumn. The onset of shorter days and cooler tem- peratures leads to changes in nesting location and colony productivity. There is also a general slowing of overall colony activity. Above-ground or near-ground level nests are eventually abandoned for deeper below-ground nesting sites. This autumn nesting site is likely different from the nest used during the summer even if the summer nest contained soil chambers [24]. By October in Missouri [24] and Connecticut, rwdzs- group ants have all moved to what will be their underground winter nests. Egg production decreases as temperatures decline, with oogenesis eventually stopping altogether. Colony activity slows [30], metabolic and development rates fall, and individuals increasingly rely on internally stored nutrients for their decreasing metabolic needs. Foraging slows and eventually ceases as the temperatures cool. 4.1.4. Winter. In winter months colonies avoid freezing tem- peratures by maintaining their nests below ground. Talbot [24] found the average depth of 5 winter colonies in Missouri to be 25 cm. Colonies in Connecticut appear to prefer deeper nests, to a depth of at least 50 cm. Developmental processes enter a diapause and worker activity within the nest is minimal. 4.2. Nesting Ecology. Downed wood provides a good nesting resource for rwdis-group ants in Connecticut and in other locations [12, 31]. Limbs and boles greater than 10 cm in diameter and slightly decaying appear to be particularly favorable. In the Connecticut study sites almost all of these suitable nesting sites were occupied by a thriving rudis- group colony. Wooden nesting structures are often in short supply relative to the high density of nests (see below). The combination of the relative scarcity and suitability of Psyche 5 Table 2: The average number of workers per colony for various populations of rudis- group ants. Population study N Mean SE Talbot 1951 [24] 71 352 38 Headley 1949 [25] 46 266 28.8 Mohegan SF 2003 17 613 53.5 Lubertazzi and Adams 2010 [14] 65 507 32.3 Morales ScHeithaus 1998 [12] Fed colonies 24 457 59 Control colonies 27 360 46 Heithaus et al. 2005 [13] 36 601 56.1 wooden nest sites [31] likely contributes to the attractiveness of artificial wooden nests. In mature hardwood forests in eastern Connecticut soil nests were common in the summer. Below-ground nest chambers are not likely to reach temperatures as conducive to larval developmental as can be found in ground surface structures or among the leaf litter [23]. Regardless of their limitations soil nests may be the only option available for many colonies. Headley [25] described the structure of soil nests sur- veyed from central Ohio. A typical ground nest had a single entrance, which was an inconspicuous circular hole ~6mm in diameter. A few nests had multiple entrances but in every case all the entrances for a single colony were located within 10 cm of one another. A central shaft, or a few bifurcating shafts, lead down from the entrance and connected the underground nest chambers. Chamber number averaged 6.5 and ranged from 2 to 17. The chambers’ dimensions averaged 12 mm high, 12 cm wide, varied in length from 18 to 50 mm and were found from just below the surface to a depth of 84 cm. Similar ground nests were reported from Missouri [24] with notable differences being slightly shallower chamber depths and, on average, fewer nest chambers. In both studies some colonies were found inhabiting various co-opted cavities such as areas between rocks and in downed wood. Ants of the rudis group maintain a concentrated central nest chamber regardless of where their nest is situated. In Connecticut the majority of the colony’s biomass (brood, nurse workers, and idle foragers) was found within 20 cm of the queen. The average density of nests was 0.5 nests/m^ across three Connecticut populations, 1.3 nests/m^ in Missouri [32], and 0.5nests/m^ in Ohio [12]. Aphaenogaster species are also known to be common in other forests [33, 34]. 4.3. Colony Life Cycle. The following provides a few details about nest founding, nest size, and productivity. The paucity of known details shows the need for further study and that there remains much to be learned about the basic biology of rudis- group ants. 4.3.1. Colony Founding. Two lines of evidence, genetic and observational, suggest that new colonies are begun claustrally by single queens. Genetic studies show that rudis- group nests contain workers produced from a single queen that has mated with one male (see the reproductive biology section). A few incipient colonies found in Connecticut contained a single queen and 25-35 minim workers. These colonies were discovered during mid-summer and were presumably founded the previous fall. 4.3.2. Productivity. Colony productivity can be highly vari- able among nests within and among populations. Worker number in mature colonies has been surveyed in a number of populations and ranges from a mean of 266 to 613 workers per nest (Table 2). Colonies with less than a hundred to more than a thousand workers can produce new sexuals (Figure 2(a)) but large colonies are more likely than small colonies to allocate energy towards reproduction (Mann- Whitney (7-test = 460, P < 0.04). The number of reproductives produced is also highly variable among nests (Table 1 and [14, 24, 25]). Some large colonies produce no sexuals (Figure 2(b)), suggesting that mature colonies do not produce new sexuals every year. 5. Foraging Ecology 5.1. Foraging. Foraging distances (Figure 3) for three eastern Connecticut populations were similar among populations (F’ 2,61 = 0.04, P = 0.9) and collectively averaged 57 cm (SD = 31). Despite this short foraging range the high density of nests provides for the abundant presence of rudis-group foragers across the forest floor. The running speed of individual workers returning to the nest with food has been found to vary with the number of workers in a colony [35]. Laden foragers returned to their nest faster in colonies with 140-150 workers than in colonies with 30-40 workers. Talbot [32] estimated that total worker density of a rudis-group ant, above and below ground, averaged ~425 workers m^ in a Missouri woodland. In a Maryland forest rudis-group workers occupied 27% of ground baits [22]. In Connecticut more than half of the ground baits placed on the forest floor were typically found by these ants within 30 minutes. Solid food is primarily brought back to the nest by individual foragers. Some recruitment of nestmates does occur at concentrated food finds (Lubertazzi, personal observation). A trail pheromone used for recruitment to food has been isolated from the poison gland of a rudis-group ant species [36]. The pheromone is a mixture of N isopentyl- 2-phenylethylamine, anabasine, and anabasiene, and 2,3'- bipyridyl. Even with recruitment rudis-group ants do not maintain more than eight nestmates at a food item at any given time [22, 36]. Ants of the rudis group are timid when encountering workers of other species and do not defend foraging territories. These ants are readily displaced at large food items by a number of co-occurring ant species [22, 37]. Their propensity to avoid confrontations is also evident in their intraspecific interactions. It was not unusual to 6 Psyche 'o o u rGl G G 'o o rGl G Colony size (number of workers) Colony size (number of workers) (a) (b) Figure 2: Distribution of the number of workers present in colonies that produced (a) or did not produce (b) sexual adults. Data are from Mohegan population trap nests collected in August of 2003 (17 colonies) and 2004 (65 colonies) [14]. Foraging distance (cm) Figure 3: Distribution of the straight line distance, in cm, from a food item that attracted foragers to the nest entrance. Data were collected from three eastern Connecticut populations in July and August of 2003. find individuals from two or three colonies of a rudis- group ant foraging on the same bait in Connecticut forests. Here and in Maryland paired individuals could occasionally be found engaging in paired battles that “involve long, seemingly inconclusive “wrestling” bouts that result in few if any casualties” [22]. In Connecticut, workers were observed indifferently walking around any intertwined pair of fighting ants that they encountered. 5.2. Diet. Ants of the rudis group are general scavengers. In Connecticut the majority of food items observed being carried by foragers were small invertebrates or parts of insects. Workers have been observed preying on termites {Reticulitermes flavipes) in the field in Connecticut and Indi- ana [38] and in a laboratory study [39]. Small invertebrates are likely the staple of their diet. Other food resources are also exploited and are clearly important, but are either temporally limited or spatially uneven in their availability. For example a mushroom species is known to be foraged upon by a rudis- group ant [40]. This opportunistically encountered resource may provide nutrients that are not readily found in their typical diet but is unlikely to even be encountered within the foraging range of many colonies. These ants are keystone seed dispersers in the mesic forests of eastern North America [41, 42]. Ants of the rudis- group move a majority of the diverse myrmecochorous seeds that are produced in these habitats. The collection of eliasome-bearing seeds by rwr/is-group ants is well docu- mented, as is the floral diversity of myrmecochorous plants throughout these ants’ ranges (e.g., [13, 43, 44]). Sexual production within colonies can be altered by elaiosome consumption [12, 45] despite the fact that colonies become satiated quickly when provided with myrmecochorous seeds. Seed foraging ceases within hours of a colony being presented with elaiosome-bearing seeds and this response can persist for many days [13]. Foragers opportunistically imbibe liquid food resources and behaviorally overcome morphological limitations in how much liquid can be held in their crops [46]. Foragers recruit nestmates to particularly rich finds and can also use absorbent objects to collect liquids [37]. Saturated materials are brought back to the colony and the liquids they hold are consumed within the nest. Workers can store an average of 0.13 mg of liquid in their crop but can transport up to 10 times this amount of liquid using an absorbent tool [37]. 6. Reproductive Biology 6.1. Intra Colonial Social Structure. Colonies of rwdis-group ants have a simple reproductive and social structure [47, 48]. There is one singly mated queen that is the sole reproductive in her nest. Young workers may have functional ovaries but worker eggs are either not produced or eliminated in queenright colonies [49]. Although Talbot [24] and Headley [25] both found some rudis- group ant colonies with more than one dealate queen, Crozier [48] suggested these did not represent polygynous Psyche 7 colonies. This was inferred from his genetic findings and observations that unmated rwdis- group queens may sponta- neously remove their wings in their natal nest. This dealation behavior was also noted by Haskins and Enzmann [50] . A few mature field colonies collected in Connecticut were found to contain numerous dealate queens after they had settled into artificial nests in the laboratory. In every case there were other winged queens present and unattached wings of queens were found in the sorting bin or in the artificial nest. Dealation was clearly occurring after the nests were collected from the field. This same behavior was observed in a few laboratory nests, originally collected in Connecticut, that had produced female reproductive. In a few colonies the unmated dealates were left in the laboratory maintained nest. Behavioral differences between a colony’s reproductive queen and her unmated dealate daughters were evident. When a reproductive queen moved within the nest and antennated a worker, the worker would typically lower her head and/or flee. Workers that initiated antennating a reproductive queen’s body typically continued to investigate the queen with their antenna. Such attention lead to the queen attracting a retinue of workers when she remained in one part of the nest. Worker-to -worker interactions within the nest produced no alarm and always quickly lead to disinterest. By contrast, dealates spend considerable time outside of the nest and appeared to be subordinate to the queen in their interactions. Antenna-to-antenna contact between workers and dealates always led to the dealates fleeing. Dealates overall were more worker-like in their actions. They were seen outside the nest, moved brood and never attracted a retinue. The one dealate behavior that was queen-like was the propensity to seek cover and remain motionless under an object when the nest was disturbed. Workers varied in their response to disturbance but were as likely to run around excitedly, pick up brood, or move outside of the nest into their foraging arena. Worker-like behaviors in the dealates become more pronounced weeks after dealation, and, when left in the nest until the following spring, dealates no longer ran as quickly or as persistently from disturbances. In worker- dealate interactions the dealate still tended to move away from worker contacts. Workers reacted with greater alarm when they come in contact with a dealate than when contacting another worker but these differences were subdued when compared to similar interactions that occurred in the fall. The reason(s) for the spontaneous dealation of gynes and any potential adaptive value of this behavior are unknown. It has been observed in species from other genera, for example, Pogonomyrmex (Bob lohnson, personal communication) and Myrmica, [51]. The aggressive reactions of rudis-group workers to dealates, the propensity of dealates to be found outside the nest in laboratory colonies, the usual absence of supernumerary queens in field colonies in Connecticut and Crozier’s genetic data suggests that if dealation occurs in a naturally occurring colony these unmated gynes are likely driven out of their natal nest. Regardless, it is not possible to rule out that spontaneous dealation is the first part of a secondary reproductive strategy. Inbreeding within the nest. Investment sex ratio Figure 4: Distribution of colony-level sex ratios (female) for field- collected colonies without a queen based on data from Table 3. Colonies with a sex ratio of 0 produce only male reproductives. dealates walking out of their natal nest and mating on the forest floor or colonies allowing unrelated males into the nest to mate with a newly dealate queen are all possibilities. 6.2. Sex Ratios. Sex ratios have been calculated for a number of rudis-group populations. The first such estimate, calculated to test hymenopteran sex ratio theory [5], was improperly derived. The 14-colony data set combined colony data from two states collected in two different years hence does not represent a population sex ratio. The first rigorous study of rudis-group sex ratios was a study examining the potential benefits of elaiosome food resources in an Ohio population [12]. Naturally occurring colonies presented with elaiosome-bearing seeds increased their reproductive allocation and had a more female biased sex ratio than control colonies. A follow-up study suggested the treatment response was a quantitative effect of adding more food rather than a result of specific nutrients contained in the elaiosomes [45]. A field study of a Connecticut Mohegan population also involved food supplementation [14]. No differences were found between control colonies and colonies provided with extra protein. Sex ratios were split (most colonies specialize in mostly male or mostly female investment) and the population sex ratio was estimated to be 0.86 (95% Cl: 0.81 to 0.91). Colonies that produced larger broods invested slightly more in males. The sex ratio of queenless field colonies was similar to those of queenright colonies (Figure 4, Table 3). Naturally occurring queenless colonies overall invested much more in female than male production. Workers in these colonies appear to be raising their recently lost mother’s brood rather than raising their sons and nephews. 6.3. Mating. Mating flights have been described for a congener [52] but have never been described for any rudis- group ant. It is not known how far queens fly from their natal nests or if mating occurs on the ground, or elsewhere. 8 Psyche Table 3: The production of sexual adults in queenless field colonies. Data are from 4 populations: M; Mohegan SF (2004); N: Nipmuck SF (2001); O: Ohio (1949) [25]; P: Pachaug SF (2001). Source Workers Males Females M 386 53 12 M 190 2 9 M 282 3 41 M 424 31 51 M 325 32 9 M 89 0 1 M 432 0 45 M 196 83 43 M 303 14 32 M 225 35 15 N 730 86 0 N 555 108 0 N 1211 6 35 N 387 33 0 0 138 6 0 0 119 6 0 0 328 3 0 P 327 20 20 P 587 0 17 P 556 0 14 P 472 28 0 In Connecticut it appears that populations have synchronous mating. Collecting whole nests from 3 different forests over numerous years revealed that winged reproductives disappeared from all the nests within a given population at the same time. New sexuals were typically gone from the Connecticut colonies, depending on the population and the year, sometime between late July and mid-August. 7. Caste Attributes 7.1. Larvae. G. C. Wheeler and J. Wheeler [53] studied and described the morphological and developmental characteris- tics of the egg and larval stages of rwr/is-group ants. There are four larval instars. First instar larvae were found to subsist on worker-provided liquid foods. Subsequent instars were also able to ingest solid foods [53, 54]. 7.2. Workers. Fielde [54] found the developmental period for workers, from egg to eclosion, averaged 64 days (time for eggs to hatch: median = 19.5 days, N = 22; larval stage: median = 28.5 days, N = 26; pupal stage: median = 16 days, N = 68). Southerland [55] examined the influence of temperature on development time. She compared the productivity of artificially created rudis-group nest fragments (a queen and 50 workers) maintained at 15°C and 25° C. During 150 days at the cooler temperature no workers were produced. Brood were present and survived but no pupation occurred. Nests maintained at the warmer temperature were able to produce new workers. The dry weight of Connecticut workers averaged 0.8 mg (SD = 0.16, N = 200; Figure 5(a)) and head width averaged slightly less than 1 mm (mean = 0.91, SD = 0.06, N = 975; Figure 6). Connecticut and Vermont [30] head width data showed workers form a single monomorphic caste. Worker head width in Connecticut did vary significantly among colonies within populations (Nipmuck population Fi8,456 = 13.4, P < 0.01; Pachaug population Fi 9, 480 = 13.1, P < 0.01) and between populations (^973 = 5.96, P < 0.01). These differences are presumed to be caused by environmental variation in food availability and temperature differences experienced among colonies. A regression of average colony worker head width on colony worker number was not significant {N = 27, P > 0.3). Southerland [55] found that worker mortality was higher in laboratory nests maintained at 30° C than in nests maintained at 15°C. Field colonies collected in Connecticut and maintained in the laboratory contained workers that survived for more than a year. The average life span of a worker in a natural setting, where there are many risks, is undoubtedly less. Workers possess functional ovaries but do not lay eggs when their colony has a healthy, fertilized queen [49]. Worker-produced males are also presumed to be uncommon in naturally occurring queenless nests (as discussed in the reproduction section). Workers of the rudis-growp exhibit little division of labor and can perform a total of 41 different behavioral acts [30]. In laboratory nests it was found that 75% percent of a workers’ time is spent in nonsocial behaviors and most individuals are inactive most of the time. Worker activity levels and brood tending rates were higher in the summer relative to autumn [30]. 7.3. Gynes. Low temperature and a sustained drop in metabolic rate are presumably necessary to induce gyne development, as has been found for other temperate ants [56] . New queens are thus produced from a subset of overwintered female brood. Workers are likely to play a role in determining which females develop into queens by altering the diet and/or temperature environments of select larvae [57] . Early-instar gynes resume development in early spring and in Connecticut eclose in mid-June. Fielde reported a single developing queen she observed spent 17 days in a pupal state [54]. Once eclosed gynes are presumably fed by their nestmates to increase their fat stores, which is typical for ant species in which queens found nests independently. Gynes collected from the Pachuag and Nipmuck forests averaged a dry weight of 6.5 mg (SD = 0.5, N = 25; Figure 5(b)). Flaskins [58] observed the survivorship schedule of 11 laboratory-housed queens, finding a median lifespan of 8 years and a maximum of 13. A number of eastern Connecticut colonies, mature when found and therefore at Psyche 9 45 o u 3 30 15 0.4 0.6 0.8 1.2 o u /n a Worker dry weight (mg) Female dry weight (mg) (a) (b) Male dry weight (mg) (c) Figure 5: The dry weights of three adult A. rudis castes, (a) workers, n = 200; (b) gynes, n = 25; (c) males, n = 25. Worker headwidth (mm) Figure 6: The distribution of the head width of 975 workers from 39 Nipmuck and Pachaug colonies. least a few years old when collected, were maintained in the laboratory from 2001 through 2005. 7.4. Males. Males can be produced from unfertilized eggs of queens or workers but in queenright nests there is no worker reproduction [49]. Males collected from the Pachuag and Nipmuck populations averaged 0.6 mg dry weight (SD = 0.1, N = 25; Figure 5(c)). Fielde [54] reported that the median duration of the pupal stage for 3 males was 19 days. Males in queenright nests are thought to be produced from overwintered brood, passing through conditions similar to those experienced by gynes [24, 25]. Adult males leave the nest within a month of eclosing and their adult lifespan is likely brief. Once leaving the nest to mate, even if they avoid being killed by a predator, they will eventually succumb to starvation. 8. Conclusion Ants of the rudis-group are an abundant component of the hardwood forests of eastern North America. Their ability to forage early in the spring, to readily move their nests, and to feed upon a wide range of resources plays a part in their success. These ants can serve as a useful study system for investigating ecological and evolutionarily questions, and for learning more about basic ant biology. Ants of the rudis group are easy to locate and collect; the use of artificial wooden nests can facilitate their study in the field, colonies 10 Psyche can be readily maintained and studied in the laboratory, their colony size is such that many characteristics of whole colonies are amenable to direct measurement and the workers are not aggressive. Finally, with numerous aspects of their basic biology having been investigated this knowledge forms a solid foundation for planning future studies. Acknowledgments Eldridge Adams’ kind and thoughtful assistance, in all aspects of this work, is much appreciated. Figure 1 was cre- ated by Neil McCoy. Lab technicians Scott Soricelli and Dawn DeFreitas helped measure ant heads and with the counting of individual ants within colonies. 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Wheeler, “Developmental and physiological determi- nants of caste in social Hymenoptera: evolutionary implica- tions,” Amen’can Naturalist, vol. 128, no. 1, pp. 13-34, 1986. [58] C. P. Haskins, “Note on the natural longevity of fertile females of Aphaenogaster picea,” Journal of the New York Entomological Society, vol. 68, no. 2, pp. 66-67, 1960. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 185312, 6 pages doi:10.1155/2012/185312 Research Article Coexistence and Competition between Tomicus yunnanensis and T. minor (Coleoptera: Scolytinae) in Yunnan Pine Rong Chun Hong Bin Wang,^’^ Zhen Zhang, John A. Byers, ^ You Ju Jin,^ Hai Feng Wen,^ and Wen Jian Shi^ ^ Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 1 00091, China ^ The Key Laboratory of Forest Ecology and Environment, State Forestry Administration, Beijing 1 00091, China ^ College of Urban Construction, University of Shanghai for Science and Technology, Shanghai 200093, China ^ USDA-ARS, US. Arid-Land Agricultural Research Center, 21881 North Cardon Lane, Maricopa, AZ 85138, USA ^College of Plant Sciences, Beijing Forest University, Beijing 100083, China Correspondence should be addressed to Hong Bin Wang, wanghb@caf.ac.cn Received 6 October 2011; Accepted 12 December 2011 Academic Editor; Qing-He Zhang Copyright © 2012 Rong Chun Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Competition and cooperation between bark beetles, Tomicus yunnanensis Kirkendall and Faccoli and Tomicus minor (Hartig) (Coleoptera: Scolytinae) were examined when they coexisted together in living Yunnan pine trees (Pinus yunnanensis Franchet) in Yunnan province in Southwest China. T yunnanensis bark beetles were observed to initiate dispersal from pine shoots to trunks in November, while the majority of T minor begins to transfer in December. T yunnanensis mainly attacks the top and middle parts of the trunk, whereas T minor mainly resides in the lower and middle parts of the trunk. The patterns of attack densities of these two species were similar, but with T yunnanensis colonizing the upper section of the trunk and T minor the lower trunk. The highest attack density of T Yunnanensis was 297 egg galleries/m^, and the highest attack density of T minor was 305 egg galleries/m^. Although there was significant overlap for the same bark areas, the two species generally colonize different areas of the tree, which reduces the intensity of competition for the relatively thin layer of phloem- cambium tissues where the beetles feed and reside. 1. Introduction A new species of pine bark beetle, Tomicus yunnanensis Kirk- endall and Faccoli (Coleoptera: Scolytinae), was recently dis- covered, and which formerly had been confused with Tom- icus piniperda (L.) [1]. T. yunnanensis is an important forest pest since it has caused extensive mortality of Yunnan pines, Pinus yunnanensis Franchet, in the southwest of China [2- 5]. More than 200,000 ha of Yunnan pine forests were killed by the bark beetle by 2005 [4-6]. In addition to Yunnan pines, T yunnanensis also feeds on Simao pines, P. kesiya var. langbianensis, and Gaoshan pines, P densata, as well as some other pine species [4, 5]. T. yunnanensis is frequently joined by T. minor (Har- tig) in attack of Yunnan pine trees in the southwest of China, Yunnan province [2, 7]. In most of the cases, the two species live in the same area and feed on the same host trees [5, 7]. Several studies have reported that there is a general competition between T. minor and T. yunna- nensis [2, 5, 7-10]. However, the specific mechanisms and the extent of the competition are unclear. An investiga- tion on the cooperation and competition between these two bark beetle species to obtain food may indicate new approaches of integrated control of pest bark beetles, especially T yunnanensis and T. minor which are very difficult to control and monitor [11-14]. Thus, the goals of the present studies were to investigate the changes in distribution on the host tree of the beetles during the autumn dispersal from shoots to trunks and to determine the mutual influence of the two Tomicus species on each other’s attack distribution and reproduction within the tree. Additional knowledge about the two pest bark beetle species’ attack distributions and densities, colonization sequen- ces, migratory movements, and competitive interactions 2 Psyche -o— T. yunnanensis ~0— T. minor Figure 1: Average attack (egg gallery) density (±SE) and distribu- tion of T. yunnanensis and T. minor along the trunks of Yunnan pines in November, 2005 (Qujing, Yunnan province, China). may provide insights into more efficient management prac- tices. 2. Materials and Methods 2.1. Study Site Conditions. A series of field studies were conducted from November to March in 2005-2006 in a 300 ha plantation of Yunnan pines, located in the mountain near Qujing city in Yunnan province (25° 14' N, 103°50' E, and 1700-1800 m above sea level). Some broad-leaved trees were scattered inside the plantation. Most of the Yunnan pines were infested with both T. yunnanensis and T. minor. The trees were 30-45 years old and ranged from 10 to 15 m in height and 10 to 15 cm in diameter. 2.2. Experimental Design. Beginning in November 2005, two to three Yunnan pines were selected at random and cut down every week through March, or approximately 44 trees in total. Every shoot was carefully examined, and any bark beetles that were found were collected and distinguished by species with a binocular microscope. In addition, one to two pine trunks that had been colonized by T. yunnanensis or T. minor were selected and cut down every week, or approximately 17 trees. The tree trunks were divided into 0.5 m long logs, and each log was carefully peeled of bark to reveal egg galleries. The entire trunk was cut into two sections (upper section and lower section) from the middle; and the number and length of all egg galleries over the surface of each log were recorded. The bark beetles within galleries were also T. yunnanensis T. minor Figure 2: Average attack (egg gallery) density (±SE) and distribu- tion of T. yunnanensis and T. minor along the trunks of Yunnan pines in December, 2005 (Qujing, Yunnan province, China). collected and distinguished with a binocular microscope. The heights and diameters of all the cut trees were measured. 2.3. Data Presentation and Analyses. Data are presented as mean ± standard error (±SE). The correlations between T. yunnanensis and T. minor were calculated with bivariate methods. These analyses were conducted using SPSS 11.5 (SPSS Inc., Chicago, IE, USA). 3. Results 3.1. Egg Gallery Distribution in Trunks. Most T. yunnanen- sis began to move from shoots to trunks in November (Eigure 1), and most T. minor began in December (Figure 2). In November, there were fewer T. minor egg galleries compared with T. yunnanensis, and all of the T. minor egg galleries were distributed in the lower half of the tree’s trunk (Figure 1). T. yunnanensis egg galleries were distributed over the entire trunk in some trees, but with different densities in different sections. The highest density of T. yunnanensis was in the upper half of the tree trunks and was about 198 galleries/m^, while the highest density of T. minor was only about 19 galleries/m^. In December, the attack density of T. yunnanensis and T. minor was in both higher than in the previous month, with T. minor increasing significantly (Figure 2). However, in the lower section of the trunks, the attack density of T. yunnanensis decreased a little compared to earlier. T. minor was still mostly distributed in the lower section of the trunk. In January, the attack density (egg galleries) of T. yunna- nensis remained highest in the upper section of the trunks Psyche 3 T. yunnanensis T. minor Figure 3; Average attack (egg gallery) density (±SE) and distribu- tion of T. yunnanensis and T. minor along the trunks of Yunnan pines in January, 2006 (Qujing, Yunnan province, China). and also remained low in the lower section of the trunk (Figure 3). The highest gallery density of T. yunnanensis was about 297 galleries/m^. The attack density of T. minor increased further, especially in the lower section of the trunks; and the egg galleries of T. minor were now distributed nearly over all areas of the trunks (Figured). The highest attack density of T. minor was about 305 galleries/m^. The measurements of egg gallery lengths show that in November these lengths for T. yunnanensis and T. minor were relatively short (Figure 5); T. yunnanensis egg galleries were about 3. 6-4. 9 cm long, while T. minors egg galleries were about 3. 2-4. 3 cm long. In December, the egg gallery length increased, especially for T. minor (Figure 6). The egg galleries of T. minor were about 5. 9-6. 2 cm long, and T. yunnanensis egg galleries were about 4. 6-6. 0 cm long. In January, egg gallery length continued to increase (Figure 7), T. minor egg galleries lengthened to about 8. 2-8. 9 cm, and T. yunnanensis went up to about 5. 2-6.6 cm long (Figure 8). 3.2. Bark Beetle Distribution in Tree Crowns. About II T yunnanensis were found in shoots per tree in November, and this number decreased to about nine by January, but the differences were not significant. Fewer T. minor (about four) were found per tree in November, and the number appeared to increase to five in December and then returned to four in January, but these differences were not significant. 3.3. Relationship between T. yunnanensis and T. minor. The two bark beetle species had different relationships in different parts of the tree during the November to January period -0— T. yunnanensis ■ T. minor (November) T. yunnanensis ■■*■■■ T. minor (December) -□- T. yunnanensis ■ ■ • ■ ■ T. minor (January) Figure 4: Progression of average attack (egg gallery) density and distribution of T. yunnanensis and T. minor along the trunks of Yunnan pines from November to January, 2005-2006 (Qujing, Yunnan province, China). T. yunnanensis T. minor Figure 5: Average egg gallery length (±SE) of T. yunnanensis and T. minor along the trunks of Yunnan pines in November, 2005 (Qujing, Yunnan province, China). (Figures 4 and 8). In November, there was no significant correlation between the two bark beetle species either with respect to trunks or to shoots (Table I). In December, the two bark beetle species in shoots had no significant correlation either, but there was a significant negative correlation (P < 0.0 1) between T. yunnanensis and T. minor 4 Psyche Average gallery length (cm) — O- T. yunnanensis — O— T. minor Figure 6; Average egg gallery length (±SE) of T. yunnanensis and T. minor along the trunks of Yunnan pines in December, 2005 (Qujing, Yunnan province, China). ~0— T. yunnanensis — O — T. minor Figure 7: Average egg gallery length (±SE) of T. yunnanensis and T. minor along the trunks Yunnan pines in January, 2006 (Qujing, Yunnan province, China). in trunks (Table 1). In January, the two bark beetle species had significant negative correlations in shoots and in trunks (Table 1). 4. Discussion The appearance of bark beetle galleries show that most T. yunnanensis begin to transfer from shoots to trunks in November, a little earlier than T. minor. Most of T. minor begin to move in December. This result is consistent with earlier reports [15, 16]. The transferring period lasts from November to January, or even longer, and there is no clear peak in transferring time. In Yunnan province, the weather is much drier than other times in winter from November to April. When winter approaches, the weather has little rainfall and the soil becomes dry. Some trees are weakened because of the shortage of water. These weak trees are the first choice for bark beetles to attack and feed on. As dry weather conditions persist, more and more trees are weakened and become suitable for bark beetles to colonize. At the study site, the colonized trees were cut down when they were found. However, as the colonized trees were cut down, newly colonized trees could always be found until February. When T. minor begins to transfer from shoots to trunks, and the density of T. minor increases in the lower trunk, the newly colonizing T. yunnanensis will no longer feed on lower sections, indicating the latter species is avoiding competition with T. minor (Figure 4). Thus, the colonized trees cut down later were found to have fewer T. yunnanensis in their lower sections. Similarly, T. minor were found mainly in the lower trunk in areas with less T. yunnanensis, thus both species Average gallery length (cm) T. yunnanensis (November) T. minor (November) T. yunnanensis (December) ■ 21 minor (December) 21 yunnanensis (January) ■ 21 minor (January) Figure 8; Progression of average egg gallery length of T. yunnanensis and T. minor along the trunks of Yunnan pines from November to January, 2005-2006 (Qujing, Yunnan province, China). appear to be avoiding competition by selecting areas of the host with lower densities of the opposite species (Figure 4). The egg gallery length of T. yunnanensis and T. minor not only increased during the three-month period, but also changed with the height of the trunks (Figures 5-8). The egg gallery length of T. yunnanensis increased, and the egg gallery Psyche 5 Table 1: The correlation between T. yunnanensis and T. minor in trunks and shoots of Yunnan pines. Month Bark beetles Pearson correlation in trunks Sig. (2 tailed) Bark beetles Pearson correlation in shoots Sig. (2 tailed) 11 -0.491 0.179 -0.126 0.729 12 -0.956 1.52E- 05** 0.169 0.642 1 -0.981 3.5E- 09** 0.768 0.009** Correlation significant at the 0.01 level (2-tailed). length of T. minor decreased with the height of the trunks in different months. We found that there are mainly two ways for T. yunna- nensis and T. minor to transfer from shoots to trunks. If the host trees were already weakened, the beetles would feed on the host tree trunks directly; if the host trees were initially more healthy, beetles would feed together on the shoots of the tree to weaken it and then transfer to the trunks of these trees. These insects use this strategy to weaken the tree’s defensive ability and allow better survival of the individual bark beetles. The relationship between T. yunnanensis and T. minor changed with time and location on the tree. When the time for transferring came, T. yunnanensis would feed on the top of the host tree’s trunk to make their host weaker or to kill it. Occasionally we found live trees with a dead top that had been killed by T. yunnanensis; this indicates that host trees generally have a strong resistance and were not easy to kill [17, 18], but when T. minor joined in to feed on the lower section of the host trunk, the resistance of the tree was further weakened which often led to its death. It is well known that the egg galleries of T. minor are perpendicular to the trunk length [19]. This would probably cut more transporting tissues and might be more harmful to trees than T. yunnanensis which have galleries aligned with the trunk. In November, the attack density was relatively low in trunks, especially for T. minor, so the relationship between the two bark beetle species was predominately cooperation. With more and more bark beetles transferring to trunks; however, the competition between the two species intensified, and the cooperation between them diminished. However, they could coexist very well since both species had a separate colonization location on the trunk to avoid competition as much as possible. In fact, we scarcely found two egg galleries of different families joined together, no matter whether the galleries were from the same or the other species, as has been observed in other species [12, 13, 20, 21]. When two egg galleries almost touched, bark beetles would not proceed ahead, and in most of the cases, T. yunnanensis would change the egg gallery direction or abandon their egg gallery, thus making some egg galleries of T. yunnanensis not straight or with deviations. T. minor also would stop when closely approaching another egg gallery and often turned and excavated in the opposite direction, because there are only two directions for T. minor to bore their egg galleries perpendicular to the grain of the trunk. In the tree crowns, the behavioral relationship between the two species was more complicated. It was not uncommon that two or three bark beetles tunneled in one shoot. But in most cases, the beetles were of the same species and usually consisted of one male and one female, or one male and two females. This suggests that there was competition between T. yunnanensis and T. minor in shoots. At the beginning of the transferring period, their relationships were dominated by competition in shoots; but with more and more bark beetles transferring from shoots to trunks, their relationship changed. In other words, the competitive relationship between the beetles changed as they transferred from branches to trunks. Thus, the competitive relationship between T. yunnanensis and T. minor occurs during their entire life cycle but alters in intensity and type with the life phase and location on the host tree. Knowledge about the movement from shoots to trunks (transferring) and trunk colonization process, as well as competition between the two species, will be useful in designing control strategies that take advantage of the vulnerability of the beetles and maximize resistance of the tree. Acknowledgment The authors thank the Forest Pest Control Station of Qu- jing City, Yunnan province for logistical support and field assistance. This study was funded by the Importing In- ternational Advanced Agricultural Science and Technology Research (2002-38) and International Technological Coop- eration Research (2006DFA31790). References [1] L. R. Kirkendall, M. Faccoli, and H. Ye, “Description of the Yunnan shoot borer, Tomicus yunnanensis Kirkendall & Fac- coli sp. n. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 818490, 10 pages doi:10.1155/2012/818490 Research Article Gerris spinolae Lethierry and Severin (Hemiptera: Gerridae) and Brachydeutera longipes Hendel (Diptera: Ephydridae): Two Effective Insect Bioindicators to Monitor Pollution in Some Tropical Freshwater Ponds under Anthropogenic Stress Arijit Pal, Devashish Chandra Sinha, and Neelkamal Rastogi Insect Behavioural Ecology Laboratory, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi 221 005, Uttar Pradesh, India Correspondence should be addressed to Neelkamal Rastogi, neelkamalrastogi@yahoo.co.in Received 30 July 2011; Accepted 29 October 2011 Academic Editor: Matilda Savopoulou-Soultani Copyright © 2012 Arijit Pal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The abundance patterns of two insects, Gerris spinolae and Brachydeutera longipes, were found to be affected by abiotic aquatic factors including free carbon dioxide, dissolved oxygen, BOD, and phosphate concentrations prevailing in four tropical freshwater ponds, three of which being anthropogenically stressed. Regression analysis between each individual-independent water quality variable and insect abundance demonstrated a significant positive correlation in each case between B. longipes abundance and BOD, phosphate, free CO 2 , and algae dry weight, while a significant negative correlation of each of these variables was found with Gerris spinolae abundance. Moreover, a significant negative correlation of B. longipes abundance was calculated with dissolved oxygen concentration, while G. spinolae abundance exhibited a positive correlation with the same. Thus, G. spinolae appears to be a pollution sensitive, effective bioindicator for healthy unpolluted ponds, while B. longipes has potential as a pollution-resistant insect species indicative of pollution occurrence. 1. Introduction Freshwater bodies in urban ecosystems are under stress due to anthropogenic pressures. Pollution of inland water habi- tats, both lotic (running water) and lentic (lakes and ponds), impacts pollution of soil and ground water and thereby affects the essential basic (drinking water supply) and social requirements (aesthetic, religious, etc.) of human societies. Monitoring and maintaining the water quality of wetlands is also important since these recharge the groundwater and also affect the plant diversity in its vicinity. Environmental monitoring of inland freshwater bodies is an essential prerequisite for their management. Biological indication is, therefore, increasingly being advocated. Biological indication or bioindication is the process of using a species or group of species that readily reflects the abiotic and biotic states of an environment, represents the impact of environmental change on a habitat, community, or ecosystem, or is indicative of the diversity of a subset of taxa or of the entire diversity, within an area [1, 2]. Bioindicators or ecological indicators are taxa or groups of animals that show signs that they are affected by environmental pressures due to human activities or the destruction of the biotic system [3]. Bioindicators also provide information about the cumulative impact of the various pollutants in an ecosystem [1, 4-6]. An ideal taxon must respond predictably, in ways that are readily observed and quantified to environmental disturbance [7]. Aquatic bioindicators used so far are plants [8-10] including diatoms [4, 11]; vertebrates, mainly fish [5, 12, 13] and macroinvertebrates [1, 6, 7, 14, 15]. Among invertebrates, insects are good candidates [16-21]. However, not all insects respond in a predictable manner to environmental pollution. Insects widely utilized as bioindicators include larval Chi- ronomids (Diptera: Chironomiidae) [22] and water striders (Hemiptera: Gerridae), the latter being particularly well known for indicating heavy metal pollution, [18, 23-26] which is also characterized by oxygen stress. Insects offer a number of advantages as bioindicators. These include the 2 Psyche availability of a wide range of insects from various insect orders which (i) exhibit high sensitivity and the degree of sensitivity gives a series of choice of bioindicators depending upon the needed resolution, (ii) involve the entire trophic levels, thus ecosystems can be monitored from functional point of view, (hi) exhibit high fecundity, greater breeding potential reduces the chances of the potential bioindicator getting destroyed entirely from the ecosystem, and finally (iv) involve less ethical problems. While some studies have been carried out on bioindi- cators of lotic ecosystems [19, 27-29] studies of potential insect bioindicators of lentic ecosystems are scanty [30, 31], especially those of the tropical regions [15]. The present study focuses on assessing the potential of two insect species: the water strider, Gerris spinolae (Lethierry and Severin) and the shorefly, a semi-aquatic dipteran, Brachydeutera longipes (Hendel). Studies pertaining to the genus Brachydeutera are scanty although its occurrence is reported from lentic habi- tats [32]. While water striders, common in freshwater water bodies of temperate and tropical water bodies, are preda- ceous [33, 34], the genus, Brachydeutera, is documented to include species such as B. hebes, B. argentata, and B. neotropica the larval stages of which scavenge upon dead and decaying plant and animal tissues and also consume algae [32]. Since B. longipes is reported to be an algal feeder [35] and algal blooms are characteristic feature of polluted ponds, this species merits further investigation to examine its poten- tial as a bioindicator. The impact of abiotic aquatic factors on the ecological response of the two focal insect species was investigated and the relationship between the abundance pattern of each species and the degree of pollution was determined. 2. Materials and Methods 2.1. Study Sites. The investigations were carried out from January to March, 2011 (3 months), in Varanasi, Uttar Pradesh, India. Four man-made ponds presently under anthropogenic stress were selected for the present study. While the pond located in the Botanical garden of Banaras Hindu University (not being under any anthropogenic stress) was considered as the control and the three ancient ponds, about 200 years old [36] under anthropogenic stress (due to human activities such as bathing, washing clothes, dumping organic wastes in the form of flowers, and so forth, in the ponds, particularly during religious ceremonies and festivals) located in a thickly populated urban ecosystem, were taken as the experimental. The Kurukshetra (Krk), Sankuldhara (Skd), Durgakund (Dgk), and Botanical garden (Btg) ponds are located in Assi, Khojwa, Durgakund, and Banaras Hindu University Campus areas, respectively. All the ponds except Btg have 1-2 old temples around them. The dimensions of Krk pond are about 20 m X 25 m X 6 m. Its four banks are bounded by stone tiles from all around. Due to dense human inhabitation around it, there is heavy anthropogenic pressure on it. The dimensions of Skd pond are about 30 m x 30 m x 7 m. Its parapets are also bounded by stone tiles and, in addition it is surrounded by iron grid fencing. The dimensions of Dgk pond are about 40 m X 40 m X 10 m. Its parapets are also bounded by stone tiles and in addition, it is surrounded by an iron grid fencing. Human activities including occasional bathing, washing of clothes, and dumping of organic wastes in the form of flowers, and so forth during religious and social ceremonies occur in all these three ponds. The dimensions of Btg pond are about 10mx8mx2m. It was constructed for the purpose of watering garden plants. It is free from all anthropogenic pressures. 2.2. Water Quality Assessment. Transparency was determined for each pond by using the Sechhi disc method while total solids were assessed by the standard dry weight method [37]. All the other physical and chemical parameters were monitored twice a week {n = 15) at each study site. DO and BOD (5 days, 20° C) were determined by the modified Winkler s method [37] . Free CO 2 level was assessed by titrating the samples with 0.05 N NaOH solution in the presence of phenolphthalein indicator. Phosphate ion concentration was determined by the standard spectroscopic method [37]. 2.3. Insect Diversity of the Ponds and Selection of Insect Species for Investigation of Aquatic Bioindicator Potential. The study revealed that each of the ponds supported a variety of aquatic insects from different orders, including water striders, back swimmers, water bugs (Order: Hemiptera), flies, mosquito larvae (Order: Diptera), and damselfly, dragonfly (Order: Odonata). Among these, two insect species, namely, Gerris spinolae, Lethierry and Severin, (Hemiptera: Gerridae; det. NPIB) RRS No. 1116-1117/11 and Brachydeutera longipes, Hendel (Diptera: Ephydridae; det. NPIB), and (RRS No. 1118-1124/11), were selected for further studies. These were identified by experts of the Network Project on Insect Biosystematics (NPIB), Division of Entomology, Indian Agricultural Research Institute, New Delhi. These two species were selected to study the impact of specific abiotic factors prevailing in the three anthropogenically stressed ponds, on the basis of the preliminary field observations regarding their differential habitat preferences. 2.4. Abundance of Adult Stages of the Two Insect Species: Gerris spinolae and Brachydeutera longipes. Insect abundance was monitored twice a week {n = 15) per pond. Quadrat sampling was done from sixteen different sites of each pond (four sites per side per pond), n = 240 quadrats per pond. The following formula was used to calculate the abundance: abundance total number of individuals of the focal species in all the sampling units number of sampling units in which the species occurred ( 1 ) Psyche 3 2.5. Life Cycle of B. longipes. Brachydeutera longipes was cultured under laboratory conditions by carefully adding about 10 mg of fresh algae, Microcystis sp. (which was carefully layered on the water surface), to 1 liter pond water contained in a 5 liter glass jar (n = 3). Thereafter, 2 pairs of B. longipes were introduced in each jar. Small fractions of the algae were examined daily under the Stereobinocular microscope and the various life cycle stages and feeding behaviour of the larval stages were recorded. 2.6. Statistical Analysis. Variation in the abiotic factors, that is, temperature, pH, free CO 2 , dissolved oxygen (DO), biological oxygen demand (BOD), phosphate ion concen- tration, and a biotic factor-concentration (dry weight/m^) of the algae. Microcystis sp. in each of the four ponds was analysed by using one-way analysis of variance (ANOVA) followed by Dunnett s post hoc test by using SPSS-PC soft- ware. Regression analysis for calculation of the correlation between the abundance of each of the two insect species, Brachydeutera longipes and Gerris spinolae with each of the above-mentioned seven water quality parameters considered individually in each case, was carried out by using SPSS-PC software. 3. Results 3.1. Life Cycle of B. longipes. Examination of the surface of the algal vegetation under laboratory conditions showed the presence of pale brown, cigar-shaped operculated eggs. Three larval instars were recorded, the duration of each stage was found to be approximately 2-3 days with that of the pupal stage being about 3-4 days. The larvae were observed to feed voraciously on Microcystis sp. Adults were recorded to have an approximate life span of 2-3 months. 3.2. Water Quality Assessment. A significant variation in water transparency was found in the four ponds: Btg pond (182.5 cm), Krk pond (63.8 cm), Skd pond (52.67 cm), and Dgk pond (29.67 cm). There was also variation in the amount of total solids present in each pond, with Btg pond having least amount of total solids including dissolved (275.8 mg/L) and suspended (3.9 mg/L) in comparison to the solids present in the other three ponds. The amount of dis- solved solids were 321.7 mg/L, 537.2 mg/L, and 873.9 mg/L and suspended solids were 4.1 mg/L, 4.6 mg/L, 7.3 mg/L in Krk, Skd, and Dgk ponds, respectively (Ligure 1). Six abiotic parameters, namely, temperature, pH, free CO 2 , dissolved oxygen (DO), BOD, phosphate ion concen- tration, and one biotic parameter, that is, food availability of B. longipes larvae in terms of the dry weight of Microcystis sp. per square meter, were monitored in all the four ponds. Sig- nificant variation was found in case of each parameter except temperature, for all the four ponds: ANOVA- temperature (F3,56 = 0.01; P > 0.05), pH (^ 3,56 = 9.307; P < 0.001), free CO 2 (^ 3,56 = 41.667; P < 0 . 001 ), dissolved oxygen (^ 3,56 = 437.235; P < 0.001), biological oxygen demand (^ 3,56 = 188.284; P < 0.001), concentration of phosphates (^ 3,56 = 32.839; P < 0.001), and food availability of B. Botanical garden Durga kund Kurukshetra Sankuldhara Ponds I Suspended solids □ Dissolved solids Figure 1; Concentration (mg/L) of total solids, suspended solids and dissolved solids in the control (Botanical garden) and anthro- pogenically stressed (Kurukshetra, Sankuldhara and Durgakund) ponds. longipes larvae in terms of dry weight of Microcystis sp. per square meter (^ 3,55 = 604.686; P < 0.001) Table 1 . Post hoc tests revealed significant differences (Dunnett’s test, P < 0.001) in case of each parameter under study (except free CO 2 level which was not found to be significantly different (P > 0.05 in the Krk pond), in all the three experimental ponds in comparison to the control. 3.3. Abundance of Adult Stages of Insects, Gerris spinolae and Brachydeutera longipes, in the Four Ponds. The abundance of adult stages of Gerris spinolae and B. longipes in the four ponds varied significantly: one-way ANOVA: F^se = 11.124; P < 0.001, for G. spinolae, and p 3,56 = 17.327; P < 0.001, for B. longipes (Ligures 2(a) and 2(b)). Post hoc tests revealed significant differences in the abundance of B. longipes in all the three experimental ponds in comparison to the control pond, the lowest being in Dgk pond (Dunnett’s test, P < 0.001), with abundance being in the increasing order in Skd and Krk ponds (Dunnett’s test, P < 0.001, for both). The two experimental ponds Dgk and Skd differed significantly from the control (post hoc test: Dunnett’s test, P < 0.001, for both) in exhibiting significantly lower abundance of the G. spinolae. However, Krk pond did not show significant deviation from the control pond in this respect (Dunnett’s test, P > 0.05). Regression analysis between each individual independent water quality variable: temperature, pH, BOD, DO, free CO 2 , phosphate, dry weight of algae, with the abundance of adult stage of each of the two insect species, Brachydeutera longipes and Gerris spinolae (dependent variables) reveals the following: a significant positive correlation (P < 0.001) between B. longipes abundance and BOD (r = 0.528), PO 4 (r = 0.587), free CO 2 (r = 0.473), and dry weight of algae 4 Psyche Table 1: Physical, chemical, and biological parameters of water quality in the four ponds (Botanical garden — control; Kurukshetra, Sankuldhara and Durgakund — anthropogenically stressed) located in different parts of Varanasi, India. Water parameters In Botanical garden pond In anthropogenically disturbed ponds (Control) Kurukshetra Sankuldhara Durgakund Temperature (°C) (Mean ± SEM ) 16.19 ± 2 16.35 ± 2.0D^ 16.53 ± 2.02"* 16.67 ± 2.02"* pH (Mean ± SEM) 6.89 ± 0.11 9.16 ± 0.015*** 8.85 ± 0.013*** 6.402 ± 0.016*** Dissolved oxygen (mg/L) (Mean ± SEM ) 8.84 ± 0.50 7.61 ± 0.42*** 4.98 ± 0.33*** 4.13 ± 0.45*** Eree CO 2 cone. (mg/L) (Mean ± SEM) 1.8 ± 0.25 1.07 ± 0.16'^® 5.18 ± 0.38*** 5.38 ± 0.5*** BOD (mg/L) (Mean ± SEM) 5.68 ± 0.42 6.49 ± 0.47*** 8.03 ± 0.53*** 8.47 ± 0.62*** Phosphate ion cone. (mg/L) (Mean ± SEM) 0.16 ± 0.02 0.43 ± 0.05*** 0.47 ± 0.05*** 0.63 ± 0.09*** Dry weight of algae. Microcystis sp.(g/sq m) (Mean ± SEM) 00.00 10.81 ± 0.35*** 10.94 ± 0.29*** 30.58 ± 0.36*** Where *P < 0.05, **P < 0.01, and < 0.001, ns — not significant. Botanical garden Durga kund Kurukshetra Sankuldhara Botanical garden Durga kund Kurukshetra Sankuldhara Ponds Ponds (a) (h) Figure 2: Abundance (No./sq. m) of adults of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) in the control (Botanical garden) and anthropogenically stressed (Kurukshetra, Sankuldhara and Durgakund) ponds, where *P < 0.05, **P < 0.01, and ***P < 0.001, ns — not significant. (r = 0.519) and a significant negative correlation (P < 0.001) with DO (r = 0.527). On the other hand, G. spinolae abundance exhibited a significant positive correlation (P < 0.001) with DO (r = 0.780) and temperature (r = 0.60) and a significant negative correlation (P < 0.001) with BOD (r = 0.686), PO4 (r = 0.604), free CO2 (r = 0.829), and dry weight of algae (r = 0.547) Table 2. Brachydeutera longipes abundance showed less significant positive correlation with pH (r = 0.346, P < 0.01) and a negative correlation with temperature (r = 0.305, P < 0.05) whereas G. spinolae abundance demonstrated no significant correlation with pH (r = 0.115, P > 0.05) Figures 3(a), 3(b); 4(a), 4(b); 5(a), 5(b); 6(a), 6(b); 7(a), 7(b); 8(a), 8(b); and 9(a), 9(b). 4. Discussion Our study clearly reveals that the abundance of adult stages of the two insect species, G. spinolae and B. longipes in the three ponds under anthropogenic stress is affected (although in a contrasting manner) due to differences in the levels of organic pollution and the resulting impacts of abiotic and biotic aquatic components of the ponds. Durgakund, Sankuldhara, and Kurukshetra ponds exhibit pollution in a decreasing order with higher concentrations of total dis- solved and suspended solids, free CO 2 levels, phosphate ion concentration, and amount of Microcystis sp. being more prevalent in the most polluted Durgakund pond and less in the remaining two anthropogenic stressed ponds. Psyche 5 Table 2: Regression analysis output obtained by corelating each variable (water quality parameter) independently with the abundance of each of the two insect species, Brachydeutera longipes and Gerris spinolae. Parameters Insect species Regression coefficient (j5) r Significance BOD Brachydeutera longipes 18.29 0.528 * * * Gerris spinolae -0.613 0.686 * * * DO Brachydeutera longipes -10.067 0.527 * * * Gerris spinolae 0.384 0.780 * * * PO 4 Brachydeutera longipes 134.317 0.587 * * * Gerris spinolae -3.563 0.604 * * * Free CO 2 Brachydeutera longipes 7.946 0.473 * * * Gerris spinolae -0.359 0.829 * * * Dry weight of algae Brachydeutera longipes 1.835 0.519 * * * Gerris spinolae -0.050 0.547 * * * Temperature Brachydeutera longipes -1.584 0.305 * Gerris spinolae 0.081 0.602 * * * pH Brachydeutera longipes 11.314 0.346 ** Gerris spinolae -0.97 0.115 ns Where *P < 0.05, **P < 0.01, and < 0.001, ns— not significant. Figure 3: Relation between BOD and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. Temperature and pH were higher in the polluted ponds in comparison to the control while transparency was much reduced. Thus, the greater the pollution level in the pond, the lesser is the abundance of G. spinolae as demonstrated by its low abundance in the Durgakund pond. Regression analysis between each individual independent water quality parameter with the abundance of B. longipes revealed a significant positive impact of BOD, free CO 2 , phosphate concentration, and dry weight of algae (characteristic of polluted aquatic conditions) and a negative impact of DO concentrations. On the other hand, a significant positive influence of dissolved oxygen concentration (characteristic of unpolluted aquatic conditions) was found on G. spinolae abundance with the correlation being negative with BOD, free CO 2 , phosphate concentration, and dry weight of algae (characteristic of polluted aquatic conditions). There- fore, higher abundance of B. longipes appears to indicate greater aquatic pollution. Since the maintenance of integrity between the physico-chemical and biological components of an ecosystem determines its health status [5, 38], it is abundantly clear that G. spinolae prefers unpolluted, while the semiaquatic shore fly prefers polluted lotic water bodies. Earlier studies demonstrate that physical, chemical, and biological parameters of an aquatic ecosystem are found to be correlated [39, 40]. Since each parameter in an aquatic ecosystem regulates the others, a freshwater pond supports 6 Psyche (a) (b) Figure 4; Relation between DO and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. Figure 5: Relation between free carbon dioxide and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. complex dynamics. It has been reported that pH decreases with the increase in temperature [41]. Moreover, increasing turbidity of water increases heat retention capability of water [42]. Hence, ponds having turbid water exhibit relatively high temperature and slightly low pH as is evident in the Durgakund pond. Physical parameters also regulate the concentration of several ions, content of free CO 2 , dissolved oxygen, even BOD [43]. However, anthropogenic stress in the three experimental ponds is apparently due to the dump- ing of organic wastes [44]. Increasing organic degradation initially results in nutrient enrichment and finally in colo- nization of the various algae and “algal bloom” formation resulting in “eutrophication.” The extent of organic pollution in terms of increase in BOD, free CO 2 , and heavy oxygen stress can be monitored conveniently by using G. spinolae and B. longipes as bioindicators. The extent of pollution in ponds can be assessed by the abundance of B. longipes which may be predicted to increase and that of G. spinolae to decrease with increasing pollution levels. The reason behind the contradictory responses of the two insects under study is due to differences in the habitat requirements of their life- history stages. Gerris spinolae lays eggs on the submerged vegetation at depths of 2-3 meters from the surface [ 14] . This submerged oviposition is regulated by the level of dissolved oxygen and male presence [ 14] . After emergence, the nymphs respire using dissolved oxygen of the water, though the adults “skate” on the pond surface. This explains the negative correlation of their abundance with parameters indicative Psyche 7 Figure 6: Relation between phosphate ion concentration and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. Figure 7; Relation between dry weight of algae (g/sq. m) and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. of higher level of pollution. Consequently, reduction in the abundance of G. spinolae may be a good indication of oxygen deficiency of water. Contrastingly, B. longipes does not rely on dissolved oxygen for respiration. The surface- living maggots feed on some species of algae like Microcystis sp., while the adults are free flying and are reported to feed on particles floating on the pond surface by rapidly extending and retracting their proboscis [32] , so their number increases with eutrophication. The study clearly demonstrates that G. spinolae and B. longipes are good positive and negative indicator taxa for healthy fresh water ponds. We, therefore, conclude that occurrence of higher G. spinolae population level indicates a positive correlation with healthy unpolluted pond conditions while enhanced abundance of B. longipes indicates higher pollution level of the pond. Since insects exhibit high fecundity, are fast breeding, easy to sample, and ethical constraints are not involved, Gerris spinolae (Lethierry and Severin) and Brachydeutera longipes (Hendel) appear to be suitable insect bioindicator candidates for assessing pollution in fresh water bodies. Utilisation of insect bioindicators would be an inexpensive method for monitoring pollution and for carrying out pre- liminary assessments of the water quality of inland ponds and lakes. This would avoid direct assessment of water quality involving expensive analytical methods, particu- larly at the preliminary stages. Integration of inexpensive 8 Psyche Figure 8: Relation between temperature of water and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. (a) (b) Figure 9: Relation between pH of water and abundance of (a) Brachydeutera longipes (Hendel) and (b) Gerris spinolae (Lethierry and Severin) as shown by regression analysis. biomonitoring methods with chemical-specific assessment methods would facilitate the restoration of the biological integrity and ecological health of freshwater bodies. Acknowledgments A. Pal and D. C. Sinha thank the Head of the Department of Zoology, Banaras Hindu University, for providing laboratory facilities for carrying out the research. The authors appreciate the advice of Dr. B. P. Singh and the help provided by Sudha Kumari for carrying out the statistical analysis. The authors also acknowledge the kind help of Dr. V. V. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 981475, 6 pages doi:10.1155/2012/981475 Research Article Effect of Larval Density on Development of the Coconut Hispine Beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) Mika Murata,'’^ Dang Thi Dung,^ Shun-ichiro Takano,^ Ryoko Tabata Ichiki,^ and Satoshi Nakamura^ ^ Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuha, Ibaraki 305-8686, Japan ^ Biodiversity Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba 305-8604, Japan Correspondence should be addressed to Satoshi Nakamura, s.nakamura@alfrc.go.jp Received 30 September 2011; Accepted 19 December 2011 Academic Editor: Donald Mullins Copyright © 2012 Mika Murata et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The coconut hispine beetle, Brontispa longissima shows, aggregation in the field. To elucidate the effect of aggregation on larval developmental aspects, we examined the effects of larval density on various aspects of larval development and on survival rates. Recently we found that B. longissima was divided into two monophyletic clades by genetic analysis. Therefore, we also compared the results between two populations, from Ishigaki, Japan (ISH) and Papua New Guinea (PNG), which were representative of the two monophyletic clades of B. longissima. In both ISH and PNG, the larval developmental period was shorter and the survival rate higher with rearing under high-density conditions than under isolated conditions. Similarly, fewer instars were required before pupation under high-density conditions than under isolated conditions. Brontispa longissima therefore developed better under high-density conditions, and the trends in the density effect were similar between two monophyletic clades. 1. Introduction The coconut hispine beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) is one of the most serious insect pests of Cocos nucifera (L.) and other palms [1, 2]. This beetle is considered to be native to Papua New Guinea and Indonesia [3], but since the 1930s it has gradually invaded Australia and the Pacific Islands including Vanuatu and Samoa [3, 4]. Since heavy infestations of B. longissima were first found in the Mekong Delta of Vietnam in 2003, it has been spreading rapidly and widely throughout Southeast Asian countries such as the Maldives, Thailand, Cambodia, and the Philippines [1, 5, 6]. In these countries, the coconut palms are very important for food, fuel, and industrial materials, and as part of landscape at tourist destinations [7, 8]. It is therefore important to establish a method of protecting coconut palms in Southeast Asian countries from the damage caused by B. longissima. However, there is insufficient information on the fundamental ecology of this species. For these reasons, we have been studying the beetle’s ecological properties. Phylogenetic analysis based on the mtDNA sequences has revealed two monophyletic clades in B. longissima [9]. One group is referred to as the Pacific clade and is distributed in Australia, Papua New Guinea, Samoa, and Sumba Island in Indonesia. The other is the Asian clade and is distributed over a wide area of Asia and the Pacific region. Morphological traits do not differ between the two clades [9], but little has known whether there are any differences in their ecological properties. In the field, B. longissima aggregates within the folded leaflets of the coconut palm [6, 10]. We therefore assumed that development at high density is promoted within the populations of this insect. Here, to clarify developmental dif- ferences in B. longissima during the immature developmental stages under different density conditions, we investigated the effects of changes in larval density on the length of the insect’s developmental period and on the number of instars required before pupation, the survival rate, and body size. 2 Psyche Furthermore, we examined whether the effects of density differed between the two monophyletic clades. 2. Materials and Methods 2.1. Insects. Brontispa longissima populations were collected from Ishigaki Island, Okinawa Prefecture, Japan, and from East New Britain Province, Papua New Guinea. The popu- lations from Ishigaki (ISH) and Papua New Guinea (PNG) have been, respectively, categorized according to genetic analysis into the Asian group and the Pacific group [9]. The two populations under the present experiment were same as the populations used in Takano et al. [9]. Larvae and adults were provided with fresh leaves of C. nucifera. The pieces of leaves (15 cm length) were bundled with elastic bands, because this beetle prefers to hide between the leaves. Until pupation, larvae were maintained with a bundle of leaves in a plastic container (15.5 cm long, 1 1.5 cm wide, 5.0 cm high) covered with a screened ventilated lid. Pupae were placed in a Petri dish and the adults were reared in a plastic container in the same way as the larvae. Rearing was conducted at 25 ± 1 ° G under a 12 : 12-h (L/D) photoperiod and 65% ± 5% relative humidity. 2.2. Experiments. ISH hatchlings less than 24 h after emer- gence were transferred into a Petri dish (5.5 cm diameter, 1.5 cm high) containing a bundle of fresh-cut leaves of C. nucifera. Some Petri dishes contained 1 individual (isolated conditions) and some contained 10 (crowded conditions). We considered a density of 10 individuals per dish to be crowded, but not overcrowded, because a preliminary experiment had shown that at this density there was still enough food for larvae of final instar which is the lifecycle stage at which the greatest amounts of food are ingested. The treatment for isolated condition was replicated 30 times (i.e., 30 individuals) and for crowded condition 10 times (i.e., 100 individuals). We transferred the larvae to a new Petri dish containing fresh leaves every day, and we checked the lengths of developmental periods of the larvae and the pupae. Survival rates and the occurrence of molting were recorded every day until adult emergence. Molting was checked by counting exuviae and measuring the head width of larvae under a stereomicroscope. Emerged adults were sexed and the length from the head to the tip of abdomen was measured under the stereomicroscope. The same procedures were followed with B. longissima PNG as with ISH. Experiments were conducted at 28 ± 1°G under a 12 : 12-h (L/D) photoperiod and 65% ±5% relative humidity. 2.3. Statistical Analyses. Differences in survival rate were statistically compared by using Fisher’s exact probability test. The chi-squared test was used to examine differences between the two rearing densities in terms of the proportions of instars each number immediately before pupation. These analyses were conducted with version 2.11.0 of R software [ 1 1 ] . A f-test was used to compare the differences in length of the developmental period and body size. These analyses were conducted with version 5.1 of JMP software (SAS Institute Inc. Gary, NG, USA). Developmental period length and body size were compared between isolated and crowded conditions within the same monophyletic group and between ISH and PNG under each density condition. 3. Results In both populations the survival rate tended to decrease with developmental stage, and from the third instar larval stage onward it was higher under crowded conditions than under isolated conditions (Figure 1). The adult emergence rate under crowded conditions was 86% in both populations. In contrast, under isolated conditions the adult emergence rate was 70.0% in ISH and 53.3% in PNG (Figure 1). We examined the lengths of the developmental periods in ISH and PNG populations at the two different densities (Table 1). The larval development period was significantly shorter under crowded conditions than under isolated conditions in both ISH and PNG. The length of the pupal development period did not differ significantly between the two density conditions in either population. We then investigated the variations in the larval instar number at which pupation occurred (Figure 2). ISH larvae pupated at the fourth or fifth instar. In contrast, PNG larvae pupated at the fourth to sixth instars. The proportion of each of the instar numbers at which pupation occurred differed significantly between the two density conditions in each clade (chi-squared test, ISH: = 16.961, P < 0.001; PNG: = 9.122, P = 0.010). At pupation, the proportion of fourth instars under crowded conditions was greater than that under isolated conditions in both ISH and PNG. The heads of last- instar larvae were significantly wider under isolated conditions than under crowded conditions (Table 2). In both ISH and PNG, body length in adult males was significantly greater under isolated conditions than under crowded conditions, whereas female body length did not differ significantly. Within each density condition, the larval development period was significantly shorter in ISH than in PNG (Table 1). Body length in adult males under isolated condi- tions was significantly greater in PNG than in ISH (Table 2). The other data did not differ significantly between the two clades (Tables 1 and 2). 4. Discussion Our results revealed that larval density influenced various ecological aspects in B. longissima and that the overall trends in the density effect were similar between the two populations, ISH and PNG. Survival rates were higher under crowded conditions than isolated conditions. This trend has been observed in many other species of insects, including the leaf beetle, Chrysolina aurichalcea Mannerheim (Goleoptera: Ghrysomelidae), and the pleasing fungus beetle Dacne picta Grotch (Goleoptera: Erotylidae) [12, 13]. Our results support the findings of these studies. Utida [14] described that low density causes stress in individuals, and in our study, the survival rates when only 1 individual was present were low. Psyche 3 1 individual -A- 10 individuals Hatch 3nd instar Pupation Adult emergence Stage 1 individual -A- 10 individuals (a) (b) Figure 1; Survival rates from hatching to adult emergence in B. longissima reared under isolated conditions (1 individual) or crowded conditions (10 individuals), (a) ISFl (individuals obtained from a stock culture initiated from insects collected on Ishigaki Island), (b) PNG (obtained from a stock culture of insects collected in Papua New Guinea). Values with the same letters do not differ significantly (Fisher’s exact probably test, P > 0.05). Table 1: Developmental periods (mean ± SE, days) of B. longissima reared under the density of one individual or 10 individuals. Developmental stage Density Population ISH PNG f-value P 1 individual 22 24.8 ± 0.7 16 28.1 ±0.8 -2.85 0.0073 Larva 10 individuals 86 20.5 ± 0.3 88 25.8 ±0.8 -13.88 <0.0001 t-value -6.83 -3.07 P <0.0001 0.0027 1 individual 21 5.1 ±0.1 16 4.9 ±0.1 1.42 0.1647 Pupa 10 individuals 86 4.9 ±0.1 86 4.8 ± 0.1 1.20 0.2300 t-value -1.73 -0.75 P 0.0873 0.4563 One reason is might therefore be that extremely low density is a stressor for B. longissima larvae. The length of the larval development period was clearly influenced by the density conditions, but larval density had no effect on the length of pupation in B. longissima. This effect of crowding on larval development is found in some other species of insects such as Diploptera punctata (Eschscholtz) (Blattodea: Blaberidae), and Mythimna sep- arata (Walker) (Lepidoptera: Noctuidae) [15, 16]. Apple- baum and Fleifetz [17] described that food consumption, metabolic rate, and general activity are enhanced by crowded conditions in the insects that show density- dependent responses. We did not examine the quantities of leaves ingested in each dish. Possibly, larvae under crowded condi- tions might grow more quickly than isolated larvae because of competitive need to feed more actively. Moreover, during the experiments, we found that several larvae overlapped with, or touched, each other, even though there was enough space for them to eat on the leaves while apart. In B. longissima, this body-touching behavior and/or the presence of feces from other individuals might shorten the larval development period under crowded conditions. However, we cannot make this inference from our current data. In some species of insects, body size diminishes with increasing population density [18]. Besides, Goulson and Cory [19] explained that this phenomenon occurs unrelated to a direct shortage of food. Also, here, we provided enough leaves for larvae every day, but we observed an inverse relationship between larval density and body length of adult males. By comparison, female body length was not influenced by density. These findings suggest that males of B. longissima, but not females, are susceptible to density effects. 4 Psyche Density ■ 6th □ 5th □ 4th Density ■ 6th □ 5th □ 4th (a) (b) Figure 2: Variations in the proportions of pre-pupation larval instars (fourth, fifth, and sixth) in B. longissima reared under isolated conditions (density, 1 individual per Petri dish) or crowded conditions (10 individuals) (a) ISH, (b) PNG. Table 2: Head width of last- instar larvae and body length of adults (mean ± SE, mm) of B. longissima reared under the density of one individual or 10 individuals. Site Density Population ISH PNG t-value P 1 individual 22 1.24 ± 0.02 16 1.25 ±0.01 -0.51 0.6098 Head width of last- instar larvae 10 individuals t-value P 86 1.19 ±0.01 -3.08 0.0027 88 1.20 ±0.01 -2.67 0.0089 -0.96 0.3375 1 individual 11 8.6 ±0.1 8 9.1 ±0.2 -2.47 0.0246 Male adult body length 10 individuals t-value P 44 8.3 ± 0.0 -2.02 0.0487 45 8.3 ± 0.0 -6.44 <0.0001 0.33 0.7423 1 individual 10 9.2 ±0.1 8 9.3 ±0.1 -0.70 0.4940 Female adult length 10 individuals t-value P 42 9.3 ± 0.0 1.50 0.1391 41 9.3 ± 0.0 0.30 0.7628 -0.10 0.9205 Regarding head width, it seems that the wider heads under isolated conditions were due to the size of male larvae. The last-instar larvae of B. longissima included more 5th or 6th instars under isolated conditions than under crowded conditions. Generally, insects initiate to pupation when the larvae have reached a critical body size [20, 21]. Some species of insects increase the number of prepupation instars as a form of compensatory growth when the larvae fail to reach their threshold size for metamorphosis under adverse conditions [22]. In our experiments, the head width of the penultimate instar under isolated conditions was smaller than that of the final instar under crowded conditions (data not shown). We therefore consider that B. longissima larvae under isolated conditions needed to undergo more Psyche 5 larval molts to reach the threshold size for pupation. The number of pre-pupation instars in B. longissima has been reported as five to six by Waterhouse and Norris [3] and four to six by Yamauchi [23]. In many species of insects, the number of pre-pupation instars is affected by rearing conditions, including food quality, density, temperature, and humidity [22] . Therefore, any differences between our results and these past reports are likely associated with differences in environmental factors, including temperature and food. Furthermore, we observed the differences in proportions of last instars (fourth, fifth, and sixth) between ISH and PNG. We therefore think that the number of pre-pupation instars varies among not only density conditions but also monophyletic clades in B. longissima. Our findings suggest that B. longissima reared under high-density conditions has a survival advantage, and there- fore high-density conditions allowed B. longissima to increase the number of its generations in the field. In other words, once B. longissima invades into the coconut palm field, it might be able to increase acceleratingly as the population increases and spread in the field. The density-dependent phenomena have the potential to influence fecundity or dispersion during the adult stage. In future, we need to investigate the effect of population density on ecological aspects in B. longissima adults. Our findings will help to elucidate the factors involved in the rapid spread of B. longissima and its damage, in the coconut palm field in Southeast Asian countries. Acknowledgments The authors sincerely thank Wiwat Suasa-ard, Sopon Uraichuen, Phueng Chomphukhia, and Jun Tabata for providing experimental devices and insects. They thank Aki Konishi for technical assistance. References [1] S. Nakamura, K. Konishi, and K. Takasu, “Invasion of the coconut hispine beetle, Brontispa longissima: current situation and control measures in Southeast Asia,” in Proceedings of the International Workshop on Development of Database (APASD) for Biological Invasion, T. Y. Ku and M. Y. Chiang, Eds., vol. 3, pp. 1-9, Taiwan Agricultural Chemicals and Toxic Substance Research Institute, Taichung, Taiwan, Food and Fertilizer Technology Center (FFTC) for the Asia and Pacific Region, Taipei, Taiwan, 2006. [2] P. Rethinam and S. P. Singh, “Current status of the coconut beetle outbreaks in the Asia-Pacific region,” in Develop- ing an Asia-Pacific Strategy for Forest Invasive Species: the Coconut Beetle Problem — Bridging Agriculture and Forestry, S. Appanah, H. C. S. Sim, and K. V. Sankaran, Eds., pp. 1- 23, Food and Agriculture Organization of the United Nations Regional Office for Asia and the Pacific, Bankok 2007, RAP publication, Bankok, Thailand, 2007. [3] D. F. Waterhouse and K. R. Norris, “Brontispa longissima (Gestro),” in Biological Control Pacific Prospects, pp. 134-141, ACIAR. Inkata Press, Melbourne, Australia, 1987. [4] J. M. Voegele, “Biological control of Brontispa longissima in Western Samoa: an ecological and economic evaluation,” Agriculture, Ecosystems and Environment, vol. 27, no. 1-4, pp. 315-329, 1989. [5] W. Fiebregts and K. Chapman, “Impact and control of the coconut hispine beetle, Brontispa longissima Gestro (Coleoptera: Chrysomelidae),” in Report of the Expert Con- sultation on Coconut Beetle Outbreak in APPPC Member Countries, FAO, Ed., pp. 19-34, Regional Office for Asia and the Pacific, FAO, Bangkok, Thailand, 2004. [6] K. Takasu, S. Takano, K. Konishi, and S. Nakamura, “An invasive pest Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) attacks an endemic palm in the Yaeyama Islands, Japan,” Applied Entomology and Zoology, vol. 45, no. l,pp. 137-144, 2010. [7] F. Guarino, “The coconut in the Pacific: the role of the secretariat of the pacific community, coconut revial: new possibilities for the “tree of life”,” in Proceedings of the International Coconut Forum Held in Cairns, Australia (ACIAR ’06), S. W. Adkins, M. Foale, and Y. M. S. Samosir, Eds., no. 125, pp. 28-30, 2006. [8] P. Naka, “Appropriate processing technologies for value addi- tion in coconut,” Indian Coconut Journal, vol. 37, no. 3, pp. 11-20, 2006. [9] S. Takano, A. Mochizuki, K. Konishi, and K. Takasu, “Two cryptic species in Brontispa longissima (Coleoptera: Chrysomelidae): evidence from mitochondrial DNA analysis and crosses between the two nominal species,” Annals of the Entomological Society of America, vol. 104, no. 2, pp. 121-131, 2011. [10] E. S. Brown and A. H. Green, “The control by insecticides of Brontispa longissima (Gestro) (Coleopt., Chrysomelidae- Hipinae) on young coconut palms in the British Solomon Islands,” Bulletin of Entomological Research, vol. 49, no. 2, pp. ISO-Ill, 1958. [11] R Development Core Team, “R: a language and environment for statistical computing,” R Foundation for Statistical Com- puting, Vienna, Austria, 2010, http://www.R-project.org/. [12] S. Fujiwara and K. Miyachi, “The effect of larval density on survival rate and development of Chrysolina aurichalcea Mannerheim (Coleoptera: Chrysomlidae),” Bulletin of Envi- ronmental Conservation, vol. 7, pp. 61-65, 1985. [13] T. Sato, N. Shinkaji, and H. Amano, “Effects of larval density on larval survivorship and imaginal fecundity of Dacne picta (Coleoptera: Erotylidae),” Applied Entomology and Zoology, vol. 39, no. 4, pp. 591-596, 2004. [14] S. Utida, The Theory of Animal Population, NHK, Tokyo, Japan, 1972, (in Japanese). [15] S. Iwao, “Phase variation in the armyworm, Leucania unipuncta Haworth II. “Effect of population density on the larval growth pattern”,” Japanese Journal of Applied Entomology and Zoology, vol. 2, no. 4, pp. 237-243, 1958 (Japanese), (with English summary). [16] A. P. Woodhead and C. R. Paulson, “Earval development of Diploptera punctata reared alone and in groups,” Journal of Insect Physiology, vol. 29, no. 9, pp. 665-668, 1983. [17] S. W. Applebaum and Y. Heifetz, “Density-dependent physio- logical phase in insects,” Annual Review of Entomology, vol. 44, pp. 317-341, 1999. [18] T. M. Peters and P. Barbosa, “Influence of population density on size, fecundity and developmental rate of insects in culture ,” Annual Review of Entomology, vol. 22, pp. 431-450, 1977. [19] D. Goulson and J. S. Cory, “Responses of Mamestra brassicae (Lepidoptera: Noctuidae) to crowding: interactions with dis- ease resistance, colour phase and growth,” Oecologia, vol. 104, no. 4, pp. 416-423, 1995. 6 Psyche [20] G. Davidowitz, L. J. D’Amico, and H. F. Nijhout, “Critical weight in the development of insect body size,” Evolution and Development, vol. 5, no. 2, pp. 188-197, 2003. [21] H. F. Nijhout, “A threshold size for metamorphosis in the tobacco hornworm, Manduca sexta (L.),” Biological Bulletin, vol. 149, no. 1, pp. 214-225, 1975. [22] T. Esperk, T. Tammaru, and S. Nylin, “Intraspecific variability in number of larval instars in insects,” Journal of Economic Entomology, vol. 100, no. 3, pp. 627-645, 2007. [23] S. Yamauchi, “Some biological notes of the Brontispa longis- sima Gestro (Coleoptera: Chrysomelidae),” Okinawa Nogyo, vol. 20, no. 1-2, pp. 49-53, 1985 (Japanese). Hindawi Publishing Corporation Psyche Volume 2012, Article ID 926089, 6 pages doKlO.l 155/2012/926089 Research Article Tatuidris kapasi sp. nov.: A New Armadillo Ant from French Guiana (Formicidae: Agroecomyrmecinae) Sebastien Lacau,^’^’^ Sarah Groc,^ Alain Dejean, Muriel L. de Oliveira,^’ and Jacques H. C. Delabie^’^ ^ Laboratorio de Biossistemdtica Animal, Universidade Estadual do Sudoeste da Bahia, UESB/DEBI, 45700-000 Itapetinga, BA, Brazil ^ Departement Systematique & Evolution, UMR 5202 CNRS-MNHN, Museum National d’Histoire Naturelle, 75005 Paris, Erance ^Programa de Pos-Graduagao em Zoologia, Universidade Estadual de Santa Cruz, UESC/DCB, 45662-000 Ilheus, BA, Brazil ‘^Ecologie des Eorets de Guyane, UMR-CNRS 8172 (UMR EcoEoG), Campus Agronomique, BP 319, 97379 Kourou Cedex, Prench Guiana ^ Universite de Toulouse, UPS (Ecolab), 118 Route de Narbonne, 31062 Toulouse Cedex 9, Prance ^Laboratorio de Mirmecologia, CEPLAC/CEPEC/SECEN, CP 07, km 22, Rodovia, Ilheus-Itabuna, 45600-970 Itabuna, BA, Brazil Correspondence should be addressed to Sebastien Lacan, slacau@cepec.gov.br Received 11 September 2011; Revised 3 November 2011; Accepted 21 November 2011 Academic Editor: Fernando Fernandez Copyright © 2012 Sebastien Lacau et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tatuidris kapasi sp. nov. (Formicidae: Agroecomyrmecinae), the second known species of “armadillo ant”, is described after a remarkable specimen collected in French Guiana. This species can be easily distinguished from Tatuidris tatusia by characters related to the shape of the mesosoma and petiole as well as to the pilosity, the sculpture, and the color. 1. Introduction As a result of the constant acquisition of new morphological and molecular data, combined with a big increase in the collection of biological material, the last decades have seen a true revolution in ant systematics and phylogeny [1, 2]. In spite of this, some rare genera of ants still remain unusually mysterious. A long time after their original description, they continue to reveal a low taxonomical diversity and are rarely collected in the field. Frequently, their phylogenetic relation- ships also remain poorly understood. The Neotropical genus Tatuidris (Formicidae: Agroecomyrmecinae) was described by Brown and Kempf in the 1967 issue of Psyche [3] after a peculiar new species collected in El Salvador. Until now, this genus has remained monotypic and very isolated in the Fam- ily Formicidae. The type species, Tatuidris tatusia, is known only from the very distinctive morphology of the worker that combines some primitive and derived characters, while its biology is completely unknown [4]. In addition, morpholog- ical and molecular studies have caused some authors to hold differing points of view regarding the phylogenetic position of Tatuidris in the family Formicidae. Thus, based on its morphology, the genus was initially placed in the subfamily Myrmicinae [3], within the tribe Agroecomyrmecini. The genus was then transferred to the Agroecomyrmecinae [5], a new subfamily proposed by Bolton, who has suggested that this taxon might be the sister group to all Myrmicinae. More recently, the genus was again combined in the Myr- micinae [6] and then returned to the Agroecomyrmecinae as a poneroid [7] and more latterly as a poneromorph [8]. However, based on morphological characters, in a very recent paper, Keller [9] corroborated Boltons former proposal [5] in considering that Tatuidris is the sister group to the Myr- micinae. Recently, some new molecular data have led other authors to argument that Tatuidris may be the sister group to the subfamily Paraponerinae in some rooted trees but placed it next to Amblyoponinae in some other analyses [10-12]. In such a context, the search for new species of Tatuidris and the study of their morphology represents an important challenge for better understanding the phylogenetic relation- ships of this genus within the Formicidae. Here we report the recent finding in French Guiana of a remarkable single 2 Psyche specimen of Tatuidris that differs from T. tatusia in several distinctive morphological characters, and this paper aims to describe it. 2. Material and Methods Morphological examination of specimens was completed at various magnifications using a light stereomicroscope Olym- pus SZX7. Morphometric measures were made with a Carl Zeiss measuring microscope and recorded to the nearest 0.01 mm. All measurements are given in millimeters, using the following definitions and abbreviations: Cl: Cephalic Index: HW*100/HL, EL: Eye Length: the maximum diameter of the eye, GL: Caster length: the length of the gaster in lateral view from the anteriormost point of first gastral segment (fourth abdominal segment) to the posterior most point (sting omitted), HEL: Hind Lemur Length: maximum length of hind femur in anterior view, HL: Head Length: the length of the head proper, excluding the mandibles; measured in full-face view from the midpoint of the anterior clypeal margin to a line drawn across the posterior margin from its highest points, HW: Head Width: the maximum width of the head in full face view, LA7: Length of Antennal segment 7: maximum length of the seventh (apical) antennal segment, ML: Mandible Length: length of a mandible mea- sured in ventral view from its basal articulation to its apex, PeNI: Petiole Node Index: (PeNW* lOO/PeNL), PeL: Petiole Length: the maximum length of the petiole in lateral view, PeNL: Petiolar Node Length: the maximum length of the petiole node in dorsal view, PeNW: Petiolar Node Width: the maximum width of the petiole node in dorsal view, PpL: Postpetiole Length: the maximum postpetiole length in lateral view, PpNL: Postpetiolar Node Length: the maximum length of the postpetiole node in dorsal view, PpNW: Postpetiolar Node Width: the maximum width of the postpetiole node in dorsal view, PrW: Pronotum Width: the maximum width of the pronotum in dorsal view, PrpW: Propodeum Width: the maximum width between the propodeum angles as seen in dorsal view, SL: Scape Length: the maximum straight line of the antennal scape, excluding the condylar bulb, TL: Total Length (HL -h ML -h WL -h PL -h PPL -h GL), WL: Weber’s Length: diagonal length, measured in lateral view, from the anterior margin of the prono- tum (excluding the collar) to the posterior extremity of the metapleural lobe. The microphotographs were made using the following sequential process: the specimen was first filmed using a video camera (Sony Lull HD 1080 AVCHD, 10.2 Mp) mount- ed on a light microscope (Zeiss lena), while the resolution was continuously scanned from the top to the bottom of the holotype specimen; the videos (in format.mts) were pro- cessed using the free software ImagGrab 5.0 (available at http://paul.glagla.free.fr/imagegrab.htm) in order to extract the sharpest images referable to differing focal points, and composite pictures were then assembled using the free software Combine ZM (available at http://www.hadleyweb .pwp.blueyonder.co.uk/index.htm). Linally, each optimum microphotograph was improved using Adobe Element Pho- toshop software (version 6.0). The terminology for the external morphology and the surface sculpturing follows [13-15]. In the description and the diagnosis of this new species, our terminology referring to the pilosity describes the variation in size of the setae observed in T. tatusia and Tatuidris kapasi sp. nov. Thus, we recognize four setal types depending on their length: that is, very short (about 0.016 mm), short (about 0.05 mm), inter- mediate (about 0.11mm), and long (about 0.3 mm). Also, the characters of the tribe and the genus are not mentioned (for a complete summary of the taxonomic characters, see [3,5]). Depository of the Holotype. The unique known specimen of the taxon is deposited in the collection of the Laboratorio de Mirmecologia at the Cocoa Research Center at CEPLAC (Itabuna-BA, Brazil), referred to by the CPDC acronym [16]. Comparative data for Tatuidris tatusia were obtained from the literature [3] and direct observations on micropho- tographs of high-resolution available in AntWeb site [17]. 3. Results 3.1. Tatuidris kapasi Lacau and Groc: New Species. See Ligures 1, 2, 3, 4, 5, and 6. 3.2. Type Material. Holotype worker: specimen deposited at CPDC and labeled “Guyane Lran^aise, Montagne de Kaw, N04°38.217W052° 17.36', Alt. 260 m., ix.2008, Winkler trap. Col. S. Groc, A. Dejean, and B. Corbara”. 3.3. Etymology, “kapasi” is the Wayanas’ Amerindian (Lrench Guiana, Surinam, and Brazil) word for “armadillo”, a mammal belonging to the Order Cingulata. The generic and specific names of the first described species [6] referred to the same animal group. 3.4. Diagnosis. The worker of Tatuidris kapasi exhibits all the diagnostic characters of the tribe Agroecomyrmecini and the genus Tatuidris. It differs from the worker of T. tatusia in Psyche 3 view. Figure 4: Tatuidris kapasi: holotype worker. Mesosoma: dorsal view. Figure 2: Tatuidris kapasi: holotype worker. Head: full-face view. Figure 3: Tatuidris kapasi: holotype worker. Detail of head: left side view. the following characters (states for T. tatusia indicated in brackets): occipital border a little more concave (nearly straight); about 5-6 facets in each eye (about 10 facets); cly- peus with the free margin medially straight and laterally concave (free margin concave overall); pronotum with the ventral sector of lateral faces smooth and shining (with longitudinal rugulae); dorsum of mesosoma mostly sculp- tured with concentric rugulae and carinulae (mostly smooth and shining); mesosoma as seen from above with lateral margins moderately converging backward (Propodeum width/Pronotum width = 0.55) (lateral margins more con- verging backward (Propodeum width/Pronotum width = 0.45)); mesopleuron with anterior crest wider and ventrally truncated (crest narrower and not truncated); mesopleuron smooth and shining, except for punctuations and areo- lae on its ventral margin (with longitudinal rugulae and areolations); metapleuron punctate and areolate, and with longitudinal rugulae around the metapleural gland orifice (metapleuron with areolations); the bulla of the metapleural gland (visible through the integument when observed in profile), forming a ring whose posterodorsal margin is fused with the posterolateral margin of the propodeum (the bulla of the metapleural gland forming a ring that is distinctly separated from the posterolateral margin of propodeum); propodeal declivity less concave in lateral view (more con- cave); propodeal spiracle separated from declivitous margin of propodeum by two diameters (separated by no more than one diameter); viewed dorsally, petiolar node twice as wide as long (viewed dorsally, shape of petiolar node subrectangular, the node no more than about 1.5 times as wide as long); viewed dorsally, shape of postpetiolar node rectangular, and not wider behind than in front (viewed dorsally, shape of postpetiolar node subrectangular, and a little wider behind than in front); and dorsum of the petiolar and postpetiolar nodes with superficial concentric rugulae and carinulae (smooth and shining). Moreover, the pilosity is markedly more dense all over the body (more scattered) and does not include any long suberect setae (long suberect setae present). 4 Psyche Figure 5: Tatuidris kapasi, holotype worker. Detail of petiole and postpetiole, left lateral view. Figure 6: Tatuidris kapasi, holotype worker. Detail of petiole and postpetiole, dorsal view. Furthermore, despite the fact that T. kapasi is known only by a single specimen and T. tatusia by two specimens for- mally described, the following comparative measurements suggest an overall size differential between the two species: the head shape is a little wider in T. kapasi (Cl: 125,78) than in T. tatusia (Cl: 118,2 ± 1.56, min-max: 117.07-119.28 {n = 2)), the scape is slightly shorter in T. kapasi (SL 0,32) than in T. tatusia (SL 0,4 (n = 1)), the pronotum is narrower in T. kapasi (PrW 0,64) than in T. tatusia (PrW 0,79 {n = 1)), and the shape of the petiole node in dorsal view is more noticeably rectangular in T. kapasi (PeNI 200) than in X tatusia (PeNI 153.64 (n = 1)). 4. Description Worker. Measurements (Holotype): TL 3.42, Cl 125.80, EL 0.05, GL 1.00, HFL 0.50, HL 0.70, HW 0.93, LA7 0.27, ML 0.39, PeL 0.21, PeNI 200, PeNL 0.18, PeNW 0.36, PpL 0.28, PpNL 0.25, PpNW 0.45, PrW 0.64, SL 0.32, WL 0.80. Except for the diagnostic characters, the external mor- phology of Tatusia kapasi sp. nov. is very similar to that of T tatusia. In our discussion, the shared characters are regarded by ourselves as sufficient for the two species to be placed within the same genus. The spatial distribution and patterns of the sculpture and pilosity as well as the body color of this new species are described hereafter. Sculpture. It includes head dorsum wholly smooth and shin- ing, except for occipital sector covered with transverse car- inulae; outer surface of mandibles smooth and shining except for longitudinal superficial striae on ventral (external) margin; ventrolateral sector of the head longitudinally carin- ulate; antennal scape shagreened and superficially areolate; pronotum ventrolaterally smooth and shining; dorsum of mesosoma with concentric rugulae and carinulae; meso- pleuron smooth and shining except for punctuations and areolae on ventral margin; metapleuron with punctuations and areolae and longitudinal rugulae around the metapleural gland orifice; propodeal declivity mostly smooth and shining with fine striae and reticulate; petiolar and postpetiolar nodes laterally finely longitudinally carinulate. Pilosity. It includes dorsum of head, mesosoma, petiole, and postpetiole with abundant setae, all fine, flexuous, and de- cumbent, varying in size and distribution as follows: some very short and relatively dense (i.e., those on clypeus and the outer surface of mandibles); others short and dense (those on dorsum of head, clypeus, mesosoma, petiolar, and postpetiolar nodes, and gaster); and others intermediate and dense (namely, setae on dorsum of head, mesosoma, petiolar, and postpetiolar nodes, dorsum of gaster and tibia). No long setae present. Color. Body brownish-ferruginous, thick margins often ap- pearing more blackish; legs brownish-yellowish. Gyne and Male. They are unknown. Geographic Range. This new species is known only from the type locality in French Guiana, situated at 260 m altitude in the Kaw Mountains, on a side exposed to the trade winds, near a great cave sheltering a big bat community. The local vegetation is typical of Amazonian lowland rainforest that is never flooded. New records of this species will probably occur in other localities of the Guiana Shield in the near future. However, the fact that no other specimen of Tatuidris has been recorded yet in the recent studies on ant biodiversity in the Guiana Shield, even using Winkler traps or other methods at a large scale [18, 19], suggests that this genus is genuinely rare in the Guianas, its members possibly spatially separated in small, isolated populations. Biology. The biology of this species is unknown, but the fact that the type specimen was found in a leaf-litter sam- ple, using a Winkler trap, suggests that it nests in some microhabitats of the leaf-litter or more or less deeply in the soil. It is also noteworthy that the leaf-litter sample (surface of 1 m^) in which T. kapasi was caught was characterized by a very high specific richness: a total of 20 other ant species belonging to 12 genera was recorded (Groc et al., unpublished information). Such richness in a unique leaf- litter sample is uncommon in Neotropical forests [19]. Psyche 5 5. Comments and Discussion The description of this new species of Tatuidris is an impor- tant event for Myrmecology, since the genus has remained monotypic for over 40 years. However, as noted by Longino [4], the advent of litter sifting and Winkler extraction as a popular method of ant collecting in the last decade led to the discovery of new species belonging to genera previously considered as rare and poorly diversified. This is the case for the new species here described. This genus has been revealed to be not as rare as it was believed to be since several new specimens were recently collected in various Neotrop- ical countries: Brazil, Colombia, Costa Rica, El Salvador, Ecuador, Erench Guiana, Mexico, Nicaragua, Panama, and Peru (see specimens imaged on the Websites: Ants of Costa Rica [4] and AntWeb [17]). In this context, D. Donoso is currently performing a first revision of this genus based on the new material deposited in myrmecological collections in the world. While T. kapasi exhibits a distinct morphology from that of T. tatusia, it possesses all the diagnostic characters of Agro- ecomyrmecinae and Agroecomyrmecini. The next step will consist in studying the whole biology of these ants for which literature is particularly scarce. Tatuidris kapasi has peculiar mandibular brushes and a powerful elongated sting similar to that of T tatusia. Brown and Kempf suggested that such adaptations indicates that armadillo ants might be specialist predators of active or slippery arthropod prey [3]. Also, we note that the flat pencil of stiff, curved yellow setae borne at the extensor angle on the forelegs, an apomorphy of this genus, may be used by these ants in order to clean the massive brush of heavy setae present along the inner surface near the masticatory margin of the mandible, through a movement directed forward. Thus, these characters could potentially represent an adaptation to feed on prey bearing a defensive pilosity. Moreover, the morphology of the gyne and the male of Tatuidris has never been described. However, microphotographs of a gyne and a male, together winged, are offered in the site Antweb, suggesting that a normal sexual reproduction by swarming occurs in this genus. Acknowledgments The authors thank Dr. Brian Heterick for his kind help in reviewing the English of this manuscript, and Bruno Corbara and Olivier Roux for field assistance. Einancial support for this study was provided by (1) the Programme Amazonie II of the Erench CNRS (project 2ID), (2) the Programme Convergence 2007-2013, Region Guyane from the European Community (project DEGA), (3) the EPVI European-funded Integrated Infrastructure Initiative Grant SYNTHESYS (S. Groc), (4) the PRONEX Program EAPESB/ CNPq (PNXOOl 1/2009, Brazil), and (5) the Program CAPES/ CNPq/MCT (PROTAX 52/2010, Brazil). I. H. C. Delabie acknowledges his research grant from CNPq. In accordance with Section 8.6 of the ICZN’s International Code of Zoo- logical Nomenclature, printed copies of the edition of Psyche containing this article are deposited at the following six pub- licly accessible libraries: Green Library (Stanford University), Bayerische Staatsbibliothek, Library — ECORC (Agriculture & Agri-Eood Canada), Library — Bibliotheek (Royal Belgium Institute of Natural Sciences), Koebenhavns Universitetsbib- liotek. University of Hawaii Library. References [1] P. S. Ward, “Phylogeny, classification, and species-level taxon- omy of ants (Hymenoptera: Formicidae),” Zootaxa, no. 1668, pp. 549-563, 2007. [2] P S. Ward, “Integrating molecular phylogenetic results into ant taxonomy (Hymenoptera: Formicidae),” Myrmecological News, vol. 15, pp. 21-29, 2011. [3] W. L. Brown Jr. and W. W. Kempf, “Tatuidris, a remarkable new genus of Formicidae (Hymenoptera),” Psyche, vol. 74, pp. 183-190, 1968. [4] J. T. Longino, “Ants of Costa Rica,” 2011, http://academic.ever- green.edu/projects/ants/AntsofCostaRica.html. [5] B. Bolton, “Synopsis and classification of Formicidae,” Mem- oirs of the American Entomological Institute, vol. 71, p. 370, 2003. [6] C. Baroni-Urbani and M. L. de Andrade, “The ant tribe Dacetini; Limits and constituent genera, with descriptions of new species,” Annali del Museo Civico di Storia Naturale Giacomo Doria, vol. 99, pp. 1-191, 2007. [7] B. Bolton and G. D. Alpert, “Barry Bolton’s Synopsis of the Formicidae and Catalogue of Ants of the World, Version 3 January 2011,” http://gap.entclub.org/. [8] B. Bolton and G. D. Alpert, “Barry Bolton’s Synopsis of the Formicidae and Catalogue of Ants of the World, Version 1 July 2011 ,” http://gap.entclub.org/. [9] R. A. Keller, “A phylogenetic analysis of ant morphology (Hymenoptera: Formicidae) with special reference to the poneromorph subfamilies,” Bulletin of the American Museum of Natural History, vol. 355, pp. 1-90, 2011. [10] S. G. Brady, T. R. Schultz, B. L. Fisher, and R S. Ward, “Evaluating alternative hypotheses for the early evolution and diversification of ants,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 48, pp. 18172-18177, 2006. [11] C. S. Moreau, C. D. Bell, R. Vila, S. B. Archibald, and N. E. Pierce, “Phylogeny of the ants: diversification in the age of angiosperms,” Science, vol. 312, no. 5770, pp. 101-104, 2006. [12] C. Rabeling, J. M. Brown, and M. Verhaagh, “Newly discovered sister lineage sheds light on early ant evolution,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 39, pp. 14913-14917, 2008. [13] B. Bolton, Identification Guide to the Ant Genera of the World, Harvard University Press, Cambridge, Mass, USA, 1994. [14] R. D. Eady, “Some illustrations of micro sculpture in the Hymenoptera,” Proceedings of the Royal Entomological Society of London, vol. 43, pp. 66-72, 1968. [15] R. A. Harris, “A glossary of surface sculpturing,” Occasional Papers on Systematic Entomology, vol. 28, pp. 1-31, 1979. [16] C. R. E. Brandao, “Major regional and type collections of ants (Eormicidae) of the world and sources for the identification of ant species,” in Ants: Standard Methods for Measuring and Monitoring Biodiversity, D. Agosti, J. D. Majer, L. E. Alonso, and T. R. Schultz, Eds., pp. 172-185, Smithsonian Institution Press, Washington, DC, USA, 2000. [17] B. L. Eisher, “Ant Web,” 2011, http://www.antweb.org/descrip tion.do?Subfamily=agroecomyrmecinae8cgenus=tatuidris& name=tatusia&rank=species8q)roject=worldants. 6 Psyche [18] J. S. Lapolla, T. Suman, J. Sosa-Calvo, and T. R. Schultz, “Leaf litter ant diversity in Guyana,” Biodiversity and Conservation, vol. 16, no. 2, pp. 491-510, 2007. [19] H. L. Vasconcelos and J. H. C. Delabie, “Ground ant commu- nities from central Amazonia forest fragments,” in Sampling Ground-Dwelling Ants: Case Studies from the World’s Rain Forests, D. Agosti, J. D. Majer, L.T. Alonso, and T. Schultz, Eds., vol. 18, pp. 59-70, Gurtin University, Perth, Australia, 2000, School of Environmental Biology Bulletin, no. 18. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 854045, 7 pages dohlO.l 155/2012/854045 Review Article Abundance of Sesamia nonagrioides (Lef.) (Lepidoptera: Noctuidae) on the Edges of the Mediterranean Basin Matilde Eizaguirre' and Argyro A. Fantinou^ ^ Department of Crop and Forest Sciences, University ofLleida, 25198 Lleida, Spain ^ Laboratory of Ecology, Agricultural University of Athens, 11855 Athens, Greece Correspondence should be addressed to Matilde Eizaguirre, eizaguirre@pvcf.udl.cat Received 5 September 2011; Revised 17 November 2011; Accepted 21 November 2011 Academic Editor: Matilda Savopoulou-Soultani Copyright © 2012 M. Eizaguirre and A. A. Eantinou. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Organisms inhabiting seasonal environments are able to synchronize their life cycles with seasonal cycles of biotic and abiotic factors. Diapause, a state of low metabolic activity and developmental arrest, is used by many insect species to cope with adverse conditions. Sesamia nonagrioides is a serious pest of corn in the Mediterranean regions and Central Africa. It is multivoltine, with two to four generations per year, that overwinters as mature larva in the northern of the Sahara desert. Our purpose was to compare the response of the S. nonagrioides populations occurring in the broader circum-Mediterranean area, with particular attention to the diapause period and the different numbers of generations per season. To this end, we tried to determine whether populations in the area differ in their response to photoperiod and whether we can foresee the number of generations in different areas. We present a model for predicting the occurrence of the critical photoperiod according to latitude and temperature and the spread of S. nonagrioides in the circum-Mediterranean countries. Responses of populations to short-day length suggest that the spread of the species is associated with a gradual loss of diapause in the southern areas, and that diapause incidence is positively correlated with latitude. 1. Introduction 1.1. Host Plants and Distribution. The corn stalk borer, Sesa- mia nonagrioides, is a polyphagous species with a fairly wide range of host plants, including corn, sorghum, millet, rice, sugar cane, grasses, melon, asparagus, palms, banana, and the ornamental plant Strelitzia reginae [ 1-9] . The population levels of this species, which has considerable potential to establish itself in an area and become abundant, may therefore depend on the abundance of these hosts. The occurrence of S. nonagrioides, including S. nonagri- oides botanephaga, has been reported in Portugal [10, 11], Spain [12-14], the Canary Islands [15], France [16-18], Italy [19], Greece [20, 21], Cyprus [22], Turkey [23, 24], Morocco [25], Israel [26], Iran [27-29], Syria [30], Ethiopia [6], Ghana [31], and several other African countries [32]. S. nonagrioides has been considered the most important pest of maize in Spain since 1929 [12]. Nye [33] observed that S. nonagrioides was morphologically very close to one of the new sub-Saharan species that had been described {Sesamia botanephaga) and indicated that Sesamia nona- grioides nonagrioides and Sesamia nonagrioides botanephaga should be regarded as two subspecies distributed to the north and south of the Sahara, respectively. Esfandiari et al. [34] stated that African S. botanephaga (or S. nonagrioides botanephaga) do not occur in Iran and that it seems that S. nonagrioides is native to SW Iran rather than an exotic pest, having adopted sugarcane as a host after it began to be cultivated there about 70 years ago. Leyenaar and Hunter [31] reported that S. n. botanephaga can cause 63% loss in maize yield in the coastal savanna of Ghana. In Kenya, S. nonagrioides has been commonly recovered in maize fields [35] . The same authors report that these species and other stem borers that are currently restricted to wild hosts may have the potential to shift to cultivated cereals in cases of serious habitat fragmentation. Moyal et al. [36] recently concluded that there is a single species of S. nonagrioides but with three different, isolated conspecific populations: one in East Africa, one in West Africa, and a Palearctic one in the circum-Mediterranean countries. 2 Psyche In the circum-Mediterranean countries, S. nonagrioides has been designated as the most important pest of corn. Many researchers have attempted to document the economic losses that it causes in Spain, but the results are not clear because the damage is not distinguishable from that caused by Ostrinia nubilalis. Arias and Alvez [37] indicated that damage caused by maize borers could range from 5% to 30% of the yield depending on the date of sowing and the cultural cycle of maize. In Greece, the existence of the species was first reported by Stavrakis [38], The pest increased steadily in the 1980s as a result of the use of new single hybrids and improved culture practices [21], This increase was followed by problems caused by S. nonagrioides, especially in the late- sown crop (sown in early luly after the harvest of small cereals) [39], According to a pilot survey in October 2005, the dominant pest in sweet sorghum. Sorghum bicolor (L.) Moench, was S. nonagrioides [40], 1.2. Number of Generations and Diapause. The number of generations is marginally governed by the onset of diapause at various latitudes and there are fewer generations in the northern region than in the southern one. Three to four gen- erations are completed per year in Greece [39] , Stavrakis [38] reported that the pupation of the overwintering population in Greece takes place in April-May. It seems that the fourth generation is partial since some of the late progeny of the third generation will not make it through [39], In Spain and Portugal, the borer completes 2 generations and a partial third one per year [37, 41, 42], with the third one having a low population size in northern Spain [43], The existence of two generations, in May and luly- August, has been reported in France [44], In Israel, the borer is at least a bivoltine wetland species, flying in March to July and in October [45], In Iran, it completes 4 generations during the active season, with a partial 5th generation in second plantings [46], It has also been referred to as multivoltine, with three to four generations per year in southern Portugal [47] and three generations per year in the Izmir area of Turkey [48], Diapause of this species has been studied extensively [20, 49-52], Eizaguirre and Albajes [49] and Fantinou et al. [51] reported that larval diapause is induced by the length of photoperiod and that constant temperature modifies the diapause response curve from type III to type I, According to previous studies, the early induction of diapause can be explained by the limited tolerance of insects originating in the tropics to low temperatures, and it could be a mechanism enabling the insect to extend its range into northern regions. It seems that temperature plays a double role in the oc- currence of a supplementary generation by increasing the developmental rate and delaying the onset of diapause, Gillyboeuf et al, [53] observed that survival of diapausing larvae at low temperature may be related to the microclimate of the overwintering site and not to their freeze tolerance capacity. However, they argue that the freezing tolerance of S. nonagrioides may be a factor favoring northern expansion. Fantinou et al, [20] and Eizaguirre et al. [54] stated that photoperiods longer than 12:12 h (L:D) terminate diapause and that field-collected larvae complete diapause spontaneously. Under a temperature similar to the natural field temperatures, diapause terminates in approximately 4 months, ensuring that the larvae reach the middle of winter without pupation. When diapause terminates, temperatures in the field are very low and larvae go into quiescence, allowing them to survive and to synchronize their cycle with that of the host plant. The fact that the temperature thresh- olds for diapause and postdiapause development are 3 or 4 degrees lower than that for continuous development [20, 41] explains the phenological model of S. nonagrioides described by Lopez et al. [41]. Moreover, Fantinou and Kagkou [55] reported that under natural conditions the increase in nighttime temperature in late winter and early spring could function as a signal eliciting diapause development. This is ecologically important because in temperate regions insects are exposed to daily photoperiods and thermoperiods in which the long nights coincide with low temperatures. The specific role of low temperature exposure in regulating diapause development is not entirely clear, beyond the fact that exposure to low temperatures is not a prerequisite for diapause termination in this species [20]. The intensity of cold stress reflected in the level of mortality occurring in larvae suggests that the northern boundary of the species’ expansion is defined by low temperatures. 2. Key Aspects for the Existence of the Species 2.1. Latitude and Critical Photoperiod. Figure 1 shows the latitude lines of the Mediterranean basin countries. S. non- agrioides can be found in northern, mainly European, coun- tries between 35° and 46° N and in southern countries, such as Morocco, Iran, Syria, and Israel, between 31° and 35° N. Spain is located between 36° and 43° N, whereas Greece is located between 35° and 41.5° N. In all the circum- Mediterranean countries where S. nonagrioides has been found, including Morocco, the species overwinters as dia- pausing larvae [2, 21, 44, 56], but there is no evidence of diapause in the populations of warmer and more southern countries. The Sahara desert probably delimits the popu- lations of the borer that diapause in the north from those that complete development without diapause in the south. Masaki [57] suggested that variations in the incidence of diapause might be due to the varying threshold of external stimuli that trigger diapause. If an insect has an extremely low threshold, it will enter diapause in a very wide range of environmental conditions, whereas if its threshold is extremely high, the conditions which induce its diapause might be nonexistent in the ordinary range of environment. Between these extreme thresholds, there is an intergraded series of the reaction thresholds. According to Eizaguirre and Albajes [49], under lab- oratory conditions the critical photoperiod (that which induces 50% diapause) is reduced from 13 h 52 min at 18°C to 13 h 15 min at 25°C; this means that a 1°C decrease in temperature corresponds to an increase in the critical photoperiod of about 5.3 minutes. This range of the critical photoperiod corresponds closely to the day length on 15 August in regions where S. nonagrioides diapauses. In these regions, the duration of the day on 15 August, from sunrise to Psyche 3 Figure 1: The latitude lines of the Mediterranean basin countries (from Google maps). sunset, is graduated approximately from 13 h 18 min at 31° N to 13 h 57 at 43° N, an increase in day length of approximately 3.16 minutes for each degree of latitude increase (Figure 2). Figure 2 shows the critical photoperiod for each latitude and temperature. In view of this, on 15 August in the north- ern regions of Europe, the longer photoperiods induce lower percentages of diapause, whilst the shorter ones occurring in southern Europe induce higher percentages. These results may seem contradictory, because in the northern regions 5. nonagrioides larvae enter diapause earlier than in the south- ern regions, but the explanation could be that temperature has been reported to play a significant role in diapause induction [20, 50, 55, 58]. Estimation of the critical photoperiod by Eizaguirre and Albajes [49] allows us to design a model that could help to predict the occurrence of the critical photoperiod according to the latitude and the temperature in various countries (Eigure 2). This model could help us to estimate the percentage of the live larvae of a generation that will be induced to diapause, the larval proportion that may develop towards adulthood, and therefore the trend of the population density of the next (last) generation. Eigure 3 shows the variation in the climate of 6 cities of the area where S. nonagrioides is distributed. The critical pho- toperiod arrival in these cities based on the data of Eigure 2 corresponds to 27 August in Bordeaux, 6-7 September in Teheran, 20 August in Milan, 31 August in Zaragoza, 20 August in Athens, and 6 September in Marrakech. Therefore, the differences in the onset of the critical photoperiods in the various areas do not seem to be significant. 2.2. Freezing Days and Number of Generations Per Year. Although the differences in the critical photoperiod are not very obvious, greater differences can be observed in the range of prevailing temperatures in each region. Milan is the city with most days with a mean minimum tem- perature below -1°C, whereas Teheran is the city with most days with a mean minimum temperature above 10° C and a mean maximum temperature above 27° C, although temperatures below -1°C may occur on a few days each year. S. nonagrioides seems to be to some extent susceptible to high temperatures in summer [56] and the endophytic larval behavior may protect the species from the extreme temperatures of some regions. Eigures 2 and 3 provide data on the factors affecting the number of generations of S. nonagrioides in the different regions of the circum-Mediterranean countries. In Northern Italy, S. nonagrioides is not present because it is very sus- ceptible to the low winter temperatures [13, 44, 56] and the short period of time with mean minimum temperatures above 10° C (close to the threshold temperatures for the pest [40, 59]). In contrast, in Iran the species completes 4 to 5 generations that can be attributed to the long period of time with prevailing mean minimum temperatures above 10° C (Eigure 3) and to the delayed onset of the critical photoperiod in September (Eigure 2). Generally, warmer temperatures tend to be associated with a higher number of generations of the insect. The number of generations in a region depends on the early appearance of the first generation derived from overwintering larvae. Galichet [44], Lopez et al. [60], and Eantinou et al. [20] demonstrated that diapause terminates by the end of February, so the occurrence of the first genera- tion will depend on the prevailing temperatures throughout March, taking into account that the threshold temperatures for postdiapause development are lower than those for normal larval development [54]. Once the first generation has occurred, the accumulation of heat units, degree days, will determine the number of generations completed per season before the arrival of the photoperiod that initiates diapause. The degree days (DG) necessary for the completion of one generation in maize are 616 DG according to Hilal [61] and 730 DG according to Lopez et al. [41]. The number of generations will also determine the population size of the pest of the last generation: the population density of the last generation of S. nonagrioides is usually higher than that of the previous one because the host crop is available [21, 35]. 4 Psyche 3 47 879 877 875 873 871 869 46 876 874 872 870 868 866 45 872 870 868 866 864 862 44 869 867 865 863 861 859 43 865 863 861 859 857 855 42 862 860 858 856 854 852 41 858 856 854 852 850 848 40 855 853 851 849 847 845 39 852 850 848 846 844 842 38 848 846 844 842 840| ■ 37 845 843 84l| 837 835 36 84l| 837 835 833 831 35i w 836 834 832 830 828 34 835 833 831 829r827| 825 1 2 3 4 5 6 836 865 863 861 862 860 858 858 856 854 855 853 851 851 849 847 848 846 844 844 842 840| 841IIH 837 8381^834 834 832 830 831 829’ 827 827 825 823 824 822 820 821 819r8T7| 8 9 10 859 857 856 854 852 850 849 847 845 843 842 840| 19 836 835 833 832 830 828 826 825 823 821 819 818,816 815 813 11 12 855 853 852 850 848 846 845 843 831836 834 832 831 829 828 826 824 822 821 819 817 815 814 812 811 809| 13 14 851 849 848 846 844 842 84l||| 837 835 834 832 830 828 827 825 824 822 820 818 817 815 813 811 810 808| ■ 805 15 16 847 845 843 844 842 840| 840P?a 836 837 835 833 833 831 829 830 828 826 826^824 822 823 821 819 820 818 816 816 814 812 813 811 809| 809H805 ■m 802 803 801 799 17 18 19 84pg 834 832 831 829 827 825 824 822 820 818 817 815 814 812 810 808| ■ 805 803 801 800 798 797 795 20 21 837 835 833 834 832 830 830 828 826 827 825 823 823 821 819 820 818 816 816 814 812 813 811 809| 810 808| ■ 804 802 803 801 799 799 797 795 796 794 792 793 791 789 22 23 24 831 829 828 826 824 822 821 819 817 815 814 812 810 808] 805 804 802 800 798 797 795 793 791 790 788 787 785 25 26 827 825 823 824 822 820 820 818 816 817 815 813 813 811 809| 810 808| ■ 804 802 803 801 799 800 798 796 796 794 792 793 791 789 789 787 785 786 784 782 783 781 779 27 27 29 821 819 818 816 814 812 811 809| 805 804 802 800 798 797 795 794 792 790 788 787 785 783 781 780 778 777 775 30 31 817 815 814 812 810 808| ■ 805 803 801 800 798 796 794 793 791 790 788 786 784 783 781 779 777 776 774 773 771 1 2 811 805 803 801 799 797 795 793 791 789 808| 804 802 800 798 796 794 792 790 788 786 804 802 800 798 796 794 792 790 788 786 784 782 801 799 797 795 793 791 789 787 785 783 781 779 797 795 793 791 789 787 785 783 781 779 in 775 794 792 790 788 786 784 782 780 778 776 774 111 790 788 786 784 782 780 778 776 774 111 770 768 787 785 783 781 779 777 775 773 771 769 767 765 784 782 780 778 776 774 772 770 768 766 764 762 780 778 776 774 772 770 768 766 764 762 760 758 777 775 773 771 769 767 765 763 761 759 757 755 773 771 769 767 765 763 761 759 757 755 753 751 770 768 766 764 762 760 758 756 754 752 750 748 767 765 763 761 759 757 755 753 751 749 747 745 4 5 6 7 8 9 10 11 12 13 14 15 August September 16°C ■ 24° C 18°C 26° C 20° C ■ 28°C 22° C Figure 2; Variation in the length of the day, in minutes, from 1 August to 15 September according to latitude. Length of the day in color indicates the day of the critical photoperiod inducing 50% diapause for this temperature and latitude. Bordeaux, 44° 50.1 min Average high/low temps Record high/low temps tin too 80 60 40 20 0 ,1 ,;n v/iii. ^ LA I'l A I ( irf'V 1 /•’' 1 ij^ k 1 1 nTTiTT 1..' 1.'. 1 1 I'vW d/ %'(7l i^rr, , ,, 1 y t\h k. M * ' -• — 'At -.Viil ■ — - — ’ 'll, V'l' >■ — ■ ’iiTiuoytiiUmi w ' rn Tn □ -18 (a) Zaragoza,41°39.7 min Average high/low temps Record high/low temps 100 80 g 60 40 20 0 it I i .y' 1 [ ,pl1 pjvr m. 1 i \ -I 6, ■■ - i Lj'iiLI LVV '■ A if'i , - ' JT, MSI , ' — ' — ■--- -.y :'J A I'nV^ — — Vf. TJ f- 38 27 16 ij o 4 -7 -18 Milan, 45° 28.2 min Average high/low temps Record high/low temps 38 100 27 80 16 ^ ^ 60 O tin 4 ^ "" 40 -7 20 ^ O ^ (c) Aw ^3 ill r’'", / 1' ( sy 1,/ v"i' ,LV'\ I. IhI, ‘■•s, L'hJl '■ TjT |V.jM 1 p It'" I'V, Mm MR ''1 38 27 16 4 -7 -18 O ^ ^ s < s ^ ^5. 3’ A! o A C ^ o 12 Q (b) Athens, 37° 58.3 min Average high/low temps Record high/low temps 120 105 90 75 S 60 "" 45 30 15 0 ,lfl Li , I'l 1, • * S'J fw7 "''h V'i jA, _ — , Vii' “ — r-i, . ‘■■Wl “ 1 .1 r n.- 1 ^ ^ s < s ^ Oh 49 41 32 24 16 U o 7 ^ -1 -9 -18 U s ^ <; O 12 Q (d) 130 110 90 g 70 50 30 10 Marrakech, 31° 36.41 min Average high/low temps Record high/low temps , , . j' .. 1 1,^ N' M'-'A 'A\ Mf J 'nt- . A > V w lit. I'l A 4 1 ' 'k‘ 1 Aij — 4'.T tj 1 — Mr M ) 1 1 54 43 32 100 80 21 U o _ 60 tin 10 "" ^ 40 -1 20 -12 0 Teheran, 35°41.9min Average high/low temps Record high/low temps (e) (f) Figure 3: Maximum and minimum temperatures of six cities in the area of distribution of Sesamia nonagrioides. The curves, from bottom to top, show the record minimum temperatures, the mean minimum temperatures, the mean maximum temperatures, and the record maximum temperatures. Days with mean maximum temperatures higher than 27° C are colored in red, days with mean minimum temperatures higher than 10° C are colored in green, and days with temperatures below -1°C are colored in blue. Psyche 5 Consequently, like many multivoltine species that undergo a state of diapause, S. nonagrioides may complete as many generations as temperature and photoperiod conditions will allow, assuming that there is an available food source. 2.3. Winter Mortality. The overwinter mortality of S. nona- grioides in the Mediterranean is not only determined by the number of freezing days in winter but may also be associated with the percentage of the larval population that “escape” the critical photoperiod in autumn. If the weather remains warm, it is likely that many larvae will avoid diapause because of the high temperatures. Therefore, a further generation will lay eggs on a suitable green crop if it is available, and the neonate larvae will successfully develop only in those regions where relatively mild autumn temperatures can occur. However, the young larvae that are subsequently exposed to the later winter temperatures are destined to die. Therefore, the higher the percentage of larvae that escape from diapause during autumn, the higher the mortality of the next generation of young larvae. 3. Summary Field populations of S. nonagrioides in the Mediterranean region display winter diapause. Voltinism in this species is a seasonally plastic trait dependent on early emergence of adults of the overwintering generation. The abundance of the species in a given region depends on the number of freezing days of the winter and the heat units accumulated from diapause termination until the arrival of the critical photoperiod for diapause induction in late summer. The species relies on latitudinal gradients in temperature and photoperiod for the induction of diapause, and the effect of environmental cues on diapause and adaptation to local environmental conditions is, therefore, variable. References [1] L. Oliveira and J. Tavares, “Contribui^ao ao estudo de Sesamia nonagrioides Lef (Lep., Noctuidae) na cultura de Strelitzia reginae Ait. (Sci., Musaceae) na ilha de S. Miguel (Azores),” Arquipelago, Serie Ciencias da Natureza, vol. 2, pp. 165-176, 1982. [2] M. J. Fazeli, “Biology and control of Sesamia nonagrioides botanephaga in the fars province,” Applied Entomology and Phytopathology, vol. 59, pp. 13-14, 1992. [3] L. Sannino, B. Espinosa, and B. Campese, “Melon: a new host of Sesamia nonagrioides (Lefebvre),” Informatore Fitopa- tologico, vol. 54, pp. 32-34, 2004. [4] N. Uygun and A. Kayapinar, “A new pest on banana: corn stalk borer, Sesamia nonagrioides Lefebvre (Lep. 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Hilal, “Etude du developpement de Sesamia nonagrioides et etablissement de moddes poru la prevision de ses populations dans la nature,” Bulletin OEPP, vol. 11, pp. 107-112, 1981. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 590619, 9 pages doi:10.1155/2012/590619 Review Article Hylastes ater (Curculionidae: Scolytinae) Affecting Pinus radiata Seedling Establishment in New Zealand Stephen D. Reay,' Travis R. Glare, ^ and Michael Brownhridge^ ^ Silver Bullet Forest Research, P.O. Box 56-491, Dominion Rd, Auckland, New Zealand ^Bio-Protection Research Centre, Lincoln University, PO. Box 84, Lincoln 7647, New Zealand ^ Vineland Research and Innovation Centre, 4890 Victoria Avenue N, P.O. Box 4000, Vineland Station, ON, Canada LOR 2E0 Correspondence should be addressed to Travis R. Glare, travis.glare@lincoln.ac.nz Received 5 August 2011; Revised 20 November 2011; Accepted 22 December 2011 Academic Editor: John A. Byers Copyright © 2012 Stephen D. Reayet al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The introduced pine bark beetle Hylastes ater has been present in New Zealand for around 100 years. The beetle has been a minor pest on pines. Research was undertaken to control the pest in the 1950s-1970s, with a biological control agent introduced with limited success. Following a reasonably long period with minimal research attention, renewed interest in developing a better understanding of the pest status was initiated in the mid to late 1990s. Subsequently, a significant amount of research was undertaken, with a number of studies exploring the role of this pest of exotic forests in New Zealand. These studies ranged from attempting to quantify damage to seedlings, evaluate the role of the beetle in vectoring sapstain fungi, explore options for management, and evaluate the potential for chemical and biological control. From these studies, a number of findings were made that are relevant to the New Zealand exotic forest industry and shed new light onto the role of secondary bark beetles globally. 1. Introduction The introduced pine bark beetle, Hylastes ater (Paykull) (Curculionidae: Scolytinae), is a pest of reestablished Pinus radiata D. Don forests in New Zealand. First recorded in New Zealand in 1929 [1], it has become a problem in second and third rotation forests where it breeds under the bark of stumps and other similar logging waste (log sections). Both adults and larvae feed on the phloem. Adults lay eggs in galleries, and larvae may take up to 300 days to develop to maturity. Subsequent emergence of adults from stumps is not necessarily immediate, and some adults continue to feed for longer periods [2]. Emerging adults feed on seedlings that have been planted in the immediate area. This maturation feeding characteristically involves the adult beetle eating the bark around the root collar of a seedling below the ground. In severe cases, seedlings may be completely ring barked and will die. Beetle feeding also commonly causes considerable sublethal damage, and feeding wounds may serve as a point of entry for soil-borne pathogens. Despite initial concerns, historically H. ater was not regarded as a significant forest establishment pest in New Zealand. More recently, surveys have indicated that attacks on P. radiata seedlings by H. ater may be more common than previously documented and not evenly distributed across forest estates [3] . Hylastes ater usually attacks seedlings within the first year after planting [3]. Consequently, mor- tality surveys that are undertaken much later may fail to detect dead seedlings that are difficult to see, or death is attributed to other causes. In cases where dead seedlings are observed, they must be removed from the soil for inspection around the root collar region to confirm feeding damage by H. ater as a potential cause of mortality. Forest establishment practices currently focus on lowering initial stocking rates and planting higher quality (and more expensive) seedling material. This means that low amounts of damage by H. ater may be more significant to forest establishment operations than previously experienced [3]. 2. Damage of Pine Seedlings by H, ater In New Zealand, large areas of mature P. radiata forest are harvested all year round. The resulting stumps create a con- tinuous supply of breeding habitat allowing H. ater and/or 2 Psyche other beetle populations to continuously persist at epidemic levels compared with a natural forest environment [4, 5]. Adults emerge from stumps following larval development and may begin maturation feeding on seedlings that were planted following harvesting operations [3]. In New Zealand, H. ater does not build up high popula- tions in all areas. Surveys where live and dead seedlings were destructively sampled in 60 compartments in the central North Island showed that seedling mortality due to severe H. ater damage in most compartments was less than 5% [3]. However, seedling mortality was occasionally higher (up to 30%). These surveys revealed relatively few dead seedlings without evidence of severe feeding damage (i.e., root collar region completely ring barked), suggesting that seedling mortality due to severe H. ater damage was more likely than other factors (e.g., drought, poor planting). These other factors may have contributed to seedling death. There was evidence of some seedling attack by H. ater in most compartments. Sublethal damage by H. ater was identified by destructively sampling live seedlings along transects and was observed to be greater than 30% in half of all the compartments sampled over the two-year period (and was over 80% in some areas) [3]. Seedlings were occasionally found to have survived severe attacks (multiple feeding attempts or complete girdling of the stem) by H. ater. When such seedlings were inspected at a later date (i.e., one year after damage had occurred), they were found to be alive and “growing well”, suggesting that if mortality did not occur subsequent to the H. ater feeding event, recovery was likely. Overall, this study suggested that considerable amounts of feeding damage might have previously been undetected as surveys were undertaken to identify areas of seedling mortality, and dead trees were often not removed from the ground for inspection. It is unlikely that in such surveys live trees were destructively removed. The time trees were harvested was identified to be an important factor in determining whether seedlings are likely to be attacked by H. ater [3]. Sites harvested during autumn and planted the following winter were at the greatest risk, and the risk of damage decreased with increasing time between harvesting and planting. Sites harvested prior to spring and planted the following winter were seldom found to contain seedlings that were attacked by H. ater. The relationship between harvesting history and the likelihood of seedling damage was related to the life history of H. ater, including flight activity and competition for brood sites by other bark beetles. Hylurgus ligniperda (Fabricius) (Curculionidae: Scolytinae) was found to be the dominant species in stumps during summer months [6]. Hylurgus ligniperda was first discovered in New Zealand in 1974 and, like H. ater, breeds in the stumps and logs of Pinus spp., and is found throughout New Zealand, but is not a threat to seedlings [7, 8]. Sites harvested during spring to late summer were colonised predominantly by H. ligniperda suggesting that this species is able to outcompete H. ater during this period. While H. ater has a spring flight, sites harvested in late summer-autumn contained the largest populations of H. ater, relative to H. ligniperda. The subsequent H. ater populations resulting from these autumnal colonisation events emerge during the following late spring/summer and attack seedlings [6]. Experimentation in forest establishment practices by some forestry companies in New Zealand resulted in areas being replanted outside of the traditional winter replanting times using containerised tree stocks. Essentially, this means the planting season was extended so re-establishment could, in theory, occur year round. In reality, planting was not undertaken during the driest months of summer. The implications of this replanting approach, with regard to seedling damage by H. ater, was not fully assessed, but, in theory, trees could be planted immediately (within a month) following harvesting. While harvested land was traditionally left “fallow” for extended periods to allow weeds to germinate and be controlled, this practice was being challenged by at least one forestry company during the early 2000s in an attempt to minimise the period between harvesting events. Consequently, areas previously considered of low risk due to the emergence of H. ater populations before planting occurred may be more at risk if populations of H. ater larvae are present in stumps when seedlings are planted. 3. The Relationship between H, ater and Sapstain Fungi Bark beetles are known worldwide as vectors of fungi, largely due to interactions between aggressive bark beetles and fungi. These fungi were thought to play an important role in the tree killing by bark beetles (e.g., members of the genus Dendroctonus) [9-13]. Six and Wingfield [13] have recently challenged this view and have presented several arguments against a pathogenic role by these fungi. Firstly, tree-killing bark beetles do kill trees in the absence of virulent pathogens. Secondly, the growth of fungi follows beetles colonisation and is relatively slow until colonisation by beetles has resulted in tree heath deteriorating beyond the point where the tree might survive. Thirdly, virulent fungi are found to be associated with bark beetles that do not typically kill trees, and many tree-killing beetles carry weak or nonpathogenic fungi. Finally, most bark beetles do not kill trees and still carry fungi similar fungi to their tree killing relatives, which indicates that fungi play important roles other than killing trees. Instead, Six and Wingfield [13] suggest that fungal phytopathogenicity may be important for fungi that exhibit this characteristic to compete with other fungi and/or survive in living trees. Staining fungi are a significant economic concern to the P. radiata forest industry [14-17], due to the high susceptibility of P. radiata wood to staining [18]. While some saprophytic, pathogenic, and endophytic fungi cause sapstain in wood, it is generally the saprophytic fungi that invade timber after the tree has been harvested [19]. The staining effect only becomes evident when conditions are favourable for fungal growth. In New Zealand, this is normally after harvesting when the sapwood dries and the aerobic sapstain fungi are able to grow in the wood cells. In some instances, wood may have to be discarded prior to processing [15, 19]. Psyche 3 Sapstaining fungi are commonly recorded from less aggressive bark beetles. In particular, the fungal species of Leptographium, Graphium, and Ophiostoma have been found on H. ater in Britain, South Africa, and Australia [ 18, 20, 21 ] . In New Zealand, Leptographium sp,, L. lundhergii Largerburg and Melin, and Ophiostoma ips (Rumb.) Nannf. were isolated from H. ater [22-24], The presence of L. procerum (Kendrick) Wingfield and O, huntii (Robins-Ieff) DeHoog and Scheffe in New Zealand is likely to be due to its introduction with either H. ater or H. ligniperda [18, 25], Ophiostoma ips is commonly associated with bark beetles [26-29] , Species of LIylastes are known vectors of fungal root diseases in other parts of the world [30-32], In these cases, Hylastes adults attack the roots of stressed or diseased adult trees and vector root disease fungi [30-32], Reay et al, [24, 34] described a strong relationship between the sublethal attack of P. radiata seedlings by H. ater and invasion by sapstain fungi. The presence of sapstain fungi was found to increase as severity of damage increased. Half of severely attacked seedlings were found to contain sapstain fungi, indicating the potential for large numbers of seedlings throughout forests to be infected [24, 34], The sapstain fungi were from the Ophiostomataceae [15, 29], Most frequently isolated were O, huntii and O, galeiforme (Bakshi) Math-Kaarik [24, 34], Ophiostoma huntii has been isolated from many parts of the world [25] and has been associated with several species of bark beetles, including H. ater [25, 26] and H. porculus Erichson, and may be an important species in red pine decline [10], Ophiostoma galeiforme is a European species, which has been found with Hylurgops palliatus (Gyllehan) on larch in Scotland [35, 36] and Hylastes cunicularius (Erichson) in Sweden [37] , Mathiesen-Kaarik [37] describes O, galeiformis as a “secondary” staining fungus, Ophiostoma galeiforme may have been introduced into New Zealand with H. ater [24], The remaining sapstain species isolated from seedlings by Reay et al, [24, 34] are commonly found in New Zealand pine plantations [15, 17], Fortunately, the fate of fungi following feeding damage appears to be limited. When areas of damaged seedlings were revisited three years following planting, Reay et al, [38] failed to isolate any sapstain fungi species from the previously damaged trees that were sampled. However, Sphaeropsis sapinea (Fr,) Dyko and B, Sutton (which was not isolated from seedlings in initial sampling following seedling damage) were isolated from 10-16% of seedlings at the three- year after beetle attack sampling, Sphaeropsis sapinea is an important opportunistic fungal pathogen of P. radiata (and other conifers) in New Zealand, While there was a possibility that colonisation by the bark beetle vectored fungi may have had some influence on the health, growth, and long-term fate of the trees, this was not investigated [38], Hylastes ater has been suggested as the mechanism by which a number of species of fungi have been introduced into New Zealand, Therefore, it is possible that future introductions of H ater (or other bark beetles) may establish new fungal species (or other organisms). If new fungal pathogens were introduced into New Zealand by other means, there is potential for H ater to vector these throughout forests. Therefore, continued treatment of bark beetles as biosecurity threats to New Zealand is imperative, despite the establishment of several species, 4. Molecular Characterisation of Hylastes ater and Associated Species Hylastes ater is currently the only example of the genus found in New Zealand, but other Coleoptera can colonise similar environments, Hylurgus ligniperda is found under the bark of pine stumps, often with H. ater. Another bark beetle beetle, Pachy cotes peregrinus, (Chapuis) (Scolytinae) and a native pinhole borer. Platypus apicalis White (Platy- podinae) are also found in pine stumps, Hylastes ater may be confused with P peregrinus [2] by inexperienced forest management personnel and is morphologically similar to closely related European species, such as H. brunneus Erichson, As biosecurity incursions are a constant threat to New Zealand exotic plantation forestry, identification of new occurrences of bark beetles is important, Earval stages are difficult to identify with morphological characteristics, so we investigated molecular identification of available species. This preliminary data is not intended as a full phylogenetic analysis, but rather to provide, through GenBank, reference sequences for each species for future researchers, 4.1. Methods. A number of individuals of H. ater, H. ligniperda, P. peregrinus, Treptoplatypus caviceps (Broun) (Platypodinae), Platypus apicalis, and P. gracilis Broun (Platypodinae) were collected from various sites through- out New Zealand (Table 1), In addition, specimens of H. brunneus, H. cunicularius, Hylobius abietis L, (Molyti- nae), and Austroplatypus incompertus (Schedl) (Platypodi- nae) were obtained from outside New Zealand and were included (Table 1), Sampling and collection of beetles were not systematic. Using DNA extracted from the heads, elytra, and legs (DNeasy Plant Kit, Qiagen), PGR was used to amplify the terminal region of the 28S rRNA domain 2 region was performed using the primers 28S- F (5'-AGAGAGAGTTGAAGAGTAGGTG-3') and 28S-R (5'- TTGGTGGGTGTTTGAAGAGGGG-3') [39], Amplifications were carried out using 30 cycles of 15 sec at 98°C, 30 sec at 48° G, 40 sec at 72° G, PGR products were cleaned using an Eppendorf Perfect Prep Gel Gleanup Kit and sequenced directly (AWCGS Sequencing Facility, Massey University, New Zealand), Resulting sequences were aligned and com- pared using Bayesian inference (Figure 1), Sequences were aligned using GlustalX [40], Phylogenetic analysis using Bayesian inference was conducted using MRBAYES version 3,1,2 [41, 42], Models of nucleotide substitution were selected using the Akaike Information Griterion (see [43]) in MrModelTest v2 [44] implemented in PAUP* 4,0b 10 [45], The model selected was GTR + G, which is a general time reversible model [46, 47] with a gamma-shaped rate variation across sites and a proportion of invariable sites. Two runs of four chains saving trees every 100 generations were conducted. After 1,000,000 generations, the two runs had converged close to the same value (determined by when 4 Psyche Table 1: Beetle isolates and GenBank sequence data used in this study. Isolate Species Location, date, collected by GenBank number Hylg 1 Hylurgus ligniperda Auckland, NZ. 2007 Reay JN544556 Hylg2 Hylurgus ligniperda Auckland, NZ. 2007 Reay JN544555 Hylg3 Hylurgus ligniperda Auckland, NZ. 2007 Reay JN544554 Hyls96 Hylastes ater Canterbury, 2004 Reay JN544548 Hyls99 Hylastes ater New South Wales, Australia 2004 Reay, D Kent JN544549 UKl Hylastes brunneus Galway, Ireland 2005 Reay, Walsh JN544550 UK12 Hylastes brunneus Galway, Ireland 2005 Reay, Walsh JN544551 UK8 Hylastes cunicularius Northumberland, England 2005 Reay, Glare JN544552 UK9 Hylastes cunicularius Northumberland, England 2005 Reay, Glare JN544553 UK6 Hylobius abietis Galway, Ireland 2005 Reay, Walsh JN544547 Pla90 Austroplatypus incompertus New South Wales, Australia 2004 D Kent JN544546 Platyl Platypus apicalis Auckland, NZ. 2007 Reay JN544557 Platy2 Platypus apicalis Auckland, NZ. 2007 Reay JN544558 Platy3 Platypus apicalis Auckland, NZ. 2007 Reay JN544559 Platy4 Platypus gracilis Canterbury, NZ. 2007 Reay JN544561 Platy5 Platypus gracilis Canterbury, NZ. 2007 Reay JN544562 Platy6 Platypus gracilis Canterbury, NZ. 2007 Reay JN544563 Platy7 Treptoplatypus caviceps Canterbury, NZ. 2007 Reay JN544568 PlatyS Treptoplatypus caviceps Canterbury, NZ. 2007 Reay JN544569 Platyl 0 Platypus gracilis Westland, NZ. 2007 Reay JN544564 Platy20 Treptoplatypus caviceps Westland, NZ. 2007 Reay JN544570 Pla47 Platypus gracilis Canterbury, NZ. 2007 Reay JN544560 Pac6 Pacyhcotes perigrinius Dunedin,NZ. 2002 S Reay JN544566 Pac7 Pacyhcotes perigrinius Tokoroa, NZ. 2004 Reay JN544567 Pac8 Pacyhcotes perigrinius Tokoroa, NZ. 2004 Reay JN544565 the standard deviation of split frequencies fell below 0.005) and the first 25% of trees were discarded as burn-in. The consensus tree, with the posterior probabilities for each split and mean branch lengths, was visualised using Treeview 1.6.6 [48]. 4.2. Molecular Identification Using 28S rRNA. Using this short sequence of the 28S rRNA, it was possible to distinguish between all species (Figure 1). As stated above, this is not a phylogenetic study and Figure 1 is provided simply for visual reference of the separations seen between species using this DNA segment. Clear separation was achieved between Platypodinae and Scolytinae, as would be expected, but also between the species of Scolytinae. The three Hylastes species were separated into a group with the related species, H. ligniperda. The results of this analysis show the potential for the 28S region of RNA to be used for the identification of Cur- culionidae and may be a useful biosecurity tool, particularly if larvae of the Curculionidae are intercepted at the border. 5. Mitigating Impacts of if. ater Early efforts to reduce the impact of H. ater in New Zealand included importation and release of three species of preda- tory Rhizophagus beetles, as no native predators were known [2]. These were originally imported in 1933 from Britain and released but did not establish. Further importations and release from Europe of natural enemies were made in 1975 and 1976. A parasitic wasp, Rhopalicus tutele (Walker) (Pteromalidae), and the predatory beetle, Thanasimus formi- carius L. (Cleridae), were released but had little impact. Following the work of Reay and Walsh [3, 49], manage- ment practices that could reduce likelihood of attack were recommended. As discussed above, high-risk sites could be planted later in the season in spring/early summer (rather than during winter) when late instar larvae are present allowing little time for seedlings to establish and grow prior to beetle emergence and may result in seedlings being more vulnerable to damage. In New Zealand, chemical insecticides are rarely used in plantation areas to control H. ater. A carbosulfan insecticide was shown to protect seedlings from damage by H. ater but is not currently in operational use [49]. 6. The Potential Role of Biocontrol of Hylastes ater Using Insect-Pathogenic Fungi Currently, site management is the only economically viable option for minimising impacts to regenerative plantings due to H. ater damage in commercial operations. This results Psyche 5 -Hylohius abietis (UK6) - Pac 8 ■ Pac 6 - Pac 7 Pachycotes peregrinus Austroplatypus incompertus (Pla 90) ■ Platy 6 ■ Platy 5 - ■ Platy 4 ■ Platy 10 ■ Pla 47 ■ Platy 1 Platypus gracilis 0.1 rHylg3 Hylg2 Hylgl r Hyls 96 ■ Hyls 99 Uk 1 Uk 12 -Uk8 -Uk9 Hylurgus ligniperda Hylastes ater ■ Hylastes brunneus Hylastes cunicularius Platy 2 Platy 3 ■ Platy 8 r Platy 20 ■ Platy 7 Platypus apicalis Treptoplatypus caviceps Expected changes per site Figure 1: Representation of the species divergence using comparison of partial 28S rDNA sequences. in land out of production and open to colonisation by weeds and erosion for a significant period. In addition, the increased use of containerised cuttings (in addition to bare root stock) has meant that planting seasons are extended, resulting in more sites at higher risk from H. ater. Alternative management options that would protect and promote the overall health and establishment of pine seedlings while reducing pest threat would benefit the forest industry. Moreover, improved control options are needed for use against any new species incursions. Biosecurity incursions are a constant threat to New Zealand plantation forests. Entomopathogenic microbes have been developed as commercially available biopesticides for some pests. For example, the bacterium Bacillus thuringiensis Berk has been used as the active agent in numerous biopesticides used in forestry for control of lepidopteron pests. Over a number of years, we have been investigating entomopathogens of H. ater in New Zealand and the potential for developing biopesticides. Entomopathogenic fungi are important mortality factors in bark beetle populations, although the natural infection rate and impact on beetle populations is estimated to be relatively low [50]. Fungi in the genus Beauveria (Balsamo) Vuillemin are the most common species reported attacking bark beetles [51]. This genus contains a number of species, all of which are pathogenic to arthropods, including insects and Acari [52, 53], and occupy diverse habitats above and belowground [54-56]. Beauveria caledonica Bissett and Widden was isolated from H. ater and H. ligniperda in New Zealand and subsequently shown to be pathogenic to these two species in laboratory bioassays [57]. Previous to this, B. caledonica was not known to be pathogenic to insects. In the UK and Ireland, B. caledonica was isolated by concentrating on the major forestry pest, the large pine weevil, H. abietis [57]. Hylohius abietis is a serious pest of spruce and pine plantation trees, with an average of 33% and up to 100% of new plantings being killed per annum when untreated in some regions [58]. A survey of Beauveria spp. in substrates (soil, stumps, bark and grass from insect galleries) associated with bark beetles in P. radiata cutover forests was undertaken to identify what fungal isolates might be present in these forest systems [59]. Beauveria spp. were commonly isolated from all substrates sampled and were recovered from all but one of the six sites surveyed. However, there was considerable variation within and between sites in the relative prevalence of the fungi across all substrates and within substrate types, and three species of fungi were isolated {B. caledonica, B. bassiana (Balsamo) Vuillemin andP. malawiensis Rehner and Aquino de Muro) [59]. Beauveria caledonica was isolated from all substrates in this study, including beetle and larval cadavers. It was not isolated from live insects [59]. In total. 6 Psyche 13 Beauveria isolates representing the three species recovered were selected and found to be pathogenic to both H. ater and H. ligniperda in laboratory bioassays [59]. Thus, in spite of the lack of B. bassiana-mfected cadavers recovered in the field, the fungus is clearly able to infect and kill both bark beetle species and has been demonstrated for other bark beetles in the laboratory [60-65]. However, no epizootics have been reported in field populations. This may simply be due to natural inoculum levels being too low to initiate an epizootic, or due to inhibition of the fungi in the field. Hylastes ater was found to be less susceptible to all of the isolates tested than H. ligniperda^ although the reasons for this are unclear. However, it is likely to have implications for any control programme using fungal entomopathogenic fungi. While entomopathogenic fungi from Beauveria are pre- dominant, fungi from other genera have been recovered from H. ater cadavers [66]. These include Metarhizium flavoviride var. pemphigi Driver and Milner and Hirsutella guignardii (Maheu) Samson, Rombach and Seifert. While some Metarhizium anisopliae (Metsch.) Sorokin, isolates are known to be pathogenic to bark beetles in laboratory bioassays, Metarhizium spp. do not appear to have been previously isolated from field-collected specimens [51]. Similarly for H. guignardii, this record is therefore not only a first for H. ater in New Zealand but may be the first record from a bark beetle [66]. Recovery of the fungi from the cuticles of bark beetle adults clearly demonstrates the capacity for insect-mediated movement of the fungi in a pine forest [59]. The recent research into entomopathogenic fungi represents recent attempts to obtain new ways to mitigating the impacts of H. ater in New Zealand. Differences in the natural prevalence of different species suggests that some isolates may be better suited as biocontrol agents as they persist in the environment better, while the high levels of inoculum detected in frass indicate that virulent, environmentally competent isolates must be selected and formulation and application technologies to efficiently target specific stages of the pest developed to effectively utilize these pathogens in bark beetle management. The use of entomopathogenic fungi as biopesticides has been considered (e.g., [59]), but our estimates of production costs of Beauveria spp. could be prohibitively expensive for broadcast application against an occasional pest in pine plantations. This, coupled with the difficulty of applying fungi to larvae and adults in cryptic habitats, makes use of biopesticides unlikely without a major appli- cation development. Interestingly, further investigation of entomopathogenic fungi in New Zealand pine plantations found that B. bassiana also exists as an endophyte in some trees. A survey by Reay et al. [67] found that B. bassiana could be recovered from needle samples, as well as roots and seed from approximately 15% of 125 trees sampled over the country. Further research has demonstrated that the fungus can be established as an endophyte in seedlings M. Brownbridge et al. (then pers comm). Beauveria bassiana has been found as an endophyte of a number of plant species around the world, and the presence of the fungus has been shown to impact feeding in some insects [68]. The fungus, as an endophyte, may also offer protection against phytopathogens [69]. We are currently researching the potential of endophytic Beauveria in New Zealand pines as a method to reduce bark beetle populations. Endophytic entomopathogenic fungi would provide cost-effective meth- ods to inoculate trees against bark beetles as we have shown seedlings can be infected with the fungus and Beauveria is carried in seed [67]. 7. Conclusion Hylastes ater may not have been considered an important pest of pine plantations in New Zealand during the early years of establishment in New Zealand, but more recently has been acknowledged as a pest of new second-generation plantings. Investigation of biological control options has included predators, parasites, and entomopathogenic microbes. No introduced predator or parasite has yet had an impact on the populations of the beetles. Entomopathogenic fungi, especially Bcawvcna spp., are common in H. ater populations in New Zealand, but development as biopesticides is unlikely to be successful for H. ater due to the cost of any product and application to the cryptic environments being difficult. 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Ownley, K. D. Gwinn, and F. E. Vega, “Endophytic fungal entomopathogens with activity against plant pathogens; ecol- ogy and evolution,” BioControl, vol. 55, no. 1, pp. 113-128, 2010 . Hindawi Publishing Corporation Psyche Volume 2012, Article ID 235840, 1 1 pages dohlO.l 155/2012/235840 Research Article Properties of Arboreal Ant and Ground-Termite Nests in relation to Their Nesting Sites and Location in a Tropical-Derived Savanna B. C. Echezona,^ C. A. Igwe,^ and L. A. Attama^ ^ Department of Crop Science, University of Nigeria, 410001 Nsukka, Nigeria ^ Department of Soil Science, University of Nigeria, 410001 Nsukka, Nigeria Correspondence should be addressed to B. C. Echezona, chezbon2001@yahoo.co.uk Received 11 July 2011; Revised 5 October 2011; Accepted 19 December 2011 Academic Editor: Panagiotis Milonas Copyright © 2012 B. C. Echezona et al. This is an open access article distributed under the Creative Commons Attribution Eicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ecosystem engineers such as ants and termites play an important role in the fertility of tropical soils. Physicochemical analyses were thus carried out on some arboreal ant nests collected from mango {Mangifera indica), bush mango (Irvingia gabonensis), kola (Cola nitida), newbouldia plant {Newbouldia laevis), and oil bean plant {Pentaclethra macrophylla) and on ground nest of termite, Odontotermes sudanensis Sjost. (Isoptera: Termitidae) in Nigeria. Arboreal nests, particularly those of M. indica, were significantly richer in the chemical constituents sampled, compared to those of ground-termite nests or adjacent unaffected soils. Available water capacity of nests from M. indica (60.0%) was significantly higher than those of other sites or locations sampled. While biogenic structures were sandy-loamy in texture, their corresponding adjacent soils were either sandy or sandy-loamy. Soils worked by ants and termites had greater proportions of silt-sized (17.9 versus 9.7) and clay-sized (19.2 versus 9.3) to the detriment of coarse-sized particles (51.2 versus 60.9) and fine-sand-sized particles (11.7 versus 20.1) relative to the adjacent soils. Generally, biogenic structures were about 348% richer in P than their corresponding adjacent soils; an attribute, which holds a strong promise in bioremediation and biofortification of soils especially during amendment. 1. Introduction Tropical-derived savannah ecosystems are often dominated with patches of arboreal ant and epigeous termite nests (?:i5 mounds/m^). Ants are ubiquitous, diverse, and abun- dant in tropical ecosystems and represent up to 80% of animal biomass [1]. Tree crops are of great economic significance in the forests of Nigeria, and the ants which inhabit those trees profoundly affect the ecosystem dynamics through the modification, maintenance, and/or creation of habitats for other organisms in the forest ecosystem [2]. Ecosystem engineers such as ants and termites play an important role in the fertility of tropical soils. Physico- chemical analyses were thus carried out on some arboreal ant nests collected from mango {Mangifera indica), bush mango {Irvingia gabonensis), kola {Cola nitida), newbouldia plant {Newbouldia laevis), and oil bean plant {Pentaclethra macrophylla) and on ground nest of termite, Odontotermes sudanensis Sjost. (Isoptera: Termitidae) in Nigeria. Arboreal nests, particularly those of M. indica, were significantly richer in the chemical constituents sampled, compared to those of ground-termite nests or adjacent unaffected soils. Available water capacity of nests from M. indica (60.0%) was significantly higher than those of other sites or locations sampled. While biogenic structures were sandy-loamy in texture, their corresponding adjacent soils were either sandy or sandy-loamy. Soils worked by ants and termites had greater proportions of silt-sized (17.9 versus 9.7) and clay- sized (19.2 versus 9.3) to the detriment of coarse-sized particles (51.2 versus 60.9) and fine-sand-sized particles (11.7 versus 20.1) relative to the adjacent soils. Generally, biogenic structures were about 348% richer in P than their corresponding adjacent soils; an attribute, which holds a strong promise in bioremediation and biofortification of soils especially during amendment. Ants and termites as decomposers have been reported as the basis for soil formation [3]. Some of the outcome of this decomposition is nutrient release to the soil worked upon by these organisms. Extensive works have been done on the nutrient recycling, soil formation, and soil structural 2 Psyche modification by these animals. Lee and Foster [4] observed that the activities of ants and termites together with their abiotic physical and chemical processes regulate the soil fertility and counteract the physical and chemical processes of soil degradation. Leprun and Roy-Noel [5], Boyer [6], and Mahaney et al. [7] reported a remarkable increase in the mineralogical properties of mounds built by ants and termites. Jouquet et al. [8] and Holt and Lepage [9] found enrichment in mineral nutrients (e.g., NH4, NO3) and exchangeable cations (e.g., Ca^"^, Mg^^, and Na'^) on biogenic (nest) structures of termites as compared to that of the surrounding soils. Konate et al. [10], Macmahon et al. [11], Kristiansen et al. [12] and Jouquet et al. [13] also observed that through the impact on soil by termites, their biogenic structures can constitute patches in the landscape where the availability of soil nutrients for plants is improved. Nests of the harvester ant Pogonomyrmex harhatus typically contain higher concentrations of organic matter, nitrogen, and phosphorus than surrounding soils [14]. Comparative studies by Jouquet et al. [15] on nests made by ants and termites revealed some changes in their soil nutrient properties. They suggested that these changes could be due to greater litter associated with ant nest relative to termite nests, which also could be responsible for changes in the morphology and performance of plants and the composition of plant communities in any agroecology. Very special biogenic structures created by ants are the so-called ant gardens [2]. Jouquet et al. [2] also indicated that a clear agreement has been established between plant preferences for soil altered by termite activity and termite preferences for plant species growing on their own nests. Ants also have been reported to be capable of building nests of carton materials constructed around epiphytic roots thereby developing aggregation of artificial soil [16, 17]. Buckley [18] noted that direct positive effect of the engineering activity of ants lies in the development of a mutualistic relationships between the ants and the epiphytes, whereby the ants profit from the roots forming an integral part of the nest and increasing its structural stability and an abundant food source close to the nest. It could therefore be suggested that these soil engineers indirectly invade their own availability through the increase in colony fitness (i.e., better nourishment of nymphs, higher alate production, and survival) [2]. While the biogenic structures of these organisms have been shown to influence soil quality, microorganism activ- ities, and plants, very little information is available about the differences in the nutrient status of the nest soils based on their nesting sites, biota, and soil locations. We therefore wanted to test the hypotheses that nest substrate (sites) does not influence nest soil properties and that nest soil properties are not a reflection of the initial soil properties when it was unaffected by soil engineers. The aims of this study therefore were to determine the physicochemical properties of the biogenic structures in relation to their nesting sites and biota nests and to compare the characteristics of these nests and their adjacent surface soil. Particle-size analyses of soils were used to assess the physical properties of the soils. The chemical properties of the soils were determined by estimating their C, N, organic matter, base saturation, cation exchange capacity (CEC), exchangeable bases, and P-contents, as well as their pH in both water and in KCl. To determine the availability of the chemical nutrients to plants and thus assess the relative importance of the various soil characteristics in the ecosystem, we also estimated the available water capacity (AWC) and the dispersion ratios (DR) of the various soils, in addition to the dispersibilities of their clay and silt fractions in both calgon and water. The specific objectives of this study thus were to (i) determine the effect of different tree hosts on the physiochemical properties of nests they inhabit, (ii) compare the physiochemical properties of biogenic structures with that of their adjacent unmodified soils, (iii) ascertain the interaction effects of the nesting sites and soil location on the physiochemical properties of their soils. 2. Materials and Methods 2. 1 . Study Site and Species Studied. Field samples of arboreal- ant and ground-termite nests were collected at Orba Nsukka in Udenu Local Government Area of Enugu State in South- east Nigeria (06° 52'N, 07° 24'E; altitude 442 m above mean sea level). Nsukka is situated in a derived savannah belt with some relics of rainforest distributed in patches [19]. The soil is well-drained reddish-brown Typic Paleustult [20]. The annual bimodal rainfall is 1800 mm and spans from April to November of each year [21], with peaks around July and September. The mean monthly temperatures vary between 25° C and 32°C [22]. The study site is a grassy humid savannah with sparse shrub vegetations intermingled with palm trees and some other tree plants forming patches of thickets. 2.2. Field Sample Collection. Six biogenic structures (com- prising five from different trees and one from ground soil termite nest — termitarium) were sampled for this study. The five trees were mango {Mangifera indica), bush mango (Irvingia gahonensis), kola {Cola nitida), newbouldia {New- bouldia laevis), and oil bean plant {Pentaclethra macrophylla) . Biogenic structures (nests) collected from ants and termite nests and the adjacent surface soils (control) were regarded as two soil locations. This is to be able to compare the constituents of nest and unmodified surface soil. Ant arboreal nests were built by Camponotus acvapimensis Mayr. (Hymenoptera: Eormicidae), while the termite nest was built by Odontotermes sudanensis Sjost. (Isoptera: Termitidae). Each ant arboreal nest sample was collected from two trees of the same tree species and about the same age and height to represent the two replications of each treatment. Ground- termite nest samples were taken in pairs from the same soil type (Typic Paleustult) also. Termite mounds were collected and excavated from 0 to 10 cm depth from actively forming nests from the open field. Adjacent soils (without any visible termite — or ant — activity) were sampled at the same depth Psyche 3 (0-10 cm depth) and 6 m away from each habitat tree and termite mound. Sampling from adjacent unaffected soils was conducted in pairs from their surrounding environment. Nest samples were randomly collected from actively forming mound samples. The entire arboreal carton ant nests were pried from their host trees, placed in plastic bags and taken to the laboratory for analysis. The nests were dissected by segmental shaving after being air-dried for seven days to remove the ants. The corresponding termite nests collected were also placed inside plastic bags and taken to the same laboratory after being air- dried for seven days. All samples were sieved with 2 mm mesh and used for laboratory analysis. Each laboratory result was read in triplicate for each sample. 2.3. Laboratory Analysis. The particle-size analyses of soils, termite, and carton nests were determined by the hydrometer method as described by Gee and Bauder [23] using sodium hexametaphosphate (Calgon) dispersant while dieonised water was used separately to disperse soil mechanically only after soaking for 24 hours and distilled water separately as dispersants. The percentage clay-sized and silt-sized particles obtained using calgon were regarded as the total clay and total silt while those obtained with water alone were assumed to be water dispersible clay and silt. Soil pH for soils and ant/termite affected soil in both 1:2.5 soil: 0.1 M KCl suspension and in a soil/water sus- pension ratio using a Beckman Zeromatic pH meter were determined. The soil organic carbon was determined by a modified acid dichromate oxidation procedure according to Walkey and Black [24] as described by Nelson and Sommers [25] . The percentage organic matter was calculated by multiplying values obtained by “Van Benmelin” factor of 1.724. The exchangeable cations and acidity were determined by the method described by Thomas [26]. The cation exchange capacity (CEC) was calculated as the total of all the exchangeable cations. Total (Kjeldahl) nitrogen was measured with a block digester [27] and distilled using NaOH. Available P was determined using Bray and Kurtz [28] method. AWC was calculated by Klute [29] method. Dispersion ratio (DR) was calculated as (WDSi -f- WDC)/(Tsilt + Tclay), where WDSi is the water dispersible silt, WDC is the water dispersible clay, Tsilt is the total silt and Tclay is the total clay. The laboratory analyses results were read in triplicates (three times) for each sample. 2.4. Data Analysis. Treatments comprised factorial combi- nations of six nesting sites of ants and termites nests and two soil locations (adjacent soils and biogenic structures) arranged in a completely randomized design (CRD). These treatment combinations were each replicated two times. The laboratory results were read in triplicates (three times) for each sample making a total of six replications on the whole. Treatment effects were tested through analysis of variance (ANOVA) and differences between means were tested with Duncans New Multiple Range Test (DNMRT). Differences were only considered significant when P values were lower than or equal to 0.05. Percent values were subjected to angular (inverse sine) transformation (arcsin^T), before analyses of variance were carried out on them. 3. Results The AWC of ant nests from Cola, Irvingia, Newbouldia, and termitaria did not differ (P > 0.05) with one another statistically, but were significantly (P = 4.08; d.f = 5; P < 0.05) lower than the AWC of ant nests collected from Mangifera (Table 1). The AWC of M. indica hosted nests (60%) was significantly higher than the AWC obtained from C. nitida (42.3%), I. gabonensis (30.4%), N. leavis (43.3%), termitaria (44.6%), and P. macrophylla (46.7%). Differences amongst the nesting site effects on dispersion ratios (DRs) of nest soils, calgon and water dispersible clay, clay + silt, and silt were not significant. However, there were evident trends of higher calgon dispersible clay + silt and silt and water dispersible clay and clay-silt on arboreal ant nests than on epigeous termite ground mounds. Similarly the AWC (53.8%), calgon dispersible clay (19.2%), clay + silt (36.5%), and silt (15.9%) and water dispersible clay (22.1%), clay + silt (32.8%), and silt (7.7%) were found to be significantly higher on biogenic structures compared to their corresponding adjacent surface soil values of 36.0%, 9.3%, 19.4%, 8.8%, 9.5%, 12.7%, and 3.3%, respectively (Table 2). Although the differences produced by the effect of soil location amongst the dispersion ratio (DR) were not statistically significant, the DR of the biogenic structure was relatively higher compared with those of their adjacent soils. Except for the total dispersible silt which differed signif- icantly when the effect of the nesting sites was combined with the soil types, the AWC, DR, and other calgon or water dispersible soil fractions did not show such statistical differences (P > 0.05) under comparable combined treat- ment effects (Table 3). This is such that ant nests of biogenic structure on I. gabonensis contained significantly (P = 3.75; d.f = 5; P < 0.013) higher calgon dispersible silt (28.6) than other nest structures or adjacent soil, while the adjacent soil around C. nitida (5.6), I. gabonensis (6.6), M. indica (13.6), N. leavis (8.5), P macrophylla (9.6), and the termitaria contained significantly (P = 3.75; d.f = 5; P < 0.013) lower calgon dispersible silt than other soils. However, there were glaring trends of numerically higher AWC, DR, and soil dispersibilities amongst the nest structures collected from any of the substrates than their corresponding adjacent soils. The percent C and N, SOM, base saturation, exchange- able bases, and pH values differed significantly amongst the various soil engineering host substrates (Table 4). The carbon contents of kola (7.32%), bush mango (9.14%), mango (6.98%), newbouldia (7.01%), and oil bean (8.27%) were significantly (P = 1L94; d.f = 5; P < 0.001) higher than the C content of termitarium (1.23%). The same trend also followed in Mg^"^. N was significantly (P = 15.28; d.f = 5; P < 0.001) higher in oil bean compared to samples, while Ca^"^ was higher in Kola (5.15 meq/100 g sample) and newbouldia (6.0 meq/100 g sample) compared to others. 4 Psyche Table 1: Main effects of soil engineers nesting substrates (sites) on the mean available water capacity (AWC), dispersion ratio (DR), and calgon and water dispersibilities of the biogenetic structure’s clay, clay and silt, and silt. Soil engineer Nesting Site AWCi (%) DR Clay Calgon dispersed Clay + silt Silt Clay Water dispersed Clay + silt Silt Ant C. nitida 42.3ab 0.72a 12.5a 23.5a 10.6a 15.2a 17.8a 2.5a Ant I. gahonensis 30.4a 0.71a 16.0a 34.0a 17.6a 20.7a 24.8a 4.1a Ant M. indica 60.0c 0.76a 17.5a 35.0a 14.5a 16.2a 24.8a oo bo Ant N. leavis 43.3ab 0.70a 10.5a 25.0a 11.1a 10.4a 19.8a 5.6a Ant P. macrophylla 46.7b 1.16a 15.5a 27.5a 11.6a 21.7a 33.7a 7.1a Termite Ground 44.6ab 0.71a 13.5a 22.5a 8.6a 10.7a 15.8a 5.1a F- value 4.08 2.11 2.10 0.94 1.16 0.91 0.93 1.60 d.f. 5 5 5 5 5 5 5 5 P value 0.005 0.087 0.088 0.469 0.348 0.485 0.471 0.201 AWC: available water capacity and DR: dispersion ratio. ^ Results of angular transformed data presented in the original scale. Values within a column followed by the same letter are not significantly different. Table 2: Main effect of soil locations on the mean available water capacity (AWC), dispersion ratio (DR), calgon and water dispersed clay, clay/silt, and silt of different soil locations. Soil location mo (%) DR Clay Calgon dispersed Clay + silt Silt Clay Water dispersed Clay + silt Silt Adjacent soil 36.0a 0.69a 9.3a 19.4a oo bo 9.5a 12.7a 3.3a Biogenic structures 53.8b 0.89a 19.2b 36.5b 15.9b 22.1b 32.8i 7.7a t-value 961.23 6.07 39.68 108.0 87.6 287.41 164.28 480.00 d.f. 1 1 1 1 1 1 1 1 P value <0.001 0.220 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 AWC: available water capacity and DR: dispersion ratio. ^ Results of angular transformed data presented in the original scale. Values within a column followed by the same letter are not significantly different. Table 3: Interaction effects of soil engineers’ nesting substrates (sites) and soil locations on the values of some soil properties of biogenic structures and their adjacent soils. Soil engineer Nesting site Soil location mo (%) DR Calgon dispersed Clay Clay + silt Silt Water dispersed Clay Clay + silt Silt Ant C. nitida Adj. soil 30.3a 0.65a 6.5a 12.0a 5.6a 7.2a 7.8a 0.56a B. structure 54.2a 0.68a 19.6a 13.0a 15.6b 23.2a 27.8a 4.56a Ant I. gahonensis Adj. soil 31.0a 0.68a 6.5a 13.0a 6.6a 7.2a po bo 1.56a B. structure 29.7a 0.74a 26.5a 55.0a 28.6c 34.2a 40.8a 6.56a Ant M. indica Adj. soil 40.0a 0.75a 14.5a 33.0a 13.6ab 15.2a 20.8a 6.06a B. structure 83.9a 0.75a 21.5a 37.0a 15.5b 17.2a 28.8a 11.56a Ant N. leavis Adj. Soil 36.2a 0.63a 6.5a 15.0a 8.6a 7.2a 8.8a 1.56a B. structure 50.9a 0.77a 16.6a 35.0a 13.6ab 13.7a 30.8a 9.56a Ant P macrophylla Adj. soil 39.9a 0.73a 13.5a 23.0a 9.6ab 11.2a 16.7a 5.56a B. structure 53.5a 1.60a 18.5a 32.0a 13.6ab 32.2a 50.8a 8.56a Termite Ground Adj. soil 38.4a 0.72a 11.5a 20.0a 8.6ab 9.2a 13.8a 4.56a B. structure 50.8a 0.70a 16.5a 25.0a 8.6ab 12.2a 17.8a 5.56a F- value 0.92 2.44 0.68 2.62 3.75 0.55 0.89 2.14 d.f. 5 5 5 5 5 5 5 5 P value 0.489 0.066 0.641 0.053 0.013 0.737 0.502 0.099 AWC: available water capacity; DR: dispersion ratio; B. structure: biogenic structure; Adj. soil: adjacent soil. ^ Results of angular transformed data presented in the original scale. Values within a column followed by the same letter are not significant different. Psyche 5 c/5 a> u Hi c/5 *s a> b^) O *a> 41^ c/5 ^a> a> Dh O Dh u (i> 44 U o .y *C/5 Dh a> O c/5 C/5 a> cu a *C/5 a> r3 c/5 :is c/5 biD c/5 a> !4 ‘^C/5 a> a> g ’bb a a> O c/5 <-•-( o c/5 U bK a> 3 w 4 CQ ^ c/5 o T3 !=1 03 CO U \p .Eh *C/5 (U ■M CO cb 44 u bO fXi 'Bb a K CL, (/3 c/D ^ CO O ^ CO u . p p p 04 td o 1— 1 hH 4— H 4 — H 0 . Pi 04 eel 0 eel OC CO 00 o CO rO oj 04 3 rO 0 04 0 04 CO L3 CO u -Q bO -Q o u LO Pi eel p (N hH LO 1— 1 04 4— H 3 4— H e — 1 04 ’ ON 'rt -O Pi eel 00 bO CO 4— H bO OC 04 CO bO p e— 1 LO O O o o e— 1 0 4— H -o -o -o pi Pi eel 04 bO 4— H 10 rO p CO hH a^ p P p 1 I a\ 3 td bO e— 1 4 — H e"eb3 ’'G 'S Sb 3 . no o §3 s 0 til ►-0 < < < < < LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO rO CO rO LO o o 0\ bO o (N o o o o o o V o o o V bO 0\ o o o o o V o o o V o o o V cs cs 1— H o o o o o V o o o V o o o V > Oh ‘0 Clh u b/D 4 cb 44 4 .2 cb u U W u 4 O 4 O 'g cb u o 3 o x CL C/) o PC Cl tC Cl 03 Lh 03 O o "d c 03 CO u C 0/ Lh 'd . c (U 03 '5 ^ l3 .SP _C cn ‘ob Lh O OJ PC o c 0/ S-i 03 Li (U a C3 S' 4 cb 1/ (U a cb hJ 4 C3 0/ •M -l-t cb a c cb b/D O C4 jH cb ^ I ol c2 ,p c/5 4 cb u 4 a L ^ 03 O 3 g bC ^ C C 03 3 o :d o ■ : stj 4 (U c/5 ^ 0/ 4= - > 6 Psyche was significantly higher (F = 47.66; d.f. = 5; P < 0.001) in Irvingia (1.74 meq/lOO g sample), mango (1.69 meq/100 g sample), and oil bean (1.78 meq/lOO g samples) relative to other soil engineer nests. The soil pH in H 2 O was lower in kola (6.38), Irvingia (6.75), mango (6.28), and termitaria (6.60) compared to other nesting sites. Although SOM, the base saturation, CEC, P, and particle-size distributions did not differ between ant and termite nests, there were clear trends of numerically higher values in the ant nests compared to the termite nests. Based on Black [30] classifications, the values of organic C obtained amongst the ant nests were high (between 6.98 and 9.14%) compared to termite nests (1.23%). Similarly, SOM content of N. leavis was significantly higher (F = 9.66; d.f. = 5; P < 0.001) than other nests, except those from L gabonensis (15.8%). Base saturation of C. nitida was significantly (F = 3.85; d.f = 5; P < 0.012) higher than those of other nests, except those of Irvingia (36,3%). The N-contents of between 0.37 and 1 .28% obtained amongst ant nests were classified as very high compared to 0.19% obtained from termite nests according to Metson [31] classification. Again, the SOM of between 12% and 16.5% from ant nests was considered high compared to SOM of 2,5% obtained from termite nests according to Metson [31]. Similarly, by FAO [32] classification, all the tree- ant nests except those of P macrophylla with base saturations of between 23 and 41% would be regarded as having medium fertility as opposed to ground-termite nests and F. macrophylla nests with base saturation of 20% each and classified as belonging to soils of low fertility. Similarly, both the tree-ant nests and ground-termite nests have high phosphorus content (23.6-54.2 mg/kg) judging by Enwezor et al. [33] classification. The exchangeable ranged from low to moderately high in tree-ant nest when compared to ground-termite nest with very low [30]. Also was high in tree-ant nests relative to the very low value obtained in the ground-termite nests using the classification of Black [30]. Conversely, Mg^"^ content of tree-ant nests was moderately high compared to the termite nest which was very low after Black [30] classification. Except for N. leavis ant nests with neutral (pH = 7.28) soil reaction, all the ant- and termite-nest soils were slightly acidic with a pH range of between 6.2 and 6.8. The particle-size distribution of the various soil fractions showed that the ecosystem engineers’ nests were all of sandy-loamy soil, while their corresponding adjacent soils were predominantly sandy soils. The result of the soil location effect on chemical compo- sitions showed that biogenic structures contain significantly higher percentages of organic carbon (12.6%), nitrogen (1.10%), organic matter (23.3%), base saturation (33.9%), CEC (51.4%), Ca2+ (4.92%), Mg2+ (5.2%), K+ (2.21%), Na^"^ (0.53%), and phosphorus (44.1%) than their respective adjacent soil nutrient contents of 0.74%, 0.10%, 1.30%, 25.4%, 1.67%, 1.70%, 0.13%, 0.46%, and 16.1% (Table 5). Similarly, both the pH and particle-size distribution of clay-sized (19.20%) and silt-sized (17.9%) particles in the structures were significantly higher for the nest structures compared with those of their adjacent surface soils of 9.3% and 9.7%, respectively. Both the particle sizes of coarse and fine sand fractions as well as the soil reactions in both H 2 O and KCl of the nest structures did not differ significantly with those of their adjacent soil. Differences in the combined effects of the nest substrates of soil engineers and soil locations differed significantly with respect to all the physiochemical properties assessed, except Na"^ content and coarse sand distribution (Table 6). There was a consistent trend of these constituent being higher amongst biogenic structure of ants and termites in the different substrates as opposed to their corresponding adjacent soils. Conversely, in all the chemical properties assessed, the unmodified adjacent soils did not show signif- icant differences amongst the different nest substrates as the nest soils to their microhabitat. Furthermore, the interaction effects of nest sites and soil locations were only significant for C, N, SOM, base saturation. CEC, exchangeable bases (Ca^"^, K"^, and Mg^"^), soil-pH, clay, and silt contents. This is such that they were significantly higher in the various ant-tree nests (biogenic structures) than in ground-termite mound nest and their corresponding surrounding adjacent soils, except amongst the termitaria (Table 6). 4. Discussion Soil engineers, notably ants and termites, have been reported to play important roles in the soil fertility in tropical ecosystems because of their impact on the soils they work on [2, 9, 13] . In the present study, however, efforts were made to ascertain whether these modifications significantly differed between biogenic structures of different soil engineers (ants and termites) and also if the modifications within biogenic structures were different for different nesting sites (trees) or locations. Results of the laboratory assays however showed that the physicochemical properties of the ant biogenic structures were glaringly higher than those of the termites. Tree ants generally are exposed to more diverse range of plant litter diets and foraging materials than ground termites due to their proximity to dense litter falls from tree hosts. There is therefore always an increased resource access associated with ant nest construction relative to termite nest construction. This could therefore be harnessed in biofortifications of our impoverished tropical agricultural soils. Again, not only the soils of nests worked upon by the ecosystem engineers precisely contained higher values of the constituents assessed than their corresponding adjacent soils, but also their nesting sites played a major role in their constituents. Nesting sites differed as to their AWC, DR, and their dispersibilities in both calgon and water but their differences were not significant except for the AWC. Ant nests on M. indica consistently had higher AWC relative to other ant plant nests or epigeous termite ground nest. The higher AWC of ant nests from M. indica biogenic structures suggest higher tendency of ensuring more water in available form for crop development than those of other soils locations or nesting sites, which makes it still a good promise for biofortification. 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G OJ co 5 ^ J2 c l3 .2P G t" !§> O O S <\J u 42 •M ;_, G S • ^ ^-> ii CD s i t/5 C3 ^ Psyche 9 of the soil organic matter content [34] and an index of nutrient availability to plants did not differ either between the nesting sites or between ant nests and termite nests. Soil texture on the other hand was not significantly affected in the termite nest compared with the ant nests, but was considerably modified in the biogenic structures compared to their corresponding adjacent unmodified soil. Thus, the nests and their corresponding adjacent soils differed as to their textural classes. The soil engineers ensured greater mineralization of clay and silt which was found to be greatly higher in the biogenic structures than in the adjacent soils resulting in differences in their textural classes. All the soils affected by ants and termites had a greater proportion of clay and silt to the detriment of coarse and fine sand. This finding suggests that the ecosystem engineers prefer the selection of finer materials such as clay and silt for building of its structures, thus supporting the result of the ability of termites and ants to select building materials when presented with different physical size materials but not clay type [2, 35, 36]. Termites have been observed to favour finer particles in their mound constructions which match their ecological, physiological, and behavioural needs [35]. In this study, however, there was a clear distinction between the physical size distribution of soils worked by ants and those by termites. The greater proportion of clay and silt as opposed to sand on soils worked by ant and termites showed the greater impact of clay and silt than sand on soils worked upon by ants and termites in the course of their building activities. Laboratory analyses of the various samples also showed that both the biota nesting sites and the soil locations richly influenced the SOM, base saturation, CEC, and soil- pH and significantly modified C- and N-contents and their exchangeable bases. It suggests therefore that there was more enrichment of C and N and exchangeable bases (Ca^''', K^, Mg^'*', and Na^) in antnests most especially in antnests built on 1. gahonensis and P. macrophylla compared with the termitarium, as nest structures generally were to unaffected adjacent soils. Tree antnests were richer in C, N, SOM, and exchangeable bases (Ca^^, IC, Mg^"^, and Na"^) compared with the termite nests. This result counteracts our first hypothesis as nesting sites have been shown to influence some of the nest soil properties. Therefore, better nest soil enrichment will be assured when nests are harnessed from 1. gahonensis, N. leavis, and P. macrophylla trees. The ability of ants and termites to work and move through the soil and to build organomineral structures with specific physical, chemical, and microbiological properties has been well documented [15, 37]. The higher mineral contents of the ant-nests relative to the termite nests and the surrounding soils could be explained by (i) the relative intensities with which the two organisms impact on the soil, (ii) the differential efficiencies of the two organisms in the mineralization of the various nutrients, (iii) the quantum of food (organic matter or litter material) available, or their diversity (structural, biochemical, or biological attributes), and (iv) the soil conditions or fertility where the nest soils were formed. That the adjacent soils of the different nest hosts did not differ significantly in this study goes to excluding soil conditions as possible explanation for the differences in the ant and termite nest s chemical content and thus supports the hypothesis that nest soil properties are not a reflection of the initial soil properties when they were unaffected by soil engineers. Jouquet et al. [8, 13] suggests availability of more diversified litter materials at the disposal of the ants than at the disposal of termites as the probable cause of this difference. The quantity of litter materials to be found deposited around trees were likely to be higher than the sparse grassy vegetations reminiscence of a derived savannah ecosystem from where the termite mounds were excavated. Although it has been reported that tropical ant diversity positively correlates with plant structures [38], it was still not clear if litter diversity affects litter nesting assemblages and compositions [39]. From the stand point of this result, ants may be considered to be more efficient in the mineralization of organic nutrients than termites. Enhancement of soil nutrient concentrations according to Wagner [40] maybe of general importance in understanding how plants benefit from interaction with ants, especially if ants are more likely to nest near plants bearing extrafloral nectaries. Also the less abundance of organic matter in soils worked by termites may be attributed to the microenvironment where the mounds are located. Jones et al. [41] observed that termite mounds are usually exposed to intensive sunlight which may reduce the activity of microbes involved in the decompositions of organic matter they contain. All the ant-nest soils irrespective of tree host contain very significantly higher phosphorus content than termite nests. Also the biogenic structures of either ant or termites irrespec- tive of their nesting sites were found to contain significantly higher P-content than those of their corresponding adjacent surface soils. Ant tree nests were between 81 and 130% richer in P-content than termite ground nests. Similarly, the biogenic structures generally were about 348% richer in P- content than their corresponding adjacent soils. The richer P-content of ant- nested structures of trees relative to the ground termite mounds could still be explained by the higher SOM content of the former as opposed to the latter and the efficient mineralization potential of ants over termites. The rich P-content of the nest soil especially the tree nests could enable them be recommended in the biofortification of acidic tropical soils deficient in phosphorus induced by Fe- and/or Al-toxicity problems. 5. Conclusion The physicochemical properties of arboreal ant nests, the ground-termite nests, and their corresponding adjacent soils were studied in addition to the influence of nesting sites on nest soil characteristics. Nesting site influenced nest soil char- acteristics. Ant nests pried from tree tops especially those from M. indica were rich in most of the chemical properties assessed compared to those of epigeous ground-termite nests or the adjacent surface soil samples. Holistically, soils altered by soil engineers, notably ants and termites, were also predominantly greater in these basic nutrients relative to 10 Psyche their adjacent unmodified soil, whereas soils collected from trees and ground nests were either sandy-loam or sandy-clay- loam in texture; those of their corresponding adjacent surface soils were mainly sandy for soils near trees and loamy-sand for soils near termitaria. The higher influence of ants than termites on the maintenance of ecosystem heterogeneity through its soil bio- perturbation effects was therefore revealed in this work. Ants in addition to termites have also been seen to influence soil properties by making resources available for other organ- isms. These findings and more could therefore be harnessed by culturing these organisms and protecting some of these prospective nesting sites like M. indica, R macrophylla, and L gabonensis capable of hosting nests of high mineral content. Such nests could be harnessed for a variety of bio-remediation and biofortification purposes for human use. Purposes where the soil engineers could be utilized include landfills to decompose waste, improvement of soils by composting materials, detoxification of hazardous sub- stances, and the production of biomass of animal feed and biochemical. Besides being fed upon by animals, solid wastes constituting environmental hazards are decomposed and readily converted into useful forms for soil improvement. The soil engineers -mediated chemical changes of soil, com- monly represented mainly by a shift in pH towards neutral from acidity and an increase in nutrient content, helps in soil detoxification. References [1] B. Holldobler and E. O. Wilson, The Ants, Springer Verlag, Berlin, Germany, 1990. [2] P. Jouquet, R Barre, M. Lepage, and B. Velde, “Impact of subterranean fungus-growing termites (Isoptera, Macrotermi- tiane) on chosen soil properties in a West African savanna,” Biology and Fertility of Soils, vol. 41, no. 5, pp. 365-370, 2005. [3] M. Anderson, “Australian termites and nutrient recycling,” Biology, vol. 394, pp. 1-11, 2005. 14] K. E. Lee and R. C. Eoster, “Soil fauna and soil structure,” Australian Journal of Soil Research, vol. 29, no. 6, pp. 745-775, 1991. 15] J. C. Leprun and J. 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Wagner, “The influence of ant nests on Acacia seed production, herbivory and soil nutrients,” Journal of Ecology, vol. 85, no. 1, pp. 83-93, 1997. [41] C. G. Jones, J. H. Fawton, and M. Shachak, “Organisms as ecosystem engineers,” Oikos, vol. 69, no. 3, pp. 373-386, 1994. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 503543, 5 pages dohlO.l 155/2012/503543 Research Article Effect of Plant Characteristics and Within-Plant Distribution of Prey on Colonization Efficiency of Cryptolaemus montrouzieri (Coleoptera: Coccinellidae) Adults Folukemi Adedipe and Yong-Lak Park Department of Entomology, West Virginia University, Morgantown, WV, USA Correspondence should be addressed to Yong-Lak Park, yopark@mail.wvu.edu Received 2 October 2011; Accepted 20 December 2011 Academic Editor: Martin H. Villet Copyright © 2012 F. Adedipe and Y.-L. Park. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cryptolaemus montrouzieri (Coleoptera: Coccinellidae) has been widely used in classical and inundative biological control of mealybugs, including the long-tailed mealybug. Pseudococcus longispinus (Hemiptera: Pseudococcidae). This study was conducted to investigate colonization and establishment efficiency of C. montrouzieri to manage P. longispinus on three different ornamental plant species {Ficus elastica, Lilium longiflorum, and Dieffenhachia seguine). Within-plant distribution pattern ofP. longispinus and the colonization ecology of adult C. montrouzieri were investigated. Significantly more P. longispinus were found on the upper parts of the plants regardless of plant species, and C. montrouzieri adults discovered P longispinus significantly faster when they were released on the top of the plants than on the bottom. Choice tests revealed that C. montrouzieri adults preferred smaller P longispinus nymphs. The implications for utilization of C. montrouzieri for biological control of mealybugs on various ornamental plants are discussed. 1. Introduction The long-tailed mealybug, Pseudococcus longispinus (Targioni-Tozzetti) (Hemiptera: Pseudococcidae), is a key pest of fruit trees and ornamental plants, P. longispinus feeds on various plant parts including roots, trunks, cordons, canes, leaves, and fruits, causing aesthetic damage on ornamental plants or yield loss of crops [1]. Fungal patho- gens that grow on the honeydew excreted by P longispinus can cause further damage. For example, high P longispinus densities often cause leaf drop and reductions of crop quality and yield; Uygun [2] reported that yield loss of citrus due to P longispinus could be up to 80-90%. Also, P longispinus can transmit viral diseases in grapevines [3]. Chemical management of P longispinus is difficult because it produces thick layers of protective wax and can hide in bark crevices, spurs, or canes. In general, chemical control is only effective when P longispinosus is in the crawler stage and when host plants do not afford physical refuges from chemical sprays [4], Therefore, biological control using natural enemies has been a major alternate method to manage P longispinus [5]. Natural enemies utilized to manage P longispinus include lady beetles, parasitic wasps, and lacewings [6]. Among the natural enemies, the mealybug destroyer, Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae), is one of the key natural enemies of P longispinus. C. montrouzieri is native to Australia and has been introduced to manage many mealybug species throughout the world [7, 8]. In the United States, C. montrouzieri was first imported in the late 1800s to manage mealybugs in California [9]. Since then, well- defined and efficient rearing techniques were developed [ 10] , and thus C. montrouzieri has been commercially available to growers throughout the United States. Cryptolaemus montrouzieri has been used for different biological applications: classical biological control [11] and augmentative biological control [12, 13]. In an established population, immature stages of C. montrouzieri dominate the stable age distribution, and most prey is consumed by 2 Psyche the larvae [14]. However, adult stage of C. montrouzieri is released when biological control of P. longispinus is initiated because of their ability to disperse and colonize. Also, it is assumed that adults will lay eggs in suitable locations and give rise to another generation that provides the majority of pest suppression when they are in the larval stage. Effectiveness of natural enemies is dependent upon the ability of the organism to establish populations in a given environment and find prey rapidly [15]. Previous studies showed that natural enemies’ ability to establish and search for prey was affected by plant structure and size [16-20]. Garcia and O’Neil [15] showed that plant size and variega- tion affected the searching efficiency of C. montrouzieri, and Merlin et al. [21] found that oviposition of C. montrouzieri was stimulated by wax filaments produced by its prey. Also, these studies indicated that successful biological control of P. longispinus would be affected by how efficiently newly released C. montrouzieri adults search for P. longispinus. Specifically, there is a high chance for C. montrouzieri adults to successfully establish when they can start to search and find the suitable prey as soon as they are released. In addition, prey-size choice could affect successful colonization of predators [22] . Therefore, key factors influencing prey search efficiency of C. montrouzieri may include release location of C. montrouzieri based on within-plant distribution of P. longispinus, plant characteristics, and stages of P. longispinus that C. montrouzieri adults prefer. This study was conducted to investigate colonization efficiency of C. montrouzieri to manage P. longispinus on three different types of ornamental plants. The objectives of this study were (1) to investigate within-plant distribution of P. longispinus, (2) to quantify the searching time of C. montrouzieri related to release location, and (3) to determine preference of C. montrouzieri adults for the size of P. longispinus. 2. Materials and Methods All experiments were conducted in the greenhouse and the entomology laboratory of West Virginia University, Morgantown, WV, U.S.A. 2.1. Within- Plant Distribution ofP. longispinus . We obtained three common species of ornamental plants from the greenhouse at West Virginia University (Monongalia County, WV, USA). The ornamental plants in this study include Ficus elastica (Urticales: Moraceae) (86-94 cm in height), Lilium longiflorum (Liliales: Liliaceae) (42-61 cm in height), and Dieffenbachia seguine (Alismatales: Araceae) (23-27 cm in height). These plants were selected because they are very common ornamental plants produced in the greenhouse. These plants had been infested with P. longispinus for at least one year before experiments to obtain moderate-to- high density of P. longispinus. Five plants of each plant species with similar infestation levels were selected, and the total numbers of P. longispinus nymphs and adults were counted on the upper and lower halves of each plant. Densities of P. longispinus on upper and lower parts of each plant species were compared using two-way AN OVA at 5% error rate [23] . 2.2. Prey Searching Time of C. montrouzieri Adults on Three Different Plant Species. C. montrouzieri adults were maintained in ventilated cages with P. longispinus and a honey- water solution under laboratory conditions of 25° C and 16 : 8 (L : D) h photoperiod. C. montrouzieri were reared on P. longispinus. All C. montrouzieri adults were denied prey but had access to water for the 12 h period preceding all experiments. One randomly chosen C. montrouzieri adult was introduced onto either top (i.e., top shoot) or bottom (i.e., bottom part of stem within 2 cm above the soil line) of a plant. Once one C. montrouzieri adult was introduced to a plant, searching time of C. montrouzieri was measured. Searching time was measured as the duration between introduction and finding the first prey. This experiment was replicated five times for each plant species and each release location. Each C. montrouzieri adult was used for the test only once. The searching time of C. montrouzieri was recorded and analyzed with two-way (i.e., releasing locations and plant species) ANOVA at 5% error rate (SAS Institute, 2008). Because we used the same plants for repeated release of different C. montrouzieri adults in the experiment, any leftover chemical cues by previously used C. montrouzieri adults could affect the next adults introduced to the plant. Therefore, we examined the effect of leftover chemical cues by previously used C. montrouzieri adults on the next adults introduced to the plant by dividing the data into two groups: first ten and last ten introductions of C. montrouzieri. The searching time of the two groups was compared ANOVA at 5% error rate [23]. 2.3. C. montrouzieri Preference to Prey Body Size. A total of 20 C. montrouzieri adults were denied prey but had access to plain water for the 12 h period preceding the experiment. Preference of C. montrouzieri for three different sizes of P. longispinus (0.3 ± 0.07, 1.3 ± 0.10, and 3.0 ± 0.14 mm) was investigated using an empty 9-cm-diameter petri dish (EAB- TEK Division Miles Eaboratories, Inc., Naperville, IE, USA) containing an excised leaf of F. elastic on the bottom of the Petri dish. Three P. longispinus with different body sizes were randomly placed on the leaf for each replication. One C. montrouzieri adult was placed in the center of the Petri dish and allowed to search for P. longispinus. C. montrouzieri adults’ choice among the three different sizes ofP. longispinus and handling and cleaning time was recorded. Handling time was measured from the start of first contact of P. longispinus by C. montrouzieri to cessation and included feeding. Cleaning time was measured as duration of waxy residue removal from the body. This choice test was run until the first nymph was consumed and replicated 20 times. Searching, handling, and cleaning times were compared using ANOVA at 5% error rate [23], and the first choices by C. montrouzieri adults to feed on the three different body sizes of P. longispinus were compared using Chi-square test [24]. Psyche 3 Table 1: Mean (±SD) number of P. longispinus on the upper and lower parts of three different plants. Note that there were no significant differences within columns. Plant species Upper part of plant Lower part of plant Ficus elastica 415 ± 264.5a* no ± 93.2b Lilium longiflorum 564 ± 172.4a 51 ± 26.8b Dieffenbachia seguine 441 ± 154.1a 51 ± 40.4b * Means within rows followed by the same letter are not significantly different {F test, P < 0.05). 3. Results 3.1. Within-Plant Distribution ofP. longispinus. There were no significant differences in the total number ofP. longispinus among the plants used in this study {F = 1.21; df = 2,57; P > 0.05). However, significantly {F = 57.4; df = 1,29; P < 0.0001) more P. longispinus were found on the upper parts of the plants regardless of the plant species (Table 1). There were no significant differences in P. longispinus densities among the three plant species when P. longispinus densities on the upper and lower parts were compared: upper parts of plants {F = 0.46; df = 2,14; P = 0.650) and the lower part of plants (P = 0.94; df = 2,14; P = 0.443). 3.2. Prey Searching Time of C. montrouzieri Adults on Three Different Plant Species. The time C. montrouzieri spent to find the first P. longispinus was significantly different among the three different plant species (P = 7.9; df = 2,24; P = 0.002) and the two different release points (top and bottom) (P = 29.73; df = 1,24; P < 0.001). Because interactions between plant species and release points (P = 7.37; df = 2,24; P < 0.001) were significant, we separately compared the time C. montrouzieri spent to find the first P. longispinus between two release locations for each plant height category. We found that C. montrouzieri spent significantly more time searching for P. longispinus when they were released from the bottom of the plants regardless of plant species (Figure 1). This indicates that prey-searching time for C. montrouzieri adults to find the first P. longispinus can be reduced by releasing them from the top of the plant. We found that there were no differences in the searching (P = 1.93; df = 1,18; P > 0.05), handling, and cleaning time (P = 1.21; df = 1,58; P > 0.05) between the first ten C. montrouzieri introduced to the plants and the next ten introduced. This indicates that there were no significant effects of chemical cues left, if any, on the searching behavior of C. montrouzieri adults in this study. 3.3. C. montrouzieri Preference to the Body Size ofP. longispi- nus. The results of the preference test of C. montrouzieri adults for three different sizes of P. longispinus showed that there was significant = 9.109; df = 2; P < 0.05) preference of C. montrouzieri adults for smaller P. longispinus compared to medium (;(^ = 7.619; df = 1; P < 0.01) and larger = 8.314; df = 1; P < 0.01) sizes. Although there were no significant differences in handling time of C. montrouzieri Figure 1: Effects of C. montrouzieri release locations and plant species on the time C. montrouzieri spent to find the first P. longispinus. Note that there were significant differences (P test; P < 0.05) between two release locations regardless of plant species. Table 2: Handling and cleaning time (minutes ± SD) of C. montrouzieri adults feeding on different sizes of mealybug. Mealybug size (mean ± SD) Handling time Cleaning Time Total Small (0.3 ± 0.07 mm) 6.9 ± 2.18a* 3.5 ± 2.03b 10.4 ± 4.21b Medium (1.3 ± 0.10mm) 10.3 ± 4.35a 4.5 ± 2.08b 14.8 ± 6.43ab Large (3.0 ± 0.14 mm) 12.0 ± 7.00a 8.7 ± 1.53a 20.7 ± 8.53a * Means within columns followed by the same letter are not significantly different {F test, P < 0.05). feeding on different sizes ofP. longispinus (P = 3.25; df = 2,17; P = 0.064), there were significant differences in cleaning time among C. montrouzieri feeding on different sizes of P. longispinus (P = 8.14; df = 2,17; P = 0.003) (Table 2). This result indicates that C. montrouzieri adults choose smaller P. longispinus more frequently and spend significantly more time to clean after feeding on larger P. longispinus. 4. Discussion Within-plant distribution of pests is key information for determining where to release natural enemies. To maximize efficiency of biological control, prey-searching time could be reduced depending on where natural enemies are released [18, 25]. In this study, we observed that significantly more P. longispinus inhabited the upper part of plants regardless 4 Psyche of plant species. This observation is in agreement with the finding by Flaherty et al. [ 1 ] who showed higher population and movement of mealybugs toward the top of grapevines. Because C. montrouzieri is known to be more effective when P. longispinus populations are high [8, 26], releasing C. montrouzieri adults from the top of the plants could reduce prey-searching time because P. longispinus is abundant on the upper part of plants. In addition, plant species could affect prey-searching efficiency by natural enemies [ 15] . The results of our study indicated that effectiveness and establishment of C. montrouzieri increased when they were released on Dieffenbachia seguine, the smallest plant tested in this study. Therefore, the effectiveness of C. montrouzieri managing P. longispinus could be maximized when C. montrouzieri are released from the top and on smaller plants. This study demonstrated that C. montrouzieri chose to feed on smaller P. longispinus. When C. montrouzieri fed on larger nymphs, they spent longer time handling and cleaning after feeding and before searching for another prey. This result is congruent with Merlin et al. [21] who found that C. montrouzieri consumed smaller P. longispinus nymphs first and then fed on larger nymphs or adults. Because establishment of natural enemies after release determines the success of augmentational biological control (i.e., inoculative release), finding the first prey by the natural enemies could increase the chance of establishment. Although a difference of 5-15 minutes in time to initial prey encounter may not make much of a difference to the final biological control outcome in a greenhouse, it could influence the chance of establishment because we frequently observed that C. montrouzieri adults could fly away to escape from the green- house when they cannot find the first prey in a reasonable period. Therefore, C. montrouzieri adults have higher chance to establish when the period for finding the first prey is shorter. The results of this study suggest a major consideration for the use of C. montrouzieri to manage P. longispinus. The efficiency of P. longispinus management by C. montrouzieri depends on the location of C. montrouzieri release and the plant species. Our study showed that effectiveness and establishment of C. montrouzieri managing P. longispinus could be maximized when C. montrouzieri were released from the top of the plants and on the smaller plants, C. montrouzieri adults can reduce prey search time when they are released where P. longispinus is abundant. Future study needs to investigate the effect of plant age and stage on vertical distribution of P. longispinus on the plant. Acknowledgments The authors thank Vicki Kondo and JB White at West Vir- ginia University for their valuable support and suggestions related to this research. They also thank Sue Myers at West Virginia University for her help in identifying plant species used in this research. This project was partially supported by the Division of Plant and Soil Science and the Faculty Senate Grant at West Virginia University. References [ 1 ] D. L. Flaherty, L. Christiensen, and W. T. Lanini, “Mealybugs,” in Grape Pest Management, D. L. Flaherty, L. P. Christensen, W. T. 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Ereeman and Gompany, New York, NY, USA, 1998. [25] Y. Jinsong and G. S. Sadof, “Variegation in Coleus blumei and the life history of citrus mealybug (Homoptera: Pseudococ- cidae),” Environmental Entomology, vol. 24, no. 6, pp. 1650- 1655, 1995. [26] D. Moore, “Agents used for biological control of mealybugs (Pseudococcidae),” Biocontrol News and Information, vol. 9, pp. 209-225, 1988. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 794683, 5 pages dohlO.l 155/2012/794683 Research Article Attraction of Tomicus yunnanensis (Coleoptera: Scolytidae) to Yunnan Pine Logs with and without Periderm or Phloem: An Effective Monitoring Bait Rong Chun Lu,^’^ Hong Bin Wang,' Zhen Zhang,' John A. Byers,^ You Ju Jin,^ Hai Feng Wen,^ and Wen Jian ShP ^ Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry and The Key Laboratory of Forest Ecology and Environment, State Forestry Administration, Beijing 100091, China ^ College of Urban Construction, University of Shanghai for Science and Technology, Shanghai 200093, China ^ US. Arid-Land Agricultural Research Center, USDA-ARS, 21881 North Cardon Lane, Maricopa, AZ 85138, USA ‘^College of Plant Sciences, Beijing Forest University, Beijing 100083, China Correspondence should be addressed to Hong Bin Wang, wanghb@caf.ac.cn Received 6 October 2011; Revised 13 December 2011; Accepted 13 December 2011 Academic Editor: Qing-He Zhang Copyright © 2012 Rong Chun Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Yunnan pine shoot beetle, Tomicus yunnanensis Kirkendall and Faccoli (Coleoptera: Scolytinae) is an important pest of Yunnan pine {Pinus yunnanensis Franch) in China. Experiments with host log baits were done to develop a pest monitoring system using host tree kairomone. Five Yunnan pine logs (each 10-15 cm diam. x 30-cm long) in a trap-log bundle were treated by peeling periderm (outer bark) off to expose the phloem, and half of each log was covered with sticky adhesive to capture any attracted adult beetles. Significantly, more beetles were attracted and caught on the periderm-peeled logs (ca 30 beetles/m^ log surface/ day) than on untreated control logs with adhesive (ca 2.5/m^/day). No significant differences were observed between catches on logs taken from lower or upper halves of Yunnan pines. T yunnanensis flies mostly during the afternoon according to trap catches throughout the day. Attraction to the periderm-peeled logs decreased considerably when they were peeled further to remove the phloem, indicating phloem volatiles play a role in selection of the host by the beetle. The readily- available log baits appear useful for monitoring pine shoot beetle populations in integrated pest management programs. 1. Introduction Tomicus yunnanensis (Coleoptera: Scolytinae) is a newly discovered and aggressive species of pine shoot beetle [1, 2]. It was confused with T. piniperda (L.) in the past [1-3]. Recent studies show there are clear morphological, genetic, and ecological differences between these two species [2-4]. Compared with T. piniperda, T. yunnanensis is more harmful because it not only causes great growth losses, but also kills healthy pines by mass attack [4-6]. It has caused extensive mortality of Yunnan pines, Pinus yunnanensis Franch, in Yunnan province of China [1, 7]. Since the 1980s, more than 200,000 ha Yunnan pine forests have been killed [3, 4, 7, 8] . T. yunnanensis attacks trunks during maturation feeding in Yunnan province [5, 7]. Trunk attacks last six to seven months from December to May, and peak attacks occur from January to March. Attacks on trunks begin in the crown and then spread down the bole. New adults fly to the shoots starting in March, the peak flight emerging in mid June. Maturation feeding lasts six to eight months [3, 5, 7, 8] . The extensive damage by T. yunnanensis indicates that as in other pine systems, effective monitoring and control are necessary [9-11]. However, there are no effective methods to manage the insect so far. It is reasonable to suspect there may be plant compounds from Yunnan pines that are attractive [7, 8, 12] . However, it is not known whether the beetles use a kairomone of Yunnan pines to locate hosts. The most popu- lar method in the past has been to use trap log bundles to see if they are attractive to the beetles. This method is simple to try, but requires detection of odor signals (kairomone) from 2 Psyche host or associated organisms that can be difficult to observe with catches of beetles using many logs [8, 13, 14] . Our objec- tive was to design experiments that tested whether Yunnan pines release an attractive kairomone from various periderm- and phloem-peeled bait logs that could serve as an effective method for monitoring and potential control of T. yun- nanensis. The research should provide methods that avoid unnecessary work and improve bait log efficiency [15, 16] . 2. Materials and Methods 2.1. Study Site Conditions. A series of experiments were con- ducted in a 300 ha plantation of Yunnan pines located on the mountain near Qujing city in Yunnan province (25°14'N, 103°50', and 1700-1800 m above sea level). The field site mainly consisted of Yunnan pines, Pinus yunnanensis L., and a small proportion (< 10%) of broad-leaved trees. Most of the Yunnan pines were infested with T. yunnanensis. The trees were 30-45 years old and ranged from 10-15 m in height and 10-15 cm in diameter. 2.2. Experimental Design. Two experiments were conducted from December to February in 2006 and 2007. In the first experiment, to avoid any sex pheromone effects, six healthy Yunnan pines were cut whose trunks were free of bark beetle attacks (no obvious beetle entrance holes). All the tree trunks were cut into 30-cm long logs. Among all the logs, 30 cut logs were randomly selected and the surface area of each log mea- sured, then the periderm (outer bark) of the logs was peeled off leaving the phloem exposed (Figure 1(a)), and every five treated logs were placed in a trap log bundle. Another 30 cut logs were treated the same, but the phloem was also peeled from the logs (Figure 1(b)). The last 30 cut logs had only their surface area measured and were not peeled (Figure 1(c)). Half the surface area of each log (15 cm) was covered with sticky adhesive supplied by Hebei Academy of Forestry to catch any bark beetles that landed. Finally all the cut logs in the 18 bundles were randomly hung at about 1.5- 1.7 m height in trees. The distance between one trap log bundle to another was about 100-120 m. In addition, six healthy Yunnan pines were randomly chosen and the trunk periderm was peeled off for 1.5 m of the trunk upward beginning 0.6 m above the ground, but the phloem was left in good condition, then half of the peeled trunk surface was covered with the adhesive every other 15-cm height interval (Figure 1(d)). All six trees remained standing. The distance between each of these trees was also about 100- 120 m. The trap catches of the bark beetles were collected daily and species distinguished with a binocular microscope. This experiment was repeated monthly for three months (December to February in 2006 and 2007); with results of all months pooled to obtain a daily mean trap catch. In the second study, four healthy Yunnan pines were cut down, each tree divided into two sections from the middle (lower half and upper half). Then a 1.5 m long log was cut from each section, and all the periderm peeled off with the phloem remain in good condition. Finally, each log was divided into five 30 cm long bolts and these were put in one trap log bundle; half the surface of each piece was covered with adhesive. The bundles from the top -half section and the bottom-half section were arranged in pairs (to give almost equal diameter) and hung up in trees at 1.5- 1.7 m height. The distance between each trap log bundle was about 100- 120 m. The experiment was replicated each of three months for two years as in the first experiment. The trap catches of the bark beetles were collected each day and species distin- guished with a binocular microscope. 2.3. Data Presentation and Analyses. Data were checked for normality and presented as mean ± standard error. Variance analysis (ANOVA) was conducted with SPSS 11.5 (SPSS Inc., Chicago, IL, USA). 3. Results The trapping efficiency of log bundles for T. yunnanensis was greatly affected by the log treatment method (Figure 2). Logs with the periderm peeled off and the phloem left were most attractive to T. yunnanensis, attracting roughly 30 T. yunnanensis per day per square meter log surface. There were significant differences between the trap catches of T. yunna- nensis among the other log treatment methods (P < 0.05, Tukey’s multiple comparison). The other two methods of treating the trap log bundles attracted much fewer T. yunna- nensis. However, the logs with both periderm and phloem peeled off attracted slightly, but not significantly, more T. yunnanensis than the logs not peeled. The uncut pine trees attracted the fewest T. yunnanensis. Dissection of the trunk (results not shown) indicated that the distribution of the entrance holes of T. yunnanensis is mainly in the mid and upper sections of Yunnan pine trees. Whether this pattern is affected by variations in a kairomone of Yunnan pine is unknown. To investigate this, logs from dif- ferent parts of Yunnan pine trees were compared for attrac- tion of T. yunnanensis. We found that logs from the upper half of Yunnan pine trees attracted a little more T. yunnanen- sis than sections from the lower half of the tree, but there was no significant difference (P < 0.05, Tukey’s paired-samples test. Figure 3). The trap catches of T. yunnanensis during the day show that T. yunnanensis was trapped in the afternoon, the peak capture rate occurred from 14:00 to 18:00, while a few were trapped from 7:00 to 14:00. The peak time of capture was strongly affected by weather conditions; occurring a little ear- lier or later, or not at all under poor weather conditions. Figure 4 shows results under good weather conditions. 4. Discussion The periderm is important to trees; the outer bark slows the release of water and organic volatiles from inside the tree. Once logs were peeled, their volatiles were released in much larger amounts, allowing the peeled logs to attract more T. yunnanensis than the unpeeled ones. Yunnan pine bark is known to contain many different monoterpenes (primarily a-pinene and 3-carene) somewhat similar to Scots pine [17]. Psyche 3 (c) (d) Figure 1; (a) Trap log bundles with periderm peeled olF. (b) Trap log bundles with periderm and phloem peeled off. (c) Intact (not peeled) trap log bundles, (d) Standing Yunnan pine tree with periderm peeled off and every other 15 cm section covered with sticky adhesive (Qujing, Yunnan, China). These volatiles may comprise a kairomone of various monot- erpenes that was shown attractive to another Tomicus species, T. piniperda, in Sweden that colonizes Scots pine [12]. Inter- estingly, the peeled logs that retained the phloem were much more attractive to T. yunnanensis than the logs peeled of both periderm and phloem. This is surprising since monoterpenes should exude from the xylem tissues and cause attraction. However, this was not the case. Observations during the experimental study revealed that all the beetle galleries were found in the phloem, which means the phloem is the main food of the beetle, as in most bark beetles [ 18, 19] . The results suggest the odors released from this part of the tree are used as the kairomone by the beetles when searching for food. The phloem is the tree’s main transport channel of photosyn- thetic nutrition. Perhaps the energy from phloem is needed to continue releasing resin (and kairomone). In any case, the experimental results from the logs treated by peeling off the phloem shows that once the log has lost its phloem, its ability to attract beetles is greatly weakened. Trunk attacks by T. yunnanensis are a major cause of tree mortality in Yunnan province [20]. It is reasonable to use kairomone of Yunnan pine trees to monitor and aid in con- trol of the beetles. Natural kairomone would be expected to be as effective as the best synthetic blends that require much research to elucidate. As an alternative to synthetic baits, one of the most popular methods is to use trap log bundles to monitor population levels of beetles. This method is simple and uses readily available resources, but does require signif- icant labor and many logs. We peeled logs to improve the quantities of volatile chemical compounds released, making it possible to use fewer logs and trap more bark beetles. In addition, the peeled logs make it easier to count beetles that 4 Psyche .Vi 5-( a K ^ K ^ 3 (L> aj O 3 tn CT ^ C/5 >- c (u S &H t3 w Cl, Cl, iS Lh 3 4 Figure 2: Daily mean trap catches (±SE, N = 180) of T. yunna- nensis attracted to: (1) periderm peeled logs, (2) periderm and phlo- em peeled logs, (3) intact (unpeeled) logs, and (4) standing peri- derm-peeled Yunnan pines (Dec.-Feb. 2006, 2007; Qujing, Yunnan, China). Figure 3: Daily mean trap catches (±SE, N = 180) of T. yunna- nensis on logs from lower-half (Down) and upper-half (Up) sections of Yunnan pines (Dec.-Eeb. 2006, 2007; Qujing, Yunnan, China). Eigure 4; Mean trap catches (±SE) per hour of T. yunnanensis on periderm-peeled log bundles during the day (Dec.-Eeb. 2006, 2007; Qujing, Yunnan, China). Most T. yunnanensis were trapped in the afternoon; may- be because T. yunnanensis was more flight active in the after- noon. Consequently, the afternoon is the best time to study bark beetle behavior in regard to host selection and coloniza- tion. In nature, most of the T. yunnanensis attacks are found in the top section of the tree [25] but when we used different sections of the tree to attract and trap the beetle, there were no significant differences among the sections. Thus, there appears to be no obvious differences in kairomone release among the different sections from the lower and upper halves of the tree. Flere we present an efficient method to determine wheth- er the kairomone of the host trees can be used to trap pest bark beetles, and whether it is possible to produce attractive baits from the host tree. Pine logs peeled of periderm as baits are easier and more effective compared with traditional log bait methods that use logs with intact bark. are attracted and caught, especially when the surface of the peeled logs is covered with sticky adhesive. Our experimental results show the logs peeled of periderm (outer bark) trapped the most T. yunnanensis. While the standing trees peeled in the same way attracted the fewest bark beetles. The standing trees should have not only had constitutive defenses but also inducible defenses [21-24], The periderm was the most important defensive factor that protected the trunks. When the periderm of trees was peeled off, the constitutive defenses lost effectiveness to some extent, but this damage activated the strong inducible defenses [23] . If the trees were cut down, they lost the inducible defenses. The periderm is important because it not only retards water loss from trunks, but also prevents volatiles from rapidly evaporating from the phlo- em’s resin canals. When the periderm was peeled off, the re- lease rate of volatiles likely increased greatly and enhanced attraction. Logs which were not peeled trapped only a few bark beetles as did the logs peeled to remove phloem. This may happen because most of the kairomone came from phlo- em, so when the phloem was lost, the attractive ability was reduced, indicating that the phloem was the most important source of the kairomone. However, whether the kairomone is synthesized in phloem is uncertain. Acknowledgments The authors thank the Forest Pest Control Station of Qujing City, Yunnan province, for logistical support and field assis- tance. This study was funded by the Importing International Advanced Agricultural Science and Technology Research (2002-38) and the International Technological Cooperation Research (2006DFA31790). 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 401703, 10 pages doi:10.1155/2012/401703 Research Article Case Study: Trap Crop with Pheromone Traps for Suppressing Euschistus servus (Heteroptera: Pentatomidae) in Cotton P. G. Tillman^ and T. E. CottrelF ^ USDA, ARS, Crop Protection and Management Research Laboratory, P.O. Box 748, Tifton, GA 31793, USA ^ USDA, ARS, Southeastern Fruit & Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA 31008, USA Correspondence should be addressed to P. G. Tillman, glynn.tillman@ars.usda.gov Received 14 September 2011; Revised 9 November 2011; Accepted 23 December 2011 Academic Editor: Antonio R. Panizzi Copyright © 2012 P. G. Tillman and T. E. Cottrell. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The brown stink bug, Euschistus servus (Say), can disperse from source habitats, including corn, Zea mays L., and peanut, Arachis hypogaea L., into cotton, Gossypium hirsutum L. Therefore, a 2-year on-farm experiment was conducted to determine the effectiveness of a sorghum (Sorghum bicolor (L.) Moench spp. bicolor) trap crop, with or without Euschistus spp. pheromone traps, to suppress dispersal of this pest to cotton. In 2004, density of E. servus was lower in cotton fields with sorghum trap crops (with or without pheromone traps) compared to control cotton fields. Similarly, in 2006, density of E. servus was lower in cotton fields with sorghum trap crops and pheromone traps compared to control cotton fields. Thus, the combination of the sorghum trap crop and pheromone traps effectively suppressed dispersal of E. servus into cotton. Inclusion of pheromone traps with trap crops potentially offers additional benefits, including: (1) reducing the density of E. servus adults in a trap crop, especially females, to possibly decrease the local population over time and reduce the overwintering population, (2) reducing dispersal of E. servus adults from the trap crop into cotton, and (3) potentially attracting more dispersing E. servus adults into a trap crop during a period of time when preferred food is not prevalent in the landscape. 1. Introduction Agronomic crops across the southeastern US face significant economic losses from stink bugs (Hemiptera: Pentatomidae), mainly the southern green, Nezara viridula (L.), the brown, Euschistus servus (Say), and the green, Chinavia hilaris (Say) [I]. For example, in cotton (Gossypium hirsutum L.), eradication of the boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), along with adoption ofBf-crops has decreased use of broad-spectrum insecticides leading to the emergence of stink bugs as major pests [2] . Stink bugs are generalist feeders that exhibit edge- mediated dispersal from early-season crops into subsequent adjacent crops as adults forage for food and sites for ovi- position [3-10]. Each year in Georgia, corn (Zea mays L.) is one of the earliest agronomic host plants available to stink bugs [II, 12], with peanut (Arachis hypogaea L.) and cotton being mid-to-late-season hosts for these pests [13, 14]. Where these three crops are closely associated in farm- scapes, E. servus disperses from corn into peanut and cot- ton and from peanut into cotton at the common boundary, or interface, of the source crop and cotton [15, 16]. One strategy for managing dispersing pests is trap crop- ping where an attractive plant species is used to arrest the pests and reduce their likelihood of entering a crop field [ 17] . Trap crops have been shown to effectively manage stink bugs in conventional and organic crop production systems [18-22]. Grain sorghum (Sorghum bicolor (L.) Moench spp. Bicolor) is an important host plant for panicle-feeding stink bugs in Georgia [23], and it can suppress populations of N. viridula in farmscapes in Georgia [24]. The pheromone of Euschistus spp. is attractive to males, females, and nymphs of E. servus and other Euschistus spp. [25]. A pyramidal trap [26] was modified [27] to facilitate stink bug capture. When these capture traps contain lures of 2 Psyche Table 1: Planting date (PD) and variety for cotton in sorghum trap crop with pheromone traps (STC/PTs), sorghum trap crop (STC), and control (CO) fields in 2004 and 2006. Year Treatment Rep Variety PD 1 &2 Deltapine 449 5/6 STC/PT 3 Fibermax 960 5/10 4 Deltapine 555 5/8 1 Deltapine 458 5/6 STC 2 Fibermax 960 4/23 3 &4 Deltapine 555 5/6 2004 5 Deltapine 555 5/15 1 Deltapine 444 5/31 2 &3 Deltapine 555 5/6 CO 4&5 Deltapine 555 5/8 6 Fibermax 960 5/12 7 Fibermax 960 4/29 8 Deltapine 555 5/15 1 Deltapine 555 5/4 STC 2 Deltapine 555 4/28 2006 3 Deltapine 555 5/10 1 Deltapine 555 5/4 CO 2 Deltapine 555 5/26 3 Deltapine 555 5/1 a specific stink bug pheromone, they effectively capture E. servus, N. viridula, and C. hilaris [27, 28]. The addition of an insecticidal ear tag improved trap captures of Euschistus spp. in pecan orchards by preventing escape of the bugs [29] . Our hypothesis was that sorghum planted in a narrow strip along the length of the interface of a source crop and cotton would attract E. servus adults. Additionally, capture traps baited with Euschistus spp. pheromone within the sorghum would help reduce dispersal out of sorghum by cap- turing and killing the pests in the sorghum. Thus, a full- scale field experiment was conducted to determine the ef- fectiveness of sorghum with Euschistus spp. pheromone traps to suppress E. servus in cotton. 2. Materials and Methods 2.L Study Sites. Twenty- three commercial cotton fields were sampled during this 2-year study (Table 1). These cotton fields were located in Irwin County GA and ranged from 5 to 15 ha in size. Overall, five cotton varieties were planted (Table 1). Sorghum, variety DK E57, was planted in a 4-row- wide strip along the length of the edge of a cotton field next to a source crop (corn or peanut) on May 5 2004 and on April 14 2007. Growers followed recommended agricultural practices for production of sorghum [30] and cotton [31]. Row width was 0.91 m for each crop, and rows of adjacent crops ran parallel. 2.2. Stink Bug Pheromone Traps. A pheromone trap consisted of a 2.84-liter clear plastic poly-ethylene terephthalate jar (United States Plastic Corp., Lima, OH, USA) on top of a 1 .22 m-tall yellow pyramidal base [27] . An insecticidal ear tag (Saber Extra, Coppers Animal Health, Inc., Kansas City, KS, USA) was placed in the plastic jar at the beginning of a test. Active ingredients in the ear tag were lambda- cyhalothr in (10%) and piperonyl butoxide (13%). Rubber septa, each loaded with 40 pL of the Euschistus spp. pheromone, meth- yl (£,Z)-2,4-decadienoate (CAS registry no. 4493-42-9) (Degussa AG fine Chemicals, Marl, Germany), were used as lures [32]. Lures were changed weekly for the duration of a test. Insects from weekly collections were taken to the labo- ratory for identification. 2.3. 2004 Experiment. Two treatments at the edge of cotton fields were examined for their ability to suppress stink bugs dispersing from an adjacent source crop into cotton: a sorghum trap crop with Euschistus spp. pheromone traps (STC/PTs) and a sorghum trap crop only (STC). Control fields had no sorghum or pheromone traps, for STC/PT fields, 21 pheromone traps were placed 12 m apart in sorghum on the row next to the source crop. At the beginning of the study, 17 cotton fields were selected, and each treatment was assigned randomly to various fields (four fields for STC/PT, five fields for STC, and eight fields for control) similar to a completely randomized design. Individual fields were used as replicates because the sorghum trap crops were planted along the full width between the cotton field and source crop. 2.4. 2006 Experiment. Only the STC/PT treatment and a control, both as explained above for the 2004 experiment, were used. At the beginning of the study, six cotton fields were selected, and each treatment was assigned randomly to three fields similar to a completely randomized design, for Psyche 3 the STC/PT treatment, 25-28 pheromone traps (depending on field width) were placed 12 m apart in the first row of sorghum closest to the source crop. 2.5. Insect Sampling. Each year of the study, cotton, sor- ghum, and pheromone traps were examined for the presence of stink bugs on a weekly basis: from the week of 16 June to the week of 28 July in 2004 and from the week of 28 June to the week of 23 August in 2006. Due to time constraints of sampling these large fields, not all fields were sampled on the same day of the week, but crops and/or pheromone traps within a field were sampled on the same day. For each sorghum sample, the aerial parts of all plants within a 1.83 m length of row were visually checked thoroughly for all stink bugs. For each cotton sample, all plants within a 1.83 m length of row were shaken over a drop cloth and the aerial parts of all plants were visually checked thoroughly for all stink bugs. Voucher specimens are stored in the USDA, ARS, Crop Protection & Management Research Faboratory in Tifton, GA, USA. For sampling purposes, the edge of a cotton field adjacent to a source crop was referred to as side A, and in a clockwise direction the other 3 sides of a field were referred to as side B, C, and D. In 2004, samples were obtained in each cotton field at two different distances from the field edge along each of the 4 sides of the field. The first edge location was 0- 3.66 m from the outside edge of the field, and the second edge location was 3.67-7.31 m from the outside edge of the field. The interior of the field was subdivided into 9 equally sized blocks. During weeks 3-6, samples were collected in each field as follows: 2 samples from each side at the 0- 3.66 m location, 2 samples from each side at the 3.67-7.31 m location, and 1 sample from the center of each interior block. During week 7, samples were collected as follows: 2 samples from the center of each interior block and 2 samples from each side at the 3.67-7.31 m location, but at the 0-3.66 m location, 6-12 samples (depending on length of field edge) were collected from each field edge. In 2006, samples were obtained at 3 distances from the edge on side A (i.e., at rows 1, 2, and 5 from the edge of the cotton field), and from 6 interior locations down the length of the field near to side C (i.e., rows 16, 33, 100, 167, 233, and 300 from the edge of the field on side A). For sides B- D, samples were taken from 2 edge locations, rows 1 and 5 from the edge of the field. The numbers of samples from each field on each date were as follows: 9 from each row on side A, 3 from each row on sides B-D, and 6 from each interior location. During 2004, the 4-row strip of sorghum was sampled by taking five random samples from rows 1 and 2 and four random samples from rows 3 and 4. In 2006, 9 random sam- ples were obtained from each of the 4 rows. 2.6. Statistical Analysis. For cotton, trap crop treatments in 2004 and 2006 were analyzed using PROC MIXED [33]. For both years of data, preliminary analyses revealed that there was only a significant treatment X week X field location in- teraction; there were no significant differences among fields. So, when trap crop data were analyzed, the fixed effect was treatment by week by field location, and random effects were replicate within treatment and residual error. Feast squares means were separated by least significant difference (FSD) [33] where appropriate. In 2004, two cotton fields (one STC/PT field and one control field) were not included in the data set for sampling week 7 because the grower treated for stink bugs after sampling on week 6. In 2006, one STC cot- ton field was not included in the data set for sampling during week 9 because it was treated for stink bugs after sampling on week 8. Chi-square analyses were used to com- pare frequencies of E. servus, N. viridula, and C. hilaris for each trap crop treatment by week in 2004 and 2006 (PROC FREQ, [33]). For 2004 data, numbers of E. servus per sample in the pheromone traps, sorghum with and without pheromone traps, cotton with sorghum trap crops with and without pheromone traps, and control cotton were plotted over time. Feast squares means from the above analyses were used for number of E. servus per sample for cotton, and only data for side A were used because statistical differences in E. servus density were detected among trap crop treatments mainly on this field edge. Means were obtained for number of E. servus adults per pheromone trap using PROC MEANS [33] . The numbers of E. servus adults per sample per week in the sorghum trap crop, with and without pheromone traps, were compared using t-tests; the means were used to plot number of E. servus adults per sample in sorghum over time. For 2006 data, numbers of E. servus per sample in phero- mone traps, sorghum with pheromone traps, cotton with sorghum trap crops with pheromone traps, and control cot- ton were plotted over time. Means were obtained for num- ber of E. servus adults per sample for sorghum and phero- mone traps using PROC MEANS [33]. Feast squares means from above analyses were used for number of E. servus per sample for cotton, and only data for side A, rows 1 and 2, were used because statistical differences in E. servus density were detected between trap crop treatments at these field locations. 3. Results and Discussion 3.L Stink Bug Species Composition. Eight species of stink bugs species, that is, E. servus, N. viridula, Oebalus pugnax pugnax (F), Euschistus quadrator (Rolston), Euschistus icteri- cus (F.), C. hilaris, Euschistus tristigmus (Say), and Piezodorus guildinii (Westwood), were found in sorghum over both years of this on-farm study in Georgia. These stink bugs species were also captured in Euschistus spp. pheromone traps (Table 2). As expected, more Euschistus spp., especially E. servus, were captured in traps baited with the Euschistus spp. pheromone than any other stink bug species. N. viridula was the predominant species in sorghum, whereas C. hilaris was rarely found in sorghum or captured in the pheromone traps. Thyanta custator custator (F.) was captured in the pheromone traps but was not found in sorghum even though this species has been collected from other sorghum plots (first author, unpublished data). E. servus nymphs were rarely captured in the pheromone traps; only 0.4% of all E. servus in these traps were nymphs. Also, for both years of the study. 4 Psyche heading flowering milking soft dough soft dough FR FR FR FR FR Week/sorghum development /cotton development STC/PT Control cotton STC STC/PT cotton PT STC cotton Figure 1: Mean number of E. servus per sample in sorghum trap crop with pheromone traps (STC/PTs), sorghum trap crop without pheromone traps (STC), and pheromone traps (PTs), and least squares means for number of E. servus in STC/PT cotton, STC cot- ton, and control cotton in 2004. FR; cotton with fruit. Number of stink bugs in pheromone traps was divided by 10. Only data on side A were used for cotton. Date refers to middle of sampling week. a range of 60-70% of the E. servus killed in these pheromone traps were females. Incorporating pheromone traps in a sor- ghum trap crop may decrease nymphal development of E. servus in the trap crop by reducing oviposition. 3.2. 2004 Experiment. For the first two weeks of the study, the number of E. servus per pheromone trap was relatively high, but density of the pest remained relatively low in sor- ghum with or without pheromone traps (Figure 1). These results indicate that E. servus was attracted to pheromone traps and uninterested in feeding on sorghum in the heading and flowering stages. Once sorghum finished flowering and reached the milking stage, E. servus began feeding on developing seeds in sorghum heads. Similarly, in an earlier study E. servus was observed on sorghum heads soon after completion of flowering [23], and in an additional study, the milking stage of sorghum heads was the most preferred stage for feeding by E. servus [22] . The number of E. servus adults was statistically higher in sorghum with pheromone traps compared to sorghum without these traps on weeks 2 and 3 (Table 3) indicating that the pheromone traps attracted more dispersing E. servus adults into the trap cropping system during a period of time when preferred food was not prevalent in sorghum. As E. servus density increased in sorghum, the number of E. servus dropped in pheromone traps (Figure 1). A similar response was observed for E. servus in peanut-cotton farm- scapes as fruit became available on cotton [28]. There are at least two possible explanations for this observed response: (1) once E. servus has dispersed into sorghum and fruit are available in the crop, these insects may begin to become more Table 2: Phytophagous stink bugs in Euschistus spp. pheromone traps and in sorghum trap crops. Species % in pheromone traps % in sorghum trap crops E. servus 69.1 10.9 N. viridula 10.6 71.5 0. p. pugnax 8.2 13.2 E. quadrator 7.4 2.8 E. ictericus 2.8 0.1 C. hilaris 1.1 1.3 E. tristigmus 0.6 0.1 P. guildinii 0.1 0.1 T. c. custator 0.1 0 interested in feeding than in responding to the aggregation pheromone, or (2) the attractiveness of the pheromone in the capture traps may decrease as pheromone from E. servus males aggregating on sorghum heads disperses throughout sorghum. These results also suggest that the number of E. servus in pheromone traps can be more a reflection of dispersal activity of E. servus into a crop rather than the density of the pest in a specific crop. Similarly, for the con- sperse stink bug, Euschistus conspersus Uhler, migration of sexually mature females from overwintering sites results in an elevated pheromone {Euschistus spp.) trap response relative to the surrounding field population early in the growing season before fruit are available in processing tomatoes, Eycopersicon esculentum Miller [34-36]. Also, the synthetic pheromone of the Neotropical brown stink bug, Euschistus heros (F.), is attractive to this pest in the field [37], and pheromone-baited traps are more efficient than field sampling mainly during the colonization of soybean [38]. For this trap crop experiment, factorial analyses revealed a significant treatment X week X field location effect {E = 1.76; df = 83,406; P = 0.0002) for number of E. servus per 1.83 m of row in cotton (Table 4). Density of E. servus adults per sample on side A was lower in cotton fields with sorghum trap crops (with or without pheromone traps) compared to control cotton fields on weeks 4 and 5. Indeed, for control cotton, density of E. servus was significantly higher on side A on weeks 4 and 5 compared to all other locations for those two weeks indicating that there was an edge effect in distribution of stink bugs in these farmscapes. There was no significant difference in density of E. servus adults between cotton fields with a sorghum trap crop, with or without pheromone traps, at this location on these two weeks. So, even though sorghum trap crops with pheromone traps may attract more E. servus than trap crops without pheromone traps, this increase in attraction does not translate into higher densities of the pest moving into cotton. A total of 4042 and 1406 E. servus adults were captured and killed in pheromone traps in these farmscapes in 2004 and 2006, respectively. Evidently, the pheromone traps effectively capture E. servus, but not all of them at any point in time, probably because they continue to disperse into the trap crop. E. servus was first observed in control cotton with fruit on week 3 (Figure 1). In control cotton, E. servus density Psyche 5 Table 3: Number (mean ± SE) of E. servus adults per 1.83 m of row in sorghum trap crops with pheromone traps (STC/PTs) and sorghum trap crops alone (STC) in 2004. Week STC/PT STC \t\ df P 1 0.0833 ± 0.0467 0.0444 ±0.0311 0.69 63 0.4908 2 0.1806 ±0.0636 0.0333 ± 0.019 2.43 160 0.0163 3 0.3889 ± 0.0852 0.1556 ±0.0497 2.47 160 0.0144 4 0.2639 ± 0.0559 0.2778 ± 0.057 0.17 160 0.8642 5 0.25 ± 0.0585 0.2444 ± 0.0507 0.07 160 0.9427 Table 4: Least squares means for number of all E. servus per 1.83 m of row in different locations in cotton for ; sorghum trap crop with pheromone traps (STC/PTs), sorghum trap crop (STC), and control (CO) fields in 2004. Location Week STC/PT STC CO 2 0 l,a,A 0 l,a,A 0 3,a,A side A^ 3 0 l,a,A 0 l,a,A 0.125 3,a,A 4 0.041 l,b,A 0.0103 l,b,A 0.4292 2,a,A 5 0.0476 l,b,A 0.0677 l,b,B 0.7015 l,a,A 2 0.125 l,a,A 0 l,a,A 0 l,a,A side B 3 0 l,a,A 0.15 l,a,A 0 l,a,A 4 0.0478 l,a,A 0.2 l,a,A 0.1651 l,a,B 5 0.0329 l,a,A 0.0254 l,a,B 0.0095 l,a,B 2 0 l,a,A 0 l,a,A 0 l,a,A side C 3 0.0625 l,a,A 0.05 l,a,A 0.0003 l,a,A 4 0 l,a,A 0.05 l,a,A 0.0818 l,a,B 5 0 l,a,A 0.2494 l,a,AB 0.1224 l,a,B 2 0 l,a,A 0 2,a,A 0 l,a,A side D 3 0.125 l,a,A 0.05 2,a,A 0 l,a,A 4 0 l,a,A 0 2,a,A 0 l,a,B 5 0 l,b,A 0.3315 l,a,A 0.0836 l,ab,B 2 0 l,a,A 0 l,a,A 0.0833 l,a,A block 1 3 0.0833 l,a,A 0.0667 l,a,A 0 l,a,A 4 0.1346 l,a,A 0.0667 l,a,A 0 l,a,B 5 0.2444 l,a,A 0.1504 l,a,AB 0.0095 l,a,B 2 0.0833 l,a,A 0 l,a,A 0 l,a,A block 2 3 0 l,a,A 0.0667 l,a,A 0.0833 l,a,A 4 0.0774 l,a,A 0.0667 l,a,A 0 l,a,B 5 0.0774 l,a,A 0.1028 l,a,B 0.1206 l,a,B 2 0 l,a,A 0 l,a,A 0 l,a,A block 3 3 0.0833 l,a,A 0 l,a,A 0.0833 l,a,A 4 0 l,a,A 0.0667 l,a,A 0 l,a,B 5 0.2444 l,a,A 0.0551 l,a,B 0.0095 l,a,B Least squares means within a column followed by the same number are not significantly different between weeks within a location for a single treatment. Least squares means within a row followed by the same lowercase letter are not significantly different between trap crop treatments within a location for a single week. Least squares means within a column followed by the same uppercase letter are not significantly different between locations within a treatment for a single week. (PROC MIXED, LSD, P > 0.05, for all E. servus, n = 1550, SE = 0.0938, df = 1040). ^Cotton edge along the common boundary of a cotton field and a field of another crop that was a source of stink bugs. was significantly higher on week 4 compared to week 3 and higher on week 5 compared to week 4 on Side A (Table 4). Thus, E. servus density increased in control cotton from week 3 to 5 as depicted in Figure 1. During this time, the pest probably dispersed from the source crops into control cotton as previously reported in other cotton farmscapes in Georgia [15, 16]. E. servus first occurred in cotton with sorghum trap crops (with or without pheromone traps) on week 4. For both trap crop treatments, density of E. servus in cotton was similar on weeks 4 and 5 (Table 4). As E. servus density increased in control cotton, it remained relatively low in the two treatments with sorghum trap crops (Table 4, Figure 1). Thus, both trap cropping systems effectively arrested E. servus reducing dispersal of this pest into cotton. Edge effects occur on other field edges, as expected, and not just at the interface of the source crop and cotton. For the sorghum 6 Psyche Table 5: Frequency (%) of E. servus, N. viridula, and C. hilaris in cotton by week for sorghum trap crop with pheromone traps (STC/PTs), sorghum trap crop (STC), and control (CO) fields in 2004 and sorghum trap crop (STC) and control (CO) fields in 2006. E. servus N. viridula C. hilaris Year Wk Trt n % n % n % df P Control 7 15.91 37 84.09 0 0 3 STC/PT 5 20.83 8 33.33 11 45.83 STC 8 53.33 5 33.33 2 13.33 Total 20 20.83 8 33.33 11 45.83 36.4 4 0.0001 Control 11 8.09 121 88.97 2 2.94 2004 4 STC/PT 5 11.36 38 86.36 1 2.27 STC 8 11.43 57 81.43 5 7.14 Total 24 9.6 216 86.4 10 4.0 3.5 4 0.4852 Control 22 5.45 373 92.33 9 2.23 5 STC/PT 13 6.4 182 87.89 8 3.94 STC 41 11.68 287 81.77 23 6.55 Total 76 7.93 842 87.89 40 4.18 20.9 4 0.0003 f, Control 8 18.18 29 65.91 7 15.91 STC/PT 1 14.29 0 0 6 85.71 Total 9 17.65 29 56.86 13 25.49 16.2 2 0.0003 2006 7 Control 19 32.2 37 62.71 3 5.08 STC/PT 2 3.08 26 40.0 37 56.92 Total 21 16.94 63 50.81 40 32.26 44.4 2 0.0001 8 Control 43 32.82 76 58.02 12 9.16 STC/PT 3 2.42 34 27.42 87 70.16 Total 46 18.04 110 43.14 99 38.82 107.5 2 0.0001 trap crop alone treatment, E. servus density was significantly higher on side D on week 5 than on side A and B and block 2 and 3 (Table 4). Three species of stink bugs, E. servus, N. viridula, and C. hilaris, were observed feeding on cotton fruit in both years (Table 5). In 2004, E. servus in cotton comprised 5-53% of the stink bug species over the three treatments. N. viridula was the predominant stink species in cotton except during week 3 when C. hilaris was predominant (with trap crops and stink bug capture traps). Also, during this same week in STC cotton, E. servus was the predominant species on cotton, and frequency of this pest was higher in these cotton fields than in STC/PT and control fields. On week 5, frequency of E. servus in cotton with sorghum trap crops alone was twice that for cotton with the STC/PT and control treatments. Apparently, incorporating the pheromone traps with the sorghum trap crop reduced dispersal of E. servus from the trap crop at least two out of three weeks. 3.3. 2006 Experiment. While sorghum was flowering, E. servus was captured in pheromone traps, but pest density was relatively low in sorghum (Figure 2), There was a slight drop in the number of E. servus per pheromone trap as the pest peaked during the milking stage of sorghum. Apparently, E. servus was drawn into the pheromone traps and then was arrested on sorghum when it was available as food. E. servus first appeared when small fruits were available in control cotton fields during week 3. Trap capture of E. servus increased from week 6 to week 7, and E. servus density increased in control cotton on week 7 and 8. This major influx of E. servus into pheromone-baited traps and control cotton indicates a significant dispersal of adults from the source crop as has been previously reported for peanut- cotton farmscapes in Georgia [15]. The pest occurred in cotton with sorghum trap crops and pheromone traps for the first time on week 7 indicating that the trap cropping system had effectively stopped E. servus from dispersing into cotton for 4 weeks, from weeks 3 through 6. For this trap crop experiment, factorial analyses revealed a significant treatment X week X field location effect (F = 4.68; df = 71, 779; P = 0.0001) for numbers of E. servus per 1.83 m of row in cotton (Table 6). There was an edge effect in distribution of stink bugs in control cotton; density of E. servus was significantly higher on row 1 on side A compared to all other locations except row 2 on week 7 and on rows 1 and 2 on side A compared to all other locations on week 8. In control cotton, E. servus density was significantly higher on week 7 compared to week 6 and higher on week 8 compared to week 7 so density of the pest increased in cotton over time probably due to continual dispersal of E. servus from the source crop into the adjacent cotton rows as has been observed in other peanut-cotton farmscapes in Georgia [15]. The trap cropping system, though, effectively suppressed this pest in these farmscapes, for density of E. servus per sample was higher in control cotton fields compared to cotton fields with sorghum trap crops with pheromone traps on rows 1 and 2 of side A on weeks 7 and 8. Psyche 7 Table 6; Least squares means for number of all E. servus per 1.83 m of row in different locations in cotton for sorghum trap crop with pheromone traps (STC/PT) and control (CO) fields in 2006. Side Row Week STC/PT CO A" 1 6 0 l,a,A 0.037 3,a,A 2 0 l,a,A 0.0926 l,a,A 5 0.0185 l,a,A 0.0185 l,a,A 16 0 l,a,A 0 l,a,A 33 0 l,a,A 0 l,a,A 100 0 l,a,A 0 l,a,A 167 0 l,a,A 0 l,a,A 233 0.0006 l,a,A 0 l,a,A 300 0.0006 l,a,A 0 l,a,A B 0 l,a,A 0 l,a,A C 0 l,a,A 0 l,a,A D 0 l,a,A 0 l,a,A A 1 7 0.0142 l,b,A 0.1667 2,a,A 2 0.0003 l,b,A 0.1111 l,a,AB 5 0.0003 l,a,A 0.0370 l,a,B 16 0.0003 l,a,A 0 l,a,B 33 0.0003 l,a,A 0 l,a,B 100 0.0003 l,a,A 0 l,a,B 167 0.0419 l,a,A 0.0556 l,a,B 233 0.0008 l,a,A 0 l,a,B 300 0.0008 l,a,A 0 l,a,B B 0.0003 l,a,A 0 l,a,B C 0.0003 l,a,A 0 l,a,B D 0.0003 l,a,A 0.0185 l,a,B A 1 8 0 l,b,A 0.4259 l,a,A 2 0.0550 l,b,A 0.1852 l,a,B 5 0 l,a,A 0.037 l,a,C 16 0 l,a,A 0.0556 l,a,C 33 0 l,a,A 0 l,a,C 100 0 l,a,A 0 l,a,C 167 0 l,a,A 0 l,a,C 233 0.0002 l,a,A 0 l,a,C 300 0.0002 l,a,A 0 l,a,C B 0 l,a,A 0.0741 l,a,C C 0.0273 l,a,A 0.0185 l,a,C D 0 l,a,A 0.037 l,a,C Least squares means within a column followed by the same number are not significantly different between weeks within a location for a single treatment. Least squares means within a row followed by the same lowercase letter are not significantly different between trap crop treatments within a location for a single week. Least squares means within a column followed by the same uppercase letter are not significantly different between locations within a treatment for a single week. (PROC MIXED, LSD, P > 0.05, for all E. servus, n = 2539, SE = 0.0342, df = 1811). ^Cotton edge along the common boundary of a cotton field and a field which was a source of stink bugs. In 2006, N. viridula was the predominant stink bug species in control cotton, and C. hilaris was the predominant species in cotton with sorghum trap crops (Table 5). On weeks 7 and 8, the frequency of E. servus was higher in control cotton than that in cotton with sorghum trap crops with pheromone traps. Apparently, incorporating the pheromone traps with the sorghum trap crop reduced the dispersal of E. servus into cotton. 3.4. Effectiveness of Trap Cropping System for Suppression ofE. servus . An ideal trap cropping system should include a host plant which is strongly preferred by the pest over the cash crop and should be able to reduce the likelihood of the pest dispersing into the cash crop [17]. In 2004, when sorghum alone was utilized as a trap cropping system, E. servus adults strongly preferred sorghum (from the milking stage through the soft dough stage) to cotton (with fruit). Furthermore, 8 Psyche flowering flowering milking soft dough soft dough hard dough mature BD FL FR FR FR FR STC/PT PT Week/sorghum development /cotton development Control cotton STC/PT cotton Figure 2: Mean number of E. servus per sample in sorghum trap crop with pheromone traps (STC/PTs), pheromone traps (PTs), STC/PT cotton, and cotton control in 2006. BD: cotton with buds; FL: cotton with flowers; FR: cotton with fruit. Number of stink bugs in pheromone traps was divided by 10. Only data for side A, rows 1 and 2, are used for cotton. Date refers to middle of sampling week. over both years of the study a sorghum trap crop with or without pheromone traps effectively attracted E. servus in sorghum reducing dispersal of this pest into cotton. Strategic placement of the trap cropping system in time and space also apparently was essential to the success of this suppression tactic in these farmscapes. Corn is an early summer source of stink bugs dispersing to peanut and cotton, and peanut is a mid-to-late summer source of stink bugs moving to cotton, especially at the interface of these farmscapes [15, 16]. Thus, in this study, a trap cropping system was established at the interface of a source crop (i.e., corn or peanut) and cotton, and the trap crop was planted in time to provide preferred food to stink bugs dispersing from the source crop into cotton. In farmscapes where stink bugs are active throughout the season, a season-long trap cropping system may be needed to protect cotton. Season- long trapping of stink bugs should reduce stink populations throughout the seasonal succession of host plants, possibly eliminating the need for additional control measures in cotton. A season-long stink bug trap cropping system that includes triticale, hairy vetch, and crimson clover during the spring followed by sunflower, buckwheat, sorghum, and pearl millet during the summer and fall has been developed to effectively manage stink bugs in organically grown soy- bean [22]. The system is economical to culture and manage provides a range of physical practices, including ratooning (mowing), and a range of maturity dates. All of these could be used alone or together by growers to customize the sys- tem for general use. Stink bugs are well-adapted opportunists that will take advantage of available food resources at crop interfaces with both sorghum and soybean [13] being preferred over cotton. Flowever, some stink bugs will still move into cotton near the preferred trap crop. In the current study, even though sorghum trap crops arrested E. servus and subsequently re- duced dispersal of this pest into cotton, some E. servus ap- parently dispersed from sorghum into cotton. Indeed, in the 2004 experiment, some E. servus moved into cotton even though the sorghum heads were still in the attractive developmental stage. In a preliminary on-farm test, E. servus adults were significantly higher in a soybean trap crop than in adjacent fruiting cotton, but adult stink bugs still fed on some cotton fruit in the first two rows adjacent to soybean, over the period of attractiveness of soybean (first author, unpublished data). Stink bug pheromone traps containing lures with Euschistus spp. pheromone (and insecticidal ear tags) have been shown to effectively capture and kill E. servus [27, 28], and thus they have great potential to suppress this pest in agricultural landscapes. One of the questions to be consider- ed on how to utilize these traps as a management tool is whether they have the ability as a single tool to suppress E. servus in crops. In two separate experiments, establishing Euschistus spp. pheromone traps at the interface of peanut- cotton plots did not inhibit dispersal of E. servus when cotton fruit became available as a food source [28]. In another small-plot trap cropping experiment in peanut-cotton plots, Euschistus spp. pheromone traps captured E. servus adults, but E. servus density was equally high in cotton in plots with only the pheromone traps and control plots when cotton fruits were present (first author, unpublished data). These results indicate that as the sole management tool, Euschistus spp. pheromone traps cannot effectively stop dispersal of E. servus from peanut into fruiting cotton. However, in the second experiment, it was also determined that density of E. servus in cotton was statistically lower in cotton in plots Psyche 9 with a soybean trap crop with pheromone traps compared to control plots suggesting that pheromone traps are more effective in suppressing E. servus when used in combination with a trap crop. Apparently, stink bugs require a source of preferred food to remain in a location. Interestingly, though, Euschistus spp. pheromone can attract E. servus adults, dispersing from an adjacent crop within the agricultural landscape, into a sorghum trap crop even when sorghum heads have not yet developed to the preferred feeding stage. Perhaps, pheromone traps should be established in sorghum before heads develop seeds and remain in the trap crop throughout the period cotton that is susceptible to economic damage. Initially pheromone traps would attract and kill E. servus dispersing from a source crop and then pheromone traps would capture and kill E. servus attracted to sorghum. Even with incorporation of pheromone traps, some E. servus still dispersed from sorghum into cotton. During 2004, preferred food was still available in sorghum when stink bugs moved into cotton. In this experiment pheromone traps were placed only on the sorghum row adjacent to a source crop. Perhaps, placing pheromone traps on every row of sorghum would decrease dispersal from sorghum into cotton. During 2006, E. servus probably began dispersing from sorghum into cotton because the seeds were no longer in the preferred feeding stage for the pest. Ratooning the sor- ghum heads or providing multiple plantings of the trap crop could extend the length of time preferred food available to the pest. In a small-plot trap cropping experiment, E. servus density was significantly lower in cotton plots with a stink bug barrier (1.83 m tall plastic wall) than in cotton plots with a soybean trap crop (first author, unpublished data). Thus, planting a tall crop such as Sudan grass {Sorghum bicolor (L.) Moench spp. drummondii (Nees ex Steud.) de We & Harlan) between a trap crop and cotton could possibly further decrease opportunistic movement from the trap crop into the cash crop. The question remains whether a trap crop with phero- mone traps is more effective in suppressing E. servus in cotton than a trap crop alone. Even though there was no sig- nificant difference in density of E. servus in cotton between the two trap crop treatments in 2004, incorporation of pher- omone traps in trap crops can provide additional benefits including the following: (1) reducing the density of E. servus adults in a trap crop, especially females, to possibly decrease the local population over time and reduce the overwintering population, (2) reducing the dispersal of E. servus adults from the trap crop into cotton, (3) potentially attracting more dispersing E. servus adults into a trap crop during a period of time when pre- ferred food is not prevalent in the landscape. A multifunctional habitat with a combination of trap crops to detract stink bugs from feeding and ovipositing on cash crops and pheromone traps with insecticidal ear tags to capture and kill stink bugs has the greatest potential for suppressing stink bugs in cotton. In Georgia, N. viridula and C. hilaris, along with E. servus, can cause economic damage to cotton fruit. Thus, a trap cropping system established to protect cotton from stink bugs must provide host plants preferred for feeding by all three stink bugs. Unfortunately, C. hilaris rarely occurs in sorghum, but because they readily feed on soybean {Glycine max (E.) Merr.) pods (first author, unpublished data), this plant may be a more suitable trap crop for this pest. However, N. viridula is highly attracted to grain sorghum heads in the milk stage through the hard dough stage [39], and N. viridula adults prefer sorghum to cotton [24]. Thus, a combination of grain sorghum and soybean could serve as an effective trap cropping system for these three stink bug species. In addition, other host plant species could be added to the trap cropping system to extend its longevity. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 480520, 8 pages dohlO.l 155/2012/480520 Research Article Insects of the SubfamUy Scolytinae (Insecta: Coleoptera, Curculionidae) Collected with Pitfall and Ethanol Traps in Primary Forests of Central Amazonia Raimunda Liege Souza de Abreu, Greicilany de Araujo Ribeiro, Bazilio Frasco Vianez, and Ceci Sales-Campos Department of Forest Products, National Institute for Amazon Research, Av. Andre Araujo, 2936. 69060-001 Manaus, AM, Brazil Correspondence should be addressed to Raimunda Liege Souza de Abreu, raiabreu@inpa.gov.br Received 30 September 2011; Revised 28 November 2011; Accepted 29 November 2011 Academic Editor: David G. James Copyright © 2012 Raimunda Liege Souza de Abreu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. An experiment was conducted in a primary forest area of the Tropical Forest Experimental Station, 45 km from Manaus-Boa Vista Elighway, in order to compare the insect fauna of the subfamily Scolytinae, in flight activity and on the ground. Five impact traps of the type Escolitideo/Curitiba, with ethanol baits, were installed at the height of 3 m above the ground, and five pitfall traps were buried in the same area of the above ground traps. The data collections were evaluated through abundance, richness, and Simpson diversity index, and, to compare these data with the pitfalls and the months collection, the ANOVA was used. The Pearson correlation test was also carried out to evaluate the meteorological factors (temperature and rainfall). From the total of 2,910 Scolytinae, 2,341 were captured in pitfall traps representing 80.45% and 569 with Escolitideo/Curitiba traps representing 19.55%. The most abundant species in the collections were Xyleborus volvulus Fabricius and Xyleborus affnis Eichhoff, and this was classified as constant in both habitats. The result of the analysis indicates that the Simpson’s index was high and that the abundance of insects was affected by the types of trap and by the month of collection. The analysis of correlation with meteorological factors showed that only Xyleborus spinulosus species presented significant correlation with temperature. 1. Introduction The Scolytinae are mostly secondary predators by developing under natural conditions in trees injured, hit by lightning, fire, plants nutritionally deficient, drooping, and so forth, but can attack healthy plants also [1]. This subfamily presents species phyllophagous (bark beetles), which feed on the phloem tissues, that is, the inner part of the bark of the tree, and xylomycetophagous (beetles of ambrosia) which have as their main food symbiotic fungi, which introduce and culti- vate in the host plant [2-4]. The beetles xylomycetophagous attack preferably sapwood which is richer in nutrients, but in some species of wood, the attack also happens in the heartwood [5]. The Scolytinae attack usually begins within twenty-four hours after the tree is cut and each forest species has greater or lesser resistance, but none is totally free of infestation by these insects. The resistance presented by each species is probably related to the attractive substances and the wood hardness which undoubtedly influences the speed of pene- tration and the general importance of the attack. These insects have been captured in various regions of the world. In the Amazon region they were also found attacking several hosts, such as forest and fruit trees, as shown in some studies carried out by Mendes [6], Abreu and Dietrich [7], Abreu [8], Abreu and Bandeira [9], Barbosa [10], DalEOglio and Peres-Filho [11], and Mafias and Abreu [ 12] . Studies involving biology and ecology of this subfamily have also been conducted in the Amazon, using mainly flight intercept traps, in which an attractive substance is used [13- 15]. These insects have also been found in other substrates, as shown by surveys conducted by Schubart and Beck [16], Penny and Arias [13], Morais [17, 18], and Rodrigues [19], in samples collected from trees, leaf litter and on the ground. 2 Psyche where various types of traps were used, including the pitfall trap. Therefore, the capture of these insects on the ground and in flight activity is important for the complete knowledge of their life cycle, and at the same time, to know if the species caught in flight are the same found on the ground. 2. Material and Methods This work was carried out with beetles of the subfamily Scolytinae collected in the Tropical Forestry Station, 45 km from Manaus, on the BR-174 highway, with an area of 21,000 ha and geographical coordinates 02° 35' 55.5" South and 60° 02' 14.8" West of Greenwich. In this area the average minimum annual temperature is 20° C and the maximum 26° C, with an average relative air humidity of 77% [20] . The soil of the region is clayey, and it can be classified as Oxisol and Ultisol [21]. Five modified impact traps of the type Escolitideo/ Curitiba (EC) were used for this survey, using 100% com- mercial alcohol as attraction [22], for the capture of the insects in flight, and five pitfall traps for capture of those with activity on the ground. The collection period happened from luly 2005 to luly 2006. The impact trap consists of a conical cover, a panel of impact, a funnel, and a bottle for collection, containing alcohol 30% with detergent. The bait is placed inside a glass bottle attached to the panel with a pierced cap to allow volatilization. The traps were installed at a height of 3 m above the ground, and with, the aid of a nylon rope, they were tied to two trees at a distance of 30 m from each other (Figure 1(a)). The pitfall trap consists of a 500 mL glass bottle, an acrylic sheet, 25 cm X 25 cm, and four PVC tubes with 40 cm in length. During the assembly, the bottles were buried, leaving the openings at the ground level to allow the insects to fall inside them. Picric acid at 0.003% concentration, which is considered neutral, that is, does not attract or repel the insects, was used to preserve the insects. The acrylic sheets were used as coverage, which were supported by four PVC tubes, buried to half of their lengths (Figure 1(b)). The samples were collected weekly, when the renewal of the bait and liquid preservatives were made. The collected beetles were identified by means of direct comparison with specimens of the Invertebrate Collection of the National Institute for Amazon Research. Taxonomic identification keys were also used [1, 23-28]. The faunistic analysis was obtained through absolute and relative abundance, constancy, species richness, and the calculation of the Simpson diversity index referring to each month of collection. Absolute abundance was done by direct count of the individuals and the relative abundance, by the calculations of the percentages of individuals of each species in relation to the total number of captured individuals [29]. The constancy was determined by the percentage of occurrence of the species in the collections. According to the obtained percentages, the species were separated into the following categories: (a) constant species (W) present in more than 50% of the collections, (b) accessory species (Y) present in 25 to 50% of the collections; (c) accidental species (b) Figure 1: Traps used to capture insects, (a) Escolitideo/Curitiba trap-EC and (b) Pitfall trap. (Z) in less than 25% of the collections [30]. The Simpson diversity index was calculated according to the formula: D = 1 “ Z(^!(^i “ 1)/N(N - 1)), where n is the number of sampled individuals for each i species and N is the total number of sampled individuals. The analysis of abundance, the Simpson s index, and the richness of the species as a function of the type of trap and the months of collection was done by AN OVA. For these analyses data were transformed into log (x -H 1), and the significance level was P < 0.01. The monthly fluctuation analysis of the four most abundant species was also carried out, relating the number of collected insects in each type of trap with the data of temperature and rainfall by Pearson Correlation [31]. 3. Results and Discussion According to the data shown in Tables 1 and 2, 2,910 Scolyti- nae specimens were captured, from which 2,341 with the pitfall trap, representing 80.45% and 569 with the EC trap, representing, 19.55%. The data show the existence of nine genera and 26 species. The Xyleborus genus stood out from the others because it represented 97% of the collection in the pitfall trap and 57.14% in the EC trap. The predominance of this genus has already been observed in the work carried out also in primary forest by Abreu [ 14] . Erom all the species captured with the pitfall trap, the ones that stood out are the species Xyleborus volvulus Psyche 3 Fabricius, representing 50.11% (1,173 individuals.); Xyle- borus affinis Eichhoff, with 38.02% (890 individuals); Xyle- borus ferrugineus Fabricius with 4.36% (102 individuals); Xyleborus spinulosus Blandford, with 4.06% (95 individuals). The others represented 3.46% (81 individuals). In the EC trap, X affinis represented 34.62% (197 individuals); X. volvulus, with 18.28% (104 individuals); Premnobius cavipen- nis Eichhoff, with 11.6% (66 individuals); Hypothenemus eruditus Westwood, with 9.67% (55 individuals); H. obscurus (Fabricius) with 5.98% (34 individuals). The remaining accounted for 19.85% (113 individuals) (Tables 1 and 2). Quantitatively there was some numerical superiority for the pitfall trap because it was responsible for almost 90% of the collected specimens, but, in richness, there was some advantage for the EC trap. While this trap was responsible for the capture of 25 species, the pitfall trap captured only 13. From the studied species, X. affinis, X. volvulus, X. ferrugineus, X. spinulosus, Theoborus solitariceps, X. flavus, P. cavipennis, H. obscurus, H. eruditus, Monarthrum sp. 2, Amphicranus sp. 1 and Coccotrypes palmarum were captured with the two traps (Tables 1 and 2). In relation to the analysis of richness, abundance, and the Simpsons index, the results indicate that the of Simpsons index {P? = 0.098; Fii ,96 = 0.953; P = 0.4939) was not af- fected neither by the type of trap, nor by the month. On the other hand, the abundance {R^ = 0.4361; Fii ,96 = 6.75; E < 0.001) is related to the month of collection, and the type of trap and richness {R? = 0.337; Fii ^96 = 4,436; P < 0.001) is only affected by the month of collection. The result of these indexes for each sampled point and month of collection is represented in Tables 3 and 4. The greatest abundance of species in pitfall traps was recorded in the months of August and November 2005 and May 2006, while, in the EC trap, it was in the months of January and July 2006. In the analysis of richness with the EC trap, the largest and smallest numbers of species were registered in the months of October and December 2005, respectively. For the pitfall trap, that happened in the months of August and July 2005. In general, the Simpson diversity index was high, reflect- ing less diversity and the dominance of two species, X. volvulus and X. affinis. For the EC trap, this index varied from 0.12 (trap 4, January 2006) to 1 (traps 2, 3, and 5, December 2005) . For the pitfall trap, it varied from 0.170 (trap 3, March 2006) to 1 (trap 1, July 2005; trap 5, March 2006; trap 5, July 2006; trap 1, 3, 5, July 2006). Regarding the constancy, in the pitfall trap, three species have been considered constant, two accessory and 6 acci- dental. In the EC trap, three species have been considered constant, five accessory and 16 accidental. It was also observed that the X. affinis species was constant for the two traps, becoming evident the importance of this species in the studied environment. The analysis of correlation of the number of insects with rainfall and temperature indicates that only the X. spmw/osMS species was affected by the temperature (r = 0.443; P = 0.039), since the number of individuals increased as a function of the temperature. Although the temperature is considered one of the most important climatic factors in the Xyleborus volvulus Xyleborus affinis Xyleborus ferrugineus Xyleborus spinulosus — Rainfall Figure 2: Monthly rainfall and total number of four of the most abundant species of Scolytinae subfamily collected with pitfall traps from July 2005 to July 2006, in primary forest of Central Amazon. development and survival of the beetles of the Scolytinae subfamily, it tends to be higher in the microclimate of the host, but less prone to sudden fluctuations in the external environment [1, 32]. This was observed in the course of this work and probably that is why this factor did not influence the activity of these beetles on external environment, that is, in flight activity and on the ground. Data from insects collected with pitfall traps and Escol- itideo/Curitiba traps relating rainfall and temperature with the number of insects of the four most abundant species are represented in figures 2, 3, 4, and 5. In these figures it can be observed that for the pitfall trap, the species X. affinis, and X. volvulus presented a population peak in August and November 2005 and May 2006. It can also be noticed that in the EC trap, X. affinis presented peaks in January, May and June 2006, while X. volvulus presented a peak only in July 2006. The months of August 2005 and July 2006 showed low values of rainfall, while, in November 2005 and May 2006 these values were high. When analyzing the rainfall data some studies suggest that this factor influences on the population and the behavior of the Scolytinae, observing that high values of precipitation affect the abundance of these insects [32, 33]. Other authors presented the opposite, as shown in a study carried out by Flechtmann et al. [34], where the capture peak of Scolytinae coincides with high intensity of rainfall. In this context and based on the outcome of this work, it can be affirmed that temperature and precipitation did not affect the activity of most of these insects, both in flight and on the ground. These results agree with Hulcr et al. [35] claiming that circumtropical species, like X. affinis and X. volvulus, are usually not affected by climatic factors, since they are found in rainy and dry weather. In this work it has been observed that, despite the 25 species captured with the EC trap using alcohol as attraction, the number of specimens is considered small if compared to the pitfall trap. In forest plantations in Brazil with a single forest species, the capture of these insects using this kind of attraction has been more efficient [22, 36-38]. In a primary or secondary forest, the substrate is very rich, due to 4 Psyche Table 1: Absolute and relative abundance of insects of the subfamily Scolytinae collected with pitfall traps during the period of July 2005 to July 2006, in primary forest of Central Amazon. Species Jul Aug Sep Oct Nov Dec Jan Months Feb Mar Apr May Jun Jul Total % Xyleborus volvulus 0 290 97 92 163 37 17 0 54 0 324 97 2 1173 50.11 Xyleborus ajfinis 1 164 79 24 325 83 30 0 23 0 116 44 1 890 38.02 Xyleborus ferrugineus 0 20 8 13 33 10 3 0 3 0 2 10 0 102 4.36 Xyleborus spinulosus 0 15 0 19 28 14 0 0 2 0 14 3 0 95 4.06 Theoborus solitariceps 0 4 0 0 18 0 0 0 0 0 0 0 0 22 0.94 Xylosandrus compactus 0 2 0 3 4 3 4 0 2 0 0 0 1 19 0.81 Xyleborus flavus 0 0 0 0 0 12 0 0 0 0 0 0 1 13 0.56 Coccotrypes palmarum 2 0 1 0 0 0 4 0 0 0 3 0 0 10 0.43 Amphicranus sp. 1 2 1 4 2 0 0 0 0 0 0 0 0 0 9 0.38 Premnobius cavipennis 0 2 0 0 0 0 0 0 0 0 0 1 1 4 0.17 Hypothenemus obscurus 0 1 1 0 0 0 0 0 0 0 0 0 0 2 0.09 Hypothenemus eruditus 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0.04 Monarthrum sp. 2 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0.04 TOTAL 6 499 190 154 571 159 58 0 84 0 459 155 6 2341 100 Table 2: Absolute and relative abundance of insects of the subfamily Scolytinae collected with Escolitideo/Curitiba traps during the period of July 2005 to July 2006, in primary forest of Central Amazon. Species Jul Aug Sep Oct Nov Dec Jan Months Feb Mar Apr May Jun Jul Total % Xyleborus ajfinis 10 17 7 8 9 1 55 0 15 0 35 28 12 197 34.62 Xyleborus volvulus 0 0 1 2 0 0 3 0 0 0 0 5 93 104 18.28 Premnobius cavipennis 2 5 13 7 4 1 4 0 6 0 16 6 2 66 11.60 Hypothenemus eruditus 1 1 3 2 3 0 1 0 2 0 7 9 26 55 9.67 Hypothenemus obscurus 1 3 8 4 3 0 0 0 0 0 10 2 3 34 5.98 Monarthrum durum 0 0 1 1 4 2 6 0 1 0 6 8 0 29 5.10 Sampsonius dampfi 0 0 5 3 2 0 0 0 0 0 6 0 0 16 2.81 Sampsonius prolongatus 0 0 0 2 1 0 6 0 2 0 2 0 0 13 2.28 Xyleborus spinulosus 0 1 0 1 3 0 3 0 0 0 0 0 3 11 1.93 Xyleborus ferrugineus 0 0 0 0 6 0 0 0 1 0 1 1 1 10 1.76 Sampsonius detractus 0 1 1 0 2 0 2 0 0 0 2 0 0 8 1.41 Coccotrypes palmarum 0 0 0 0 0 1 0 0 0 0 0 4 0 5 0.88 Amphicranus sp. 1 0 0 0 1 0 1 0 0 1 0 1 0 0 4 0.70 Theoborus solitariceps 0 2 0 1 1 0 0 0 0 0 0 0 0 4 0.70 Monarthrum semipaleans 0 2 0 0 0 0 0 0 0 0 0 0 0 2 0.35 Sampsonius pedrosai 0 0 1 0 0 0 1 0 0 0 0 0 0 2 0.35 Xyleborus flavus 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0.18 Xyleborus solitarinus 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0.18 Hypothenemus sp. 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0.18 Sampsonuis giganteus 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0.18 Cryptocarenus diadematus 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0.18 Cryptocarenus seriatus 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0.18 Cryptocarenus heveae 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0.18 Monarthrum sp. 1 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0.18 Monarthrum sp. 2 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0.18 TOTAL 15 36 42 34 38 6 81 0 28 0 86 63 140 569 100 Psyche 5 Xyleborus ferrugineus . Rainfall Xyleborus spinulosus Figure 3; Monthly rainfall and total number of four of the most abundant species of Scolytinae subfamily collected with Escolitideo/Curitiba traps from July 2005 to July 2006, in primary forest of Central Amazon. =3 <3J Xyleborus volvulus Xyleborus affinis Xyleborus ferrugineus Xyleborus spinulosus — Temperature Figure 4: Monthly temperature and total number of four of the most abundant species of Scolytinae subfamily collected with pitfall traps from July 2005 to July 2006, in primary forest of Central Amazon. 27.5 27.0 26.5 26.0 25.5 33 -t-t 25.0 u Oh 24.5 a 24.0 23.5 23.0 22.5 Xyleborus volvulus Xyleborus affinis Xyleborus ferrugineus Xyleborus spinulosus — Temperature Figure 5; Monthly temperature and total number of four of the most abundant species of Scolytinae subfamily collected with Escolitideo/Curitiba traps from July 2005 to July 2006, in primary forest of Central Amazon. Table 3: Abundance, richness, and Simpson index of the species collected with five pitfall traps from July 2005 to July 2006. (a) Month 2005 Abundance Richness Simpson 0 0 1 2 1 0 Jul 10 3 0,46 0 0 1 0 0 1 118 6 0,589 102 4 0,470 Aug 84 4 0,596 95 6 0,618 104 6 0,433 3 3 0,667 8 3 0,594 Sep 45 4 0,593 59 3 0,561 75 2 0,461 36 5 0,690 32 5 0,375 Oct 18 3 0,494 36 4 0,437 32 4 0,689 218 6 0,526 102 4 0,552 Nov 72 5 0,588 108 4 0,573 71 4 0,492 65 5 0,455 33 5 0,571 Dec 22 4 0,479 27 5 0,694 12 4 0,681 (b) 2006 Month Abundance Richness Simpson 7 1 0 15 4 0,516 Jan 11 3 0,562 2 2 0,5 23 4 0,567 14 3 0,541 5 3 0,64 Mar 32 2 0,170 33 5 0,630 0 0 1 6 Psyche (b) Continued. Month 2006 Abundance Richness Simpson 44 4 0,539 188 4 0,201 May 118 4 0,373 41 3 0,604 75 4 0,543 21 5 0,522 82 3 0,361 Jun 15 1 0 20 4 0,415 0 0 1 0 0 1 4 3 0,625 Jul 0 0 1 2 2 0,5 0 0 1 many different species of trees, producing different attractive substances when they fall, die, or decay. This may have contributed to the reduced number of specimens collected with the EC trap although the number of species was greater. The majority of the captured species belong to the Xyleborini tribes, with 13 species, distributed in the genera Xyleborus, Sampsonius, Premnobius, and Theoborus, followed by Corthylini, with the genera Monarthrum and Amph- icranus. These species are prevalent in tropical regions, with xylomycetophagous habits, that is, feed on fungi that grow inside the plant. Another tribe found was Cryphalini, with the genera Cryptocarenus and Hypothenemus, also common in tropical regions, with varied eating habits, being considered myelophagous because they feed on pith and buds, phloephagous, feed on the tissues of the phloem, and xylophagous, feed on the xylem [1, 39]. This was also observed in the work of Abreu [ 14] . The Xyleborus genus was represented by six species, but Xyleborus volvulus and X affinis were dominant and found both in flight and on ground. These species are quite common and abundant in primary forest of Amazonas State, according to the work of Abreu [14] and Matias and Abreu [12], and, from what can be seen, they also have outstanding preference for the region of the collections, since there is a large difference between them and the other species, including when compared with those that have the same eating habits. The species X. ferruginous, despite being regarded as one of the most important and abundant in tropical regions, including being the vector of Ceratocystis fimbriata (Ellis & Halsted) fungus that causes the death of several plants [1, 40, 41] presented low abundance, confirming the work carried out by Abreu [8] , Abreu and Bandeira [9] , Matias and Abreu [12], and Abreu [14]. In contrast, many works carried out in the South, Southeast and Midwest of Brazil show that this species is abundant in those regions [22, 33, 34, 36, 37], being considered fairly common in Brazil. Table 4: Abundance, richness, and Simpson index of the species collected with five Escolitideo/Curitiba traps from July 2005 to July Month 2005 Abundance Richness Simpson 6 4 0,722 2 1 0 Jul 3 2 0,444 2 1 0 2 1 0 11 5 0,744 6 4 0,667 Aug 12 7 0,708 4 2 0,375 3 3 0,667 6 3 0,667 10 5 0,76 Sep 10 7 0,78 7 3 0,571 9 4 0,617 5 5 0,8 10 8 0,86 Oct 9 6 0,741 9 6 0,790 1 1 0 14 8 0,837 11 5 0,711 Nov 4 3 0,625 4 3 0,625 5 5 0,8 5 4 0,72 0 0 1 Dec 0 0 1 1 1 0 0 0 1 (b) 2006 Month Abundance Richness Simpson 20 6 0,64 18 4 0,377 Jan 10 5 0,68 13 2 0,142 20 5 0,545 4 2 0,375 2 1 0 Mar 5 4 0,72 9 4 0,667 8 4 0,656 Psyche 7 (b) Continued. Month 2006 Abundance Richness Simpson 20 4 0,575 19 8 0,731 May 13 5 0,734 19 7 0,765 15 6 0,658 12 5 0,764 12 5 0,764 Jun 12 3 0,486 13 4 0,485 13 4 0,675 27 3 0,565 46 3 0,163 Jul 11 4 0,645 38 7 0,636 19 5 0,504 Another very representative genus was the Sampsonius, with five species, including S. dampfi. The species in this genus also feed on fungi that grow in the host. As females are unable to construct an entrance tunnel in the plant, they look for recently constructed galleries of Xyleborus, appropriate for their bodies. After entering, they wait until the tunnels are extended, cleaned, and after that they expel the lodgers [ 1 ]. Many Scolytinae are attracted by resin-oils, terpene hydrocarbons or alcohols and other substances emanating from the vascular tissues of newly felled trees, decayed and still with high levels of humidity [3, 4]. In accordance with Hulcr et al. [35], ambrosia beetles are strongly attracted by hosts that liberate high levels of alcohol. This work reinforces the theory that ambrosia beetles are common in tropical forests, because these environments present favorable climatic conditions for the development of these insects, as well as their fungal symbionts. Studies carried out by Hulcr et al. [35, 42] in forests of Thailand and Papua New Guinea confirmed this theory. 4. Conclusions The capture of Scolytinae in primary forest of Central Ama- zon shows a low diversity of these insects and the existence of two predominant species in the region. It also shows that many species, in addition to flying, also have activities on forest ground. In this area there are many lignocellulosic materials as trunks and branches of trees, where they can cultivate the fungi they feed on. The majority of the insects collected in the studied area have no correlation with the temperature and rainfall. Acknowledgments The authors would like to thank the doctoral student Fabri- cio Baccaro of the postgraduate programme at INPA, for the help with the statistical analyses and also the laboratory technicians Frank Antonio de Oliveira Campos and Janio da Costa Santos for their help in field collections. References [1] S. L. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 621934, 11 pages doi:10.1155/2012/621934 Review Article Galling Aphids (Hemiptera: Aphidoidea) in China: Diversity and Host Specificity Jing Chen^’^ and Ge-Xia Qiao^ ^ Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China ^College of Life Sciences, Graduate University of Chinese Academy of Sciences, No. 19 Yuquan Road, Shijingshan District, Beijing 100049, China Correspondence should be addressed to Ge-Xia Qiao, qiaogx@ioz.ac.cn Received 29 August 2011; Revised 2 November 2011; Accepted 10 November 2011 Academic Editor: Moshe Inbar Copyright © 2012 J. Chen and G.-X. Qiao. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gall formation is an interesting plant response to aphid feeding. This paper presents a review of galling aphids in China. Altogether, 157 species and subspecies in ten families and subfamilies are found to induce galls on their host plants. As many as 39% species are endemic to China. The Eriosomatinae include the highest percentage of gall-inducing species. The great diversity of gall morphology may be described in terms of five characteristics: type, site, size, shape, and structure. The host association and host specificity of galling aphids are also discussed. 1. Introduction Aphids (Hemiptera: Aphidoidea) are an important group of phloem-feeding insects that may limit plant productivity [1] and transmit plant viruses [2]. They feed from the phloem sieve elements by penetrating their slender stylets intercel- lularly during which specific plant responses are triggered [3, 4]. A remarkable and interesting plant response is the formation of galls. These atypical plant tissue growths, the result of interactions between the inducer and the plant [5], reflect the complex and intimate relationship between plants and insects. Galls provide abundant nutrition [6-11], a favorable microenvironment [6, 7, 12, 13], and protection against natural enemies [6, 7, 14] to the inducer and its offspring. They also mitigate clonal mixing and maintain the genetic integrity [ 15, 16] . Therefore, galling aphids have been viewed as a useful model system for studying herbivore- plant interactions. Approximately 10-20% of the 4,700 aphid species known worldwide can induce galls on their host plants [17]. Gall traits are important biological characteris- tics of galling aphids. The shape, structure, and site of galls are highly species specific [18]. Thus, galls are commonly regarded as the extended phenotype of aphids [19-21] and hence helpful for aphid identification and phylogenetic study. A total of approximately 1,100 aphid species are now known from Ghina, constituting at least 23% of the world’s aphid fauna [22]. Studies of galling aphids in Ghina focus primarily on the subfamilies Eriosomatinae and Hormaphid- inae. Zhang et al. [23] and Ghen and Qiao [24] systematically studied the diversity of the galls of these two subfamilies, respectively. The evolution of galls in the tribes Fordini and Pemphigini of Eriosomatinae was also discussed [10, 11]. In this paper, galling aphids in Ghina are reviewed with special emphasis on the diversity of gall-inducing species and gall morphology. Host association and host specificity are also considered. The aphid species information in this paper was obtained from the species records of specimens deposited in the National Zoological Museum of Ghina, Institute of Zoology, Ghinese Academy of Sciences, Beijing, China (NZMCAS) and identified by aphid taxonomists of our research group. The information about galls and host plants was taken from field collection records. The host plant species were identified by plant taxonomists of the Institute of Botany, Chinese Academy of Sciences and Beijing Forestry University. 2 Psyche The aphid classification system used in this paper follows G. Remaudiere and M, Remaudiere [25]. 2. Galling Aphid Diversity At present, approximately 157 species and subspecies (14% of the total number of Chinese aphid species) are known to induce galls on their specific host plants in China. They are restricted to ten families and subfamilies, that is, Adelgidae, Phylloxeridae, Eriosomatinae, Hormaphidinae, Aphidinae, Chaitophorinae, Mindarinae, Myzocallidinae, Phyllaphidi- nae, and Thelaxinae. Of these gall-inducing aphid species, 83 species belong to the subfamily Eriosomatinae, 37 to Aphidinae, and 19 to Hormaphidinae. The principal groups of galling aphids and the host plants bearing galls in China are listed in Table 1. Out of these galling aphids, 61 species and subspecies are endemic to China, accounting for 39% of the total galling species. Endemic species, localities (province) where galls were collected, host plants bearing galls, and gall morphology are listed in Table 2. 3. Gall Morphological Diversity 3.1. Type. Aphids can induce both pseudogalls and true galls on their host plants. Pseudogalls are commonly found on leaves and appear as leaf folds, leaf rolls, leaf curls, or leaf blisters. In the subfamilies Aphidinae, Chaitophorinae, Min- darinae, Myzocallidinae, Phyllaphidinae, and Thelaxinae, only pseudogalls are produced. Hyalopterus pruni (Geoffroy) produces noticeable leaf-roll pseudogalls on Prunus arme- niaca in the spring (Eigure l(n)). Cryptosiphum artemisiae Buckton causes leaves to swell and roll on Artemisia (Eigure l(o)). Different from pseudogalls, true galls are more diverse in shape, complex in structure, and not only located on the leaves. The Adelgidae and Phylloxeridae induce true galls on their host plants. In the Eriosomatinae and Hormaphidinae, both pseudogalls and true galls are formed. A species of Hormaphidinae, Tuber aphis takenouchii (Taka- hashi), forms broccoli-head-like galls on the twigs of Sty rax formosana, found in Taiwan. However, its congeneric species T. viscisucta (Zhang), which occurs in Yunnan, feeds on the leaves of Viscum album and causes the leaf edges to curl downward. 3.2. Site. The fundatrix (the first spring generation that hatches from overwintering eggs) of each galling aphid species induces its gall at a specific site on a specific host [26]. Galls may occur on the leaf blade, the leaf vein, the petiole, the main axis of a compound leaf, twigs, branches, and roots. Viteus vitifoliae (Eitch) causes galls on grape leaves and gall-like swellings on grape roots. Pemphigus bursarius (Linnaeus) produces galls on the petioles of Populus simonii (Eigure 1(e)). The galls of Floraphis meitanensis Tsai & Tang are located on the main axis of the compound leaf of Rhus punjabensis var. sinica. Among these galls, the leaf gall is the most common type. The galling site is closely related to the sink strength of galls and therefore has important effects on the reproductive success of the fundatrix [18, 27-29]. Eundatrices compete for galling sites. Intraspecific fights have been reported in different aphid species [28, 30, 31]. Eurthermore, one plant species can host several galling aphid species. These species coexist by attacking different organs of a single host plant. In China, Distylium chinense harbors at least four species of Hormaphidinae, that is, Asiphonipponaphis vasigalla Chen, Sorin & Qiao, Neothoracaphis yanonis (Mat- sumura), Metanipponaphis sp., and Nipponaphis sp.. They form specific galls on the leaf midrib (Figure l(j)), the leaf blade (Figure 1(1)), the petiole (Figure l(k)), and the twig (Figure l(m)), respectively. This observation indicates an adaptive radiation of galling aphids that exploit different ecological niches within a host plant. 3.3. Size. Gall size is also highly variable. Some aphids make very small galls. For example, the galls of Acanthochermes similiquercus Jiang, Huang & Qiao (Figure 1(b)) are small bean sized. Each gall contains only one aphid. In contrast, some galls are extremely large. The galls of Ceratoglyphina styracicola (Takahashi) in Taiwan can reach a diameter of 9 cm and contain 100,000 aphids [32]. It is obvious that gall size is directly linked to aphid colony size. Like the galling site, size is also an important factor influencing the gall sink strength [18, 29]. The species of Hormaphidinae on Distylium chinense serve to illustrate this principle. Large galls of Nipponaphis sp. (Figure l(m)) divert nutrients from neighboring shoots. The smaller galls of Metanipponaphis sp. (Figure l(k)) divert nutrients only from neighboring leaves on the same shoot. The smallest galls of Neothoracaphis yanonis (Figure 1(1)) use only the leaf on which they are located. 3.4. Shape. The shapes of aphid galls vary and have been described as chilli-like, cockscomb -like, bag-like, spheri- cal, cucumber-like, flower-like, boat-like, broccoli-head-like, banana-bundle-shaped, and so on. Gall shape is highly species specific. Galls produced by individuals of the same species are distinctively similar in shape [18]. Chaetogeoica foliodentata (Tao) produces cockscomb-like galls on Pistacia weinmannifolia (Figure 1(h)). Epipemphigus imaicus (Chol- odkovsky) forms silkworm-like galls on Populus cathayana (Figure 1(d)). Different species induces different shaped galls even on the same organ of the same host plant. On the leaves of Distylium chinense, for example, Asiphonipponaphis vasigalla forms vase-shaped galls (Figure l(j)), whereas the galls of Neothoracaphis yanonis are spherical with a pointed bottom and protrude from both sides of the leaves (Figure 1(1)). This observation suggests that gall shape is determined by the aphids rather than by the plants and gall is an extended phenotype of the aphids [20]. Fundatrix behavior during gall initiation is assumed to play a decisive role in regulating gall shape, especially complex and peculiar shapes [18, 29]. 3.5. Structure. Based on the number of cavities, galls can be divided into single-cavity and multiple-cavity types. The single-cavity gall is the simplest and most common type of aphid gall. The multiple-cavity gall is composed of Psyche 3 Table 1: Taxonomy of galling aphids and host plants bearing galls in China. Family Subfamily/tribe Genera Host plants Adelgidae Adelges, Pineus Picea Phylloxeridae Acanthochermes Quercus Viteus Vitis Aphididae Eriosomatinae Eriosomatini Aphidounguis, Colopha, Eriosoma, Kaltenbachiella, Tetraneura Ulmus Eordini Aploneura, Baizongia, Chaetogeoica, Forduy Namaforda, Smynthurodes Pistacia Floraphisy Kaburagia, Meitanaphis, Nurudea, Schlechtendalia Rhus Pemphigini Epipemphigus, Pachypappa, Pachypappella, Pemphigus, Thecabius Populus Prociphilus Caprifoliaceae, Oleaceae, Rosaceae Hormaphidinae Cerataphidini Aleurodaphis Sinojackia Astegopteryx, Cerataphis, Ceratoglyphina, Ceratovacuna, Ktenopteryx, Pseudoregma Sty rax Tuberaphis Sty rax, Viscum Hormaphidini Hamamelistes Betula Nipponaphidini Asiphonipponaphis, Metanipponaphis, Neothoracaphis, Nipponaphis Distylium Aphidinae Aphidini Aphis Malvaceae, Prunus Cryptosiphum Artemisia Hyalopterus Rosaceae Macrosiphini Cryptaphis Geranium Cryptomyzus, Hyperomyzus Ribes Diuraphis Pooideae Dysaphis, Myzus, Ovatus, Sappaphis, Sorbaphis, Tuberocephalus Rosaceae Hayhurstia Chenopodium Eipaphis Cruciferae Neorhopalomyzus Eonicera Rhopalosiphoninus Deutzia, Syringa Semiaphis Eonicera, Umbelliferae Chaitophorinae Chaitophorus Populus Periphyllus Acer, Aesculus, Koelreuteria Mindarinae Mindarus Abies Myzocallidinae Tinocallis Sapindus, Lagerstroemia Phyllaphidinae Phyllaphis Fagus Thelaxinae Kurisakia Quercus 4 Psyche Table 2: Endemic galling aphid species in China. Species Distribution Host plants bearing galls Gall morphology Adelgidae Adelges (Gilletteella) glandulae (Zhang) Sichuan, Yunnan Picea brachytyla, P likiangensis, P likiangensis ysLY. balfouriana, P purpurea Leaf gall, cone-like, multiple- cavity, without associated needles [33]. Adelges (Sacchiphantes) roseigallis (Li & Tsai) Gansu Picea asperata, P crassifolia Leaf gall, cone-like [34]. Pineus (Pineus) armandicola Zhang, Zhong & Zhang Yunnan Picea likiangensis Leaf gall, cone-like or torch-like, multiple- cavity [35]. Picea brachytyla. Pineus (Pineus) sichunanus Sichuan, Yunnan P likiangensis, P Leaf gall, broom-like or Zhang likiangensis var. balfouriana, P purpurea torch-like, multiple -cavity [33]. Phylloxeridae Acanthochermes similiquercus Jiang, Huang & Qiao Sichuan Quercus sp. Small spherical gall on the leaf with an opening on the lower surface [36]. Eriosomatinae Eriosomatini Eriosoma cerum Zhang Beijing, Hebei, Inner Mongolia Ulmus pumila Leaf edge curls downward [37]. Eriosoma fukangense Zhang Xinjiang Ulmus pumila Leaf rolls, screw-like [37]. Multiple -cavity gall on the Eriosoma multilocularis Zhang & Zhang Beijing, Hebei Ulmus pumila underside of leaves, with longitudinal ridges on lateral sides [38]. Eriosoma spirifolium Zhang Xinjiang Ulmus pumila Leaf curls, spiral- shaped [37]. Eriosoma togrogum Zhang Inner Mongolia, Shaanxi Ulmus pumila Leaf pseudogall [39]. Eriosoma ulmipumilicola Zhang & Zhang Hebei Ulmus pumila Leaf rolls and swellings [37]. Eriosoma usuense Zhang & Qiao Xinjiang Ulmus pumila Leaf spirally twisted [37] . Kaltenbachiella glabra Akimoto Taiwan Ulmus uyematsui Almost globular gall, without hairs, arising upward from the midrib of leaves [40]. Tetraneura (Indotetraneura) asymmachia Zhang & Zhang Liaoning Ulmus parvifolia, U pumila Leaf gall, irregular pouch-like, stalked, smooth, with secondary exit laterally [37]. Tetraneura (Tetraneura) aequiunguis Zhang & Zhang Inner Mongolia, Shandong Ulmus sp. Leaf gall [41]. Tetraneura (Tetraneura) Liaoning, Ulmus pumila Leaf gall, peach- shaped, stalked persicina Zhang & Zhang Shandong 137]. Tetran eu ra (Tetraneura) Hebei, Shandong, Ulmus pumila Irregular-shaped gall on the triangula Zhang & Zhang Tianjin upper side of leaves [37]. Tetraneura (Tetraneura) ulmicema Zhang Ningxia Ulmus glabra Leaf gall, smooth, without hairs 137]. Eriosomatinae Eordini Chaetogeoica foliodentata (Tao) Jiangsu, Shaanxi, Shandong, Sichuan, Yunnan, Zhejiang Pistacia chinensis, P weinmannifolia Cockscomb-like gall, arising from the midrib on the upper surface of leaves [37]. Chaetogeoica ovagalla (Zhang) Shandong Pistacia chinensis Elongate oval gall, arising from the midrib of leaves [42]. Psyche 5 Table 2: Continued. Species Distribution Host plants bearing galls Gall morphology Chaetogeoica sensucopia (Zhang) Shandong Pistacia chinensis Small elongate oval gall, arising from the midrib of leaves [42]. Chaetogeoica ulmidrupa Zhang Shandong Pistacia aculeata Dimidiate gall, with a longitudinal ridge [43] . Chaetogeoica yunlongensis (Zhang & Zhong) Shaanxi, Yunnan Pistacia chinensis Cucumber-shaped gall, arising from the midrib of leaves [42]. Floraphis choui Xiang Shaanxi Rhus potaninii Gall produced on leaflets, branched with every branch obconical [44] . Floraphis meitanensis Tsai & Tang Guizhou, Hunan, Sichuan Rhus punjabensis var. sinica Gall produced on the main axis of a compound leaf, flattened, branched from base [37, 45]. Kaburagia rhusicola ensigallis (Tsai & Tang) Guizhou, Hubei, Hunan, Sichuan Rhus punjabensis var. sinica Elongate jujube-like gall, with a pointed tip, arising from the main vein or secondary vein on the base of leaves [37]. Kaburagia rhusicola ovatirhusicola Xiang Hubei, Shaanxi, Yunnan Rhus potaninii Gall obovate, outer surface with prominent net-like veins, produced on leaflet [44] . Gall ovate, tip rounded, with Kaburagia rhusicola ovogallis Guizhou, Hubei, Rhus chinensis, short hairs, arising from the (Tsai & Tang) Hunan, Sichuan R. punjabensis var. sinica secondary vein on the base of leaves [37]. Meitanaphis elongallis Tsai & Tang Guizhou, Hubei, Hunan Rhus punjabensis yar. sinica Rhus chinensis, R. chinensis var. roxburghii, R. punjabensis var. sinica Gall jujube-like, outer surface with many fine longitudinal ridges, arising from the midrib on the lower surface of leaves [37, 45]. Gall ovate, somewhat flattened. Schlechtendalia peitan (Tsai & Guizhou, Hunan, with short soft hairs, arising Tang) Sichuan from the secondary vein on the base of leaves [37]. Eriosomatinae Pemphigini Gall cockscomb-like, outer surface uneven with 3 small Epipemphigus chomoensis (Zhang) Tibet Populus sp. protuberances, situated along the midrib on the upper surface of leaves, with a primary exit on the lower surface [46]. Gall cockscomb-like, outer surface uneven with 3-5 acute Epipemphigus yunnanensis Guizhou, Yunnan Populus bonatii. angled protuberances, situated (Zhang) P cathayana, P yunnanensis along the midrib on the upper surface of leaves, with a primary exit on the lower surface [46]. Pachypappella aliquipila Zhang Hebei Populus sp. Leaf folds [37]. Pemphigus circellatus Zhang & Yunnan Populus tremula var. Big opened gall on the branch Zhong davidiana, P. yunnanensis |47]. Pemphigus cylindricus Zhang Tibet Populus sp. Gylinder- shaped leaf gall [48]. Pemphigus mangkamensis Zhang Tibet Populus sp. Gockscomb-like gall on the upper side of leaves [48]. Pemphigus sinobursarius Heilongjiang, Inner Mongolia, Populus cathayana. Round pouch-like gall, arising from the base of midrib on the Zhang Liaoning, Ningxia, Yunnan P euphratica, P simonii under surface of leaves [46]. 6 Psyche Table 2: Continued. Species Distribution Host plants bearing galls Gall morphology Pemphigus tibetensis Zhang Beijing, Gansu, Hebei, Tibet, Xinjiang Populus cathayana Pomegranate-like gall, with thick wall and a primary exit, produced on the twig [46]. Pemphigus turritus Zhang Gansu Populus purdomii Sharp chilli-like or sharp horn-like gall, arising from the midrib on the upper surface of leaves, with a primary exit on the lower surface [37, 49]. Pemphigus wuduensis Zhang Gansu Populus purdomii Flattened globular gall, arising from the base of midrib on the upper surface of leaves, with a secondary exit [37, 49]. Pemphigus yangcola Zhang Yunnan Populus yunnanensis Semispherical gall on the twig, smooth, with primary exit [46]. Prociphilus (Prociphilus) gumbo sue Zhang 8c Zhang Hebei Syringa oblata Leaf curls [50]. Prociphilus (Prociphilus) ligustrifoliae (Tseng 8c Tao) Guizhou, Shaanxi, Sichuan, Yunnan Ligustrum japonicum, L. lucidum Leaf curls [51]. Thecabius (Oothecabius) sequelus Zhang Hebei, Xinjiang Populus simonii Leaf folds and swellings along the midrib, dumpling-shaped [52]. Thecabius (Parathecabius) zhongi Zhang Gansu Populus cathayana Dumpling- shaped pseudogall, formed by folding of the two halves of a leaf together [52] . Thecabius (Thecabius) beijingensis Zhang Beijing, Hebei, Heilongjiang, Liaoning, Shanxi Populus beijingensis, P cathayana, P koreana Leaf folds and rolls up along the midrib, forming a swollen sausage-shaped pseudogall [52]. Hormaphidinae Leaf curls, boat-shaped [53]. Vase- shaped gall, arising from or near the midrib on the upper surface of leaves, with a flower-shaped opening at the tip when mature [54]. Large bell-shaped gall on the branch, covered with much white wax [55]. Small conical gall on the upper side of leaves [56]. Leaf deformation [57]. Branched gall on the twig [58] . Leaf curls [59]. Aphidinae Cryptosiphum artemisiae linanense Zhang Gansu, Hebei, Artemisia argyi. Heilongjiang, Jilin, Liaoning, Zhejiang A. atrovirens, A. mongolica, A. selengensis Leaf rolls, fist-like [60]. Cryptosiphum atriplicivorum Zhang Gansu Artemisia sp. Leaf rolls and swellings [39]. Neorhopalomyzus lonicerisuctus Zhang, Zhong 8c Zhang Sichuan, Yunnan Lonicera sp. Leaf curls [61]. Aleurodaphis sinojackiae Qiao 8c Jiang Jiangsu, Zhejiang Sinojackia xylocarpa Asiphonipponaphis vasigalla Ghen, Sorin 8c Qiao Hunan Distylium chinense Cerataphis jamuritsu (Takahashi) Hong Kong, Taiwan Styrax suberifolia Hamamelistes similibetulae (Qiao 8c Zhang) Tibet Betula albosinensis Ktenopteryx eosocallis Qiao 8c Zhang Fujian, Guangxi Styrax odoratissima Tuber aphis cymigalla (Qiao 8c Zhang) Fujian Distylium racemosum (unlikely, requiring further confirmation) Tuberaphis viscisucta (Zhang) Yunnan Viscum album Psyche 7 Table 2: Continued. Species Distribution Host plants bearing galls Gall morphology Sappaphis dipirivora Zhang Beijing Pyrus sp. Leaf rolls and swellings [62]. Sappaphis sinipiricola Zhang Hebei, Henan Pyrus betulaefolia Leaf rolls [62]. Tuberocephalus ( Trichosiphoniella ) tianmushanensis Zhang Beijing, Zhejiang Prunus pauciflora Leaf curls, cylinder-shaped [60]. Tuberocephalus (Trichosiphoniella) tuberculus Su, Jiang 8c Qiao Sichuan An unidentified species of Rosaceae Pseudogall along the lateral vein of young leaves [63] . Chaitophorinae Periphyllus acerihabitans Zhang Jiangsu, Zhejiang Acer buergerianum Leaf curls [64]. several subgalls with more complex ontogeny and structure [24, 65]. The Adelgidae and many galling species in the tribe Cerataphidini of Hormaphidinae produce multiple- cavity galls (see [65]). The pineapple-like galls of Adelges sp. (Figure 1(a)) are formed from spruce buds in which the developing leaves enlarge laterally and the margins merge into each other, forming multiple chambers within which the aphids feed [66]. Galls can also be categorized into two types of structure: closed and open. Some galls are initially closed and do not open until maturity. However, other galls remain open during their entire period of development. True galls include both closed and open types. In Shandong, the galls of Tetraneura (Tetraneurella) nigriabdominalis (Sasaki) (Figure 1(c)) are closed from early May through early June and then split laterally. From these slits, the alatae leave the gall to found colonies on the roots of Gramineae. However, the galls of Pemphigus bursarius have a natural lateral opening over an extended period from late May through September (Figure 1(e)). In contrast, pseudogalls are generally open and relatively simple in structure. On Populus ussuriensis, Thecahius (Oothecabius) populi (Tao) causes the leaves to fold downward, forming open dumpling- shaped pseudogalls (Figure 1(g)). Gall shape and structure are closely related to gall fitness, the number of offspring produced within a gall. Together with gall size, gall shape and structure determine the inner surface area of the gall, the most accurate and practical mea- sure of gall fitness [67]. The inner surface area determines the feeding area available to the nymphs within the gall and is thus correlated with the aphid colony size. It would be interesting to investigate the possibility that aphid galls tend to be more complex in shape and structure (e.g., multiple- cavity galls) to support larger colonies. Aphid galls show great diversity of type, site, size, shape, and structure. However, these gall traits are also highly species specific. Therefore, they help to identify species, es- pecially species that are difficult to distinguish morpholog- ically [68]. Knowledge of the evolutionary driving forces behind the divergence of gall morphology is limited. Natural enemies [6, 7, 14, 21], competition for galling sites [26], and increasing sink strength [8, 10, 11] might have influenced the divergence of gall traits. The great variety of galls is hypoth- esized to be related to aphid adaptive radiation [5, 69]. Several case studies of selected aphid groups have been con- ducted to investigate how gall diversification has influenced aphid speciation. The gall-inducing aphid group Fordini is supposed to have radiated primarily by using different sites on the same plant organ and by enlarging the gall size [8, 10] . Its sister group, the gall-inducing Pemphigini, is assumed to have diversified by attacking different plant organs [11]. 4. Host Association and Host Specificity Gall-bearing host plants are richly abundant in Ghina. These gall-bearing plants belong to at least 64 genera and 27 fami- lies. The common gall-bearing plants include Picea, Ulmus, Populus, Styrax, and Distylium (Table 1). Gall-inducing aphids are generally heteroecious, alternate between primary host plants (woody), where galls are produced and the sexual phase of life cycle is completed, and secondary host plants (herbaceous) where only parthenogenetic generations occur. Some aphids, however, are capable of inducing galls on sec- ondary host plants, for example, Hamamelistes similibetulae on Betula albosinensis and Cryptosiphum artemisiae on Arte- misia. Galling aphids are strictly associated with their primary hosts. The host specificity is well defined and represented particularly in the Adelgidae, Eriosomatinae, and Horma- phidinae (Table 1). The pattern of host association differs among aphid lineages. The Adelgidae are restricted to Picea (Pinaceae). The subfamily Eriosomatinae includes three host-specific tribes: Eriosomatini on Ulmus (Ulmaceae), Fordini on Pistacia and Rhus (Anacardiaceae) (Fordina on Pistacia, Melaphidina on Rhus, resp.), and Pemphigini on Populus (Salicaceae). The Hormaphidinae include three tribes: Gerataphidini on Styrax (Styracaceae), Hormaphidini on Hamamelis (Hamamelidaceae), and Nipponaphidini on Distylium (Hamamelidaceae). But the galling species of Hor- maphidini in Ghina are restricted to their secondary host Betula (Betulaceae) owing to the absence of primary host in their distribution areas. In Aphidinae, the association is not so rigid. Most gall-inducing species are primarily associated with Rosaceae. Some other remotely related plant species, such as Lonicera (Gaprifoliaceae) and Ribes (Saxifragaceae), are also occupied. 8 Psyche Figure 1: (a) A gall of Adelges sp. on Picea crassifolia (Fluangzhong, Xining, Qinghai, China; 8 July 2009): pineapple-like, multiple-cavity, closed, (b) Galls of Acanthochermes similiquercus Jiang, Huang 8c Qiao on a leaf of Quercus sp. (Baoding, Panzhihua, Sichuan, China; 15 April 2005): spherical, single- cavity, some matured ones are open, (c) Galls of Tetraneura (Tetraneurella) nigriabdominalis (Sasaki) on a leaf of Ulmus sp. (Hulun Buir, Inner Mongolia, China; 1 August 2004): spindle-shaped, single- cavity, closed, (d) A pseudogall of Epipemphigus imaicus (Cholodkovsky) on Populus cathayana (Shangri-La, Yunnan, China; 27 July 2010): silkworm-like, single-cavity, open on the undersurface of the leaf, (e) A gall of Pemphigus bursarius (Linnaeus) on a petiole of Populus simonii (Miyun, Beijing, China; 25 July 2009): bag-like, single- cavity, with a natural lateral opening, (f) A gall of Pemphigus immunis Buckton on a twig of Populus sp. (Miyun, Beijing, China; 25 July 2009): long saccate, single-cavity, closed, (g) A dumpling-shaped leaf-fold pseudogall of Thecabius (Oothecabius) populi (Tao) on Populus ussuriensis (Miyun, Beijing, China; 25 July 2009). (h) A gall of Chaetogeoica foliodentata (Tao) on the leaf midrib of Pistacia weinmannifolia (Qishan, Baoji, Shaanxi, China; 12 July 2004): cockscomb-like, single-cavity, closed, (i) A gall of Tuberaphis owadai Kurosu 8c Aoki on a twig of Styrax japonica (Yuanyang, Kunming, Yunnan, China; 10 June 2009): coral-like, single-cavity, with many small openings on the projections, (j) Galls oi Asiphonipponaphis vasigalla Chen, Sorin 8c Qiao on the leaf midribs of Distylium chinense (Jishou, Hunan, China; 15 April 2010): vase-shaped, single-cavity, open on the bottom, (k) A gall of Metanipponaphis sp. on a petiole of Distylium chinense (Jishou, Hunan, China; 6 September 2009): global, single-cavity, closed. (1) Galls of Neothoracaphis yanonis (Matsumura) on the leaves of Distylium chinense (Jishou, Hunan, China; 21 May 2009): spherical with a pointed bottom, protruding from both sides of the leaves, single-cavity, closed, (m) A gall of Nipponaphis sp. on a twig of Distylium chinense (Jishou, Hunan, China; 5 September 2009): bottle- shaped, single- cavity, closed, (n) Leaf rolls caused by Hyalopterus pruni (Geoffroy) on Prunus armeniaca (Huangzhong, Xining, Qinghai, China; 8 July 2009). (o) Leaf rolls and swellings caused by Cryptosiphum artemisiae Buckton on Artemisia sp. (Miyun, Beijing, China; 25 July 2009). Psyche 9 The association of galling aphids with their specific host plants is supposed to be ancient [70]. One hypothesis about what plays an important role in shaping aphid-host plant association is “fundatrix specialization” [71-74]. This hypothesis suggests that the fundatrix is highly specialized to the ancestral host and is less able to acquire new hosts than are other morphs over long evolutionary periods. It should be noted that, with few exceptions, the fundatrix, the morph that hatches from the overwintering egg on the primary host in the spring, is the only morph that can induce a gall [18, 29]. Thus, the phylogenetic constraints on the fundatrix would in some degree explain the high host specificity of galling aphids to their primary hosts. Aphids have very intimate associations with their host plants. The aphids obtain food resources and habitat from their hosts. It is commonly assumed that host plants have a great influence on aphid diversification [75]. Two of the major hypothesized pathways of diversification in phy- tophagous insects are cospeciation with host plants [76, 77] and speciation through host shift [78]. Both hypotheses rely on some degree of host specialization and suggest that host plant diversity plays a major role in insect diversification [75, 79]. In particular, galling aphids are closely linked with their host plants and, as a whole, occupy a wide range of plant species. Primary host specificity is strong. Different aphid families, subfamilies, or tribes are strictly associated with different plant families or genera. We think that the high host plant diversity should have considerable influence on the diversification of galling aphids and the adaptation to different host plants in different galling lineages might have driven the divergence of galling aphids at higher taxonomic levels. 5. Conclusion This paper has presented a preliminary review of gall-induc- ing aphids in China. The major groups of galling aphids were surveyed. The great diversity of gall morphology was analyzed systematically according to type, site, size, shape, and structure. 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Jermy, “Evolution of insect/host plant relationships,” Amer- ican Naturalist, vol. 124, no. 5, pp. 609-630, 1984. [79] I. S. Winkler and G. Mitter, “The phylogenetic dimension of insect-plant interactions: a review of recent evidence,” in Specialization, Speciation, and Radiation: The Evolutionary Biology of Herbivorous Insects, K. J. Tilmon, Ed., pp. 240-263, University of Galifornia Press, Berkeley, Galif, USA, 2008. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 905109, 9 pages doi:10.1155/2012/905109 Review Article Specialized Fungal Parasites and Opportunistic Fungi in Gardens of Attine Ants Fernando C. Pagnocca,^ Virginia E. Masiulionis,^ and Andre Rodrigues^’ ^ ^ Centre for the Study of Social Insects, Sao Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil ^Department of Biochemistry and Microbiology, Sao Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil Correspondence should be addressed to Andre Rodrigues, andrer@rc.unesp.br Received 1 September 2011; Revised 2 November 2011; Accepted 5 November 2011 Academic Editor: Volker Witte Copyright © 2012 Fernando C. Pagnocca et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ants in the tribe Attini (Hymenoptera: Formicidae) comprise about 230 described species that share the same characteristic: all coevolved in an ancient mutualism with basidiomycetous fungi cultivated for food. In this paper we focused on fungi other than the mutualistic cultivar and their roles in the attine ant symbiosis. Specialized fungal parasites in the genus Escovopsis negatively impact the fungus gardens. Many fungal parasites may have small impacts on the ants’ fungal colony when the colony is balanced, but then may opportunistically shift to having large impacts if the ants’ colony becomes unbalanced. 1. Introduction Restricted to the New World, the approximately 230 fungus- growing ant species in the tribe Attini cultivate basidiomyce- tous fungi on freshly harvested plant substrate [ 1-3] . A well- known subset of species in this tribe, the leaf-cutting ants, are considered the most important herbivores in the Neotropics [4, 5] , due to the large amount of fresh leaves and flower parts that workers cut and use to nourish the mutualistic fungal cultivar. The attine ant-fungal symbiosis is ancient and probably originated from ancestral ants occurring in the Amazon basin about 50 million years ago [6, 7]. Since then, the tribe Attini evolved five patterns of fungiculture that are currently recognized [6]. Thus, the lower and higher attine ant genera practice different types of fungiculture, which are classified according to the type of fungus and the type of substrates used to maintain the fungal partner [6]. Therefore, the various fungiculture can be defined as (i) lower attine agri- culture performed by phylogenetically basal ant genera such as Cyphomyrmex, Mycetagroicus, and Mycetophylax which cultivate fungi in the tribe Leucocoprini [8, 9], (ii) a specific type of agriculture performed by some species of the lower attine genus Apterostigma, which cultivates fungi within the family Pterulaceae (the “coral fungi” [ 10, 1 1 ] ), (iii) a group of ants that cultivate Leucocoprini fungi in the yeast form (ants in the Cyphomyrmex rimosus group), (iv) the higher attine agriculture that encompass the derived genera Trachymyrmex and Sericomyrmex which cultivate phylogenetically derived fungi within the Leucocoprini, mostly on fallen vegetation or organic matter, and finally (v) a specific group of ants within the higher agriculture, the leaf-cutting ants, cultivate a recent clade of derived Leucocoprini fungi [12]. Evidence shows that attine ants domesticated their fungal cultivars during the evolution of the symbiosis [8]. Thus, the evolutionary history of fungus -growing ants was marked by several horizontal transfers (switches) of cultivars. Par- ticularly, within the lower attine fungiculture these cultivar switches occurred multiple times [8]. Mikheyev et al. [13] demonstrated that leaf-cutting ants cultivate the same species of cultivar, Leucoagaricus gongylophorus in an association known as “many to one.” Interestingly, it was found that in colonies of leaf- cutting ant, just a single clone of the mutualistic fungus is cultivated by workers [14, 15]. Since the detailed study by Moller [16], it is known that attine fungiculture is continuously exposed to alien microor- ganisms. As fungus -growing ants rely on the mutualistic fungi as the main food source for the colony, fungiculture 2 Psyche requires from workers several mechanisms to keep their cultivars protected from alien microorganisms that would harm the symbiosis [17]. The most important strategies applied by attine ants in order to preserve their nests from harmful microbes consist of mechanical and chemical barriers including (i) careful cleaning of the leaf fragments used as substrate for the fungal cultivar in order to put away spores and microorganisms [18, 19]; (ii) massive inoculation of the mutualistic fungus mycelium onto the clean plant fragment increasing the colonization of this substrate by the cultivar [2]; (iii) the use of antimicrobial glandular secretions [20-24] and faecal droplets [25, 26]; (iv) weeding and grooming of infected parts of the garden when an undesired microorganism is detected [18]; (v) antagonistic activity of the mutualistic fungus against alien microorganisms [14, 19]; (vi) unspecific microbial interrelationship between microorganisms which benefit the whole nest [26]; (vii) control of humidity in disposal chambers [27]; (viii) association with antibiotic- producing bacteria [28-36]. Despite such mechanisms to suppress the development of alien microbes, a plethora of bacteria, filamentous fungi and yeasts are still found in ant gardens [35, 37-41]. Fungi on the genus Escovopsis are considered specialized parasites of attine gardens while others are consistently isolated in association with attine gardens and need further studies to understand their role as symbionts. Flere, we focus on Escovopsis sp. and the additional filamentous fungi and yeasts found in attine gardens and address the few studies that have explored the role of such microorganisms in the attine ant-fungal symbiosis. 2. Escovopsis sp.: The Specialized Garden Parasite of Fungus-Growing Ants The existence of the anamorphic fungus of the genus Escovopsis (Figure 1) was observed by various researchers [2, 42] and was uniquely discussed for the first time by Moller [16]. Recently, Currie et al. [37] reported that Escovopsis sp. is associated with several genera of attine ants and is considered a parasite of the fungus cultivated by these insects [43]. Except for fungus-growing ants in the Cyphomyrmex rimosus group, this parasite has been found in most attine ant genera with frequency of occurrence ranging from 11% to 75% [37, 38, 44, 45]. Escovopsis sp. can affect fungus gardens in various man- ners: in extreme cases, the parasite grows rapidly over the colony, resulting into its total collapse [45] (Figure 2). Ac- cording to experiments conducted by Currie [45], Escovopsis can remain in the colony for an extended period of time, thereby, suppressing subsequent colony development. Such impacts on the ant colony are supposed to be due to the necrotrophic action of Escovopsis sp. towards the cultivar [43]. With respect to the occurrence of Escovopsis, so far, this parasite was not recorded from any other environmental source other than in association with attine ants, a pointer to a long history of coevolution with these ants and their mutualistic fungi [46]. This ancient evolutionary pattern resulted in broad phylogenetic associations between the var- ious types of fungiculture and specific phylogenetic lineages of Escovopsis [46-49] as it is the case of the relationship of this mycoparasite and ants in the genus Apterostigma that are naturally threatened by a specific lineage of Escovopsis sp. [44] . This lineage comprises four Escovopsis morphotypes defined on the basis of conidial colours ranging between white, yellow, pink, or brown [44] . Meanwhile, within a particular fungiculture group, the same Escovopsis sp. strain can be associated with many genera of ants and vice versa, demonstrating that the interrelationship is apparently nonspecific or weak at a finer phylogenetic level [50, 51]. Even in the same nest, different Escovopsis strains can be found as confirmed in the work of Taerum et al. [52], who verified that 67% of the colonies of Atta sp. and Acromyrmex sp. were infected by multiple strains of the parasite. Interestingly, such strains did not engage in interference competition for their hosts [52]. So far, only two species are formally recognized in this genus, namely, Escovopsis weberi [53] and Escovopsis asper- gilloides [54]. These species were originally isolated from gardens of Atta sp. (in Brazil) and Trachymyrmex ruthae (in Trinidad and Tobago), respectively. Available data indicate that there exists a high variation in the morphology and genetic characteristics among strains of the two currently known Escovopsis species [44], suggesting that putative new species in this genus may be described in the near future [44, 55]. Several aspects of the biology of Escovopsis sp. still remain undiscovered. Nothing is known about their life cycle or whether there is a teleomorphic (sexual) state. Also, the mode of transmission between colonies is unknown. Regarding this aspect, Currie et al. [37] suggest that transmission may be through other arthropods that visit or inhabit the nests, such as mites. As a matter of fact, vertical transmission (from parental to offspring colonies) of this fungus has not been observed. Considering the harmful effect and close relationship with the attine cultivar [49], it is not surprising to consider that this parasite could be used as a biocontrol agent. Accord- ingly, Folgarait et al. [56] studied the antagonistic effect of Escovopsis sp. towards three strains of the mutualistic fungus. The authors’ findings indicate that, under in vitro conditions, Escovopsis sp. retarded the growth of the mutualistic fungus of Acromyrmex lundii and this effect is Escovopsis strain dependent. Similar results were previously reported by Silva et al. [57] in in vitro bioassays using one Escovopsis sp. strain and the mutualistic fungus of A. sexdens ruhropilosa. Despite these preliminary results about Escovopsis sp. as a potential agent of biological control, the ants’ defensive mechanisms need to be considered. As a result of Escovopsis sp. infection ant colonies first mount a generalized response through a large mobilization of the individuals [58]. Second, workers physically remove and concentrate spores in the infrabuccal cavity [18, 59] (grooming); in addition, workers remove affected parts of the fungus gardens [18] (weeding). Regarding these two ant behaviours, there seems to exist caste specialization [60] and recruitment of workers to the site Psyche 3 Figure 1: Escovopsis sp. parasites from fungus-growing ants, (a) General aspect of Escovopsis sp. isolated from the leaf-cutting ant Atta sexdens rubropilosa (Corumbatai, Brazil) cultured in potato dextrose agar (PDA) for 6 days at 25° C. (b) Close view of Escovopsis sp. isolated from Acromyrmex lobicornis (Santa Fe, Argentina) in PDA after 5 days at 25° C. (c) Escovopsis sp. conidiophores from (a). Note the cylindrical vesicles covered with ampulliform phialides. (a) (b) Figure 2; Laboratory colonies of Atta sexdens rubropilosa (Corumbatai, Brazil). Colonies were treated with baits without insecticide, control (a) and formulated baits containing the insecticide Hydramethylnon (b). Escovopsis sp. (white mycelia) emerged two days after treatment from fungus gardens of the infected colony (b), which depicts the aggressive effect of such parasite on attine ant colonies. of infection [61]. An additional important factor that may impair the use of Escovopsis as agent of biological control is their low spore viability (about 3% viability [24]) and it should be considered on the development of biological control methods. Perhaps the most effective defensive mechanism against Escovopsis sp. is the association of attine ants with microbial symbionts capable of producing antifungal substances. For example, ants are associated with Pseudonocardia, a group of bacteria found in the ant’s exoskeleton that antagonize Escovopsis sp. and which is vertically transmitted during the foundation of new nests [28, 62]. Additionally, other micro- organisms may antagonize Escovopsis sp. such as Amyco- latopsis sp. [35], Burkholderia sp. [63], Streptomyces [32, 34, 35, 64], and yeasts [65]. Thus, rather than a one-to-one symbiosis between the ants and their fungi, recent work suggests that rather the ants rely on a consortium of microbes and their compounds to defend themselves against Escovopsis sp. parasites [26]. 3. Occurrence of Additional Fungi on Gardens of Attine Ants and Their Possible Role as Symbionts Evolutionary theory predicts that organisms with restricted genetic diversity are susceptible to exploitation by several parasites [66]. This can be the case of attine ants which cultivate a single strain of the mutualistic fungus [14, 15]. Since the fungus garden is an ideal environment for the growth of the fungal cultivar, it is expected that additional alien fungi would exploit this substrate. As indicated earlier, Moller [16] was the first to record the existence of filamentous fungi in attine gardens. In addition to the fungus that was latter named Escovopsis sp. and to which the author referred as the “strong state” of the mutu- alist cultivar, Moller [16] provided detailed information on the occurrence of Aspergillus sp., Mucor sp., Penicillium, and Rhizopus sp. on the fungus gardens of Acromyrmex disciger (collected in Blumenau, Brazil). This author reported 4 Psyche that such filamentous fungi covered the fungus gardens when left unattended by workers. Later, Spegazzini [67] found ascocarps of Xylaria micrura in abandoned nests of Acromyrmex lundii in Argentina and also provided detailed drawings of this structure. When working with laboratory nests of Trachymyrmex septentrionalis, Weber [68] observed that gardens were also overgrown by several filamentous fungi such as Aspergillus sp., Mucor sp., and Penicillium sp. Kreisel [42] also observed Cunninghamella sp., Fusarium sp., Rhizopus sp., and Trichoderma sp. when studying fungus gardens of Atta insularis (in Cuba) unattended by workers. Similarly, Bass and Cherrett [69] studying the roles that workers play on the fungus garden maintenance observed that Aspergillus sp., Cladosporium sp., Fusarium sp., Mucor sp. and Penicillium sp. quickly overgrow gardens when left unattended by workers for several days. Luciano et al. [70] found Aspergillus sp., Nigrospora sp., and Penicillium sp. growing on the fungus garden of laboratory colonies of Acromyrmex heyeri in south Brazil. Fisher etal. [39] demonstrated that Atta cep /za/otcs colon- ies reared in the laboratory presented a shift in the fungal species composition when offered different plant substrates, thus providing the first experimental evidence that the fungal community on attine gardens may be influenced by the type of plant substrate used in the experiments. In addition, Cur- rie et al. [37] demonstrated that gardens from diverse attine ant genera spanning all the phylogenetic diversity of the tribe Attini harbor alien fungi. These authors studied mostly attine ants from Central America and particular attention was drawn to the garden parasite Escovopsis sp. but several other fungi also occurred which were not identified [37]. Following this study, Ortiz et al. [71] reported the occurrence of Fusarium sp., Rhizopus sp., and Trichoderma lignorum when studying fungus garden fragments left unattended by workers of A. cephalotes (in Colombia). Barbosa et al. [72] and Barbosa [73] also reported a comprehensive list of species of filamentous fungi present in fungus gardens of Atta laevigata field nests in northeastern Brazil and concluded that the genus Trichoderma was prevalent in such gardens. In addition to reports about filamentous fungi several authors also recorded a variety of yeasts on nests of attine ants. For example. Craven et al. [74] provided the first evidence that attine gardens contain yeasts using scanning electron microscopy. Pagnocca et al. [75] and Carreiro et al. [40] were the first to systematically study the yeast populations on laboratory colonies of A. sexdens rubropilosa. Such authors found variations in the abundance of yeasts populations in gardens and pointed out that Candida, Cryptococcus, Pichia, Rhodotorula, Sporobolomyces, Tremella, and Trichosporon were the prevalent genera. Carreiro et al. [76] showed that yeasts found on attine gardens produce the so-called killer toxins (or mycocins), which were proposed to be involved on the regulation of yeast populations on attine gardens. In this sense, Rodrigues et al. [65] proposed that yeasts may have a protective role in attine gardens against alien filamentous fungi. Up to date, the yeast survey on attine gardens rendered the description of three new species: Cryptococcus haglerorum [77], Blastobotrys attino- rum (= Sympodiomyces attinorum [78]), and Trichosporon chiarellii [79]; however, there is evidence that additional new yeast species associated with these insects await discovery. Recently, black yeasts in the genus Phialophora were reported to live on the exoskeleton of attine ants [80, 81] and the authors pointed out that they could antagonize the protective role of their symbiotic Pseudonocardia. Polysaccharidases secreted by yeasts and bacteria [82, 83] may also be im- portant for the nest homeostasis and it is an open field for further investigation. Despite the proposed roles that yeasts may play on attine gardens, few studies focused on the potential roles that fila- mentous fungi may perform on the attine ant-fungal inter- action. Several filamentous fungi found in attine gardens are commonly found in soil or plant substrates, suggesting that these microorganisms are probably transported on the workers’ integument or introduced into gardens via the plant material collected by the foraging workers [38, 41, 84] . Thus, it has been suggested that filamentous fungi are present in the fungus gardens as transient spores and may not play significant roles in the symbiosis [85] . In agreement with this hypothesis, Currie and Stuart [18] observed that when Atta sp. gardens are experimentally infected with Trichoderma sp. spores (a generalist fungus in comparison to Escovopsis sp.), the ants groom out spores efficiently that it is apparently removed from gardens. In contrast, gardens infected with Escovopsis sp. spores sustained long-term infections. Thus, this result suggests that general fungi like Trichoderma sp. may not play any role in the symbiosis [ 18] . On the other hand, recent studies address that filamen- tous fungi may play important roles in gardens of fungus- growing ants. Considering the studies reviewed here it is clear that a common trend arises: filamentous fungi (i) are found in association with diverse genera of attine ants, (ii) are found in attine nests from different localities, and, most important, (iii) quickly develop when the fungus gardens are unattended by workers (Figure 3). In this sense, Rodrigues et al. [86] determined that the majority of microfungi found in gardens of A. sexdens rubropilosa, a leaf-cutting ant species spread all over Brazil, belong to genera commonly found in soil and plant sub- strate. Particularly, the fungus Syncephalastrum racemosum was found in 54% of gardens from laboratory nests treated with baits supplemented with the insecticide sulfluramid (commonly used in Brazil to control leaf-cutting ants). A variety of other fungi, including Fusarium solani, were found in such gardens but with fewer than 20% of prevalence. None of the laboratory nests used as control (either treated with Eucalyptus sp. leaves or baits without insecticide) had the fungus gardens overgrown by filamentous fungi. On the other hand, Fusarium oxysporum and Trichoderma harzianum were found in 23% and 38% of gardens from field nests treated with baits supplemented with sulfluramid, respectively. It is interesting to note that S. racemosum was not observed in gardens from nests treated with insecticides under field conditions [86]. In addition, Escovopsis sp. was isolated in 21% and 15% in gardens of laboratory and field nests treated with sulfluramid, respectively [86]. In another experiment, several microfungal species were observed to quickly overgrow the fungus garden of A. sexdens rubropilosa Psyche 5 (a) (b) (c) Figure 3; Fungus garden fragments of leaf- cutting ants overgrown by filamentous fungi, (a) Atta texana garden fragment (TX, USA) with green tufts of Trichoderma sp. (b) Atta hisphaerica garden fragment (Botucatu, Brazil) with green conidiation of Trichoderma sp. Workers from (a) and (b) were manually removed and garden fragments were kept in wet chambers for 5 days at 25° C. (c) Isolation plate showing fungus garden fragment on potato dextrose agar medium supplemented with 150 jWg-mL^^ of chloramphenicol. On the right note the white mycelia of the mutualistic fungus of attine ants and on the left the micro fungus Aspergillus sp. Both fungi emerged from the garden fragment. {n = 12) when workers were experimentally removed [87]. The observed species included Acremonium kiliense (42%), E. weberi (42%), Trichoderma sp. (50%), and a fungus pre- viously identified as Moniliella suaveolens (50%), which now is known to be a genus not yet described (Harry Evans, personal communication). Similarly, Carlos et al. [88] found several fungal species including PenicilUum spp. and S. ra- cemosum on A. sexdens ruhropilosa gardens when treated with a variety of formulated insecticides. In another systematic study, filamentous fungi were also reported from field nests of several species of Acromyrmex from south Brazil [38] . The authors observed a high diversity of fungi and noted that F. oxysporum, Escovopsis sp., and Cunninghamella hinariae were present in 40.5%, 27%, and 19%, respectively, out of 37 nests. In contrast with previous studies, 5. racemosum was found in 5.4% of the nests. Re- cently, Rodrigues et al. [41] showed that Cyphomyrmex wheeleri {n = 16 nests), Trachymyrmex septentrionalis {n = 16), dead Atta texana {n = 4) sampled in Texas (USA) harbor a diverse community of microfungi which varies across sea- sons and are structured, in part, by location where nests were collected, reflecting a spatial component on the structuring of fungal communities. Interestingly, both Escovopsis sp. and S. racemosum were not found in the studied nests. Moreover, many filamentous fungi are carried by the fe- male alates (gynes) of leaf- cutting ants during the foundation of a new nest [89-91]. These microorganisms were more prevalent on the integument than in pellets found in the infrabuccal pocket [37, 91]. Although such fungi may be accidentally transported by gynes, they compose the initial microbiota associated with the ant nests and might be involved in the success on the establishment of a new nest. In fact, Autuori [92, 93] reported that several incipient nests do not thrive the period following the nuptial flight. In addition to flooding and birds, this author argued that fungi were also responsible for the mortality of incipient nests [93]. An additional observation sheds light on the possible role of filamentous fungi as opportunistic antagonists. When laboratory subcolonies of A. sexdens ruhropilosa were artificially infected with spores suspensions of Fusarium solani, Trichoderma cf. harzianum, S. racemosum, and E. weberi, Rodrigues et al. [94] observed that only nests treated with E. weberi provided a persistent infection (detected up to 300 hours after infection). However, about twelve hours after treatment with S. racemosum spores, workers removed fragments of fungus gardens and dumped away from gardens. This observation parallels the weeding behavior originally described as a specific adaptation for removing germinated spores of Escovopsis sp. [18]. Dumped fragments were collected and after plating quickly revealed the presence ofS. racemosum [94]. In addition to Escovopsis sp. other filamentous fungi were thought to be used as biological control agents [95]. Thus, attempts to use fungal spores on bait formulations demon- strated the effectiveness of this approach. Formulated baits with a combination of spores of Metarhizium anisopliae (an insect pathogenic fungi) and Trichoderma viride (oppor- tunistic antagonist of the ant cultivar) controlled 100% of laboratory colonies of Atta cephalotes compared to the control (nests treated with baits without spores) [95]. Field experiments showed that baits with M. anisopliae and T viride spores achieved 100% of nest mortality when com- pared to the insecticide Pirimiphos- methyl, which caused 60% of nest mortality. However, the time necessary to achieve 100% of nest mortality using the formulated baits was more than 60 days [95], which is considered ineffective for controlling leaf-cutting ants in large areas. Despite the failure of such attempts these initiatives are desirable and perhaps will guide the development of alternative techniques to control these pest ants. 4. Conclusions and Future Directions The evidence gathered so far suggests that filamentous fungi act as opportunistic antagonists on the attine ant- fungal interaction. In comparison to the specialized fungus Escovopsis sp., filamentous fungi are considered nonspecific antagonists of the ant cultivar. The antagonistic effect of these fungi is evident in disturbed gardens (either caused by insecticides or other unknown factor), when gardens are unattended by workers and on incipient nests. Future 6 Psyche experimental studies should systematically address whether filamentous fungi also influence healthy colonies. The results of such studies will ultimately help in the development of new strategies for controlling leaf- cutting ants. Finally, despite the arguments in favour of the antago- nistic nature of filamentous fungi, we do not rule out that some may have other unknown functions in the attine ant symbiosis. For instance, Freinkman et al. [96] demonstrated that fungi may be a potential source of new compounds as it is the case of bionectriol A, isolated from Bionectria sp. derived from the fungus gardens of Aptero stigma dentigerum. Perhaps, future reports will unravel the existence of filamen- tous fungi that are beneficial to the ant colony. This aspect is totally unexplored and should also be considered when studying such microorganisms. Acknowledgments The authors are grateful to FAPESP (Funda^ao de Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for finnancial support. V. E. 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Clardy, “Bionectriol A, a polyketide glycoside from the fun- gus Bionectria sp. associated with the fungus- growing ant, Apterostigma dentigerum” Tetrahedron Letters, vol. 50, no. 49, pp. 6834-6837, 2009. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 940315, 9 pages doi:10.1155/2012/940315 Research Article Behavior of Paussus favieri (Coleoptera, Carabidae, Paussini): A MyrmecophUous Beetle Associated with Pheidole pallidula (Hymenoptera, Formicidae) Emanuela Maurizi,^ Simone Fattorini,^’^ Wendy Moore, ^ and Andrea Di Giulio^ ^ Department of Environmental Biology, University “Roma Tre”, Viale G. Marconi 446, 00146 Rome, Italy ^Azorean Biodiversity Group, University of Azores, GITA-A, Largo da Igreja, Terra Gha, 9700-851 Angra do Heroismo, Portugal ^ Water Ecology Team, Department of Biotechnology and Biosciences, University of Milano Bicocca, Piazza della Scienza 2, 20126 Milan, Italy Department of Entomology, University of Arizona, Tucson, AZ 85721-0036, USA Correspondence should be addressed to Emanuela Maurizi, emaurizi@uniroma3.it Received 21 September 2011; Accepted 8 December 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 Emanuela Maurizi et al. This is an open access article distributed under the Creative Commons Attribution Eicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Several specimens of the myrmecophilous beetle Paussus favieri were reared in ant nests of Pheidole pallidula. Their interactions were recorded and all behaviors observed are described. Duration and frequency of five behaviors of P favieri were analyzed with ANOVA and post hoc Tukey tests; these comprised rewarding, antennal shaking, antennation, escape, and “no contact”. Significant differences both in duration and in frequency among behaviors were detected. The main result is that the rewarding behavior, during which the beetle provides attractive substances to the host, is performed significantly more frequently than all others. This result strongly supports the hypothesis that the chemicals provided by the beetles and licked by the ants are of great importance for the acceptance and the full integration of P favieri in the ant society. This result also suggests that, contrary to previous findings and interpretations, the myrmecophilous strategy of P. favieri is very similar to the symphilous strategy described for P turcicus. The occasional interactions of some beetle specimens with the P pallidula queen were recorded, illustrated, and discussed, indicating the possibility of a more complex strategy of P favieri involving a chemical mimicry with the queen. In addition, the courtship performed by the beetle is described for the first time, together with a peculiar “cleaning” behavior, which we hypothesize functions to spread antennal chemicals over the body surfaces. 1. Introduction Ant nests are very attractive for many organisms, because they represent well-protected and stable environments that are rich in various resources (ants, their brood, stored food, waste materials, etc.). In particular, a large number of insects establish relationships with ants for a considerable part of their life cycle [1-3] and are classified as true myrmecophiles [4]. Insect-ant interactions range from commensalism to specialized predation, parasitism, and mutualism [1]. The most specialized myrmecophiles are able to deceive the complex communication and recognition systems of the ants, infiltrating their societies and exploiting their resources [1, 4, 5]. These ant parasites represent about 10% (~ 10,000 species) of known myrmecophilous insects and most are members of Coleoptera, Lepidoptera, Orthoptera, and Diptera [6]. They show several refined adaptations (e.g., chemical and morphological mimicry, specialized feeding behaviors, structural modifications) to avoid ant attacks, to be accepted by ants, and to develop and reproduce within ant nests [7]. All members of the ground beetle tribe Paussini (Co- leoptera, Carabidae, Paussinae) are myrmecophiles and are considered to be ant parasites [8]. Like many other parasites of ants, they show striking adaptations, such as greatly modified antennae (flattened, enlarged, lenticular, globular. 2 Psyche concave, elongate, etc.), slender or compact bodies, elongate or flattened legs, and peculiar “myrmecophilous organs” composed of trichomes (tufts of hairs) connected to exocrine glands for the release of chemical secretions. Paussini (known as “ant nest beetles”) are typically rare insects living in concealed environments which makes it difficult to observe their behavior in nature [7]. Therefore, while they have been extensively studied from a taxonomic point of view [8], information about their interactions with hosts and their life cycle is limited and largely indirect (i.e., inferred from their structural adaptations) with few ethological observations [9]. Although several attempts have been made to rear Paussini with their host ants, this has proven to be particularly difficult, and promising results have been achieved only for a few species (5 out of the currently recognised 572 Paussini species) [8]. The first observations of Paussini behaviors in captivity were reported by Peringuey [10, 11] for Paussus lineatus Thunberg, in 1781, and P. linnaei Westwood, in 1833, and, to a lesser extent, for P hurmeisteri Westwood, in 1838. Other early ethological notes were reported by Escherich [12] for P. turcicus Frivaldszky, in 1835, P. favieri Fairmaire, in 1851 [13], and P. arahicus Raffray, in 1885 [14]. These authors carefully reported their annotations mainly emphasizing the obligate association of these beetles with the ants (especially the ant genus Pheidole), their feeding strategy on larvae of the host ants, and some interactive behaviors between beetles and ants (e.g., dragging, grooming, aggressive behaviors). According to this first, though limited and speculative set of information gathered in captivity, and to previous anecdotal observations in nature reported by several authors (e.g., [15-19]), Escherich [14] tentatively categorized the strategies of the members of the genus Paussus in three main levels of interactions, referring to Wasmann’s [20, 21] myrmecophilous categories: synectrans (e.g., P. linnaei), synecoetes (e.g., P arahicus and P. lineatus), and symphilous (e.g., P turcicus). Eater, LeMasne [22-24] successfully reared P favieri, adding valuable and detailed information to the knowledge on the biology of this species which is a guest of the facultatively polygenic ant Pheidole pallidula (Nylander, 1849). Le Masne mainly focused his observations on the predatory strategy of P favieri while feeding on adults and ant larvae [22, 24], and on the mechanisms of adoption of the beetle inside the nest [23] . More recently, Escherich’s [ 14] classification has been reviewed and updated by Geiselhardt et al. [8], and three different strategies have been identified, exemplified by three Paussus species: (1) the strategy of P arahicus reported by Escherich [14] which is considered the most basal, since the initial contact with the ants triggers their aggression; however, the attacks cease after the contact with the ants [10, 12, 14, 25], and for this reason, the authors speculated that a chemical camouflage might occur in this species [8]; (2) the costly strategy of P turcicus, which is actively groomed by its host ants, to which the beetle supplies an attractive and possibly rewarding antennal secretion [12, 13]; and (3) the strategy of P. favieri, which is considered the most derived, since it has no apparent costs. According to the observations by Escherich [13] and Ee Masne [23], this beetle is readily accepted and fully integrated within the colony without hostility. It is usually ignored by the ants, only rarely touched, quickly groomed, and dragged, and it moves undisturbed within the nest, free to feed on brood and adults. Probably, an advanced chemical mimicry mediates the mechanism of this association [8]. Paussus favieri was also the object of recent researches, being one of the most common species of Paussini in North- ern Africa and one of the two species present in Europe. Cammaerts etal. [26-28] showed that P. /avicn' preferentially follows the pheromone trail produced by the poison glands of its host ant, discriminating this from pheromones of nonhost ant species. Lastly, Di Giulio et al. [7] reared and described the first instar larva of P favieri that, like other Paussus larvae, shows remarkable adaptations to a myrme- cophilous lifestyle (e.g., shortened and degenerated head capsule, reduced mouthparts, partial atrophy of legs, fused terminal disk), with specialized feeding behaviors that sug- gest that the larvae are fed by the ants through trophallaxis. To clarify the mechanisms underlying host-parasite rela- tionships between P favieri and its host ant P pallidula, we investigated the interspecific and intraspecific behaviors performed by the beetles inside the ant nests maintained in laboratory. In particular, our aims were (1) to describe the main behaviors performed by P. favieri and its host ant, (2) to analyze the duration and frequency of the behaviors per- formed by the beetles, and (3) to discuss the possible func- tional and adaptive significance of the observed behaviors. 2. Materials and Methods 2.1. Material Examined and Rearing Conditions. During an expedition to Morocco (High Atlas Mountains) in May 2010, adults of P. favieri were collected under stones, in nests of P. pallidula (Figure 1). Beetles and ants were then transported to the laboratory for behavioral experiments. Each beetle was reared with the ants from the nest in which it was found; when multiple specimens of P. favieri were found in the same nest, all specimens were reared together. Ants and beetles were housed in transparent glass boxes (32 x 22 X 15 cm) lined with a layer of plaster, and the walls were coated with fluon to prevent ants from escaping. Golonies were kept under controlled conditions (2 1-24° G; 12h: 12h light: dark; 60% humidity), following the procedures described by Detrain and Deneubourg [29], and maintained on a diet of sugar or honey, and fruit flies or moth caterpillars provided three times per week. The boxes were kept open to facilitate observations. After the ants and beetles were acclimated to these new conditions (about for 10 days), behavioral observations were made. Ten colonies were established but we used only five, well- structured colonies with at least 100 nestmates (70% minors, 30% majors and queen) for behavioral observations. 2.2. Descriptions of Behaviors. Host-parasite interactions and intraspecific behaviors (cleaning and mating) were observed under natural light. Video was recorded with an NV GS120EG Panasonic video camera for a total of 20 hours. Because manipulation could have unpredictable effects on Psyche 3 Figure 1: Paussus favieri with minor and major worker of Pheidole pallidula (photo by P. Mazzei). the host-parasite interactions, beetles and ants were not marked and beetles were not sexed. For the analysis of the host-parasite interactions, we selected 14 beetles for which recording sessions of at least 15 minutes were available. All behaviors of both the beetles and the ants were described and classified into five categories (see Sections 3.2 and 3.3). The behaviors performed by P. pallidula during the interactions with the beetles were described following the behavioral repertoire suggested by Holldobler and Wilson [1], Passera and Aron [30], and Sempo and Detrain [31]. Beetle cleaning and sexual behaviors were described after analyzing the videos in slow motion. 2.3. Statistical Analyses of Behaviors. We statistically analyzed five behaviors performed by the beetle while interacting with the host ant (see Section 3.2). Recording sessions were analyzed using the observation transcription tool EthoLog 2.2 [32] to continuously record the time that the beetle spent performing different behaviors. We tested whether different behaviors of beetles have significantly different durations, that is, if there are differences in the amount of time a beetle spends engaged in different behaviors when it interacts with ants. Differences between behavior duration were tested using a main effect AN OVA. A total of 1030 measurements of behavior duration (dependent variable) were analyzed. Because the beetles were not obtained by rearing but were collected from ant nests in the field, we have no information about possible interindividual variation due to genotypic differences, or previous experience with ants, age, days of fasting, and so forth. Thus, we combined all of these unknown factors into the concept of “individuality”. To control for this “individuality”, beetles were numbered from 1 to 14 and “beetle identity” was introduced as a second factor in the ANOVA. Therefore the identity of the beetle, which exhibited a behavior, and the type of behavior (classified into five categories, A-E, see Section 3.2) were used as categorical predictors (factors). Post hoc comparisons were performed using Tukey HSD tests. To determine whether different behaviors were performed more frequently than others, we executed analogous analyses on the recorded frequency of the behaviors. Statistical analyses were performed with Statistica for Windows version 7.2 (StatSoft Inc., Tulsa, OK, USA). 2.4. Scanning Electron Microscopy. Morphological structures of P. favieri (Figure 2) involved in the interactions with host ants and with others conspecifics were studied using a Philips XL30 scanning electron microscope at L.I.M.E. (Interde- partmental Laboratory of Electron Microscopy, University “Roma Tre”, Rome). Specimens used for morphological study were kept overnight in a detergent water solution, cleaned by ultrasounds for 15 seconds, rinsed in water, dehydrated through a series of EtOH baths of increasing concentration (70, 80, 90, 95, and 100%), critical point dried (Bal-Tec CPD 030), mounted on a stub (by using self adhesive carbon disks), and sputter-coated with gold (Emitech K550 sputter coater). 3. Results 3.1. General Morphology of Paussus favieri. The beetle is small (length ~ 4 mm), much bigger than minor workers of P. pallidula, with intermediate dimensions between majors and queen (Eigures 1 and 3). The body is slim with slender elongated legs and bulged modified antennae. The body color is light brown, similar to that of minor and major workers of the host ant, with shining, oily appearance. The head is subhexagonal with elongate palpi and dark eyes, bearing dorsally a long medial tuft of trichomes (Eigures 2(a) and 2(b)). The antennae are particularly modified, composed by three joints: (1) a cylindrical and slightly elongated scape; (2) a globular, ring-like pedicel; and (3) a single segment “antennal club” (resulting from the fusion of 9 flagellomeres) that is wide, sub-triangular, swollen, and strongly asymmetrical (Eigures 2(a) and 2(b)). The scape and the antennal club are covered by several modified trichomes and glandular pores (Eigure 2(d)), while chemoreceptors are mainly distributed apically. The antennal club has a pointed basal spur with two tufts of trichomes (myrmecophilous organs, Eigures 2(a) and 2(c)), and ventral pockets (Eigure 2(d)) where glandular secretions are stored. The prothorax is elongated, of about the same width as that of the head, strongly constricted in the middle, without tufts of trichomes. Like the other Paussus species, a stridulatory organ is present on the ventral side, composed of finely ridged pars stridens on the hind femora and a plectrum (row of cuticular spines) on the basal part of the abdomen. The elytra are parallel and covered with elongate, branched trichomes. The pygidium is truncate with short-fringed trichomes. The ventral side of the body is smooth, without trichomes. 3.2. Description of Paussus favieri Behaviors When Interacting with Host Ant (a) Rewarding. The beetle remains still, while it is anten- nated and actively licked by ant minor and major workers (Eigure 1; (see Supplementary Material 1 available online (e) (f) Figure 2: SEM micrographs of Paussus favieri: (a) anterodorsal view of head and thorax; (b) ventral view of head and thorax; (c) basal spur of the antennal club, dorsal view; (d) ventral antennal pockets with visible secretion; (e) elytra with modified sensilla chaetica; (f) modified sensilla chaetica on head with glandular pores. at doi: 10.1155/2012/940315). This behavior is generally associated with movements of the beetle’s hind legs, either singly or in combination. (b) Antennal Shaking. The beetle vibrates the antennae, quickly shaking them forward and backward in the vicinity of the ants. This behavior mostly occurs after a long period of rewarding (see above). (c) Antennation. The beetle moves its antennae in a slow, alternate, vertical way, oriented toward the object of interest. The beetle usually explores an ant’s body with the apices of the antennae, which are particularly rich in sensorial structures. (d) Escape. The beetle tries to elude the host ant in a tem- porary negative reaction. This behavior is not connected with aggression by the host, but rather in most cases it is a consequence of the presence of a high number of excited ants antennating and licking the beetle, or after an extended rewarding period. (e) No Contact. The beetle does not interact with the ants. This state includes many different activities like exploring, resting, cleaning, interacting with partners, mating, and so forth. Feeding and mating behaviors were observed rarely. The beetle feeds on ant larvae by piercing the integument with its mandibles and carrying around the victim while sucking blood and soft tissues from the abdomen. In these situations. Psyche 5 Figure 3: Interactions between Paussus favieri and queen of Pheidole pallidula (photo by P. Mazzei). the ants do not react aggressively toward the beetle. These behaviors never occurred in the movies selected for analyses. Beetles were observed directly interacting with the queen (Figures 3(a) and 3(b)). In a few cases, the beetles remained in the queens chamber for some days, antennating and rubbing against the queens body without any aggressive reaction from the queen or the workers. 3.3. Description of Pheidole pallidula Behaviors When Interacting with Beetles (a) Antennation. The ants touch the beetles with their anten- nae on all exposed parts of the body, but especially on the beetle’s antennae (Figures 2(a) and 2(b)). (b) Alarm. The ants antennate frenetically and widely open their mandibles, similarly to alarm behaviors performed during dangerous situations [1, 31]. This behavior is rarely observed against beetles, but when it is, it is not followed by biting. (c) Licking. The ants lick all exposed parts of the beetle’s body that are rich in trichomes (antennae, head, legs, elytra and pygidium) (Figures 2(a), 2(e), and 2(f)). This licking behavior is very similar to the ants’ allogrooming behavior ([31] and references therein). The ants spend most time licking the trichomes on the basal spur and the pockets of the antennae (Figures 2(c) and 2(d)). This activity can be performed simultaneously on one beetle by many ants (minor and major workers), and it is the reciprocal behavior to “rewarding” hj P. favieri (see Section 3.2). (d) Dragging. The ants, mostly minors, occasionally bite the antennal club of P. favieri and quickly drag the beetle around the nest. The beetles, though much bigger than ants, do not resist being dragged around. In two cases, we observed minor workers dragging a beetle inside the queen’s chamber. 3.4. Cleaning Behavior of Paussus favieri. The cleaning behavior is characterized by the following phases (see Sup- plementary Material 2). (1 ) Antennal Cleaning. The forelegs clean the antennae one at time, starting from ventral to dorsal side of antennal surface. In particular, the tarsus and the hairy apical part of tibia rub the apex and the posterior part of the antennal club, the ventral pockets (Figures 2(b) and 2(d)), and the posterolateral teeth (Figure 2(a)), with numerous quick movements. The tarsus also rubs against the whole antennal club, moving laterally from base to apex with slow movements. Additionally, the tibia cleans the dorsal side of antennal surface, with a single movement. During this phase, the antenna is highly movable and it is rotated according to the side to be cleaned. (2) Head Cleaning. One of the forelegs moves over the head, rubbing the apical tuft of long sensilla (Figure 2(a)). This behavior has been rarely observed. (3) Leg Cleaning. This cleaning is performed mutually by pairs of legs of the same side, the fore against the middle, and the middle against the hind legs. The tarsus and the tibia of one leg slowly rub the reciprocal leg from the base to the apex. In addition, the tarsi are rubbed together (fore-middle, middle-hind) repeatedly. (4) Elytral Cleaning. The elytra are cleaned in the antero- posterior direction with slow repeated movements of the middle and hind tibiae and tarsi of the same side. The tarsi of the middle and posterior legs also rub the lateral surface of the abdomen. 3.5. Mating Behavior of Paussus favieri. The mating behavior of P. favieri is characterized by two distinct phases: courtship and copulation (see Supplementary Material 3). Courtship. Males actively search for females, approaching them by antennal contact (antennal approach) in one of two different ways: (a) with a slow alternate vertical movements of his antennae touching the female’s antennae (frontal approach), and (b) his antennae touching laterally the side of the female’s elytron (lateral approach). After the lateral antennation, the male forelegs are moved up and down. 6 Psyche touching the female elytra and pronotum. The female replies by moving her antennae and the hind legs. After this preliminary antennal approach, the male climbs upon the female’s body, dorsally positioning himself in the opposite direction of the female, touching his antennae the apex of the female’s abdomen. This dorsal inverted phase lasted a few seconds; afterwards the male turns 180°, reaching the typical mating dorsal phase. During this phase, the partners reciprocally touch their antennae, and the female often moves her hind legs. In the 10 sequences analyzed, the dorsal phase lasts from 5 to 12 minutes and, in a few cases, it was followed by copulation attempts. Copulation. From the dorsal phase, the male of P. favieri slides backwards, bends the abdominal apex downward, extrudes the aedeagus, and tries to insert it into the female’s genitalia. The antennae of the male are frenetically moved up and down. The copulation with complete insertion of genitalia was observed only once. In fact, the female often rejects the male and avoid copulation. During mating, the ants frequently interact with the bee- tles, antennating them and/or actively licking their antennae and legs. 3.6. Analyses of the Behaviors of Paussus favieri during Interactions -with Its Ant Host. The following behaviors of P. favieri were analyzed statistically: (A) rewarding, (B) antennal shaking, (C) antennation, (D) escape, and (E) no contact. We detected significant differences in the time a beetle spends performing different behaviors (Table 1). Post hoc Tukey tests showed significant differences between E versus A, B, C, and D (P < 0.0001 in all pairwise com- parisons). Individuality was not significant, which indicates that behavioral patterns do not vary significantly among individuals. Differences in the mean duration of different behaviors are shown in Figure 4. We found that significant differences among the frequen- cies that different behaviors were performed (Table 2). Post hoc Tukey tests showed significant differences between A versus B, C, D and E (P < 0.0001) and between C versus B and D (P < 0.05). Differences in the mean values of frequencies of different behaviors are shown in Figure 5. 4. Discussion According to Wasmann [33, 34], two defensive structural types are generally recognized in myrmecophile morphol- ogy: the “protective” type, characterized by a compact body with hard and smooth surfaces, and retractable appendages; and the “symphilous” type, characterized by slim bodies with long slender appendages and many trichomes covering the body and/or crowded in myrmecophilous organs [8, 35]. These body forms suggest different strategies both for entering the host nests and for avoiding ant attacks. Both body types are present in the Paussini, sometimes with intermediate forms, with the symphilous type generally present in the most derived taxa that are considered to be the best integrated into ants’ colonies [1, 36]. Paussus favieri is Table 1: Results of a main effect ANOVA for values of times spent performing different behaviors by beetles when interacting with ants, d.f.: degrees of freedom; SS: sum of squares; MS: mean sum of squares; F: Fisher; P: probability. Effect d.f. SS MS F P Individuality 13 6183.000 475.620 0.848 0.6090 Behavior 4 27389.200 6847.310 12.201 0.0001 Error 1012 567925.800 561.190 Figure 4: Differences in the duration of behaviors performed by beetles when interacting with ants. Mean values are shown by squares, standard errors as boxes, and standard deviations as whiskers. A; rewarding; B: antennal shaking; C: antennation; D: escape; E; no contact. Table 2: Results of a main effect ANOVA for values of frequency of different behaviors performed by beetles when interacting with ants, d.f.: degrees of freedom; SS; sum of squares; MS: mean sum of squares; F: Fisher; P: probability. Effect d.f. SS MS F P Individuality 13 643.238 49.480 1.027 0.4370 Behavior 4 4498.345 1124.586 23.336 0.0001 Error 66 3180.655 48.192 clearly assignable to the latter type, showing all the distinctive characters noted previously. Our observations confirm that P. favieri is fully integrated in the host ant society since almost no aggressive behaviors against the beetles were observed. On the contrary, ants were strongly attracted by the beetle’s secretions. The results of our statistical analyses show that beetles and ants spend a significantly longer amount of time not interacting (no contact, E) than the time they spend interacting with one another in a specific behavior. The state of no contact (E) can be the effect of a temporary withdrawal of the beetle, or the absence of caring by the ants. This is an expected result, since it is reasonable that the beetle spends most time in a number of activities that do not involve host interactions (i.e., exploring, mating, cleaning, resting, etc.). Psyche 7 A B C D E Behavior Figure 5; Differences in the frequencies of behaviors performed by beetles when interacting with ants. Mean values are shown by squares, standard errors as boxes, and standard deviations as whiskers. A: rewarding; B: antennal shaking; C; antennation; D: escape; E: no contact. Concerning the behaviors performed by the beetle during the interactions with its host ants, the analysis of duration showed that rewarding (A), antennal shaking (B), antennation (C), and escape (D) are performed for similar amounts of time. However, it is notable that frequency of the rewarding behavior (A) is significantly greater than that of all other behaviors. During the rewarding behavior, P. favieri is antennated and actively licked by the ants, especially near the antennal symphilous organs (Figure 2(c)). This is consistent with the fact that the primary role of the highly modified antennae of R favieri is glandular, producing substances that are highly attractive to the ants [37]. These substances are mostly stored inside the antennal pockets (Figure 2(d)). The chemical nature of this secretion is unknown, but it seems to be important for the acceptance and survival of the beetles within the ant nest [37] and for the success of the parasitic interaction. It has been speculated that, similar to other social parasites [1, 38], the chemicals secreted by Paussini beetles may have an appeasing function [8, 34]. Another hypothesis is that these substances provide a protective or rewarding food for the ants and their brood [8] . The rewarding behavior is generally associated with movements of the beetle’s hind legs, an action possibly connected to the emission of stridulations. The high frequency of the rewarding behavior recorded in our experiments is quite in contrast with the previous observations by Escherich [13] and Le Masne [23], who reported that the ants only occasionally groom the beetle. According to our observations, the myrmecophilous strategy of R favieri seems very similar to that of P. turcicus [ 12, 13] , and the supposition that this strategy corresponds to a more derived (less costly) level of integration for R favieri [8] seems unjustified. The quick shaking of the antennae (antennal shaking (B)), not noted by Le Masne [22-24] and never recorded for any other coleopteran genera, has been occasionally observed in another species of Paussus [15]. Our observations suggest that antennal shaking might be correlated with the glandular activity of the antennae, facilitating the spread of the viscous exudates from the antennal surface, or, most probably, with the spray of volatile allomones whose presence needs to be confirmed. The antennation behavior (C) was described by Le Masne [22, 24], who interpreted it as a precursor to predation. Le Masne [24] observed that through antennation the beetle finds the ant’s abdomen. Once found, the beetle pierces the abdomen with its sharp mandibles and feeds on the ants’ hemolymph. However, in the videos analyzed for the present work, we never observed predation following the antennation behavior. The occasional observation of some beetles interacting with the queen (Figures 3(a) and 3(b)), also for a prolonged time, is particularly interesting. We hypothesize that the physical interaction could supply a queen-specific chemical camouflage to the beetle and/or that the beetle could spread some of its attractive substances on the queen’s body. In both cases, a chemical combination of beetle and queen odors could be reached, resulting in a deception of the hosts, allowing the beetle to achieve a higher social status inside the nest. The dragging of R favieri inside the nest by R pallidula minor and major workers is a behavior that this species (Maurizi and Di Giulio pers. obs.) and other Pheidole species [39] usually reserve for the queen [1, 8] and could be related to this possible mimicry. However, further research is required to confirm that this is a regular interaction, and that an exchange of cuticular hydrocarbons or other substances is involved. The cleaning and mating behaviors performed by P. favieri inside the nest of R pallidula have been observed and described in this work for the first time. Peringuey [10] mentioned a similar “brushing” behavior by fore and hind legs performed by males of R lineatus after copulation. The complex cleaning behavior of R favieri is quite different from the simple cleaning of other Carabidae [40] which mainly involves rubbing the comb organ of the forelegs (a row of spines positioned in an emargination of the inner edge of the fore tibiae) against antennae and mouthparts. In fact, the typical comb organ of ground beetles is vestigial or absent in Paussini [40, 41], In P. favieri, the antennae have primarily a glandular function [37] and secrete a large amount of attracting substance. We interpret the rubbing of the forelegs against the antennae and then against middle and hind legs, head, elytra and abdomen, as a means of spreading antennal substances all over the body. This is also supported by the fact that the ants actively lick not only the antennae but also the head, legs, and elytra, suggesting that the attractants are present also on these body parts. Little is known about the sexual behavior of Carabidae [42, 43], while no information is available for the Paussini except for a brief note of Peringuey [10] on P. lineatus. In this species, the male fixes his mandibles in the prothoracic excavation of the female and, with the hind legs, pulls the abdominal apex of the female towards him; in order to strengthen his position on the female’s back, the male passes his antennae under the females antennae, keeping this position for several hours. Serrano et al. [44] observed in 8 Psyche Portugal two specimens of R favieri in copulation in an ant nest of P. pallidula, confirming that this beetle mates inside the colony, as is reported for other myrmecophilous beetles [45]. In captivity we observed the specimens of P. favieri mating in the ant nests several times and for a long duration. Precopulatory behavior includes exchanging tactile signals with antennae and legs, though it is possible that chemical signals are also involved. Unlike observations of P. lineatus [10], in both precopulatory and copulatory behaviors the mandibles are not used by P. favieri, while the dorsal position is maintained only by the male’s legs. Of particular interest is the presence of an “inverted” dorsal phase (not noted in P. lineatus) that may be unique within the Carabidae. Our experiments also suggest that acoustic signals are probably exchanged during the precopulatory behavior, since the female has been observed repeatedly moving the hind legs, a behavior possibly connected to the emission of stridulations (see Section 3.2(a)). However, the actual role of the acoustical communication in intra- and interspecific behaviors remains unknown. In conclusion, the importance of the rewarding behavior confirms the primary role of the antennal secretions, possibly spread by a complex “cleaning” behavior, for the successful acceptance and integration of P favieri inside the host colony. The identification of the secretions would be very important to verify their appeasing/rewarding properties, providing a more complete understanding of the myrmecophilous strategy of P favieri and of other members of this tribe. Acknowledgments The authors thank Paolo Mazzei for taking the pictures of the living specimens of Paussus favieri (Figures 1 and 2). They also thank Peter Hlavac, Raffaella Bravi, and Valerio Viglioglia for their precious help in the field and two anonymous reviewers for their suggestions. They are grateful to Professor Ahmed El Hassani (Institut Scientifique de rUniversite Mohammed V-Agdal, Rabat, Morocco) for his support and facilitation in the field expeditions. References [1] B. Holldobler and E. O. Wilson, The Ants, Springer, Berlin, Germany, 1990. [2] G. W. Elmes, “Biological diversity of ants and their role in ecosystem function,” in Biodiversity Research and its Perspec- tives in the East Asia, B. H. Lee, T. H. Kim, and B. Y. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 328478, 7 pages dohlO.l 155/2012/328478 Research Article Improved Visualization of AIpbitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) — Part 1: Morphological Features for Sex Determination of Multiple Stadia J. F. Esquivel,^ T. L. Crippen,^ and L. A. Ward^ ^ Areawide Pest Management Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, 2771 F&B Road, College Station, TX 77845, USA ^Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, 2881 F&B Road, College Station, TX 77845, USA ^ Department of Entomology, Texas A&M University, College Station, TX 77843, USA Correspondence should be addressed to J. F. Esquivel, jesus.esquivel@ars.usda.gov Received 1 October 2011; Accepted 12 December 2011 Academic Editor: Russell Jurenka Copyright © 2012 J. E. Esquivel et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The lesser mealworm, Alphitobius diaperinus (Panzer), is a perennial pest of poultry facilities and known to transmit pathogens of poultry and humans. Ongoing research examining reservoir potential of A. diaperinus revealed the need for a comprehensive, user-friendly guide for determining sex of A. diaperinus at different stadia. This paper is unique in providing a comprehensive illustrated guide of characters used for differentiation of sexes in A. diaperinus. 1. Introduction The lesser mealworm, Alphitobius diaperinus (Panzer) (Co- leoptera: Tenebrionidae), is a perennial pest of poultry facilities. The species is of tropical origin from sub-Saharan Africa and has adapted to the moist, temperature controlled environments of poultry production facilities [ 1 ] . As a noc- turnal species, the low-light conditions within broiler grow- out houses are conducive for populations to thrive [2]. Peak population densities occur during the warmer summer season but the pest is present throughout the year [3]. Om- nivorous feeding habits allow A. diaperinus to feed on manure, feed, chicken carcasses, detritus, and other larvae and beetles [4-7]. Generally, 5 to 8wks are required for A. diaperinus to complete a life cycle within a broiler grow-out house; am- bient temperature and humidity will affect the life span [8]. Female beetles oviposit in sheltered areas of the house, depositing 1000 to 1800 fertile eggs over their life span [2, 9- 11]. Eggs hatch in 4-7 days and larvae live in the moist litter and along the building walls [12]. The larvae progress through 6 to 11 stadia over a span of 30 to 100 days before reaching adulthood [9, 13, 14]. Late instars nearing pupation seek a drier environment by tunneling into compacted earthen floors beneath the litter or into the insulation and crevices of the building walls [15]. Adults emerge after 7 to 8 days. Under laboratory conditions, life span of an adult can exceed a year; however, in field setting, longevity of adults and larvae is influenced by predation from poultry and abio- tic conditions. In addition to causing structural damage to poultry houses, A. diaperinus has been associated with the transmis- sion of pathogens (e.g.. Salmonella) which can be potentially fatal to poultry and, more importantly, humans [16]. Con- sequently, studies are ongoing to further determine the reservoir capacity of lesser mealworm. Factors that may affect reservoir capacity and transmission potential are sex of bee- tles and larvae and size of the respective alimentary canals. Size of the A. diaperinus alimentary canal and its respective sections are presented in a companion paper in this issue [17]. Rapid determination of sex in the field settings of the poultry facilities would enable more timely and proactive approaches to beetle management. However, during the course of examining, the reservoir potential and alimentary 2 Psyche canal capacity of A. diaperinus, it was determined that a comprehensive assessment of characters for determining sex of A. diaperinus was lacking. Schematics of genitalia for determining sex of pupae and beetles are available [18-20], but these schematics fail to clearly reflect the orientation and morphology of said features. Further, secondary sexual characteristics, such as orientation of tibial spurs within A. diaperinus adults differ and reports vary in the description and use of either mesothoracic or metathoracic tibial spurs to determine sex [21, 22]. The objective of this paper is to improve upon extant schematics and establish a definitive illustrated guide of morphological characters for determin- ing sex of A. diaperinus. 2. Materials and Methods 2.1. Insect Rearing. The Southern Plains Agricultural Re- search Center (SPARC) starter colony of A. diaperinus was obtained from a colony originally isolated from a poultry farm in Wake County, NC, maintained by Dr. D. W. Watson (North Carolina State University, Raleigh, NC). The SPARC colony has remained in production since 2004. The colony was reared in plastic containers (15 X 15 X 30 cm) with screened tops; containers held 1000 mL wheat bran (Morri- son Milling Co., Denton, TX) on the floor surface. A 6 cm^ sponge moistened with deionized water and a slice (0.5 cm thick) of apple provided a water and food source, respec- tively. Both were replenished twice per week. The moistened sponge was placed on a piece of aluminum foil to prevent contact with the bran. Also, 30 mL of fishmeal (Omega Pro- tein, Inc., Hammond, LA) were added to the wheat bran once per week; new wheat bran was added as it was depleted by dropping through the screened bottom of the cage. Layered black construction paper (6 X 6 cm) was provided as an oviposition substrate to allow collection of eggs to sustain the colony. Eggs were transferred to a separate container and resulting larvae, pupae, and adults were maintained as des- cribed above. The colony was maintained at 30° C in an 8 : 16 hr (light : dark) photoperiod. 2.2. Validity of Characters for Determining Sex. Late instars reportedly retain pygopods upon pupation and resulting pygopods on pupae reflect female pupae [18]. Three cohorts of 25, 85, and 96 larvae were sorted based on presence of pygopods and development was monitored from larvae to adulthood. Resulting pupae were sorted based on previously reported characters [18, 23] and eclosing adults were sexed to confirm relationship with pygopods on pupae. Paired pygopods in the larval stage, present ventrally on the 10th abdominal segment, aid in locomotion [24]. 2.3. Preparation of Specimens. Late-instar larvae, pupae, and adults were collected from rearing containers and placed in respective 25 dram vials. Vials containing larvae and pupae were placed in a freezer to kill the insects; adults were killed by adding 80% ethyl alcohol (EtOH) to vials. Adults in EtOH-filled vials were briefly agitated by hand to remove adhering diet and frass. Larvae and pupae were cleaned by brushing with a fine-tipped paintbrush dipped in EtOH, since full immersion increased transparency of their more weakly sclerotized, lighter pigmented cuticle. Specimens were allowed to air dry on a clean Kimwipe towel. Larvae and pupae were pinned through the thorax to prepare them for imaging. For adults, slight pressure was applied to the abdomens using fine-tip forceps to evert genitalia before pinning. Additional adults were processed to document differences and orientation of tibial spurs between sexes. Tibiae of the mesothoracic legs were excised and indi- vidually point mounted by their proximal ends for imaging apical spurs. 2.4. Imaging. A specially constructed viewing arena con- sisted of an 80 LED ring light (Model KD-200; Gain Express Holdings Ltd, Hong Kong) with a modified pinning stage located within the center of the ring. A small (7.62 X 2.54 X 1.27 cm) Styrofoam bar attached to the internal face of the ring light provided an additional, vertical pinning surface to facilitate orientation of specimens. A removable hemisphere- shaped dome with a 2.7 mm viewing aperture fit over the outer edge of the ring to uniformly distribute light within the arena. Point mounted or direct-pinned specimens of all stadia and excised tibiae were placed in the viewing arena and manipulated for best orientation under a Leica MZ16 micro- scope equipped with APO lens (Leica Microsystems, Wetzlar, Germany). A ProgRes 3008 digital camera mounted on the microscope interfaced with a Windows-driven operating sys- tem. Sequentially focused images of each specimen were cap- tured using PictureFrame 2.3 software (Optronics, Goleta, GA). Each image series was subsequently processed using Auto-Montage Pro software 5.01.0005 (Syncroscopy USA, Frederick, MD) to construct one composite image with enhanced depth of field. Adobe Photoshop GS5 (Adobe Systems, Inc., San Jose, GA) was used to improve clarity of composite images. 3. Results 3.1. Larvae. All late-instars in two cohorts {n = 110 total) possessed a pair of prominent fleshy pygopods ventral to the pygidium (Figures 1(a) and 1(b)). In the third cohort, a small percentage of larvae (12.5%; n = 96) did not exhibit prominent pygopods; instead the pygopods were unappar- ent, or much reduced (Figures 1(c) and 1(d)). A previous report suggested that the pygopods [24] were retained to the female pupal stage (discussed below). However, sex ratios of resulting pupae from sorted larval cohorts indicate that presence of pygopods does not exclusively reflect females. Of the larvae with prominent pygopods surviving to adulthood, 56.9% {n = 86) of adults were males. Similarly, adult males comprised 58.3% {n = 12) of the larvae with unapparent pygopods. Both sexes possess a urogomphus (Figure 1). 3.2. Pupae. The pupae of both sexes of A. diaperinus pos- sess a pair of urogomphus dorsally (Figure 2). The larval urogomphus are retained to the pupal stage [18], and. Psyche 3 (a) (b) (c) (d) Figure 1: External posterior characteristics of late-instar A. diaperinus: with prominent pygopods ((a) lateral view and (b) ventral view) and unapparent, or much reduced, pygopods ((c) lateral view and (d) ventral view). Pg, pygopod; Py, pygidium; Ur, urogomphus. reportedly, the additional pair of prominent processes seen ventrally in female pupae (Figures 2(a) and 2(b)) are the paired pygopods retained from the larval stage (Figure 1(a)) [19]. However, as previously demonstrated, not all larvae with prominent pygopods are females. Conversely, all pupae with prominent pygopods {n = 37) yielded female beetles; males resulted from all pupae without pygopods {n = 49) (Figures 2(c) and 2(d)). Thus, in the pupal stage, pygopod presence is a reliable indicator of sex. Newly formed pupae are typically a pearly white color [18] but the urogomphus and the pygopods, if present, darken as the pupae age [25]. 3.3. Tibial Spurs of Adults. Paired tibial spurs are present apically on the tibiae of all legs, but orientation of the spurs on the mesothoracic and metathoracic legs can aid in differ- entiation of sexes. The spurs arise from the anterior and pos- terior apical corners of the tibiae (Figure 3). In females, both spurs of the mesothoracic tibiae are parallel to each other and align along the longitudinal axis of the tibia (Figure 3(a)). Conversely, the mesothoracic tibial spurs on the male are not parallel to each other. In males, the anterior spur curves away from the longitudinal axis of the tibia without much deviation in the horizontal axis (i.e., not curved in the direction of either the anterior or posterior face of the tibia; Figures 3(b) and 3(c)). In fact, the small size and lack of color contrast often obscure the spur’s curvature when the tibia is examined directly from a dorsal or ventral perspective. In these cases, the anterior spur may superficially appear shortened relative to the posterior spur until the segment is rotated (or higher magnification/better lighting/etc. is utilized). The posterior spur remains straight and generally in line with the longitudinal axis of the male tibiae. This same orientation of the tibial spurs is present on the metathoracic tibiae of males. However, the curvature of the anterior spur on the metathoracic tibiae of the males is less extreme than the curvature of the spur on the mesothoracic tibiae [21]. Only images of mesothoracic spurs on males are provided here because their sharper curvature relative to the metatho- racic spurs enables more rapid determination of sex. 4 Psyche Figure 2: External posterior characteristics for determining sex of A. diaperinus pupae: female with urogomphus and prominent pygopods ((a) lateral view and (b) ventral view) and male with urogomphus, lacking prominent pygopods ((c) lateral view and (d) ventral view). Pg, pygopods; Ur, urogomphus. Figure 3: External characteristics of mesothoracic tibiae for determining sex of A. diaperinus adults: (a) anteroventral view of parallel apical spurs on females; (b) and (c) anteroventral and anterior views, respectively, of apical spurs on males, note curvature of anterior spur. Ant, anterior spur; scale bar applicable to all frames; tibiae from left legs shown. Psyche 5 Figure 4: Genitalia of A. diaperinus adults: female ovipositor:protracted, lateral (a), and ventral views (b, c); male aedeagus: retracted, lateral, and ventral views (d); protracted, ventral (e, f), and lateral views (g). Ad, aedeagus; Ov, ovipositor. 3.4. Adult Genitalia. Images of A. diaperinus genitalia (Figure 4) improve upon previous drawings [19, 20]. Coloration of the distal end and along the length of the ovipositor indicates sclerotization within the organ membrane (Figures 4(a)-4(c)). Two dark longitudinal lines dorsally and ventrally within the ovipositor suggest sclerotization along the length which may aid in movement of the ovipositor (i.e., protrac- tion and retraction, directionality). In fact, the dorsal lines were observed to aid in the opening and closing of the anus. The paired cerci protruding from the end of the ovipositor each possesses a solitary seta, likely to aid in site selection for oviposition. The male aedeagus is a sclerotized organ and, if not readily visible (Figure 4(d)), can be protracted by squeezing of the abdomen (Figures 4(e)-4(g)), although more pressure is required than that used on females. When protracted, the aedeagus may have a curvature to either side of the longitudinal midline of insect body (Figures 4(e) and 4(f)) and is projected away from the body (Figure 4(g)). 4. Discussion This paper is unique in providing a comprehensive pho- tographic guide for differentiation of sexes in A. diaperi- nus. Further, this paper clarifies that the prominent fleshy processes observed ventrally on female larvae are pygopods. Although general dorsal, lateral, and ventral views of A. dia- perinus larvae have been provided [9, 22], sex determination of late-instars has been previously alluded to because the pygopods were reportedly retained from late instars to the female pupal stages [18]. Visual aides were not provided to unequivocally establish the external morphology of late instars; however, our observations indicate determination of sex in the larval stages based on the presence of pygopods is unreliable. The urogomphus has been used to differentiate between species of Alphitobius [26], but this is the first record to visually demonstrate the variation of pygopods in A. diaperinus larvae. This paper clarifies the definition of the ventral paired processes in female pupae as remnants of the paired py- gopods observed in larvae. The pygopods were previously noted but incorrectly identified as “genital appendages” and “second valvifers” [18, 23]. Line drawings of external char- acters on pupae for differentiating between sexes [18] fail to show potential coloration of urogomphus and pygopods. Figure 2 allows more clear identification of these exter- nal characters, including representation of coloration for these characters. Newly formed pupae are completely white [18]. Coloration of external characters suggests these pupae 6 Psyche (Figure 2) are not newly formed; thus, coloration could po- tentially be used as an indicator of pupal age. However, the latter was outside the scope of this paper. Differences in the orientation of tibial spurs of Alphito- bius laevigatus (E) have been previously shown [21, 22], The curvatures of tibial spurs on A. laevigatus reportedly [21] resemble those of A. diaperinus but comparative illustrations of A. diaperinus tibial spurs were not provided. Viewing angle of the specimen is critical when assessing spur orientation to determine sex; more so, if using the metathoracic tibial spurs [22] because the curvature of male metathoracic spurs is “so slight as to be barely noticeable” [21]. Thus, it is recom- mended that the mesothoracic spurs be used for determi- nation of sexes for adults, in addition to examination of genitalia. Descriptions presented here regarding location and orientation of the spurs are more thorough and images more clearly delineate differences between mesothoracic spurs of respective sexes. Differing intensity of coloration was observed between ovipositors of different females. Whether coloration intensity of ovipositors reflects age remains to be determined. In sexing dead adults, either the cerci were protruding slightly from the last abdominal segment or the ovipositor was protruding altogether. If neither were evident, application of slight pressure to the abdomen caused the cerci to protrude. Generally, if the ovipositor and cerci are not immediately visible after applying pressure to the abdomen, in all likeli- hood the insect is a male. However, to confirm male gender, applying more pressure to the abdomen caused protrusion of the aedeagus for confirmation (Figures 4(e)-4(g)). Detailed descriptions and morphometries of the ovipositor and the aedeagus were previously reported [19, 20] . This comprehensive guide is user-friendly towards novice entomologists and nonentomologists (e.g., microbiologists, pathologists) and will be an invaluable tool for those entering the study area of A. diaperinus and pathogen interactions affecting poultry and humans. Acknowledgments The authors are indebted to Dr. Robert Wharton, Texas A&M University, for the use of microscopy and imaging equip- ment. Mention of trade names, companies, or commercial products in this publication is solely for the purpose of pro- viding specific information and does not imply recommen- dation or endorsement of the products by the USA Depart- ment of Agriculture. The USDA is an equal opportunity provider and employer. References [1] C. J. Geden and J. A. Hogsette, “Research and extension needs for integrated pest management for arthropods of veteri- nary importance,” in Proceedings of a Workshop in Lincoln, Nebraska, pp. 1-328, April 1994. [2] C. J. Geden and R. G. Axtell, “Factors affecting climbing and tunneling behavior of the lesser mealworm, Alphitobius dia- perinus (Goleoptera: Tenebrionidae),” Journal of Economic Entomology, vol. 80, no. 6, pp. 1197-1204, 1987. [3] R. Dass, A. V. N. Paul, and R. A. Agarwal, “Feeding poten- tial and biology of lesser mealworm, Alphitobius diaperinus (Panz.) (Gol., Tenebrionidae), preying on Corcyra cephalonica St. (Lep., Pyralidae),” Zeitschrift fur Angewandte Entomologie, vol. 98, no. 1-5, pp. 444-447, 1984. [4] R. G. Axtell, Ed., Biology and Economic Importance of the Darkling Beetle in Poultry Houses, Poultry Supervisors’ Short Gourse, North Garolina State University, 1994. [5] R. G. Axtell and J. I. Arends, “Ecology and management of arthropod pests of poultry,” Annual Review of Entomology, vol. 35, no. l,pp. 101-126, 1990. [6] D. G. Pfeiffer and R. G. Axtell, “Goleoptera of poultry manure in caged layer houses in North Garolina,” Environmental Ento- mology, vol. 9, pp. 21-28, 1980. [7] L. M. Rueda and R. G. Axtell, “Arthropods in litter of poultry (Broiler Ghicken and Turkey) houses,” Journal of Agricultural and Urban Entomology, vol. 14, no. 1, pp. 81-91, 1997. [8] L. M. Rueda and R. G. Axtell, “Temperature- dependent de- velopment and survival of the lesser mealworm, Alphitobius diaperinus,” Medical and Veterinary Entomology, vol. 10, no. 1, pp. 80-86, 1996. [9] J. G. Dunford and P. E. Kaufman, Lesser Mealworm, Litter Beetle, Alphitobius diaperinus (Panzer) (Insecta: Goleoptera: Tenebrionidae), Entomology and Nematology Department, Elorida Gooperative Extension Service, Institute of Eood and Agricultural Sciences, University of Elorida, Gainesville, Ela, USA, 2006. [10] M. Hosen, A. R. Khan, and M. Hossain, “Growth and develop- ment of the lesser mealworm, Alphitobius diaperinus (Panzer) (Goleoptera: Tenebrionidae) on cereal flours,” Pakistan Journal of Biological Sciences, vol. 7, no. 9, pp. 1505-1508, 2004. [11] K. Sarin and S. G. Saxena, “Pood preference and site of damage to preferred products by Alphitobius diaperinus (Panz.),” Bulletin of Grain Technology, vol. 13, no. 1, pp. 50-51, 1975. [12] T. A. Lambkin, R. A. Kopittke, S. J. Rice, J. S. Bartlett, and M. P. Zalucki, “Pactors affecting localized abundance and distri- bution of lesser mealworm in earth- floor broiler houses in subtropical Australia,” Journal of Economic Entomology, vol. 101, no. 1, pp. 61-67, 2008. [13] O. Prancisco and A. P. do Prado, “Gharacterization of the larval stages of Alphitobius diaperinus (Panzer) (Goleoptera: Tenebrionidae) using head capsule width,” Brazilian Journal of Biology, vol. 61, no. 1, pp. 125-131, 2001. [14] T. H. Wilson and P. D. Miner, “Influence of temperature on development of the lesser mealworm, Alphitobius diaperinus (Goleoptera: Tenebrionidae),” Journa/ of Kansas Entomological Society, vol. 42, no. 3, pp. 294-303, 1969. [15] K. G. Stafford, G. H. Gollison, J. G. Burg, and J. A. Gloud, “Distribution and monitoring lesser mealworms, hide beetles, and other fauna in high-rise, caged-layer poultry houses,” Journal of Agricultural Entomology, vol. 5, no. 2, pp. 89-101, 1988. [16] T. L. Grippen and T. L. Poole, “Lesser mealworm on poultry farms: a potential arena for the dissemination of pathogens and antimicrobial resistance,” in On-Earm Strategies to Gontrol Eoodborne Pathogens, T. R. Gallaway and T. S. Edrington, Eds., Nova Science, New York, NY, USA, 2012. [17] T. L. Grippen and J. P. Esquivel, “Improved visualization of Alphitobius diaperinus (Panzer) (Goleoptera: Tenebrionidae): IE Alimentary canal components and measurements,” Psyche. In press. [18] H. E. Barke and R. Davis, “Sexual dimorphism in the lesser mealworm, Alphitobius diaperinus (Panz.) (Goleoptera: Psyche 7 Tenebrionidae),” Journal of the Georgia Entomological Society, vol. 4, pp. 119-121, 1967. [19] J. D. Hopkins, C. D. Steelman, and C. E. Carlton, “Anatomy of the adult female lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) reproductive system,” Journal of the Kansas Entomological Society, vol. 65, no. 3, pp. 299-307, 1992. [20] J. D. Hopkins, C. D. Steelman, and C. E. Carlton, “Internal reproductive system of the adult male lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae),” Journal of the Kansas Entomological Society, vol. 66, no. 4, pp. 446-450, 1993. [21] P. S. Hewlett, “Secondary sexual characters in Alphitobius lae- vigatus (E.) and A. diaperinus (Panz.) (Col., Tenebrionidae),” The Entomologist’s Monthly Magazine, Yol. 94, p. 144, 1958. [22] A. J. Roche, The lesser mealworm, Alphitobius diaperinus (Panzer), and its role in Salmonella transmission to poultry, M.S. thesis. University of Georgia, 2007. [23] D. G. H. Halstead, “External sex differences in stored-products Coleoptera,” Bulletin of Entomological Research, vol. 54, pp. 119-134, 1963. [24] E. W. Stehr, Immature Insects, vol. 2, Kendall- Hunt, Dubuque, La, USA, 1991. [25] H. E. Barke and R. Davis, “Notes on the biology of the meal- worm, Alphitobius diaperinus (Coleoptera: Tenebrionidae),” Journal of the Georgia Entomological Society, vol. 4, pp. 46-50, 1969. [26] E. J. Priess and J. A. Davidson, “Characters for separating late-stage larvae, pupae, and adults of Alphitobius diaperinus and A. laevigatus (Coleoptera: Tenebrionidae),” Annals of the Entomological Society of America, vol. 63, no. 3, pp. 807-808, 1970. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 149572, 14 pages doi:10.1155/2012/149572 Review Article Mechanisms of Odor Coding in Coniferous Bark Beetles: From Neuron to Behavior and Application Martin N. Andersson Department of Biology, Lund University, 223 62 Lund, Sweden Correspondence should be addressed to Martin N. Andersson, martin_n.andersson@biol.lu.se Received 3 October 2011; Accepted 12 December 2011 Academic Editor: John A. Byers Copyright © 2012 Martin N. Andersson. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Coniferous bark beetles (Coleoptera: Curculionidae; Scolytinae) locate their hosts by means of olfactory signals, such as pheromone, host, and nonhost compounds. Behavioral responses to these volatiles are well documented. However, apart from the olfactory receptor neurons (ORNs) detecting pheromones, information on the peripheral olfactory physiology has for a long time been limited. Recently, however, comprehensive studies on the ORNs of the spruce bark beetle, Ips typographus, were conducted. Several new classes of ORNs were described and odor encoding mechanisms were investigated. In particular, links between behavioral responses and ORN responses were established, allowing for a more profound understanding of bark beetle olfaction. This paper reviews the physiology of bark beetle ORNs. Special focus is on 1. typographus, for which the available physiological data can be put into a behavioral context. In addition, some recent field studies and possible applications, related to the physiological studies, are summarized and discussed. 1. Introduction Bark beetles (Coleoptera: Curculionidae: Scolytinae) consti- tute some of the most destructive pests of coniferous trees throughout the world, destroying forests of great economic value. Currently, the large-scale outbreak of the mountain pine beetle, Dendroctonus ponderosae, in North America has resulted in the loss of hundreds of millions m^ timber and turned the forests into major sources of carbon release [1]. In Europe and parts of Asia [2, 3], the European spruce bark beetle, Ips typographus (Eigure 1), is considered the most destructive bark beetle of coniferous forests [4, 5] . Bark beetles, like most insects, locate their hosts mainly by means of olfactory signals. It is clear that they utilize both attractants and antiattractants that emanate from host and nonhost plants, as well as from conspecific and heterospecific bark beetle individuals [3, 6-10]. The odor molecules are transported downwind from their source of release as an odor plume with a complex structure [11-13]. Molecules are picked up by olfactory receptors (ORs) or ionotropic recep- tors (IRs) [ 14] , located mainly in the antennae and maxillary palps. Specifically, the ORs are present in the cell membrane of olfactory receptor neuron (ORN) dendrites that, in turn, are housed within olfactory sensilla [15]. The ORs are encoded by a large and diverse family of olfactory receptor genes [16]. Each ORN is generally thought to express only one member from this family in addition to the widely expressed coreceptor, Oreo [17]. IRs act in combinations of up to three subunits that are comprised of odor-specific receptors and one or two broadly expressed coreceptors [ 14] . These receptors are expressed in neurons that do not express ORs. When an odor molecule binds to a receptor, the ORN sends a neuronal signal to the primary olfactory center of the brain, the antennal lobe. Typically, the signal that is generated by an ORN is an increase in the firing frequency of action potentials (excitation), but some odorants may instead cause a decrease in firing activity (inhibition). ORNs can be divided into classes based on their odor response profiles. Often, ORNs are fairly specific and activated by only one or a few compounds, but some appear to have a broader tuning. In addition, each compound often activates more than one type of ORN, and thus, the odor input is thought to be con- structed as a combinatorial code [18]. 2 Psyche Figure 1: The European spruce bark beetle, Ips typographus. Photo: Goran Birgersson. In contrast to the well- studied chemical ecology of bark beetles, until recently, little was known about the physio- logical responses of individual bark beetle ORNs. Mainly in the 1980s, Single-sensillum recordings (SSR) were carried out, primarily identifying classes of ORNs that responded to various pheromone compounds. Some decades later, comprehensive studies on 1 . typographus have characterized additional ORNs that respond also to host and nonhost plant compounds [19] and have provided novel insights into potential odor coding mechanisms in insects in general [8]. This review summarizes the results from early and recent studies on the physiology of ORNs in conifer- feeding bark beetles. Particular focus is on L typographus, for which a sufficient amount of information has emerged in order to bridge the physiological data with previously recorded behavioral responses to several semiochemicals. In addition, some recent behavioral studies with connections to olfactory physiology are summarized and possible applications dis- cussed. First, however, a brief overview of the semiochemicals that are used by 1 . typographus in host selection is presented. 2. Host Selection by L typographus The male is the initial host seeking, or “pioneering,” sex of /. typographus. Once a male has located a suitable host material to colonize, it releases an aggregation pheromone, a mixture of (4S)-d5-verbenol and 2-methyl-3-buten-2-ol [20], which attracts individuals of both sexes. Although the olfactory- mediated host location behavior of 1 . typographus has been extensively studied, it is not known how the pioneering males locate a suitable host tree, as no primary attraction (in the absence of pheromone) to spruce volatiles has been demonstrated. However, spruce volatiles may modulate the pheromone response [9] or possibly attract beetles to a suitable habitat [2 1 ]. It is also possible that pioneering beetles land randomly on trees and assess host quality upon contact [22]. However, apart from the few pioneering males, the aggregation pheromone attracts the majority of individuals to the host. The attraction to the pheromone is modulated by other semiochemicals that appear in later attack phases. Verbenone and ipsenol are two such compounds that are believed to be used as cues to avoid heavily attacked trees [23]. In addition, volatiles that are particularly abundant in nonhost angiosperm plants (so called nonhost volatiles, NHV), such as green leaf volatiles (GLVs) [24] and compounds from the bark, such as C8-alcohols and trans-conophthorin [25, 26], have inhibitory effects on pheromone attraction. Com- bining these compounds with verbenone produces a strong synergistic effect and a potent antiattractant blend [27]. Possibly, the individual constituents in the synergistic blend represent different levels in the host selection sequence [6]. The GLVs that are common to broad-leaved plants may re- present a signal of a nonhost dominated habitat. More spe- cific plant volatiles, such as fraus-conophthorin, may indicate nonhosts at the tree species level [7], whereas the antiat- tractive pheromone components may signal unsuitability of individual spruce trees. 3. Olfactory Receptor Neurons of L typographus and Other Bark Beetles Many compounds that are either attractants or antiattrac- tants for conifer bark beetles have been identified [3, 6, 7]. Single-sensillum recordings from the ORNs of several bark beetle species have shown that many of the behaviorally active compounds elicit responses in different classes of neurons (Table 1). It is obvious that, except for/, typographus and the ambrosia beetle Trypodendron lineatum, more is known about ORN responses to pheromone components than about responses to plant odors (Table 1). In addition, several of the tested compounds (i.e., ipsdienol, ipsenol, ver- benone, ds/trans-verbenol, cxo-brecicomin, and a-pinene) elicit strong responses in the majority of species studied. For more details on ORN specificity, sensitivity, and abundance in each species, the reader is referred to the cited literature. Olfactory sensilla of L typographus are present in three areas (or bands) on the antenna (Figure 2(a)) [41]. Ander- sson et al. [19] screened 150 olfactory sensilla for respons- es to an odor panel comprised of similar numbers of syn- thetic pheromone, host, and nonhost compounds. Strong excitatory responses were obtained from 106 ORNs; 45 re- sponded specifically to various bark beetle pheromone compounds, 37 to host compounds, and 24 to antiattractive nonhost volatiles (NHVs). Based on response spectra, the 106 ORNs were grouped into 17 different classes (Figure 3). Additionally, 26 neurons (divided into 12 ORN classes) re- sponded only weakly to any test odorant, indicating that the most potent compounds for these ORNs were lacking. In addition to the ORN classes described by Andersson et al. [19], three other classes, responding specifically to {+)-trans- verbenol, phenylethanol, or campher plus pino-camphone, respectively, had been identified previously (Table 1) [28]. Furthermore, the majority of the ORN classes responding to pheromone compounds was found in both studies. Many ORN classes have been subjected to dose-response trials that indicated that the ORNs, in general, are highly sensitive and specific for only one or a few structurally related pheromone or plant compounds (Figures 4(a)-4(c)) [19, 28]. Response thresholds for the best ligand(s) were normally found around Psyche 3 Table 1: Compounds from different ecological sources that elicit strong responses in olfactory receptor neurons in eight species of Scolytinae. Species Beetle-produced compounds Host compounds Nonhost compounds References (-l-)-ipsdienol Myrcene Pine bark extract (-)-ipsdienol Campher (B) Birch bark extract (-)-ipsenol Pino-camphone (B) 1-hexanol (D) ( -)-a‘s- verbenol p-cymene E2-hexenol (D) Ips typographus (-1- )-trflns- verbenol (-)-verbenone 3-carene 1,8-cineole Z3-hexenol (D) , , , 1 [19,28-31] l-octen-3-ol 2-methyl-3-buten-2-ol (-l-)-a-pinene (C) 3-octanol Amitinol (-)-a-pinene (C) (S,S) -frans- conophthorin (A) Phenylethanol CAO-brevicomin (A)* (±)-chalcogran (A) (-l-)-ipsdienol Linalool (-)-ipsdienol Camphor Ipspini (±)-ipsenol czs-verbenol Myrcene [29, 32-34] frans- verbenol Verbenone Ips paraconfusus (-l-)-ipsdienol (-)-ipsdienol (±)-ipsenol [33] Frontalin a-pinene 3-methyl-2-cyclohexenone 3 - methyl- 2 - cyclohexenol Limonene Dendroctonus pseudotsugae 1 - methyl- 2 - cyclohexenol frans- verbenol [35,36] cfs-verbenol Verbenone Ipsenol (-)-frontalin a-pinene Dendroctonus frontalis CAO-brevicomin cndo-brevicomin 3-carene [37,38] Verbenone frans-verbenol Dendroctonus micans (-l-)-ipsdienol CAO-brevicomin [31] (-l-)-lineatin Ethanol Pine bark extract Phenylethanol Methanol Birch bark extract Trypodendron lineatum Butanol a-pinene j^-pinene [39] Spruce bark extract Tomicus destruens Compounds in pine extract Benzyl alcohol [40] * Compounds that elicit responses of similar strength in the same ORN class are indicated by the same capital letter. Odorants eliciting secondary responses are omitted for clarity. (a) (b) Figure 2: (a) Olfactory sensilla are present in three areas (A, B, and C) on the antennal club of Ips typographus. (b) Spatial distribution patterns of four classes of olfactory receptor neurons (ORNs). ORNs responding to green leaf volatile alcohols (nonhost) = green squares, myrcene (host) = yellow triangles, a‘s-verbenol (pheromone) = red circles, 1,8-cineole (host) = blue small circles (from [19], with permission from the publisher). Scale bar = 50 ^m. the 1 ng dose on the filter paper using paraffin oil as solvent [19]. A high specificity, not only among pheromone ORNs, but also among those for plant compounds, seems to be a general rule also in other bark beetle species (see especially [32, 35]). The ORNs off. typographus are not randomly distributed on the antenna. Instead, ORNs from a particular class are generally found either in both the proximal and medial bands of sensilla, or exclusively in the distal area (Figure 2(b)) [19]. This distribution pattern seems to correspond to the distribution of the two morphological types of single-walled sensilla previously identified [41]. It is common in insects that the pheromone ORNs are numerous on the antenna and that the most common ORN type is tuned to the major (most abundant) component [42, 43]. In 1. typographus, the most recurrent ORN class was tuned to (-)-ds-verbenol [19]. In contrast, there were only few cells specific for 2-methyl-3-buten-2-ol (MB) (Figure 3) [19, 28], an essential pheromone component which is pro- duced, and behaviorally active, in much larger quantities [20, 44]. This suggests that the pattern might be reversed in the bark beetle. However, the MB cells were found in a restricted area on the antenna [19], that is, on the borderline between the medial band and distal area of sensilla, which could have resulted in this cell type being underrepresented among the sampled sensilla. Alternatively, as MB is highly volatile, the low number of cells could be the result of the compound being lost from the stimulus cartridge upon stimulation. Indeed, photoionization detector measurements showed that the airborne amount of MB released from the stimulus pipette drops dramatically upon stimulation (Figure 5) [45]. However, the insect ORN still responded vigorously despite the low concentration, rendering this explanation unlikely. In contrast to Andersson et al. [19], Tommeras [28] found that the ORNs tuned to ipsdienol were the most common ones ORN class: (1) ch-verbenol (2) Ipsenol (3) IpsdienolA ^ (4) IpsdienolB ^ (5) 2-methyl-3-buten-2-ol (6) Amitinol (7) Verbenone P (8) a-pinene (9) Myrcene (10) p-cymene (11) p-cymene/ myrcene d (12) 1,8-cineole (13) A3-carene (14) GLV-OHs (15) 3-octanol (16) l-octen-3-ol d (17) Irans-conophthorin 0 42% 35% 23% Figure 3: Number of olfactory receptor neurons (ORNs) of 17 strongly responding classes of Ips typographus (data from [19]). ORN classes are labeled according to which compound(s) elicited the strongest response. As pure enantiomers were not tested, it is likely that the ipdienol ORN classes A and B correspond to the ORNs responding to (-1-)- and (-)-ipdienol, respectively [29]. Orange = bark beetle pheromone compounds, brown = conifer compounds, green = nonhost volatiles. GLV-OHs = green leaf volatile alcohols. in L typographus. This discrepancy may also be explained by the nonrandom localization of ORNs on the antenna (i.e., neurons for ds- verbenol are abundant only in the distal part of the antennae, Figure 2(b)). Although no primary attraction has been demonstrated, the high frequency of ORNs tuned to conifer- related Psyche 5 Dose (log ng) czs-verbenol Verbenone trans-verbenol 1,8-cineole Chalcogran Dose (log ng) i?,i?-trans-conophthorin exo-brevicomin S,S-trans-conophthorin (a) (b) Dose (log ng) Dose (log ng) p-cymene 3-carene (-)-limonene (+)-limonene 1 -hexanol E2-hexenol Z3-hexenol Chalcogran Hexanal E2-hexenal l-octen-3-ol (c) (d) Figure 4: Dose-response curves from four receptor neuron classes of Ips typographus, demonstrating specific primary responses to (a) the pheromone component ds- verbenol, the spruce compounds (b) 1,8-cineole and (c) p-cymene. (d) Indiscriminate response to the three green leaf volatiles 1-hexanol, £2-hexenol, and Z3-hexenol (modified from [19], with permission from the publisher). monoterpenes (Table 1, Figure 3) suggests that host kairo- mone is relevant for host location by 1. typographus. As men- tioned previously, these compounds may serve as habitat-scale attractants [21], or as modulators of phero- mone attraction [8, 9]. Perhaps the most striking finding from the bark beetle SSR studies is that almost 25% of the strongly responding ORNs were specifically tuned to anti- attractive NHV (Table 1, Figure 3) [19]. This indicates that insects may devote a lot of olfactory capacity to the detection of compounds from sources that they avoid. Similar results have not been found in any other insect studied so far, however, it is likely that many other bark beetles that show strong GC-EAD responses to NHV also have a large propor- tion of ORNs tuned to such compounds [7, 46-48]. 4. Discrimination of Enantiomers Most bark beetle pheromone compounds are chiral. Attrac- tion is typically evoked by only one of the enantiomers, while the other sometimes inhibits attraction (e.g., [20, 33]). The enantiospecific behavioral response is reflected in the specificity of the ORNs detecting the compounds (Table 1). For instance, ORNs that are specific for either the (-1-)- or the (-)-enantiomer of ipsdienol, ipsenol, verbenone, or cis- and tra ns- verbenol have been identified in several Ips species [28, 33]. Other examples are the ORNs in the Southern pine beetle, D. frontalis [37], and in the Douglas-fir beetle, D. pseudotsugae [36], that discriminate between the (-1-)- and (-)-enantiomer of frontalin (Table 1). 6 Psyche 12000 -1 Q CO r 250 +1 0 5 10 15 20 25 Stimulation number O ORN • PID Q Ln +1 c o Dh Pi O Figure 5: Response of Ips typographus olfactory receptor neurons (ORNs) and a photoionization detector (PID) to successive stimu- lations with 2-methyl-3-buten-2-ol {N = 4) (modified from [61]). In general, the sensitivity of the pheromone ORNs seems to be 10- 100- fold higher for the enantiomer that they are tuned to, as compared to the other [28, 33]. In addition, there seems to be a correspondence between the attraction to a specific enantiomer, and the frequency of ORNs on the antenna that responds to it. For instance, 1. pini is attracted by (-)-ipsdienol and has more of its ORNs tuned to (-)- ipsdienol than to (-l-)-ipsdienol. Similarly, L paraconfusus, which is attracted by (-b) -ipsdienol, has most of its ipsdienol ORNs tuned to the (+) -enantiomer [33]. Enantiospecific responses to plant compounds have been recorded in L typographus [19]. The neuron class that responded most strongly to the nonhost volatile trans- conophthorin (Table 1) was > 100-fold more sensitive to the (5S,7S)-enantiomer than to the (5R,7R) -enantiomer. In fact, other structurally related compounds (racemic exo- brevicomin and chalcogran) elicited stronger responses in this ORN than did the (5R,7R) -enantiomer of trans-con- ophthorin [19]. In another class of ORN, the natu- rally occurring (-)-l-octen-3-ol elicited a slightly stronger response than the racemic mixture, indicating that the (-)- enantiomer is the key ligand. In contrast, the neuron that is tuned to a-pinene responded similarly to both enantiomers (Table 1) [19]. 5. Olfactory Receptor Neuron Responses and Behavior The results that have been obtained from single sensillum recordings [19, 28] indicate that behavioral responses of 1. typographus to several compounds can likely be explained by the responses of the ORNs. Several volatiles from nonhost plants were previously shown to inhibit pheromone attraction of 1. typographus [24-26]. The three GLVs: 1-hexanol, T2-hexenol, and Z3- hexenol all reduced pheromone attraction to a similar extent. However, combining the three did not produce a stronger inhibition of attraction, a phenomenon defined as redundancy [27]. Interestingly, the only ORN that was sen- sitive to any of these volatiles had a more or less identical sensitivity to all three of them (Table 1, Figure 4(d)) [19]. Thus, it appears as if the bark beetle cannot differentiate between the compounds at the physiological level, which agrees well with their behavioral redundancy. In contrast, the compounds verbenone and traus-conophthorin that syn- ergize the inhibition are detected by different ORNs [19, 28] . Interestingly, the pheromone component, chalcogran, of the sympatric Pityogenes chalcographus, was primarily detected, by 1. typographus, by the same neuron as fraus-conophthorin (Table 1). Chalcogran also inhibits pheromone attraction of I. typographus [49]. Most insects house their ORNs for pheromone com- pounds in sensilla that are distinct from the ones that detect plant compounds (e.g., [50, 51]). However, in some sensilla in 1. typographus, the ORN for the aggregation pheromone component a's-verbenol (cV) is colocalized with an ORN that responds to the host plant compound 1,8-cineole (Ci) [8, 19] (Figure 2(b)). This lack of segregation between ORNs detecting pheromones and plant volatiles may suggest that host finding in bark beetles is an integrated process that involves both pheromones and plant volatiles. When the ORN for Ci responded, the colocalized cV cell was inhibited, indicative of interactions between ORNs in the periphery. In addition, Ci was found to be particularly abundant in heavily attacked spruce trees and the compound strongly reduced pheromone attraction (88% reduction in trap catch) in the field [8]. Possibly, Ci is a signal of an unsuitable (crowded) host or a well-defended tree. 6. Peripheral Modulation of ORN Responses Colocalization of insect ORNs in the same sensillum is thought to improve coincidence detection, which increases the insects spatiotemporal resolution of odor signals [52] and improves ratio detection of ecologically relevant odor mixtures [53]. In addition, the presence of two or more neurons in the same sensillum may provide opportunities for signal modulation in the periphery. Indeed, in the Douglas-fir beetle, Dendroctonus pseudotsugae, two ORNs, each specific for one of the two pheromone components 3-methyl-2-cyclohexenone or 3-methyl-2-cyclohexenol, are colocalized. When either one of the ORNs responded to its specific ligand, the spontaneous activity of the other ORN was reduced. This observation indicated reciprocal intera- ctions, either directly between the two neurons, or between the two ligands and their respective receptors [35]. In addi- tion, when another ORN type that responded to limonene (10 ng dose) was challenged with a binary mixture of limonene and 3-methyl-2-cyclohexenol (10: 1000 ng), the response to limonene was completely shut down [35]. In L typographus, not all cV neurons (large amplitude A- cell) are colocalized with the neuron for Ci (small amplitude B-cell) (Figure 6(a)). These cV neurons are instead found Psyche 7 cV 1 ng cV 1 ng + Ci 10 /^g (b) cV Ing cV 1 ng Ci 1 /^g cV 1 ng Ci 10 /^g Blank cV 1 ng cV 1 ng Ci 1 /^g cV 1 ng Ci 10 /^g Blank (c) Figure 6: (a) Schematic drawing of the two types of sensilla in Ips typographus containing the A cell (large amplitude spikes) for a‘s-verbenol (cV), accompanied either by a nonresponsive {left column) B cell (small amplitude), or a B cell for 1,8-cineole (Ci) {right column), (b) Responses of both sensillum types to 1 ng cV {upper traces), and a binary mixture of 1 ng cV and 10 |Mg Ci {lower traces). Note the inhibition of the cV response during the response to Ci in the B cell. Black horizontal bars indicate the 0.5 s stimulation period, (c) Detailed response curves to cV and binary cV ; Ci mixtures showing a Ci dose-dependent inhibition of the cV response only in sensilla that also contain the Ci cell {N = 10-12). Arrows indicate the onset of the 0.5 s stimulation period (modified from [8], with permission from the publisher). 8 Psyche together with another ORN type that does not respond to any odorant tested so far [19]. The Ci inhibited the cV cell only in sensilla in which the two neurons were colocalized, implying that the inhibition might be due to interactions between the ORNs, To test this hypothesis, Andersson et al. [8] recorded both types of cV sensilla (with or without the Ci cell) and tested responses to binary cV/Ci mixtures. They found that not only the spontaneous activity but also the ORN response to the lowest cV dose ( 1 ng) was inhibited by simultaneous stimulation with high doses of Ci (1-10 f/g). This inhibition occurred only in sensilla that also contained the Ci cell (Figures 6(b)-6(c)). In addition, the response to the higher cV dose (10 ng) was more strongly inhibited in sensilla where the Ci cell was colocalized. Thus, it seems plausible that the two ORNs interact, possibly by means of passive electrical interactions [54]. However, if or to which extent the reduction in pheromone trap catches by the presence of Ci [8] can be explained by the inhibition of the cV ORN remains unknown, as the excitatory input from the two ORNs provides the means also for central integration [55]. It seems like similar inhibitory interactions between colocalized ORNs occur also in other insects [51, 56, 57], but the phenomenon has so far only been systematically ad- dressed in bark beetles. 7. Difficulties in Comparing ORN Responses to Compounds with Different Volatility In most SSR studies, odor stimuli are prepared based on a known amount (e.g., in nano- or microgram) of compound applied to a piece of filter paper that is positioned inside a Pasteur pipette odor cartridge. Upon stimulation, the head- space in the cartridge is blown over the insect preparation. Depending on compound, solvent, and how many times the cartridge has been used, the quantity of molecules reaching the insect can be highly variable and seriously affect the ORN response [58, 59]. Indeed, different stimulation regimes, compound doses, and solvents (mostly hexane and paraffin oil) have been used in the various bark beetle SSR studies (Table 1), making it difficult to directly compare the sensitivity and specificity of ORNs characterized in different species or studies. Furthermore, the physical parameters of the odor-delivery system also affect the integrity of an airborne odor stimulus [60], which may further increase the variability among responses. Airborne amounts of different compounds have been measured with a photoionization detector [45]. A huge vari- ation among compounds was observed. For the most volatile compounds, such as 2-methyl-3-buten-2-ol (Figure 5), ca 80% of the headspace in the odor cartridge was lost at the first puff, even though paraffin oil was used as solvent. Air- borne amounts of heavier compounds, such as linalool, were reduced by only ca 50% after 50 reiterative stimulations. In addition, compounds that were dissolved in pentane were released at a much higher rate than compounds in paraffin [45]. The large variation between compounds, solvents, and successive stimulations could easily bias electrophysiological responses in insects. This was verified by reanalyzing the response of the 3-octanol ORN of L typographus [19] to two C8-alcohols (3-octanol and l-octen-3-ol) and two C6-alco- hols (Z3-hexenol and 1-hexanol) using both fresh (not used) and “old” (used 10 times) stimulus pipettes [45]. The ORN response to fresh pipettes was clearly different from the response to the “old” pipettes. In particular the re- sponse to the C6-alcohols was clearly lower when old pipettes were used. In fact, the difference in response was so large that it falsely implied that recordings were made from two distinct ORN classes. Such a finding suggests that it is absolutely necessary to use very strict experimental protocols for elec- trophysiological recordings, and that it sometimes is required to measure airborne odor amounts, especially when com- pounds of different volatility are used as stimuli [45]. 8. Odor Coding in Bark Beetles Compared to Other Insects In insects in general, neurons that detect pheromone con- stituents have a narrow tuning. Bark beetles are no exception as the ORNs that respond to aggregation pheromone com- pounds are, in most cases, sensitive to only one compound. The tuning width of insect ORNs detecting plant volatiles seems to range from narrow to broad, although ORN speci- ficity is strongly correlated to the stimulus concentration tested [ 18] . Most of the ORNs for plant volatiles in 1. typogra- phus are narrowly tuned [8]. However, some show more indiscriminate responses, such as the GLV neuron that had similar sensitivity to 1-hexanol, £2-hexenol, and Z3-hexenol. This is in contrast to the highly specific GLV neurons that have been described in, for instance, scarab beetles [50, 51], and in the Golorado potato beetle [62]. The difference may be related to the fact that these other species feed on angiosperms, which presumably requires a better resolution of angiosperm dominated volatiles (i.e., GLVs) than what is needed for a conifer specialist. Many of the bark beetle ORNs are highly selective for specific enantiomers, both in terms of pheromones and plant compounds. However, this feature is not unique for bark beetles; highly enantioselective neurons have been characterized also in other insects [63-65]. In contrast, no other insect studied so far has a comparable frequency of ORNs tuned to antiattr actants as the one found inf. typographus [19]. The co-localization of ORNs for pheromone and plant compounds in 1. typographus is not commonly found in insects. This special type of ORN pairing may be related to the fact that host colonization in bark beetles often involves both pheromone and plant-produced compounds [8]. In addition, the colocalized neurons for plant and pheromone compounds also interacted by inhibiting their neighboring neuron while responding [8]. It is difficult to say whether a similar interaction occurs also in other insects, since it has not been systematically addressed elsewhere. However, inhibition of the spontaneous activity of the large-spiking cell when the small-spiking cell responds seems to be a com- mon phenomenon [35, 51, 56, 57], indicating that the same type of modulation could be present. Indirect evidence for ORN interactions was found previously in the honeybee [66]. The 18-35 ORNs that are housed within honeybee Psyche 9 (c) (d) Figure 7; (a) Lindgren funnel traps (19-funnel size) were used in the vertical spacing tests with Ips typographus. Dispensers positioned under grey cups, (b) A Lindgren trap (5-funnel size) was attached to a wind vane in the horizontal spacing tests to ensure constant distance between plumes, (c) Pipe trap surrounded by eight nonhost volatile dispensers in the antiattractant background tests, (d) Soap bubble visualization of vertical plume overlap at a spacing distance of 48 cm. Distance between black poles = 1 m (modified from [61] ). sensilla placodea seemed to respond to odors in a coordi- nated manner, indicating that the individual ORNs do not act as independent response units. However, in that study, it was not possible to keep track of the individual ORNs. Taken together, odor coding in bark beetles is, in gen- eral, similar to odor coding in other insects, but it also exhi- bits some rare features. The coding principle seems to be con- sistent with the “combinatorial code” theory, but the olfac- tory input travels mainly through highly specific channels. 9. Detection and Behavior in Odor-Diverse Habitats Activation of an ORN by an attractant may cause an upwind flight by the insect towards the odor source. However, if repulsive compounds simultaneously trigger other ORNs to fire, the upwind flight may be aborted. Thus, in environ- ments with a high “semiochemical diversity” [27] where odor plumes from different sources intermix, localization of host plants may be hampered by the presence of odors from nonhosts [67, 68]. Thus, for bark beetles, it may be possible to reduce the risk of attacks by making the environ- ment more semiochemically diverse. Homogenous mixing of odor plumes from different sources is, however, contradicted by the partitioning of plumes into “odor packages” (or filaments) that are interspersed with pockets of “clean air” [11, 12] . This, in turn, is thought to facilitate plume discrim- ination by insects. Placing an NHV mixture inside a pheromone trap, that is, next to the pheromone bait, greatly reduces trap catch of 1. typographus [27]. However, to test the “semioche- mical diversity hypothesis,” pheromone trap catches in the presence of NHV at different vertical and horizontal dis- tances from the pheromone dispenser (Figures 7(a) -7(b)), were investigated [69]. Trap catches in response to sepa- rated pheromone components (ds-verbenol and 2-methyl- 3-buten-2-ol) were also tested (in the absence of NHV) to further investigate responses to separated baits in gen- eral. In addition, the response of the beetle was com- pared to the response of the Egyptian cotton leaf worm, Spodoptera littoralis (Lepidoptera: Noctuidae), to separated sex pheromone components and to separated pheromone and behavioral antagonist. In both species, increased spacing between pheromone and antiattractants led to increased trap catch, whereas, as expected, increased spacing between pheromone components had the opposite effect. However, 10 Psyche Spacing distance (m) [log scale] Spacing exp: Spod. Ph-ant low dose Spod. Ph-ant high dose Spod. Ph major compound in Spod. Ph major compund out Ips Ph vertical Ips Ph horizontal Ips Ph-NHV horizontal Ips Ph-NHV vertical Background exp: - 9 - Ips IT- REP low dose -O- Ips IT-REP high dose (a) (b) Figure 8: (a) The effect of spacing between attractant and antiattractant sources on trap catches of Ips typographus and Spodoptera littoralis, illustrated by measures of effect size (Hedges’ unbiased g). The effect size provides a measure of a biological treatment effect by scaling the difference between the treatment and the control means, with the pooled standard deviation for those means. Effect sizes further from zero than 0.8 are regarded as strong effects. In all experiments, the pheromone bait alone (zero distance between components) was the control. The zero cm spacing distance in experiments involving antiattractants is omitted for clarity, (b) Effect sizes in the Ips antiattractant background experiments using nonhost volatile dispensers at eight positions, or flakes around the trap. * Elakes were evenly distributed on the ground 0-2 m from the trap. Thus, this treatment is “not to scale” on the x-axis. Ph = pheromone; Ant = Spodoptera pheromone antagonist; NHV = nonhost volatiles; IT-REP = semicommercial Ips typographus repellent dispenser (from [69], with permission from the publisher). the two species differed greatly with respect to the spacing distances that affected their trap catch (Figure 8(a)). While beetle trap catches were affected by separation of some decimeters, trap catches of the moth were affected by separation distances of just a few centimeters [69]. In each species, the spacing distances affecting trap catch did not differ between the pheromone component spacing and the pheromone/antiattractant spacing experiments [69]. The bark beetle pheromone/NHV spacing experiments indicated antiattractive effects of NHV up to a distance of >lm [69]. To further investigate potential effects of NHV at even longer distances, pheromone attraction was studied in the presence of a synthetic background of NHV, either created by eight NHV point sources positioned in a ring (with 1, 2, or 3 m radius) around a central pheromone trap (Figure 7(c)), or by ca 6000 small (ca3 X 3 mm) NHV impre- gnated flakes [70] on the ground around a phero- mone trap [69]. With the eight NHV sources, bark beetle at- traction was reduced up to the 2 m spacing distance, and there was still a tendency for reduced attraction at the 3 m distance (Figure 8(b)). Similar to the eight point sources, the NHV flakes also reduced pheromone attraction [69]. The active spacing distances are in accordance with the “active inhibitory range” of NHV of at least 2 m that was estimat- ed previously [27]. The pheromone dose used by Andersson et al. [69] was comparable to that released from a mass-at- tacked tree, which is a very strong signal. Thus, it is strik- ing that volatiles from nonhost plants can inhibit attraction when they are released a few meters away from the phero- mone source. This indicates that avoiding not only nonhost species, but also nonhost habitats, likely improves bark beetle fitness. The different spacing distances that affected trap catches of the beetle and the moth may reflect differences in the size of the natural odor sources (and plumes) the insects orient to [69]. While a male moth orients towards a single calling female, bark beetles may orient to large patches of trees with hundreds of calling males. Furthermore, the moth sex pheromone communication system is highly specialized. A male moth flies towards a calling female for mating only, whereas the bark beetle aggregation pheromone can be used as a signal of mates, food, and oviposition sites. Thus, the different selection pressures that operate on these systems have likely resulted in different degrees of specialization. The ORNs for pheromone compounds in moths are housed in specific sensilla (trichodea), distinct from the ones that Psyche 11 detect plant odors [43]. In contrast, L typographus groups the ds-verbenol pheromone ORN together in the same sensilla as the ORN for the plant compound 1,8-cineole, al- though the ORNs themselves are specific in their response [ 8 ]. Similar to 1. typographus, studies on Dendroctonus bark beetles indicated synergistic interactions between phero- mone components when two baits were separated by several meters [71, 72]. The sharp response of S. littoralis to odor source spacing has been observed previously in other moths [73-75]. The most extreme example is provided by Fadamiro et al. [52], who found that 1 mm separation be- tween pheromone and antagonist was sufficient to restore upwind flight to the pheromone by male Helicoverpa zea. It was hypothesized that coincident detection of pheromone components and antagonists, achieved by colocalization of the ORNs, was the reason for this amazing ability of the males. Furthermore, synchronous detection of pheromone compounds was shown to improve the temporal spiking pat- tern by projection neurons in the antennal lobe of Manduca sexta moths [76]. Thus, it is clear that coincidence detection is of great importance in the pheromone system of moths. Soap bubble generators were used to visualize plume overlap at the different spacing distances used for L typographus (Figure 7(d)) [69]. The simulations indicated that filaments from different plumes are more likely to overlap and, thus, to be detected coincidently, when the sources are close to each other. Therefore, the lower sensitivity of I. typographus to small-scale spatial separation of odor sources might indicate that coincidence detection is of less importance for bark beetles than for moths [69]. 10. Applications Conifer pest insect infestations are typically less common in diversified habitats [67], which in part may be due to the presence of antiattractive NHV. The finding that NHV, from a distance of at least 2 m (see also [27]), can reduce attraction to a pheromone dose comparable to that released from a mass -attacked tree suggests a potential for NHV in forest protection. However, pheromone attraction was not completely shut down so it is more likely that, instead of counteracting ongoing mass attacks, synthetic or natural NHV sources may reduce the risk of spruces being attacked in the first place. Indeed, spruces were previously protected by NHV dispensers attached to every second tree, demonstrat- ing a protective effect of ca 2 m [77] . In another study, groups of ten trees were all protected by 20 NHV dispensers, and bark beetle attacks were diverted to trees >15 m away [78] . In addition, the spruce compound 1,8-cineole that strongly reduced pheromone attraction should be further tested in combination with the other active semiochemicals for possible improvement of antiattractant blends. It is pos- sible that the repression of the ORN for ds-verbenol by 1,8- cineole, adds another inhibitory mechanism by distorting the “perceived blend ratio” of the aggregation pheromone. If so, it is likely that a more effective antiattractant blend can be obtained than the one that is comprised of GLV alcohols, C8- alcohols, traus-conophthorin, and verbenone [27]. 11. Conclusions and Future Directions The recent advances in bark beetle olfactory physiology have provided a connection between the physiological and behavioral responses of 1. typographus to ecologically re- levant compounds. This connection has allowed for a deep- er understanding about how bark beetles (and possibly in- sects in general) may encode, and respond to, the odor envi- ronment. However, there are still several ORNs of I. typogra- phus (and other species) for which odor ligands have not been identified, meaning that there is yet more to be learned about its olfactory physiology. Identification of active com- pounds should be achieved by GC-coupled SSR and by testing headspace collections from, for instance, attacked and unattacked or resistant host trees. At the molecular level, Andersson and collaborators [61, 79] recently sequenced the antennal transcrip tome of 1. typographus, leading to identification of gene sequences for 40 different candidate olfactory receptors (ORs). The amino acid sequences of the receptors were compared, in a sequ- ence similarity tree, with receptors that were previously iden- tified from the genome of the flour beetle, Tribolium castaneum. Many of the Ips ORs formed a bark beetle-spe- cific branch, indicating an extension of OR function. Possibly, these receptors detect conifer- related volatiles or pheromones that are especially relevant for bark beetles. The other ORs of Ips were grouped together with ORs of T. cas- taneum, which may indicate conserved functionality of some sets of ORs within Coleoptera. Functional studies to reveal which compounds the ORs of Ips bind will be the next step in the study. Such studies will hopefully extend the connec- tion from behavior, through physiology, all the way to the level of the receptor and gene. The identification of the bark beetle ORs paves the way for the development of potential novel management strategies in the future. If the receptors for pheromone com- ponents and antiattractive NHV can be identified, it might be possible to identify ligands that pharmacologically block the pheromone receptors or hyperstimulate [80] receptors for nonhost volatiles. If such compounds are found, they might be dispensed in the forest to disrupt bark beetle pheromone communication and host tree localization. One hypothesis why insect colocalize specific ORNs in the same sensilla is that it allows for improved spatiotem- poral resolution of odor stimuli [52]. This hypothesis could be tested by comparing trap catches of I. typographus in response to spacing between pheromone and 1,8-cineole (ORNs for ds-verbenol and 1,8-cineole co-localized), with trap catches in response to spacing between pheromone and verbenone, the latter compound being detected by an ORN that is never colocalized with an aggregation pheromone neuron. Predictably, the beetle should be more “sensitive” to small-scale spacing between pheromone and 1,8-cineole than to spacing between pheromone and verbenone. In order to put the sensory physiology into a more natural context, a portable single sensillum recording device [81] should be used in the field. 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Dunphy Copyright © 2012 Samira Farahani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Effect of five soybean cultivars (Sahar, JK, BP, Williams, and El 7) on life table parameters of the beet armyworm Spodoptera exigua (Hiibner) (Lepidoptera: Noctuidae) was evaluated at 25 ± 1°C, 60 ± 5% relative humidity and a photoperiod of 16 h light/8 h dark. The highest and lowest net fecundity rates were obtained on Williams (244. 1 ± 19.4 eggs/female) and L17 (80.5 ± 6.9 eggs/female), respectively. The net reproductive rates (Rq) were highest on Williams (137.0 ± 11.2 females/ female/generation) and lowest on BP (41.3 ±4.1 females/ female/generation). The intrinsic rate of increase (r^) differed significantly among hve soybean cultivars, ranging from 0.1169 day“^ (on Williams) to 0.122 day“^ (on BP). The highest value of finite rate of increase (A) was on Williams (1.18 day“^), which was significantly different from other cultivars. The mean generation time {Tf) was significantly different on various cultivars, ranging from 32.0 ± 0.3 days (on L17) to 28.8 ± 0.3 days (on Sahar). The results are discussed with respect to the potential effect of soybean cultivars on the performance of S. exigua. 1. Introduction Soybean {Glycine max (L.) Merrill) is an economically im- portant crop and commercially produced in the north- ern region of Iran [1]. The beet armyworm, Spodoptera exigua (Hiibner) (Lepidoptera : Noctuidae) is native to Asia but has been introduced worldwide and is now found almost anywhere its many host crops are grown. It is an important pest of soybean in Iran [2] and some parts of the world [3, 4] . It is one of the most common and destructive insect pests of more than 90 plant species in at least 18 families around the world [3, 5, 6]. The wide host range of the beet armyworm includes soybean, sugar beet, cabbage, cauliflower, brussel sprouts, tomato, maize, cotton, lettuce, peanut, alfalfa, shal- lot, pastures crops, and various wild hosts [3]. The intensive use of insecticides for control of this pest has resulted in high levels of resistance to virtually all commercial insecticides in many parts of the world [4, 7]. Host plant resistance is one way of controlling insects that is not detrimental to the environment and also reduces expenses for growers [8]. Identification of host plant resistance mechanisms can enable proper selection of resistant genotypes that can be used in plants breeding programs [9]. In recent years, the beet armyworm has become a serious pest on soybean in some parts of Iran. Therefore, the objective of this research was to examine the effect of different soybean cultivars on the life table of S. exigua, which is useful for its management and potential soybean breeding for resistance. Life table parameters are important in the measurement of population growth capacity of species under specified conditions. These parameters are also used as indices of pop- ulation growth rates responding to selected conditions and as bioclimatic indices in assessing the potential of a pest population growth in a new area [10]. Fertility life tables are appropriate to study the dynamics of animal populations, especially arthropods, as an intermediate process for estimat- ing parameters related to the population growth potential, also called demographic parameters [11]. This information could be extremely valuable for the future development of IPM programs against S. exigua. Life table studies have several applications including analyzing population stability and structure, estimating extinction probabilities, predicting 2 Psyche life history evolution, predicting outbreak in pest species, and examining the dynamics of colonizing or invading species [12, 13]. Life table information may also be useful in constructing population models [14, 15] and understanding interactions with other insect pests and natural enemies [ 16] . The intrinsic rate of increase (r^) is a key demographic parameter useful for predicting the population growth potential of an animal under a given environmental condi- tion [10, 17, 18]. Under non limiting environmental condi- tions and with a stable age distribution, this parameter will be characterized by a constant population size. The -value can be estimated from life table data under standardized laboratory condition. The -value is the rate of growth of a population when that population is growing under ideal conditions and without limits [10]. A number of extrinsic and intrinsic factors have been shown to affect the -value and related demographic parameters. These include tem- perature, geographical origin of insect, and host plant cul- tivars [19-21]. The life history and population growth parameters of S. exigua have been studied on different host plants by Greenberg et al. [22], who reported that the performance of S. exigua was best on pigweed, worst on cabbage, and intermediate on cotton, pepper, and sunflower. Realized and potential fecundity and egg fertility of S. exigua under different adult diet regimes have been studied by Tisdale and Sappington [7]. Several studies have dealt with beet armyworm fecundity and growth potential, and some of the environmental factors that may influence them [23-28]. However, there is no information related to performance of this pest on different soybean cultivars. The current study is designed to evaluate the effectiveness of five soybean cultivars on the demographic parameters of the S. exigua. These five cultivars are covering the most area of soybean growing in Iran and some other parts of the world [29]. Knowledge of life table parameters of S. exigua and resistance susceptibility of different soybean cultivar may be necessary to select the most suitable cultivar or employ the appropriate control tactics and integrated crop management (ICM) for particular soybean cultivars. 2. Materials and Methods 2.1. Rearing Methods and Experimental Conditions. Seeds of the five soybean cultivars including Sahar, JK, BP, Williams, and LI 7 were obtained from the Plant and Seed Modification Research Institute (Karaj, Iran) and were sown in the research field of Tarbiat Modares University in suburbs of Tehran, Iran in May 2008. For this study, the leaves of different soybean cultivars were transferred to a growth chamber at 25 ± 1°C, 60 ± 5% RH and a photoperiod of 16:8 (L : D) hours and used for feeding of different larval stages of S. exigua. Larvae and pupae of S. exigua were originally collected from sugar beet fields in Ghafar Behi village in West Azerbai- jan, Iran (45°09'35"E and 37°31'49"N). The cultures were reared on each soybean cultivar for one generation in a growth chamber at the same conditions as above before using them in the experiments. The colony was supplemented. from time to time, with larvae collected from field to prevent any inbreeding effects. 2.2. Survival Rate and Mortality. The newly emerged adults were transferred to cages (14 cm diameter, 19 cm height) for mating. This cage was a clear and cubic Plexiglas container. A piece of wax paper was inserted in the container on which the females lay their eggs. After 12 h, the laid eggs were collected from the container and were used in the experiments. In total, 219, 200, 225, 176, and 230 eggs of S. exigua were used to collect data on Sahar, IK, BP, Williams, and LI 7 soybean cultivars, respectively. In order to calculate age- specific survival rate and daily fecundity of S. exigua, each egg was placed individually into plastic Petri dishes (8.5 cm diameter and 3 cm height) containing fresh leaves of different examined soybean cultivars. A hole (3 cm diameter) was cut at the centre of top of Petri dishes and covered by fine nylon mesh for ventilation. Larval instars were determined by the presence of newly molted exuviae and head capsules. Developmental stages were checked daily and developmental periods and mortality of eggs, larvae, pupae, and adults were recorded. The experiment was continued until the death of all individual members of each cohort. 2.3. Reproduction and Population Growth Parameters. In order to calculate reproduction and population parameters of S. exigua, a pair of newly emerged female and male adult moths (with 25 replications on each soybean cultivar) were introduced into each clear plastic container ( 1 1 cm diameter, 12 cm height), which was closed at the top with fine nylon mesh for ventilation. A small cotton wick soaked in 10% honey solution was placed in the oviposition containers to provide a source of carbohydrate for adult feeding. The number of eggs laid by each female was recorded daily until the last female died. To obtain sex ratio on each host plant cultivars, deposited eggs produced on each soybean cultivar were maintained until adult moths emerged, and sex ratio of emerged adults was determined. 2.4. Data Analysis. Life span differences among cohorts on various soybean cultivars were analyzed by Kolmogorov- Smirnov test [30] . The reproduction parameters for S. exigua including gross fecundity rate and gross fertility rate (the total number of eggs and hatching eggs by an average female that lives to the last day of possible life in the cohort, resp.), net fecundity rate and net fertility rate (the average lifetime production of eggs and fertile eggs for a newborn female, resp.), gross hatch rate (the ratio of gross fertility to gross fecundity), daily eggs laid per female, and daily fertile eggs laid per female on different cultivars were estimated using formulae suggested by Garey [14, 15]. The age-specific survival rate, daily fecundity, and sex ratio were used to construct lx life tables from which the following population growth parameters were calculated using formula suggested by Garey [14, 15]: intrinsic rate of increase (r^), mean generation time (T^), finite rate of increase (A), net reproduction rate (Ro), and doubling time (DT). Age-specific survival rates (lx) and average number of Psyche 3 female eggs per day (m^) for each age interval (x) were used to construct age-specific fertility life tables. For example, the intrinsic rate of increase (r^) of S. exigua on different cultivars was estimated using the following: = 1 . ( 1 ) x=a The Jackknife procedure was used to estimate the variance for Tm and the other population parameters [31]. The Jackknife method removes one observation from the original data set and recalculates the statistic of interest from the truncated data set. These new estimates, or pseudovalues, form a set of numbers from which mean values and variances can be calculated and compared statistically [11]. The steps for the application of the method are the following. The Vm value was estimated by considering the survival and reproduction data for all the n females, referred to as true calculation (rm(aii)). Then this procedure was repeated for n times, each time excluding a different female. In so doing, in each time, data of n - I females were taken to estimate parameters In the following step, pseudo values of r^(psv rm[i)) were calculated for the n samples using the following equation: psvr^(;) = nx rm(aii) - {n - 1) x rmay (2) After calculating ail the n pseudo -values for r^. Jackknife estimate of the mean variance, and standard error is calculated, respectively, by the following: ^m{J) Z[LiPsvrm(f) n var xti (psvr^(/) n - 1 (3) BP, Williams, LI 7, Sahar, and JK, respectively. The results indicated that the death of the last female (maximum age) occurred in the age of 39.2, 42.9, 43.3, 38.3, and 40.5 days for males and in the age of 45.1, 46.2, 46.3, 39.8, and 41.0 days for females on BP, Williams, LI 7, Sahar, and JK, respectively. The mated females began oviposition 5-6 days after emergence. The females began to lay eggs in the age of 30.5, 28.5, 32.5, 30.5, and 30.5 days on BP, Williams, L17, Sahar, and JK cultivars, respectively. The highest daily fecundity (nix) of S. exigua adults emerged from larvae reared on these cultivars was 37.5, 56.8, 44.3, 62.1, and 33.3 eggs/female/day occurred in the age of 33.5, 31.5, 40.5, 32.5, and 34.5 days, respectively (Figure 1). The lifespan of females of S. exigua was significantly longer on Williams and JK in comparison to other cultivars (P < 0.05). However, no significant difference was observed between lifespan of males on soybean cultivars (Table 1). 3.2. Reproduction Parameters. The results of the reproduc- tion parameters of S. exigua are given in Table 2. The gross hatch rate of S. exigua ranged from 26% on LI 7 to 37.5% on Williams. The gross and net fecundity rates significantly differed on various soybean cultivars (P = 4.55; df = 4,108; P < 0.01 and F = 28.40; df = 4,105; P < 0.01). The highest rates of gross and net fecundity were obtained on Williams. However, there was no significant difference between gross and net fecundity rates on other soybean cultivars. Among different soybean cultivars, the gross fertility rate was the highest on Williams. The net fertility rate varied from 21.0 to 102.5 eggs, which was significantly higher on Williams compared to the other cultivars. Both daily number of eggs and daily number of fertile eggs laid per female were significantly higher on Sahar than LI 7, Jk, and BP, but there was no significant difference between Sahar and Williams cultivars (Table 2). The mean values of n jackknife pseudo values for each soy- bean cultivar were subjected to one-way ANOVA. The similar procedures were used for the other param- eters {Ro-, A, X and DT). Effect of host plant cultivars on different parameters was analyzed using one-way ANOVA. If significant differences were detected, multiple comparisons were made using the Student-Newman-Keuls (SNK) (P < 0.01). Statistical analysis was carried out using Minitab ver.13.1 software [32]. 3. Results and Discussion 3.1. Survival Rate and Fecundity. Age-specific survival rate (X) and fecundity (m^) of S. exigua on different soybean cultivars are shown in Figure 1. Survivorship curves of S. exigua on all soybean cultivars were type III. In this type of survivorship, most of the mortality occurs early in the life(e.g., egg stage) [33]. The survival rate to the first day of adult emergence was 19.6, 37.5, 16.5, 21.9, and 24.5% on 3.3. Population Growth Parameters. The population growth parameters of S. exigua on five soybean cultivars are given in Table 3. There were significant differences between the net reproductive rates (Ro) on five soybean cultivars (P = 34.412; df = 4,103; P<0.01). The highest value of Rq was on Williams. However, no significant difference was observed between Pq - values on other soybean cultivars. The intrinsic rates of increase (r^) were also found to be significant on different cultivars (P = 8.991; df = 4,103; P < 0.01). The re- value ranged from 0.1169 to 0.1680, which was the highest on Williams. The highest value of finite rate of increase (A) was obtained on Williams, which was significantly different from other cultivars (P = 23.342; df = 4,103; P<0.01). The doubling time (DT) was not found to be significantly different on various soybean cultivars (P = 1.296; df = 4,103; P > 0.05) (Table 3). But, the mean generation time (Pc) was significantly different on host plant cultivars (P = 10.443; df = 4,103; P < 0.01), which showed longer generation time on LI 7 compared to the other cultivars (Table 3). The reproduction and population growth parameters of S. exigua vary considerably depending on various factors such as host plants and environmental conditions [7, 22]. 4 Psyche Age (days) — mx (e) Figure 1: Age-specific survivorship ilx) and age-specific fecundity (m^) of beet armyworm Spodoptera exima on various soybean cultivars, (a) JK, (b) Sahar, (c) LI 7, (d) Williams, and (e) BP. The present study demonstrated that the performance of S. exigua significantly differed on the five soybean cultivars tested. In regard to insect-plant interactions, it is useful to determine the effect of the different cultivars on the performance of herbivores. Understanding the life table parameters of S. Exigua is one of the essential components in developing an integrated pest management program for soybean in Iran. No other study has covered the effect of various soybean cultivars on survival and reproduction of the S. exigua in Iran. Differences in the number of eggs per female (gross fecundity rate) among soybean cultivars were found to be significant. Sethi et al. [28] reported that fecundity of S. exigua for Valmaine and Tall Guzmaine cultivars of lettuce was 123.2 and 383.6 eggs, respectively, which was lower than those obtained in the current study. The difference between Psyche 5 Table 1: Comparison of lifespan of Spodoptera exigua on five soybean cultivars using the Kolmogorov-Smirnov test. Soybean cultivars Female (mean ± SE) Lifespan (Days) range Male (mean ± SE) range JK 9.93 ± 1.08^^ 3-56 9.46± l.OD 3-51 Sahar 7.70 ± 0.84^ 3-45 7.86 ±0.83" 3-43 L17 7.64 ± 0.88^ 3-56 7.20 ±0.81" 3-50 Williams 14.29 ± 1.58^ 3-59 11.377±1.4" 3-64 BP 8.54 ± 0.97^ 3-60 8.06±0.86" 3-56 Means followed by the same letters in a column are not significantly different (P < 0.05). Table 2: The reproduction parameters (Mean ± SEM) of the beet armyworm Spodoptera exigua on five soybean cultivars. Parameters JK Sahar Soybean cultivars L17 Williams BP Gross fecundity rate 549.0 ± 79.8^ 558.3 ±57.9’^ 555 ± 771.3 ±49.2" 462.9 ± 34.8’^ Gross fertility rate 203.6 ± 29.6^^ 202.1 ±32.5’^ 144.5 ± 11.6'^ 292.4 ± 20.7" 134.2 ± lO.H Net fecundity rate 114.9 ± 12.5’^ 96.1 ± 12.5’^ 80.5 ± 6.9^^ 244.1 ± 19.4" 84.0 ± 8.51^ Net fertility rate 42.6 ± 4.7'^ 29.7 ± 17.0^" 21.0 ± 1.8" 102.5 ±8.2" 24.4 ± 2.5" Mean egg per day 20.3 ± 2.9’^" 31.0 ±3.2" 21.4 ± 1.7'^" 24.9 ± 1.6"'^ 14.9 ± 1.1" Mean fertile egg per day 7.5± l.W 11.2 ± 1.8" 5.6 ±0.5" 9.4 ± 0.7"'^ 4.3 ± 0.3" Means followed by the same letters in a row are not significantly different (P < 0.01). Table 3: The population growth parameters (Mean ± SEM) of the beet armyworm Spodoptera exigua on five soybean cultivars. Parameters JK Sahar Soybean cultivars L17 Williams BP Net reproductive rate (Ro) 59.0 ± 6.4^^ 44.4 ± 6.2'^ 42.0 ± 3.6'^ 137.0 ± 11.2" 41.3 ±4.H Intrinsic rate of increase (r^) 0.137 ±0.004"'^ 0.132 ±0.006^^" 0.117 ±0.003" 0.168 ±0.004" 0.122 ±0.004" Finite rate of increase (A) 1.15 ±0.0051^ 1.14±0.006''" 1.12 ±0.003*^ 1.18 ±0.005" 1.13 ±0.004""* Doubling time (DT) 5.0 ± 0.2" 5.2 ±0.2" 5.9 ±0.1" 4.1 ±0.1" 5.7 ±0.2" Mean generation time ( T) 29.7 ± 0.5’^" 28.8 ±0.3" 32.0 ± 0.3" 29.3 ± 0.3'^" 30.4 ± 0.4*^ Means followed by the same letters in a row are not significantly different (P < 0.01). our study and results of Sethi et al. [28] could be resulted of host plant differences. The higher intrinsic rate of increase (r^) on Williams were resulted from faster development (shorter generation time), higher survivorship, and higher fecundity rates. High value of Tfn indicates the susceptibility of a host plant to insect feeding, while a low value indicates that the host plant species is somewhat resistant or tolerant to the pest. Therefore, our data showed the tremendous growth capacity of S. exigua under favorable conditions. Greenberg et al. [22] investigated the life table parameter of this pest on different host plants and observed that the Vm value was the highest on pigweed (0.264) and lowest on cabbage (0.156). Some possible reasons for disagreement are due to physiological differences depending on the type of the host plant cultivar, genetic differences as a result of laboratory rearing or varia- tion in geographic populations of the pest. Furthermore, our findings led us to consider soybean as a poorer-quality host for S. exigua than those tested by Greenberg et al. [22] . Our finding indicated that the highest value of net reproductive rate of S. exigua was obtained on Williams. Greenberg et al. [22] reported that net reproductive rate of the S. exigua ranged from 139.3 to 596.0 on cabbage and pigweed, respectively. Therefore, according to our results, the net reproductive rate of S. exigua on Williams was nearly similar to cabbage. The longest generation time(Tc) was obtained on L17 (32.0 ± 0.3 days), and the shortest was on Sahar (28.8 ±0.3 days). According to the results of Greenberg et al. [22], the mean generation time of the S. exigua was longest on cabbage (31.6 days) and shortest on pigweed (24.2 days) on cabbage which was nearly similar to our estimated generation time on LI 7 soybean cultivar. Naseri et al. [34] investigated the reproductive performance of Helicoverpa armigera (Hiibner) (Lepidoptera : Noctuidae) 6 Psyche reared on thirteen soybean varieties and observed that Williams variety was more suitable, and other varieties (BP, Sahar, JK, DPX, and GorganS) showed less suitability as host plants for H. armigera reproduction which was nearly similar to our results. There are many factors affecting host suitability, includ- ing nutrient content and secondary substances of the host and the capability of digestion and assimilation by an insect [8, 9, 22, 28], For a better understanding of the insect-plant interaction, basic biochemical studies for the isolation and identification of phytochemicals, which adversly affect the buildup of S. exigua population on soybean, are required. Through this research, we may be able to determine the population dynamics of the pest on different host cultivars and use the information to manage the pest population below the economic injury level. In conclusion, knowledge of the influences of soybean cultivar quality on the life table parameters of the S. exigua can help us to understand the population dynamics and management of this insect. The results of the comparison of reproduction and population growths parameters of S. exigua on five soybean cultivars revealed that Williams was the most suitable and LI 7 and BP were the partially resistant cultivars. After laboratory studies, more attention should be devoted to semifiled and field experiments to obtain more applicable results in field conditions. Acknowledgments This study was partly supported by the Department of Ento- mology, Tarbiat Modares University and a grant from the Center of Excellence for Integrated Pests and Diseases Man- agement of Oil Crops of Iran (Tarbiat Modares University, Tehran), which is greatly appreciated. The authors also cor- dially thank the editor. Professor Gary B. 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Shurtleff and A. Aoyagi, “Histoty of soybeans and soyfoods in the Middle East (1909-2007): extensive annotated bibliog- raphy and sourcebook,” Soyinfo Center, Lafayette, La, USA, 2008. [30] D. A. Pyke and J. N. Thompson, “Statistical analysis of survival and removal rate experiments,” Ecology, vol. 67, no. 1, pp. 240- 245, 1986. [31] J. S. Meyer, C. G. Ingersoll, L. L. McDonald, and M. S. Boyce, “Estimating uncertainty in population growth rates: jackknife vs. bootstrap techniques,” Ecology, vol. 67, no. 5, pp. 1156- 1166, 1986. [32] M. A. Murphy and A. Salvador, “International subcommission on stratigraphic classification of lUGS international commis- sion on stratigraphy. International stratigraphic guide — an abridged version,” GeoArabia, vol. 5, no. 2, pp. 231-266, 2000. [33] C. J. Krebs, Ecology: The Experimental Analysis of Distribution and Abundance, Benjamin Cummings, San Erancisco, Calif, USA, 2001. [34] B. Naseri, Y. Eathipour, S. Moharramipour, and V. Hosseini- naveh, “Comparative reproductive performance of Helicov- erpa armigera (Hiibner) (Lepidoptera: Noctuidae) reared on thirteen soybean varieties,” Journal of Agricultural Science and Technology, vol. 13, no. 1, pp. 17-26, 2011. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 484765, 7 pages dohlO.l 155/2012/484765 Review Article Cleptobiosis in Social Insects Michael D. Breed, Chelsea Cook, and Michelle O. Krasnec Department of Ecology and Evolutionary Biology, The University of Colorado, Boulder, CO 80309-0334, USA Correspondence should be addressed to Michael D. Breed, michael.breed@colorado.edu Received 12 October 2011; Accepted 18 December 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 Michael D. Breed etal. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In this review of cleptobiosis, we not only focus on social insects, but also consider broader issues and concepts relating to the theft of food among animals. Cleptobiosis occurs when members of a species steal food, or sometimes nesting materials or other items of value, either from members of the same or a different species. This simple definition is not universally used, and there is some terminological confusion among cleptobiosis, cleptoparasitism, brood parasitism, and inquilinism. We first discuss the definitions of these terms and the confusion that arises from varying usage of the words. We consider that cleptobiosis usually is derived evolutionarily from established foraging behaviors. Cleptobionts can succeed by deception or by force, and we review the literature on cleptobiosis by deception or force in social insects. We focus on the best known examples of cleptobiosis, the ectatommine ant Ectatomma ruidum, the harvester ant Messor capitatus, and the stingless bee Lestrimellita limdo. Cleptobiosis is facilitated either by deception or physical force, and we discuss both mechanisms. Part of this discussion is an analysis of the ecological implications (competition by interference) and the evolutionary effects of cleptobiosis. We conclude with a comment on how cleptobiosis can increase the risk of disease or parasite spread among colonies of social insects. 1. Introduction For animals, foraging is often time consuming, risky and may not result in the discovery of food or other resources. A commonly observed alternative to searching for resources that may be dispersed in the environment is to take, either by force or deception, the resource from another animal. This theft of food or other resources from foragers or from larder caches is common enough to merit considerable interest in behavioral ecology. Many social insects act as thieves, and the focus of this paper is food theft that in some way involves a social insect species, but we also consider theft of other items, such as nesting material. We also consider how theft among social insects fits into the larger evolutionary picture of resource theft in animals. Social insects can be either the victim or the villain in food thefts. Some social insects frequently victimize other social insects, and these thieves may be members of the same species or of a different species. Social insects can also be victimized by commensals that are able to plug into social feeding mechanisms that normally direct food among colony members. From this point forward, we use the term cleptobiosis to describe food theft, or theft of other items of value such as nesting material, by one animal from another. The evolutionary roots of cleptobiosis are simple; finding food and subduing prey items is key to animal survival, and cleptobiosis provides, for some species, an alternative to foraging costs in terms of time, energy, and exposure to the possible risk of the forager becoming, itself, prey. Most cleptobionts facultatively engage in theft, when profitable opportunities appear, but obligate cleptobiosis has evolved in at least one genus of stingless bee and in inquilines residing within social insect colonies. While the subject of this paper is cleptobiotic relationships involving social insects, the evolutionary principles apply broadly across many types of animals. 2. What Is Cleptobiosis? This is an easy question with an unfortunately complicated answer. At the outset we gave a simple definition; in more formal terms we consider cleptobiosis to be “an ecological 2 Psyche relationship in which members of one species, as of ants, steal food from another” (http://universalium.academic.ru/ 93503/cleptobiosis), but we would add that the stolen items may also include nesting materials or other items of value. The term can also be spelled kleptobiosis, and, in studies of animals outside the social insects, kleptoparasitism (clep- toparasitism) is often used synonymously with cleptobiosis. However, serious confusion can arise from the different ways in which these terms have been used in the literature on insects and the literature on vertebrates, and in the remainder of this section we discuss how this terminology has been applied by investigators studying different types of organisms. Iyengar [1] presents a general review of cleptobiosis in animals. In insect studies, cleptobiosis refers to food theft (or other valuable materials), and cleptoparasitism refers to brood parasitism (Table 1). Alternatively, the term cuckoo or cuckooism is used to describe brood parasites among birds and mammals, as well as bees. Cuckoo bees are brood parasites (strictly speaking, this is family Apidae, subfamily Nomadinae, but also refers to other bees with similar lifestyles) and are often termed cleptoparasites. These bee species lay their eggs in the nests of other bee species, rather than stealing food. Their young are then reared by adults of the host species [2]. Brood parasitism of this sort is also characteristic of birds such as cuckoos (family Cuculidae) [3] and cowbirds (Icteridae). Unfortunately, the term cleptoparasitism (or kleptoparasitism) is also frequently used to describe food theft in bird and mammals [4] . Adding to the terminological confusion is the occasional use of lestobiosis to refer to more furtive (as opposed to forcible) forms of cleptobiosis. We view cleptobiosis as theft of food or other valuable items and prefer other terms to describe brood parasitism. In some cases, as in gulls stealing from one another on a beach, the theft is direct interference with immediate consumption. In other cases, such as honeybees robbing honey from a hive, the target is stored food in a nest or cache. To avoid confusion, species that lay eggs in the nests of others (either conspecific or heterospecific) are probably better termed cuckoos or brood parasites, rather than cleptoparasites. This paper focuses specifically on cleptobiosis, defined as food thievery, in social insects. Adding to the confusion, Crespi and Abbot [10] call thrips that steal galls induced by other thrip species clep- toparasites. Nest usurpation is also known in honeybees and in a broader context is similar to territorial contests in birds and mammals, in which an individual may lose a nesting site. We do not include nest usurpation in our review. Theft of brood for the purpose of employing the stolen individual’s efforts in support of the thief is dulosis (in the older literature this is called slave-making, but the preferred terms is now dulosis). Dulosis is common in ants but is only incidentally observed in other types of animals. Inquilinism is social parasitism in which a reproductive enters a host colony, lays eggs, and relies on the host colony to rear its offspring. Unlike brood parasitism, the inquiline remains within the nest and typically its brood does not outnumber the host’s brood. Inquilines are typically evolutionarily close to their hosts, as with ants living within the colonies of another ant species. Inquilines may, of course, take food from the host colony, potentially making this behavior a kind of cleptobiosis. Consumption of brood, as by corvids (family Corvidae: ravens, crows, magpies, and jays), which target eggs and nestlings of smaller birds [16], is predation rather than cleptobiosis. Before moving on to our consideration of cleptobiosis, one other type of interspecific interaction that should be discussed is “guests” in social insect colonies. This type of symbiosis is related in some ways to cleptobiosis. Ant and termite colonies, in particular, often have guests, which are termed myrmecophiles and termitophiles, respectively. Guests living with ants include other ant species, beetles, flies, and Collembola. Larvae of lycaenid butterfly species display a variety of mechanisms — chemical, morphological, and behavioral — to induce care from ants [17, 18]. Like furtive cleptobionts, many of these symbionts “gain the keys to the kingdom” by mimicking the chemical recognition signatures of the host colony, a mechanism that we discuss in more detail below. 3. Nonsocial Insect Cleptobionts Social insects are not the only members of cleptobiotic associations. Many animal species use cleptobiosis to exploit social information. This makes their search for food more efficient [19, 20]. By observing other animals’ foraging activities, an animal can take advantage of information that took another individual’s time and energy to discover. Birds in a flock may assemble, for example, to obtain social information about food [21]. In some cases such information theft leads to direct stealing of food. Heron gulls voraciously attempt to steal each other’s food discoveries [7]. Spiders steal food from each other in a similar manner [22, 23]. Interspecific theft of food items is common among carnivorous mammals, with thefts by hyenas in which wild dogs are victimized having been particularly well studied [24], although no mammalian or avian species makes their living solely by stealing food items from other species. Taking this logic a step further, targeting cached food can be a highly efficient strategy [25]. Scatter caching is a form of food storage in which single food items are placed in caches within an animal’s home range. Sometimes, as in jays or squirrels, this can result in a high number of caches of food to be retrieved in a later season. Scatter caching is also subject to pilferage, but scatter caching animals typically do not defend individual caches. Ravens, for example, seek out each other’s food caches [25]. Raven defenses against cache pilferage largely rely on clever storage, rather than aggressive defense. However, when a mountain lion caches a deer carcass, it may then defend its food. In scatter caching, cache locations are associated with where prey was killed, rather than a central nest or den. Among mammals and birds, caching seems responsive to immediate evolutionary pressures. Tree squirrels exemplify evolutionary flexibility in caching strategies, as some species are scatter cachers (e.g., the Eastern fox squirrel, Sciurus Psyche 3 Table 1: Terminology for food theft, brood theft, brood parasitism, and related phenomena. The term “cleptoparasite” has been used with such diverse meanings that it is probably best dropped from the lexicon for this area. Term Function Example Cleptobiosis Theft of food or another item of value from another animal Gulls [5], honeybees [6], Ectatomma ants [7, 8], bowerbirds (mating display materials) [9] Lestobiosis Cleptobiosis by furtive or deceptive means Ectatomma ants [7, 8] Nest usurpation Theft of a nest structure, perhaps including brood or food cached within the nest Honeybees [6], thrips [10] Brood parasitism Laying eggs in the nest of another animal, to be reared by that animal, functionally, this is theft of brood rearing Guckoos (birds) [3], cuckoo bees [2] Dulosis Theft of brood to rear as workers “Slave-making” ants [11] Inquilinism Living within a social group as a social parasite, a conspecific or heterospecific reproductive that exploits the host colony by laying brood that are cared for by the host colony Psithyrus bees in bumblebee (Bombus) colonies, Dolichovespula arctica and D. adulterina, (initially inquilines, often become usurpers) in other Dolichovespula colonies [12, 13], numerous ant species within other ant colonies [11] Guests, myrmecophiles, and termitophiles Live within a social insect colony, often adopting chemical recognition signature of host colony, may consume resources but do not represent a lethal drain on colony resources Many species representing many insect orders, as well as noninsects. Specific examples are lycaenid Lepidoptera in ant colonies [14, 15] and wax moths. Galleria mellonella, in honeybee colonies [6] Parasites Live within a social insect colony but represent a potentially lethal presence Varroa mites in honeybee colonies [6] Gorvids (ravens, crows, magpies) Brood predation Eating eggs or brood from within a nest consuming other bird’s eggs, army ants, many species of which target brood of other social insects [1] niger), and others are larder cachers (e.g., the pine squirrel, Tamiasciurus hudsonicus). The difference between these species may in part reflect differing pressures from cache pilferage. 4. Larder Caches and the Evolution of Cleptobiosis A larder cache is food stored in a central place for future use. Food stored in larder caches is particularly attractive to potential thieves, and many larder caching social insect species, such as harvester ants, most stingless bees, and honeybees, have evolved impressive modes of colony defense. The evolution of larder caching is probably driven by the value of having food in a central place during unfavorable seasons, but the threat of loss of the cache, to animals of the same species or different species, is clearly a countervailing evolutionary force. Larder caching occurs in some ants and social bees, and to a lesser extent in social wasps, and is particularly important in our discussion of cleptobiosis in social insects. The long-term survival of social insect nests containing stored food, sometimes spanning many years, may help to make their colonies particularly susceptible to repeated raids. For some individuals within a population, taking food from others can become the predominant mode of food collection, but robbing conspecifics is not an evolutionarily stable strategy for all individuals within a population — it is clearly a dead-end if all animals in a population are robbers and none are foraging independently. Fleterospecific food theft, on the other hand, can lead to obligate cleptobiotic specialization. An example of an obligate cleptobiont is the stingless bee, Lestrimellita limdo [14, 15]. Social insect species may be particularly well equipped to engage in cleptobiosis, as the victim and thief share social mechanisms, and thief workers can evolve for specialized foraging roles without conflict with reproduction, which is the province of the queen in the colony [26]. Larder caching social insects are also victimized by birds and mammals. Perhaps most famously the western honeybee, Apis mellifera, is well armed to protect itself against vertebrate cache thieves attempting to access stored honey. 5. From Foraging to Cleptobiosis Cleptobiosis often appears to have arisen from foraging behavior that is redirected to stored foods. Stored food presents some of the same sensory profiles as the food in its original state; honey is both sweet and imbued with floral scents even though it has been concentrated from nectar and is found in honeycombs rather than flowers. Western honeybee, A. mellifera, colonies commonly rob honey from 4 Psyche other colonies. The western honey bee is a feeding generalist and forages on both floral and nonfloral sources of sugars, such as extrafloral nectaries. The evolutionary switch from foraging on nectar collected at flowers and extrafloral nectaries to foraging on stored honey in other bees’ colonies is within the foraging flexibility of honeybees and may not have required any particular adaptations to allow bees to make this switch. Other social insects, such as the ectatommine ant, Ectatomma ruidum, have made similar shifts, but have mechanisms for evading detection by workers in the colony being robbed. In more extreme cases, cleptobiosis involves specific adaptations for thievery from colonies of other social insects. Members of the stingless bee genus, Lestrimellita, are good examples of this. Lestrimellita species no longer forage on floral sources, instead they have evolved as an obligate robber of other stingless bee species. These examples, E. ruidum and L. limdo, are discussed in more detail below. Cleptobiosis can be intraspecific or interspecific. The focus on food collected by the victim, rather than the victim itself, differentiates this behavior from predation. The evolu- tion of cleptobiosis appears to draw from foraging behaviors, but also may involve the evolution of specific mechanisms for overcoming defenses of the victimized colony. Guarding honey bees [27], for example, serve a primary function of preventing cleptobiosis. The number of guards present and the intensity of guarding behavior are responsive to the intensity of pressure from robbing bees from other colonies [28]. This is an interesting example of intraspecific coevolution, in which robbing behavior yields a fitness reward for their colony, but pressure from robbers results in the evolution of heightened defensive behavior [28]. A shift in foraging preferences is a reasonable hypothesis for the initial evolution of cleptobiosis. But how does obligate cleptobiosis evolve? The most likely evolutionary scenario seems to be first facultative cleptobiosis on other species, followed by the evolutionary loss of noncleptobiotic foraging preferences. Holldobler and Wilson [11, Chapter 12], give a detailed discussion of how predation and territoriality might lead to dulosis and inquilinism. Nearly all of our examples of species exhibiting cleptobiosis are facultative cleptobionts. For individuals of these species, the ability to be a cleptobiont adds foraging opportunities and often allows these animals to take advantage of public information concerning food availability. Obligate cleptobiosis enables the evolutionary loss of specializations, such as pollen carrying structures, and the evolution of special abilities for overcoming nest entrance guards; both of these evolutionary outcomes are seen in the stingless bee genus Eestrimellita. However, obligate cleptobionts are uncommon, suggesting that perhaps this is a narrow niche which can only be filled under fairly conscribed circumstances. 6. Army Ants and Predation on Social Insect Brood Army ants cross the line from cleptobiosis to predation, but they merit mention here because many army ant species are feeding specialists on the brood of other social insect species. Army ants subdue their prey by force, rather than by decep- tion, but their match in body size, social behavior, and their ability to recruit colony mates to food sources, makes their predatory behavior in some ways homologous to the brood capturing behavior of dulotic ants. The culminative act, predation or capture of labor, surely differs, but the ultimate outcome for the victimized colony is similar. Holldobler and Wilson [11, Chapter 12] give a more detailed discussion of the possible intertwining of evolutionary pathways between predation and dulosis. 7. How Do Cleptobionts Succeed? There are two basic strategies that cleptobionts can use to enter a target colony: deception and force. Deceptive entry usually involves furtive behavior, sometimes combined with manipulation of the chemical signals involved in nestmate recognition. We discuss these two strategies in more detail in the following two sections. 7.1. Cleptobiosis by Deception: Evading Nestmate Recog- nition and Guards. In some cases successful cleptobiosis is dependent on exploitation of social mechanisms. One common mechanism is evasion of a species’ nestmate recognition system. For a colony to defend itself against potential cleptobionts, workers in the colony must be able to discriminate nestmates from nonnestmates. The presence of a nonnestmate within the colony, or at the colony entrance, is typically detected through differences in surface chemistry between colony residents and nonresidents [6, 29- 31]. Residents defensively respond to perceived differences, biting and stinging (if a sting is present in the species) the intruders. Often the defenders are specialized guards, which are primed to respond to nonnestmates. The chemical cues used in nestmate recognition are typically hydrocarbons that are probably coopted from their original role as cuticular waterproofing [32]. In Polistes wasps and in honeybees, nestmate recognition cues are acquired from nesting materials and all individuals in a colony present similar chemical signatures [6]. The mode of cue acquisition varies among ant species, but in at least some ants the postpharyngeal gland serves as a “gestalt” organ for a unifying colonial odor [33]. Potential cleptobionts can evade a chemical nestmate recognition system either by mimicking the chemical signature of the target colony or by not presenting a chemical signature of their own (figuratively speaking, they are a blank slate). The threat of cleptobiosis and social parasitism may be driving forces in the evolution of efficient nestmate recognition systems. In E. ruidum, a neotropical ectatommine ant, cleptobiosis is common among colonies [7, 8, 34-36]. Ectatomma ruidum colonies are small, with typically fewer than 100 workers, and are abundant in many lowland dry, moist, and wet habitats. Ectatomma ruidum forages on small arthropods, seeds, and nectar. Where they occur they are abundant, with a mean distance of 1 to 2 meters between neighboring colonies. Foragers will repeatedly attempt to gain entrance into neighboring nests. These repeated attempts cooccur Psyche 5 with a reduction of concentration of cuticular hydrocarbons and with a convergence of cuticular hydrocarbons between the thief ant and the target colony. After repeated attempts, thieves can gain entry into target colonies without resistance and position themselves to receive food from foragers returning to the colony. They then depart and carry the food to their own colony. Ironically, colonies of equal strength, in terms of the number of workers, tend to equally rob each other, with no net effect on food flow into the colonies. Small colonies are at a distinct disadvantage and tend to lose considerably more food than they gain [37]. This is a clear example of cleptobiosis by deception. 7.2. Cleptobiosis by Force. The stingless bee, L. limao, is an obligate cleptoparasite of other stingless bees, and sometimes has been observed robbing honeybee colonies [38]. The highly sclerotized workers of L. limao [14] have a strong lemon odor due to the presence of citral. A common victim of L. limao is Tetragonisca angustula, a very common bee in the neotropics. One kind of guard in T. angustula hovers around the nest entrance [39-41]; hundreds of these hovering guards are present at any given time, making the presence of a nest of this species easily observable. When a L. limao worker enters the defensive perimeter of a T angustula colony, the guards are alerted by the flight pattern, color, and odor of the intruder. Tetragonisca angustula guards cannot outfight the larger, more heavily armored L. limao workers, but can successfully disable L. limao by biting onto a leg or wing, once the hold on the intruder is secured, the L. limao worker ability to fly is compromised by the weight and imbalance created by the attached T. angustula worker [42]. If a T angustula colony is effective against the first arriving L limao, then the attack is not serious. IfL. limao workers evade detection, then they recruit massive numbers of additional attackers, and the T angustula colony will be overrun by cleptobionts. Lestrimellita may steal plant resins used in nest construction, as well as food [43]. In the western honey bee, A. mellifera, rich food resources, in the forms of pollen and honey, are stored within the nest. This food supports survival of the colony through cold temperate winters and provides reserves that allow rapid colony buildup in the spring and investment in reproduction (swarms and drones) early in the growing season. The presence of massive nutritional reserves provides a tempting target, and honey bee colonies can target each other for theft of food, particularly honey. This behavior fits well within our definition of cleptobiosis. Guard honeybees use chemical cues to discriminate nestmates from nonnestmates and act to exclude robbers from the nest [27, 44]. Weaker honeybee colonies are more susceptible to being robbed, and if guards are unable to exclude the first few robbers, massive recruitment of additional robbers may result in the targeted colony being overwhelmed [28] . Ectatomma ants, which were described above as deceptive cleptobionts, forcibly gain food from the ant Pheidole radoszkowskii [8] . Ectatomma ants have also been observed as victims of interspecific cleptobiosis. Ectatomma tuberculatum workers collect nectar and carry droplets externally, between their mandibles, making them a potential target for clep- tobiosis. Richard et al. [45] observed Crematogaster limata parabiotica workers robbing nectar from E. tuberculatum workers. Crematogaster workers were able to enter E. tuber- culatum nests, but food theft was targeted at E. tuberculatum workers that were returning to their nest. Espadaler et al. [46] observed ant, Messor barbarus and Aphaenogaster senilis, workers robbing Euphorbiaceae seeds from workers of other species, particularly Tapinoma erraticum (referred to as T nigerrimum). Ants in the genus Messor are seed harvesters, so it is easy to see cleptobiosis of seeds by Messor workers as a fairly simple shift in foraging strategy. Aphaenogaster species show a wider range of food preferences, but A. senilis workers collect Euphorbiaceae seeds in the Mediterranean habitat in which this study was conducted. Messor capitatus workers engage in a variety of interference tactics that affect foraging by congeners, including cleptobiosis of seeds [47, 48]. Similarly, honey ant, Myrmecocystus mimicus, workers rob insect prey items from harvester ant, Pogonomyrmex, workers returning to their colonies [49]. Additional examples expand the range of species that can engage in forcible cleptobiosis. LaPierre et al. [50] observed Polybioides tabida E (Ropalidiini) wasps robbing workers of the ant, Tetraponera aethiops. The wasp, Charterginus sp., (Epiponini) robs food normally collected by mutualistic ants on acacias. Corbara and Dejean [51] reported theft of paper nest material from ants by the social wasp Agelaia fulvofasciata. In sum, cleptobiosis is exhibited by ants, bees, and wasps, giving support to the hypothesis that cleptobiosis is easily derived from preexisting features such foraging preferences, territorial behavior, and social mechanisms such as kin recognition. 8. Cleptobiosis and the Risk of Disease Spread Cleptobionts, by coming into close contact with conspecifics, put themselves at risk for exposure to any disease their victim might carry. This becomes particularly important if the victim’s weakness from disease makes them an easier target for cleptobiosis. Increased risk of disease transmission through cleptobiosis is best known in honeybees, A. mellifera [52]. Colonies weakened by the bacterial disease, American Foul Brood, Paenibacillus larvae, the intestinal parasite, Nosema, or by Varroa mites are much less well able than healthy colonies to defend themselves against robber bees from other colonies [52]. 9. Discussion Food theft — cleptobiosis — is an important form of inter- colonial interaction in social insects. In this paper we have defined cleptobiosis and discussed the difference between cleptobiosis and other types of interactions among social insect colonies, such as dulosis. We also suggest that the term “cleptoparasitism” has been used in so many different ways that it has lost its usefulness and should be avoided. We point out that brood parasitism, inquilinism, and “guests” 6 Psyche in social insect colonies all may involve mechanisms similar to cleptobiosis. Cleptobionts may be divided into deceptive and forceful types. Deceptive cleptobionts bear considerable resemblance to “guests” in the manner in which they gain entrance to target colonies. The ecological implications of cleptobiosis are clear, and the reader should refer to Holldobler’s [49] discussion of the significance of cleptobiotic ants in interference com- petition among ant colonies. 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Dejean, “Wasps robbing food from ants; a frequent behavior?” Naturwissenschaften, vol. 94, no. 12, pp. 997-1001, 2007. [51] B. Gorbara and A. Dejean, “Paper stealing on an arbori- colous ant nest by the wasp Agelaia fulvofasciata degeer (Hymenoptera: Vespidae),” Sociobiology, vol. 39, no. 2, pp. 281-283,2002. [52] D. Naug, “Disease transmission and networks,” in Encyclope- dia of Animal Behavior, M. D. Breed and J. Moore, Eds., vol. 1, pp. 532-536, Academic Press, Oxford, UK, 2010. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 814865, 5 pages doi:10.1155/2012/814865 Research Article Australian Alleculinae: New Genera, New Combinations, and a Lectotype Designation (Coleoptera: Tenebrionidae) Eric G. Matthews South Australian Museum, North Terrace, Adelaide, SA 5000, Australia Correspondence should be addressed to Eric G. Matthews, eric.matthews@samuseum.sa.gov.au Received 15 September 2011; Revised 29 November 2011; Accepted 9 December 2011 Academic Editor: Donald Mullins Copyright © 2012 Eric G. Matthews. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Litopous baehri gen. et sp. nov. and Scaphinion clavatus gen. et sp. nov. are described. New combinations (previous genus in parentheses) are Aethyssius minor (Carter) (Tanychilus Newman), Homotrysis subopaca (Carter) {Metistete Pascoe), Metistete armata Carter {Homotrysis Pascoe), M. opaca (Carter) {Tanychilus), M. punctata (Carter) {Melaps Carter), and Nocar subfasciatus Carter {Taxes Champion). A lectotype and two paralectotypes are designated for Dimorphochilus diversicollis Borchmann which is removed from synonymy, and the genus is briefly discussed. 1. Introduction Australian Alleculinae are poorly known and there are numerous unnamed species. Many species that are now included in genera such as Homotrysis Pascoe, Metistete Pascoe, and Aethyssius Pascoe apparently belong to other genera, but no consistently reliable morphological characters have been proposed to distinguish them. In the present paper two such taxa from Western Australia are included in two new genera based on the discovery of diagnostic characters. Another taxon from the same state was considered as possibly belonging to a third distinct genus with simple tarsal claws, a character very unusual in the Alleculinae. However, examination of the claws under high magnification revealed that the inner edges are minutely crenulated, and also in other respects, such as the apically unidentate mandibles, cultriform terminal maxillary palpomere and front of head not produced, this species (unnamed) falls within the current concept of Aethyssius. The terms frontal index and stomatiform punctures fol- low Matthews and Bouchard [ 1 ] , and the checklist of names included in that work gives the dates and citations of all descriptions. 2. Material Material from the following collections was examined: AM: Australian Museum, Sydney; ANIC: Australian National Insect Collection, Can- berra; ANIC(MM): Macleay Museum material on perma- nent loan to ANIC; BMNH: Natural History Museum, London; NMV: Museum of Victoria, Melbourne; QM: Queensland Museum, South Brisbane; SAMA: South Australian Museum, Adelaide; WAMA: Western Australian Museum, Perth; ZSM: Zoologische Staatssammlung, Munich. 3. New Genera 3.1. Litopous gen. nov. Type Species. Litopous baehri sp. nov. Description. Oblong, densely setose with long erect bris- tles on all dorsal surfaces and legs. Head not prolonged. Antennomeres elongate, bearing dense long bristles, only a few compound sensoria on apical 3 segments. Eyes deeply emarginate anteriorly. Mandibles bluntly bidentate. Apical maxillary palpomere roundedly securiform, symmetrical. 2 Psyche Tarsi bearing long bristles only, without lobes or cupuliform segments. Claws not pectinate, with only small teeth on basal half of inner edges (Figure 2(d)) . Intercoxal process of first ventrite acutely angular. Length 8.3 mm. Distribution (Figure 3). Known only from the type locality near Wurarga inland from Geraldton, WA. Etymology. Greek litos = plain, pous = foot. Discussion. This is the most unusual Australian alleculine examined, having no trace of lobes or cupuliform tar- someres. The penultimate segment of the protarsus bears two stout diverging acute projections, probably coalesced bristles. The teeth on the tarsal claws are very short (Figure 2(d)), not typically pectinate as in all other known Australian Alleculinae except the unrelated Hemicistela Blackburn and the simple-clawed species mentioned in the introduction. The antennae are also unusual in bearing numerous long stiff setae and very few compound sensoria. According to current classifications of the subfamily (e.g., Matthews et al. [2] ) Litopous does not fit into the subtribe Alleculina as other Australian alleculines because of the absence of tarsal lobes, and it does not conform to the diagnostic characters of any of the other higher taxa of the subfamily as discussed by Gampbell [3]. For the present, this genus will be treated as incertae sedis within the tribe Alleculini. 3.2. Litopous baehri sp. nov. (Figures 1(a), 2(d), 3) Description of Female. Elongate-oblong, uniformly dark brunneous, with dense erect setae, total length 8.3 mm, width at humeri 2.7 mm. Head. Frons and clypeus not prolonged, frontal index 0.42, edges indented at frontoclypeal suture, which is impressed. Basal membrane of labrum short, labrum very transverse bearing long stiff bristles. Eyes moderate, separate dorsally by distance equal to a little more than width of one eye. Antennomere 3 longer than 4 but shorter than 4+5, segments gradually shortening distad, terminal segment fusiform and apically acute. Antennomeres 1-7 bearing long bristles, 8-11 short stout setae, only a few compound sensoria on apical 3 segments. Mandibles short, bluntly bidentate. Prothorax. Subquadrate, 1.2 times as wide as long, sides strongly convex, maximum width a little before middle. Pronotal surface densely and coarsely punctuate, punctures bearing long erect bristles. Prosternal process about as wide as half of one procoxa. Pterothorax. Much wider than prothorax, sides of elytra straight, subparallel for anterior 2/3, roundedly tapering posteriorly. Striae deeply impressed, strial punctures round, simple, separated by distance about equal to one-puncture diameter. Interstriae with numerous punctures bearing long erect bristles. Wings fully developed. (a) (b) (c) Figure 1: Habitus, (a) Litopous baehri sp. nov. female; (b) Scaphin- ion clavatus sp. nov. male, dorsal; (c) ditto, lateral. Scale lines 1 mm. Legs. Slender, unmodified, femora projecting beyond body outline for about half their length. Tarsi a little longer than half tibial length, basal metatarsomere a little longer than next 3 combined. Abdomen. Intercoxal process of first ventrite acutely triangu- lar. Male. Unknown. Type. Holotype female: Australia, WA 06/168, 2 km SE Wurarga, 28.37913 S, 116.33105 E, 371m, 3-4.3.2006, M. Baehr. WAMA Reg. no. 82708. Etymology. The author takes pleasure in naming this species after its collector, Martin Baehr of ZSM. 3.3. Scaphinion gen. nov. Type Species. Scaphinion clavatus sp. nov. Description. Elongate-oblong, bearing long recumbent setae. Head not prolonged. Eyes large, deeply emarginate. Antennomeres sometimes greatly enlarged distally. Man- dibles small, acutely bidentate. Apical maxillary palpomere broadly securiform, somewhat asymmetrical. Occiput deeply transversely excavate and receiving prolongation of anterior part of pronotal disc. Elytral punctures uniformly large, deep, with sides thickened (stomatiform). Tarsi with small square lobes on penultimate segments. Glaws pectinate. Total length +5 mm (based on 3 available specimens of the genus). Distribution (Figure 3). The Kimberley District of north- western Australia where all three known species were col- lected. Etymology. Greek scaphe = hollowed out, inion = back of head. Discussion. Scaphinion is related to Metistete Pascoe because the elytral punctures are entirely stomatiform. All three Psyche 3 Figure 2: Outlines, (a) Scaphinion clavatus aedeagus, lateral; (b) ditto, dorsal; (c) S. clavatus sternite 8; (d) Litopous baehri, tarsal claw. Figure 3: Known distributions in Western Australia. (■) Litopous behri; (A) Scaphinion clavatus and S. sp. 1; (A); Scaphinion sp. 2. known species differ from the latter genus in having the pronotum anteriorly prolonged and received in a concavity of the occiput. The enlargement of the antennae of two of the known specimens (Figures 4(a), 4(b)) is also unknown in Metistete. The third specimen has simple antennae (Figure 4(c)). The three male specimens known of this genus each belong to a different species as discussed below. The author believes that in general it is not desirable to describe new species of known genera on the basis of single specimens, but where new genera are clearly involved, as is the case in the present paper, they should be described even though their type species are initially represented by uniques. The two species of Scaphinion which are not types of the genus are briefly characterized but not named now in order to illustrate some of the specific diversity which is to be expected in the genus. A forthcoming faunal survey of the Kimberley District by the Western Australian Department of Environment and Conservation should discover more specimens of the genus and thus permit detailed descriptions of both sexes. 3.4. Scaphinion clavatus sp. nov. (Figures 1(b), 1(c), 2(a), 2(b), 2(c), 3, and 4(a)) Description of Male. Elongate oblong, uniformly fuscous, with dense recumbent setae, total length 5.3 mm, width at humeri 1.8 mm. Head. Clypeus broadly truncate, sides diverging and form- ing a straight line with edges of canthi. Head moderately prolonged, frontal index 1.0. Basal membrane of labrum very short, labrum very transverse, bearing moderately long setae. Eyes deeply emarginate anteriorly, large, separated by a distance subequal to about 1.5 eye width as seen from above. Antennomere 3 about as long as 4, 3-9 subtriangular, gradually widening and becoming asymmetrical, 10 very transverse, wider than 9, 11 transverse, smaller than 10 and contained within a cavity of latter (Figure 4(a)). All segments bearing short setae, 3-11 with numerous compound senso- ria. Mandibles small, acutely bidentate. Prothorax. Anterior edge of pronotum strongly produced to broadly rounded apex which overhangs base of head, prono- tum widest just behind middle, sides slightly convergent to base, basal edge straight. Ratio of maximum width to length 1:1. Pronotal surface densely and coarsely punctate with long recumbent setae. Prosternal process as wide between coxae as 1/3 width of 1 coxa. Pterothorax. Much wider than prothorax, sides of elytra subparallel for anterior 2/3, broadly tapering to apices. Striae not impressed, consisting of very large and deep, close-set rounded stomatiform punctures. Interstriae with numerous small punctures bearing long recumbent setae. Wings fully developed. Legs. Moderately slender and setose, femora with about 1/2 of their length projecting beyond elytral edges. Tibiae subparallel, slightly bowed, not distorted. Tarsi about 1/3 length of tibiae. Lobes of preapical tarsomeres small. Abdomen. Intercoxal process of first ventrite acutely triangu- lar. Aedeagus (Figures 2(a) and 2(b)) simple. Female. Unknown. Type. Holotype male. 16.22S 125. 12E WA. Charnley Riv. 2 km SW Roily Hill CALM site 1311 16-20 June 1988, I.D. Naumann, at light, open forest near closed forest margin, ANIC. Discussion. The three known male specimens belong to three different species, S. clavatus and two others which remain unnamed for now for the reasons discussed above. The latter two have the following locality data: Species 1, 16.38S 125. 15E CALM site 28/3 4 km W of King Cascade, W.A., 12-16 June 1988 T.A. Weir, at light closed forest margin, ANIC; species 2, 14.39S 126. 57E Drysdale River, WA. 18- 21 Aug. 1975, l.F.B. Common and M.S. Upton, ANIC. The most obvious difference between the three is in the form of the antennae, sp. 1 having only moderately enlarged antennae (Figure 4(b)) and sp. 2 unmodified filiform ones (Figure 4(c)). In addition, in both unnamed species the eyes are larger than in clavatus, the interocular distance being equal to about half an eye width and the shape of abdominal sternite 8 is different in all three species, this being the most 4 Psyche (a) (b) (c) Figure 4: Head and prothorax of Scaphinion. (a) S. clavatus; (b) S. sp. 1; (c) S. sp. 2. Scale line 1 mm for all figures. reliable indication of specific distinctness. The aedeagi are identical. 4. New Combinations A recent examination of Carter types of Alleculinae in NMV revealed that a number of species were incorrectly assigned to genus in [ 1 ] , The following names are transferred to different genera from those listed in the latter work. The correct generic name is first and the name in [1] is in parentheses. Aethyssius minor (Carter) (Tanychilus Newman). Homotrysis suhopaca (Carter) {Metistete Fascoe) . Metistete armata Carter (Homotrysis Pascoe). Carter placed this species in the correct genus but it was wrongly assigned by [ 1 ] . Metistete opaca (Carter) (Tanychilus). Metistete punctata (Carter) (Melaps Carter). Nocar subfasciatus Carter (Taxes Champion). Also placed correctly by Carter but misplaced by [ 1 ] . 5. Previous Lectotype Designations Carter and other authors of that time did not specifically designate holotypes in their descriptions, but they always labelled one specimen of the type series as the “Type” and the others as cotypes, clearly intending that they should be considered holotype and paratypes, respectively. In all unpublished museum catalogs and lists, the specimens which bear the label “Type” are listed as holotypes and often given numbers. However, according to Article 74 of the ICZN [4] this is not sufficient for the specimens in question to be con- sidered primary types unless they were clearly identified (or indicated to be unique) in the original descriptions. On the other hand, many Carter and Macleay numbered specimens are listed in print as holotypes in AM by McKeown [5] and in this case this action is tantamount to lectotype designation according to Article 74.6 of [4]. The names involved, listed in their present genera, are Aethyssius cyaneus (Macleay), A. cylindricollis (Carter), A. major (Carter), A. mastersi (Ma- cleay), A. metallica (Carter), A. oculatus (Carter), A. piceus (Macleay), A. puncticeps (Blackburn), A. ruficollis (Macleay), A. rugosulus (Macleay), A. suturalis (Carter), A. tenuicor- nis (Carter), A. vitticollis (Macleay), Dimorphochilus pas- coei (Macleay), Euomma palpalis (Macleay), E. mastersi (Macleay), Homotrysis albolineata Carter, H. interstitialis Carter, H mastersi (Macleay), H montium Carter, H. rufi- cornis Macleay, H. rufobrunnea Carter, H. sexualis Carter, H silvestris Carter, H. subgeminata Macleay, Metistete clermontia (Carter), M. elongata (Macleay), M. illidgei (Carter), M. pascoei (Macleay), M. planicollis (Macleay), M. punctipennis (Macleay), M. rufipilis (Carter), M. subsulcata (Macleay), M. yeppoonensis (Carter), Nocar convexus (Macleay), N. depressiusculus (Macleay), N suttoni Carter, Ommatophorus mastersi Macleay, and Scaletomerus politus (Macleay). For all of these, “ST” in the checklist of [ 1 ] should be changed to LT. The remaining approximately 80 specimens listed as holotypes or the equivalent in the catalogs of ANIC(MM), BMNH, NMV, QM, SAMA, and ZSM must be considered only syntypes unless the original descriptions were based on single specimens. In the checklist of [1] they are appropri- ately listed as ST and HT, respectively. In most cases the specimens labelled “Type” by their original describers will eventually be selected as lectotypes, but this action should be deferred until the taxa in question are revised. 5.1. Dimorphochilus Borchmann, 1908. Borchmann [6] de- scribed this as a new genus with three new species, D. apicalis, D. diversicollis, and D. sobrinus, collected during the Hamburg Museum Southwest Australia expedition of 1905. The concept of this genus was uncertain because D. sobrinus should be assigned to Tanychilus, which has apically unidentate mandibles unlike Dimorphochilus. Tanychilus gouldi (Hope) was also misplaced in Dimorphochilus by Carter [7] partly for this reason. The fixation of the type species of Dimorphochilus as D. apicalis Borchmann by [1] restricts the diagnostic characters of the genus as follows: apices of mandibles bidentate, or bilobate with a longitudinal groove between lobes; dorsal surfaces entirely glabrous, the body castaneous with legs paler, often flavous; elytra with simple punctures only and a sutural gap. The latter term refers to a widening of the suture at the apices of the elytra, the space thus opened being covered with overlapping expansions of the marginal membranes of the suture. This character is illustrated for D. apicalis by [ 1 ] in Figure 80 K, which shows the extremely widened gap of a female on the right and the more normal one of a male on the left. Carter [8] erroneously synonymised D. diversicollis with D. apicalis. The former species is here resurrected following examination of the types listed below. Psyche 5 The genus Dimorphochilus as now understood includes the following five named species known from the states indicated in parentheses: apicalis (WA), diversicollis (WA), caudatus (Carter) (Qld), luctuosus (Champion) (Tas), and pascoei (Macleay) (Qld, NSW). In addition, there are two unnamed species in SA, probably others elsewhere. There is some doubt about D. luctuosus as it is entirely piceous and the elytral gap is minimal. The material collected during the Hamburg Southwest Australia expedition of 1905 was largely taken to Hamburg and subsequently destroyed during World War II. It was con- sidered that Borchmann material of Alleculinae was likewise destroyed until three female specimens of D. diversicollis agreeing with the original description and bearing the expe- dition labels were discovered among miscellaneous acces- sions in WAMA. Borchmann had five males and numerous females of the species but did not designate a holotype. The specimens discovered are here designated lectotypes of Dimorphocilus diversicollis Borchmann, 1908 as follows, with the sequence of labels indicated by numbers in parentheses. Lectotype Female. (1) (printed) Hamb. S. W. Austr. Exped. 1905. Stat. 76 Day Dawn 9.-10. VII; (2) (handwritten) Di- morphochilus diversicollis Borch. Cotype! 4238; (3) (yel- low, printed) Western Australian Museum Entomology Reg no 57620; (4) (red, handwritten by author) EECTO- TYPE Eemale Dimorphochilus diversicollis Borch. Sel. by E. Matthews. Paralectotype Females ( Two ). (I) ( 1 ) as above Stat. 7 1 North- ampton 4238a 15. VII; (2) as in 3 above Reg no. 57621; (3) (blue, handwritten by author) PARAEECTOTYPE Eemale Dimorphochilus diversicollis Borch. (II) (1) as above Stat. 77 Yalgoo 4238b 11. VIE Dimorphochilus diversicollis originally handwritten on underside; (2) as in 3 above Reg no. 57622; (3) as in 3 for previous paralectotype. Acknowledgments The author wishes to thank the curators of the collections examined: Dave Britton (AM), Tom Weir (ANIC), Ken Walker (NMV), Max Barclay (BMNH), Geoff Thompson and Geoff Monteith (QM), Brian Hanich and Terry Houston (WAMA), Martin Baehr (ZSM), and also Roland Grimm for sending the specimen of Litopous haehri and Martin Baehr for donating it to WAMA. [3] J. M. Campbell, A Revision of the Genus Lobopoda (Coleoptera: Alleculidae) in North America and the West Indies, vol. 37 of Illinois Biological Monographs, University of Illinois Press, Urbana, 111, USA, 1966. [4] International Commission of Zoological Nomenclature, Inter- national Code of Zoological Nomenclature (ICZN), International Trust for Zoological Nomenclature, 4th edition, 1999. [5] K. C. McKeown, “A reference list of types of Coleoptera in the Australian Museum,” Records of the Australian Museum, vol. 22, no. 1, pp. 95-139, 1948. [6] E Borchmann, “Alleculidae,” Die Fauna Sudwest-Australiens, vol. 1, pp. 348-358, 1908. [7] H. J. Carter, “Revisional notes on the family Cistelidae (Order Coleoptera),” Transactions of the Royal Society of South Aus- tralia, vol. 44, pp. 198-217, 1920. [8] H. J. Carter, “Check list of the Australian Cistelidae. Order Coleoptera,” Australian Zoologist, vol. 6, pp. 269-276, 1930. References [1] E. G. Matthews and P. Bouchard, Tenebrionid Beetles of Aus- tralia: Descriptions of Tribes, Keys to Genera, Catalogue of Species, Australian Biological Resources Study, Canberra, Australia, 2008. [2] E. G. Matthews, J. E Lawrence, P. Bouchard, W. E. Steiner Jr., and S. A. Slipinski, “Family Tenebrionidae,” in Handbook of Zoology, A Natural History of the Phyla of the Animal Kingdom, R. G. Beutel and R. A. B. Leschen, Eds., vol. 4, pp. 574-659, Walter de Gruyter, Berlin, Germany, 2010, Arthropoda: Insecta, part 38, Coleoptera, Beetles, vol. 2, Systematics, part 2. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 921465, 5 pages dohlO.l 155/2012/921465 Research Article Predatory Behavior of Canthon virens (Coleoptera: Scarabaeidae): A Predator of Leaf cutter Ants Luiz Carlos Forti,^ Isabela Maria Piovesan Rinaldi,^ Roberto da Silva Camargo,^ and Ricardo Toshio Fujihara^ ^ Laboratorio de Insetos Sociais-Praga, Departamento de Produgao Vegetal, Faculdade de Ciencias Agrondmicas, UNESP, PO. Box 237, 18603-970 Botucatu, SP, Brazil ^Departamento de Zoologia, Instituto de Biociencias, UNESP, 18618-970 Botucatu, SP, Brazil Correspondence should be addressed to Roberto da Silva Camargo, camargobotucatu@yahoo.com.br Received 28 October 2011; Accepted 11 December 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 Luiz Carlos Forti et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We present a detailed description of the predatory behavior of the beetle Canthon virens Mannerheim, 1829, on the leafcutter ant Atta sp. We observed 51 acts of predation, which were also recorded on film and subjected to behavioral analysis. Canthon virens exhibited 28 behaviors while predating upon Atta sp. queens. Adult beetles search for queens while flying in a zigzag pattern, 15 to 20 cm above the ground. After catching a queen, the predator stands on its back and starts cutting the queen cervix. Once the prey is decapitated, the predator rolls it until an insurmountable obstacle is reached. The distance from the site of predation to the obst- acle can vary widely and is unpredictable. The beetle rolling the queen also buries it in a very peculiar way: first, it digs a small hole and pulls the queen inside, while another beetle is attached to the prey. The burial process takes many hours (up to 12) and may depend on the hardness of the soil and the presence of obstacles. In general, one or two beetles are found in a chamber with the queen after it is buried. They make the brood balls, which serve as food for the offspring. This study contributes to the knowledge of the predatory behavior of Canthon virens, a predator poorly studied in Brazil and widespread in the country. 1. Introduction Canthon dives Harold, 1868, and Canthon virens Manner- heim, 1829, prey on queens of leafcutter ants, Atta spp., after the queen nuptial flight. For this reason, the beetles are con- sidered natural biological control agents of these ants. In 1937, Lichti [1] was the first to report the predation of leaf- cutter ant queens by Canthon dives beetles. Later, Navajas [2] reported that not only queens but also winged forms of both sexes are preyed upon by Canthon virens. Atta queens lose their wings after their nuptial flight and start looking for a place to dig. When searching for a suitable place to build their nests, and during nest digging, ant queens are most vulnerable to their beetle predators. They can be attacked by one to over six individuals simultaneously [3]. The predation rate of Atta by Canthon virens [4] alone is 7.6%, but when other species of Canthon are considered, this proportion can reach more than 50% [3]. Using the tissues of their prey, Canthon beetles build two or three brood balls for their larvae. Detailed observations of this behavior have been documented for Canthon virens: first, the beetle positions itself, with some difficulty, on the back of the queen. Then, using the clypeus as a lever and the jagged edges of the tibiae as a saw, the predator cuts the prey cervix or neck [5, 6]. Both male and female beetles prey on queens [7], but a relationship between the size of the prey (queen) and the predator (beetle) has not been found. However, there is a positive relationship between the densities of queens and beetles, suggesting that the relationship between Atta spp. and Canthon virens might be obligatory [3, 6]. The brood balls are buried 7.7 ± 2.3 cm deep. Each fe- male beetle lays one egg in each brood ball, and males and females remain in the burrow until the offspring has devel- oped [3]. Little is known about the predation sequence of Atta queens by Canthon virens. In view of this gap, we have 2 Psyche investigated and provide a detailed description of the steps from searching for Atta queens to the confection of the brood balls. 2. Material and Methods The behavior of the beetle predator C. virens was monitored for 5 years in a natural situation, in several commercial plan- tations of Eucalyptus grandis or natural vegetation (Cerrado) in the municipalities of Botucatu, Itatinga, and Anhembi, in the state of Sao Paulo, Brazil. Fifty-one cases of queen predation by beetles were filmed on VHS, using two Panasonic M-8 cameras. Filming began when the queens came to the ground after their nuptial flight. The videos were labeled and stored for later analysis. All behaviors were quantified by counting the number of times each occurred during 1 minute, at 2-minute intervals. We then used those results to calculate the frequencies of each behavior. Behaviors were then analyzed for a pattern. 3. Results 3.1. Behaviors during Predation. A total of 28 behaviors was recorded during the predation of Atta queens (Figure 1), as follows: (1) A single beetle flies over the queen in a zigzag pattern before capturing it. (2) A single beetle on the ground climbs onto the back of a walking queen. (3) Two beetles fly over a queen, but only one attacks and beheads it. (4) A single beetle flies over a queen and catches it. (5) A single beetle flies over a queen, is momentarily unable to catch it, but ends up succeeding. (6) A single beetle flies over a queen, lands on it, and then abandons it. (7) A beetle positions itself on the thorax of a queen, with its head pointing to the queen cervix. (8) A beetle attaches itself to the thorax of a queen, with its head turned to one side, that is, the time required for the beetle to turn in the direction of the queen head and start beheading it. (9) A beetle attaches itself to the queen thorax, with the head facing the queen gaster; the beetle then points its head towards the head of the walking queen, that is, the time required for the beetle to stand with its head toward the head of the queen and start cutting. (10) A beetle attaches itself to the queen thorax while the queen is digging and then changes position, standing up to kill it. (1 1) A beetle attaches itself to the queen thorax and then abandons it. ( 12) A beetle, on the ground, secures the queen abdomen, leaves its prey momentarily, and then comes back to catch it again. (13) A beetle, on the ground, secures the queen abdomen but does not turn its head toward the prey head. ( 14) A beetle cuts the queen cervix, the time needed to start and finish the process. (15) A beetle walks over a queen that is moving with diffi- culty. (16) A beetle attempts to cut the queen neck, but does not succeed. (17) After cutting the queen neck, the predatory beetle walks on the back of its prey, examining it. (18) After cutting the queen neck, the predatory beetle walks away. (19) After cutting the queen neck, the predatory beetle examines its prey, walks to the side, and comes back to roll it, that is, the time spent from cutting the queen cervix to starting to roll it. (20) After cutting the neck of the queen, the predator examines it, walks beside it, but does not continue with the process of predation. (21) Numerous beetles show up and “dispute the prey” by rolling it together. When only two beetles are left, one is attached to the queen and the other one rolls it, that is, the time from the dispute to the burial. (22) Numerous beetles dispute the dead body by rolling it to- gether. When only one beetle is left, it rolls the queen to the burial site. (23) More beetles show up and dispute a live queen (walking), which is being preyed upon by another beetle. When only three beetles are left, one goes away and two remain. (24) More beetles show up and dispute a live, yet immobile queen, which is being preyed upon by another beetle. One beetle goes away and the other stays with the original predator. (25) The beetle that killed the queen rolls it. From time to time it climbs up and down the queen, and then goes back to rolling it, that is, the time spent rolling and going up and down. (26) A beetle, after rolling its prey, begins to excavate the soil to bury it. It goes to the bottom of the hole and comes back to the surface. If two beetles are present, one digs and the other remains on the queen. (27) A beetle, after rolling the queen, digs a small hole in the ground, buries the queen only partially, and remains on the surface with the abdomen facing up, that is, the time spent to dig, to bury the queen superficially, and to position the legs up. (28) A beetle buries the queen very deep and then sits on the surface with the abdomen facing up. 3.2. Descriptive Sequence of Behaviors. Adult beetles showed up for the nuptial flight only. They were seen in greater numbers a little before and during the nuptial flight flying in a zigzag pattern, more or less 15 to 20 cm from the ground, searching for prey. They flew very fast, and periodically inter- rupted their activity to rest on the ground or on the vegeta- tion. A total of 92% of the queens (47 beetles) were captured by a beetle that was flying in zigzag and came upon the prey thorax (Figure 3). In 4% of the cases (2 beetles), however, the queen was not captured. One queen (2%) was chased by two beetles, but only one beetle finished the task. An alternative, rarer capture method observed in 4% of the cases did not involve a zigzag flight. In that case, a beetle walking on the soil surface tried to climb on the back of a queen to kill it. In some instances, a beetle that failed to succeed using this method tried again, but seldom (2%) on the same individual. An interesting result of our study is the observation that female beetles are those that capture and behead Atta queens. When a female beetle sits on the thorax of its prey, it often has the head turned to one side (40% of cases, 20 beetles) of the queen body. The predator then turns its head toward the head of the queen. Often, the female beetle sits in the correct position to cut the queen neck (Figures 1, 2(a), and 2(b)), but sometimes the female has its head turned toward the queen gaster (27% of cases, 14 beetles) (Figure 2(d)). Once the pre- dator is positioned correctly (Figure 2(b)), it immediately begins to cut the queen neck. After a queen is killed, many beetles join the killer (Figure 2(e)), but only two are left to bury the prey (Figure 2(f)). Division of labor according to sex in C. virens is well defined. The female catches, kills, rolls, and buries the prey. Psyche 3 Figure 1: Fluxogram showing the behavior of Canthon virens preying on leafcutter ants {Atta spp.). The male joins the female after the murder. In some cases, we observed females burying without a male, with her abdomen facing upward and her body partially buried in the tunnel under construction (Figure 2(g)). A beetle can roll a queen for distances of 2 to 3 m. Some beetles that roll in push feces up to 9 m. Canthon virens individuals roll their prey until they find an insurmountable obstacle. The distance between the site of predation and the obstacle can vary widely and is unpredictable. Usually a bee- tle rolling a queen will stop from time to time, go away a few inches, then return, climb on the dead body, and start rolling it again. The burial is always conducted by the beetle that pushed the queen: first, the beetle digs a small hole and pulls the queen into the pit, while the other beetle remains on the prey. The burial process takes up to 12 hours and may be dependent on soil compaction and/or the presence of ob- stacles. As the queen is buried, the soil is deposited on the surface in the form of small pellets. Up to three beetles were found buried with the queen during the first 24 hours, but the most common is to find one or two beetles per chamber with the queen (Figures 2(h) and 2(i)). 4. Discussion Predation is an unusual form of specialization in Scarabaei- nae beetles because the majority of them are coprophagous. Some species of Canthon specialize in the predation of Atta queens. Other authors have described [2, 3, 5] the specialized predatory behavior of these beetles, beginning after the nuptial flight: first the beetle approaches the queen. 4 Psyche (g) (h) (i) Figure 2: (a) A beetle flies zigzag over prey and lands on its back, (b) The beetle cuts the neck of the queen and is attached to it by its legs, (c) The queen has been killed: the beetle examines the prey, before rolling it. (d) Sometimes the beetle may attach itself to the thorax of the queen, having the head turned toward the queen abdomen, (e) When the queen is dead, many other beetles may join, (f) After a few minutes only two beetles remain to bury the queen, (g) The beetle is partially buried and has its abdomen and hind legs facing up and is partially buried; this phenomenon is observed when the beetle buries its prey by itself, (h) The nest of Canthon virens, with a channel about 1 cm in diameter and 15 cm deep, (i) Chamber at the end of the canal, with the brood balls. Figure 3: Frequency of predatory behaviors of Canthon virens. lands on its back, and then beheads it. However, previous descriptions of the predation sequence were insufficient in view of the amount of information that can be obtained. In our study, we found that 28 behaviors are performed. showing the complexity of the sequence of predation events (Figure 1). First, Canthon beetles were observed flying at about 15 to 20 cm from the soil surface in search of prey. They fly ex- tremely fast and periodically stop to rest on the ground or on the vegetation. Because queens are located by their beetle predators regardless of whether they remain still or move, we believe that individuals of Canthon virens use their sight to locate them. However, because vision is generally poorly developed in Scarabaeinae beetles, whereas their tactile sense is sharp [5], we cannot rule out the possibility that smell, such as that produced by allelochemicals, plays a role in prey location. It is well known that, when an odor current is de- tected by dung beetles, they take flight and perform a short- distance search [5]. The same could apply to Canthon virens beetles. Once a queen is found walking on the ground, the pre- dator flies in a zigzag fashion, lands on it, and later beheads it. In this study, we found that female beetles are responsible for catching the ant queens. This result contrasts with those Psyche 5 of Navajas [2] and Silveira et al. [7], who found that both males and females prey on the queen and that only the males attack it. After landing on the queen, the female beetle proceeds to decapitate it, as shown in Figure 1. According to Halffter and Matthews [5], the beetle cuts the queen cervix with the aid of its front legs and clypeus, a fact we also observed. When a female beetle lands on the thorax of its prey, it most often positions itself with the head facing sideways. The predator then turns to face the queen cervix. Frequently a beetle will land on the queen thorax with its head pointing to the head of its prey. In rare cases, the predator stands on the queen thorax with its head pointing to the queen gaster. The queen keeps walking for a while as the predator pro- ceeds to behead it. At a certain moment, the queen stops and assumes a position in which her body is sideways, arched, and immobile. Infrequently, two or three other beetles may join before the queen has been beheaded and while it is still walk- ing. Most often, however, beetles will group around a victim that is already dead. In some cases, we have observed up to 1 1 beetles surrounding a single queen. Apparently, some are there to compete for the female beetle. In the end, usually one or two beetles remain at the scene. Forti and Rinaldi [3] reported that a queen may be attacked by up to six beetles when it is looking for an ideal place to start digging its nest. We believe that their reports actually describe what happens after the queen is already dead. Normally one beetle rolls the queen, while the other is attached to it. Most Scarabaeini make balls from vertebrate feces and have developed specialized ways to roll them. The balls are rolled either by one or two beetles [5, 8]. When two beetles are involved, they usually belong to different sexes, and the roles taken by each member of the couple differ from one genus to another. In some species of Canthon, for instance, the male always rolls, while the female remains on the top of the ball. However, in C. virens, the female rolls the queen while the male is rolled along with it. As soon as the beetle finds an insurmountable obstacle, it starts digging. Similarly, Silveira et al. [7] observed that beetles roll the headless queen until they find a dead leaf and, from that moment on, start digging. In other coprophagous species of Canthon, for instance C. lituratus, either the male or the female, or both, prepares fecal balls. However, the female is the one to bury the ball, even when a male is present [9, 10]. In C. virens, the queen is buried by the beetle that was rolling it. First, the beetle digs a small hole and pulls the queen into the pit, while the other beetle remains stuck on the prey. The same behavior was observed in C. lituratus: the female buries the fecal ball while the male, when present, sits on it [9]. When a beetle excavates alone, it returns to the surface with the abdomen and hind legs facing up, remaining par- tially buried (Figure 2(g)). Silveira et al. [7] also noted that the beetle remains with the hind legs pointing up and sug- gested that this may be an indication that it is emitting sex pheromones to attract the opposite sex to the nesting site [ 11 ]. Finally, the soil is deposited on the surface at the same time that the queen is being buried. Within 24 hours, the beetles, usually one or two individuals, are buried in the chamber next to the brood ball (Figures 2(h) and 2(i)). This study contributes to the knowledge of the predatory behavior C. virens, a predator poorly studied in Brazil. Acknowledgment L. C. Forti thanks the CNPq for a research aid (301917/2009- 4). References [1] I. Lichti, “Canthon dives Harold (Col. Copridae), predador das femeas de Atta laevigata Smith (Hym. Formicidae),” Revista Entomologia, vol. 7, pp. 117-118, 1937. [2] E. Navajas, “Manifesta^oes de predatismo em Scarabaeidae do Brasil e alguns dados bionomicos de Canthon virens (Mannh.) (Col. Scarabaeidae),” Ciencia e Cultura, vol. 2, pp. 284-285, 1950. [3] L. C. Forti and I. M. R Rinaldi, “Comportamento predatorio de Canthon dives (Coleoptera, Scarabaeidae) sobre Atta laevi- gata (Hymenoptera, Formicidae),” in Congreso Latino Ameri- cano de Ecologia, p. 47, Montevideo, Uruguay, 1992. [4] L. C. Forti, I. M. P. Rinaldi, W. Yassu, and M. A. S. Pinhao, “Avalia^ao da eficiencia de preda^ao de Canthon virens (Cole- optera, Scarabaeidae),” Naturalia, vol. 24, pp. 241-242, 1999. [5] G. Halffter and E. G. Matthews, “The natural history of dung beetles of the subfamily Scarabaeinae (Coleoptera, Scarabaei- dae),” Eolia Entomologica Mexicana, no. 12-14, pp. 1-312, 1966. [6] F. Hertel and G. R. Colli, “The use of leaf-cutter ants, Atta laevigata (Smith) (Hymenoptera: Formicidae), as a substrate for oviposition by the dung beetle Canthon virens Manner- heim (Coleoptera: Scarabaeidae) in central Brazil,” Coleopter- ists Bulletin, vol. 52, no. 2, pp. 105-108, 1998. [7] F. A. O. Silveira, J. C. Santos, L. R. Viana, S. A. Falqueto, F. Z. Vaz-De-Mello, and G. W. Fernandes, “Predation on Atta laevigata (Smith 1858) (Formicidae Attini) by Canthon virens (Mannerheim 1829) (Coleoptera Scarabaeidae),” Tropical Zoology, vol. 19, no. 1, pp. 1-7, 2006. [8] R. Heymons and H. von Lengerken, “Biologische untersun- chungen an coprophagen lamellicorniern. I. Narungserwer- bund fortpflanzungsbiologie der gattung Scarabaeus L.,” Zeitschrift fur Morphologic und Oekologie der Tiere, vol. 14, pp. 513-613, 1929. [9] S. R. Rodrigues and C. A. H. Flechtmann, “Aspectos biologicos de Canthon lituratus (Germar, 1813) e Canthidium (Canthid- ium) megathopoides Boucomont, 1928 (Coleoptera, Scarabaei- dae),” Acta Zoologica Mexicana, vol. 70, pp. 1-12, 1997. [10] G. Halffter and W. D. Edmonds, “The nesting behavior of dung beetle (Scarabaeinae). An ecological and evolutive ap- proach,” Publication no. 10 Instituto de Ecologia, Mexico D. R, Mexico, 1982. [11] G. D. Tribe, “Pheromone release by dung beetles (Coleoptera, Scarabaeidae),” South African Journal of Science, vol. 71, pp. 277-278, 1975. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 134746, 24 pages doi:10.1155/2012/134746 Review Article Diversity of Species and Behavior of Hymenopteran Parasitoids of Ants: A Review Jean-Paul Lachaud^’^ and Gabriela Perez-Lachaud^ ^ Departamento de Entomologia Tropical, El Colegio de la Erontera Sur, Avenida Centenario km 5.5, 77014 Chetumal, QRoo, Mexico ^ Centre de Recherches sur la Cognition Animale, CNRS- UMR 51 69, Universite de Toulouse, UPS, 118 route de Narbonne, 31062 Toulouse Cedex 09, Trance Correspondence should be addressed to Jean-Paul Lachaud, jlachaud@ecosur.mx Received 3 October 2011; Accepted 28 October 2011 Academic Editor: Alain Lenoir Copyright © 2012 J.-R Lachaud and G. Perez- Lachaud. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Reports of hymenopterans associated with ants involve more than 500 species, but only a fraction unambiguously pertain to actual parasitoids. In this paper, we attempt to provide an overview of both the diversity of these parasitoid wasps and the diversity of the types of interactions they have formed with their ant hosts. The reliable list of parasitoid wasps using ants as primary hosts includes at least 138 species, reported between 1852 and 2011, distributed among 9 families from 3 superfamilies. These parasitoids exhibit a wide array of biologies and developmental strategies; ecto- or endoparasitism, solitary or gregarious, and idio- or koinobiosis. All castes of ants and all developmental stages, excepting eggs, are possible targets. Some species parasitize adult worker ants while foraging or performing other activities outside the nest; however, in most cases, parasitoids attack ant larvae either inside or outside their nests. Based on their abundance and success in attacking ants, some parasitoid wasps like diapriids and eucharitids seem excellent potential models to explore how parasitoids impact ant colony demography, population biology, and ant community structure. Despite a significant increase in our knowledge of hymenopteran parasitoids of ants, most of them remain to be discovered. 1. Introduction Ants are distributed all over the world, and their colonies provide both a stable food resource and numerous niches for thousands of other organisms, termed myrmecophiles, that exhibit a diverse array of relationships with their hosts [1-7]. Among myrmecophiles, numerous species of hymenopterans are associated with ants through predation, parasitism on the brood and/or adults, cleptoparasitism, parabiosis, mimetism, true symphily, or indirect parasitism through trophobionts and/or social parasites. However, in most cases, the precise nature of their relationship with their ant hosts remains obscure. A review of the diversity of parasitoid wasps attacking ants has not been attempted since the work of Schmid- Hempel [7]. In his extensive review of the parasites of social insects, he pointed out the wide variety of hymenopteran parasitoids that attack these insects but, with the exception of the family Eucharitidae (with 33 valid species really involved), his list provided very few other examples (only 10) of true parasitoidism, that is, cases where the attack of the wasp species on ants (adults or brood) has been reliably demonstrated. Knowledge has increased greatly in the intervening years, and numerous cases of parasitic associations involving wasps and ants have been reported. Moreover, changes in nomenclature and phylogeny have been numerous in the last two decades (see, e.g., [8-15]), and many species names of both the parasitoids and their ant hosts required emendations. In the present paper, we address only hymenopteran parasitoids and focus strictly on ant-parasitoid wasp asso- ciations in which parasitism has been established beyond any doubt, and where ants are proved to be the primary hosts. Therefore, no bethylid species are considered here even though various members of the genera Pseudisobrachium and 2 Psyche Dissomphalus are strongly suspected of being parasitoids of ant brood [16-18]. Neither are any species of ceraphronid, dryinid, figitid, platygastrid, proctotrupid, or pteromalid wasps considered although several species belonging to the genera Ceraphron, Conostigmus, Gonatopus, Kleidotoma, Platygaster, Exallonyx, and Spalangia are known to be associated with ants, most of them probably as parasitoids [2, 19-24] . All of these species were omitted from the present paper because wasps have not been reliably reared from ants or their brood. Moreover, according to the definition of “parasitoid” which implies the killing of a single host, asso- ciations such as those involving numerous sphecid species, particularly those of the genera. Aphilanthops, Clypeadon, and Tracheliodes, which are known to specialize with preying on and storing numerous adult ants (of the genus Formica, Pogonomyrmex, or Liometopum, resp.) [25-27] are not dealt with. Likewise, the highly interesting associations of ants with some braconid species such as Compsobraconoides sp. [28] and Trigastrotheca laikipiensis Quicke [29], which are known to consume various stages of their ant hosts {Azteca spp. and Crematogaster spp., resp.) during their development, are not covered in the present paper. In spite of such restrictions, the list of hymenopteran species reliably involved in parasitic associations with ants remains impressive and represents more than a quarter of all of the hymenopteran species known to be associated with ants [30]. Here, we attempt to provide an overview of both the diversity of the species of parasitoid wasps known to attack ants and the diversity of the interactions they have developed with their hosts. By so doing, we also call attention to this little known biodiversity. 2. Checklist of Hymenopteran Parasitoids of Ants Records of associations of hymenopteran wasps with ants involve more than 500 wasp species [30], but only a fraction have unambiguously been reported as parasitoids. The term parasitoid applies to organisms whose juvenile stages are parasites of a single host individual, eventually sterilizing, killing, or even consuming their host, while the adult parasitoid is free living [31]. With few exceptions, female parasitoid wasps oviposit on or inside the body of their host, typically another arthropod, and all stages of development of the host are susceptible to attack. After hatching, the parasitoid larva feeds on the host’s tissues, gradually killing it. A survey of the literature since 1852 and some of our own unpublished results have allowed us to identify at least 138 species (see Table 1 and Supplementary Material available online at doi: 10. 1155/2012/134746) reported as primary endo- or ectoparasitoids of larvae, pupae, or adult ants. All of these species are included in 3 superfamilies: Chalcidoidea (with 6 families concerned), Ichneumonoidea (2 families), and Diaprioidea (only 1 family) (Table 1). In 2007, Sharkey [12] estimated that there were approximately 115,000 described species of Hymenoptera (perhaps up to 1,000,000 if undescribed species — especially species of par- asitoid wasps — were included), and that Chalcidoidea and Ichneumonoidea were the most species-rich superfamilies among the parasitoid hymenopterans. So, it is not surprising that most of the parasitoid wasps attacking ants belong to these two superfamilies, especially the Chalcidoidea which alone includes more than 70% of all of wasp species parasitizing ants registered until now. In the following text, we follow Sharkey [12] for the higher-level phylogeny of the order Hymenoptera (see also [15]). The taxonomic validity of the scientific names is in accordance with Bolton [8, 9] for ants, and with different databases available on the web for other hymenopterans: Hymenoptera Name Server (version 1.5) (http://osuc.biosci .ohio-state.edu/hymDB/nomenclator.home_page) , Global Name Index (version 0.9.34) (http://gni.globalnames.org/ name_strings). Universal Chalcidoidea Database [32] (http://www.nhm.ac.uk/chalcidoids), and Home of Ichneu- monoidea (version 2011) (http://www.ichneumonoidea .name/index.php). Authors of all scientific names are given throughout the text only when they are not already reported in Table 1. 2.1. Diaprioidea. The superfamily Diaprioidea is a mono- phyletic group, with 4 recognized families [15], and accounts for more than 4000 species around the world in over 210 genera [8, 155-157], almost all in the family Diapriidae. Most diaprioids are primary endoparasitoids of dipterans (eggs, larvae, or pupae), but several species are known to attack Hymenoptera, Homoptera, or Coleoptera, and some are facultative or obligate hyperparasitoids. Some of the species attacking Diptera have been considered as potential biological control agents, but their efficiency has not been demonstrated [157, 158]. 2.1.1. Diapriidae. Despite their number, the members of this large family are relatively unknown and less than half of the 4000 species estimated to occur worldwide have been described [8, 156, 159]. Three subfamilies are currently recognized: Ambositrinae, Belytinae, and Diapriinae [15]. Their biologies are diverse, but most species are primary parasitoids ofpuparia of Diptera [156-160]. Although some diapriids have only occasionally been found in ant nests, a number of species are closely associated with ants (all belonging to the Belytinae and Diapriinae subfamilies). However, there are few behavioral data on host- diapriid myrmecophile interactions (but see [36]). These symphyles are often highly adapted to their hosts, exhibiting morphological and behavioral adaptations to living with ants (extensive morphological mimicry of the host ants — coloration, ocellus regression, convergence in sculpture — , presence of appeasement substances in specialized structures and trichomes, trophallaxis, etc., [161-166]), which presum- ably aid them in avoiding detection and/or aggression by host ants [34] . The adaptations can include secondary apterism in which the wings of the wasps are assumed to have been bitten off by either the parasite itself or its host (e.g., Mimopria, Bruchopria, Lepidopria, and Solenopsia, [156, 161, 164, 167, 168]). Most often, the presence of a diapriid in an ant nest is suspected to be just circumstantial [160] and related to its Psyche 3 Table 1: List of parasitic wasps recorded as true primary parasitoids of ants (brood or adult). As all of the eucharitids are true parasitoids of ants, all known associations with ants have been included, but see **. For further details, see text. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as Diaprioidea: Diapriidae (26) Acanthopria sp. Gyphomyrmex salvini Eorel 133] Acanthopria sp. — Trachymyrmex cf. zeteki Weber — [34] Acanthopria sp. no. 1 — Gyphomyrmex transversus Emery — 135] Acanthopria sp. no. 2 — Gyphomyrmex transversus Emery — 135] Acanthopria sp. no. 3 — Gyphomyrmex transversus Emery — [35] Acanthopria sp. no. 4 — Gyphomyrmex transversus Emery — [35] Acanthopria sp. no. 5 — Gyphomyrmex transversus Emery — [35] Acanthopria sp. no. 6 — Gyphomyrmex transversus Emery — [35] Acanthopria sp. no. 7 — Gyphomyrmex transversus Emery — [35] Acanthopria sp. no. 8 — Gyphomyrmex transversus Emery — [35] Acanthopria sp. 1 — Gyphomyrmex minutus Mayr — [36] Acanthopria sp. no. T — Gyphomyrmex rimosus (Spinola) — [36] Acanthopria sp. no. 2' — Gyphomyrmex rimosus (Spinola) — [36] Acanthopria sp. no. 3' — Gyphomyrmex rimosus (Spinola) — [36] Acanthopria sp. no. 4' — Gyphomyrmex rimosus (Spinola) — [36] Mimopriella sp. — Gyphomyrmex rimosus (Spinola) — [36] Mimopriella sp. 1 — Trachymyrmex cf. zeteki Weber — [34] Mimopriella sp. 2 — Trachymyrmex cf. zeteki Weber — [34] Oxypria sp. — Trachymyrmex cf. zeteki Weber — [34] Plagiopria passerai Huggert and Masner — Plagiolepis pygmaea (Latr.) — [37] Szelenyiopria lucens (Loiacono) Gymnopria lucens Acromyrmex ambiguus (Emery) — [38] Szelenyiopria pampeana (Loiacono) Gymnopria pampeana Acromyrmex lobicornis (Emery) — [39] Szelenyiopria sp. 1 — Trachymyrmex cf. zeteki Weber — [34] Szelenyiopria sp. 2 — Trachymyrmex cf. zeteki Weber — [34] Triehopria formicans Loiacono — Aeromyrmex lobicornis (Emery) — [40] Trichopria sp. — Acromyrmex lobicornis (Emery) — [40] Chalcidoidea: Chalcididae (2 + 2*) Smicromorpha doddi Girault Oecophylla smaragdina (Eabr.) [41,42] Smicromorpha keralensis Narendran* — Oecophylla smaragdina (Eabr.) — [43] Smicromorpha masneri Darling — Oecophylla smaragdina (Eabr.) — [44] Smicromorpha minera Girault * — Oecophylla smaragdina (Eabr.) — [42] Ghalcidoidea; Encyrtidae ( 1 ) Blanchardiscus sp. ‘?pollux Noyes Pachycondyla goeldii (Eorel) [45] Ghalcidoidea: Eucharitidae (86 + 7** + 1 ***) Ancylotropus manipurensis — Gamponotus sp.*** — [11,46] Ancylotropus sp. — Odontomachus troglodytes Santschi — [11] Athairocharis vannoorti Heraty — Anoplolepis sp. Anaplolepis sp. [11] Austeucharis fasciiventris (Brues) Psilogaster fasciiventris Myrmecia gulosa (Eabr.) — [47] Austeucharis implexa (Walker) — Myrmecia pilosula E. Smith — [11] 4 Psyche Table 1: Continued. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as Austeucharis myrmeciae (Forel) Eucharis myrmeciae Cameron Myrmecia forficata (Fabr.) — [48] Austeucharis sp. — Myrmecia pavida Clark M. atrata Clark [49, 50] — Myrmecia nigriceps Mayr M. nigriceps Smith [49, 50] — Myrmecia pilosula F. Smith — 150] Epimetagea sp. Myrmecia pyriformis F. Smith — [51] — Myrmecia tarsata F. Smith — [50] — Myrmecia vindex F. Smith M. vindex Eorel [50] Chalcura affinis (Bingham) Rhipipallus ajfinis Odontomachus ruficeps F. Smith 0. ruficeps subsp. coriarius Mayr [52] Chalcuroides versicolor Odontomachus sp. Myrmecia sp. [53, 54] Girault Chalcura deprivata (Walker) — Odontomachus haematodus (L.) 0. haematodes [55] Chalcura nigricyanea (Girault) — Rhytidoponera metallica (F. Smith) R. metallicum [11] Chalcura polita (Girault) — Rhytidoponera metallica (F. Smith) R. metallicum [11] Chalcura sp. — Eormica rufa L. — [56] Chalcura sp. nr. polita (Girault) — Rhytidoponera chalybaea Emery — [11] Dicoelothorax platycerus Ashmead — Ectatomma brunneum F. Smith — [57] Dilocantha lachaudii Heraty — Ectatomma tuberculatum (Olivier) — [58, 59] Eucharis adscendens (Fabr.) — Eormica Icunicularia Latr.** E glauca Ruzsky [60] — Eormica rufa L. — [61] — Messor barbarus (L.)** Aphaenogaster barbara L. [62] Eucharis bedeli (Cameron) — Cataglyphis bicolor (Fabr.)*** C. viaticus [63] Chalcura bedeli Cataglyphis viaticus (Fabr.) Myrmecocystus viaticus [64, 65] Chalcura bedeli Eormica rufa L.*** — [61,65] Eucharis esakii Ishii E. scutellaris Gahan Eormica japonica Motschoulski E fusca fusca japonica Mots. [66] E. scutellaris Gahan Eormica sp. — [55] Eucharis microcephala Boucek — Cataglyphis nodus (Brulle) C. bicolor ssp. nodus [67] M. barbarus r. Eucharis punctata Forster — Messor concolor Santschi** semirufus v. concolor [68] Sm. Eucharis rugulosa Gussakovskiy — Cataglyphis sp.** — [60] Eucharis shestakovi Gussakovskiy — Messor structor (Latr.)** — [69] Eucharis sp. — Eormica neorufibarbis Emery** E fusca neorufibarbis [70] — Myrmica incompleta Provancher** M. brevinodis Emery [70] Galearia latreillei (Guerin-Meneville) Thoracantha bruchi Pogonomyrmex cunicularius Mayr** P carnivora Santschi [11,71] Gollumiella longipetiolata Hedqvist — Paratrechina sp. — [72] Hydrorhoa sp. striaticeps KielFer complex — Camponotus maculatus (Eabr.) C. maculatus Mayr [11] Isomerala coronata (Westwood) Isomaralia coronata Ectatomma tuberculatum (Olivier) — [73] — Ectatomma ruidum Roger*** — [11] Kapala atrata (Walker) K. surgens Pachycondyla harpax (Eabr.) — [11] Kapala cuprea Cameron — Pachycondyla crassinoda (Latr.) — [74] Kapala floridana (Ashmead) — Pogonomyrmex badius (Latr.)** — [70] Kapala iridicolor (Cameron) K sulcifacies (Cameron) Ectatomma ruidum Roger — [75, 76] Psyche 5 Table 1: Continued. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as — Gnamptogenys regularis Mayr — [76] — Gnamptogenys striatula Mayr — [76] — Gnamptogenys sulcata (F. Smith) — [76] — Pachycondyla stigma (Fabr.) — [76] Kapala izapa Carmichael — Ectatomma ruidum Roger — [76] Kapala sp. — Dinoponera lucida Emery — [77] — Ectatomma brunneum F. Smith — [78] — Ectatomma tuberculatum (Olivier) — [79] — Gnamptogenys sulcata (F. Smith) — [80] — Gnamptogenys tortuolosa (F. Smith) — [78] — Hypoponera nitidula (Emery) — [81] — Odontomachus bauri Emery — [11] — Odontomachus brunneus (Patton) — [80] — Odontomachus haematodus (L.) — [77] — Odontomachus hastatus (Fabr.) — [11] — Odontomachus insularis Guerin-Meneville 0. haematodes insularis pollens Wheeler [66] — Odontomachus laticeps Roger — [80] — Odontomachus mayi Mann — [78] — Odontomachus meinerti Forel — [81] — Odontomachus opaciventris Forel — [80] — Pachycondyla apicalis (Latr.) — [80] — Pachycondyla harpax (Fabr.) — [81] — Pachycondyla stigma (Fabr.) — [81] — Pachycondyla verenae (Forel) — [78] — Typhlomyrmex rogenhoferi Mayr — [81] Odontomachus insularis Guerin-Meneville 0. haematodes Kapala terminalis Ashmead — insularis pollens Wheeler [66] Lophyrocera variabilis Torrens, Heraty and Fidalgo — Gamponotus sp. — [82] Mateucharis rugulosa Heraty — Gamponotus sp. — [11] Neolosbanus gemma (Girault) — Hypoponera sp. — [83] Neolosbanus palgravei (Girault) — Hypoponera sp. — [83] Obeza floridana (Ashmead) — Gamponotus floridanus (Buckley) C. abdominalis floridanus [84] Orasema aenea Gahan — Solenopsis quinquecuspis Forel — [85] Orasema argentina Gemignani — Pheidole nitidula Santschi P strobeli misera Snts. [71] Orasema assectator Kerrich — Pheidole sp. — [86, 87] Orasema coloradensis Wheeler 0. coloradensis Ashmead Diplorhoptrum validiusculum (Emery) Solenopsis molesta validiuscula [70] 0. coloradensis Gahan Pormica areas comptula Wheeler — [88] 0. coloradensis Gahan Eormica subnitens Creighton — [88] 0. coloradensis Ashmead Pheidole bicarinata Mayr P. vinelandica Eorel [70] Orasema costaricensis Wheeler and Wheeler — Pheidole flavens Roger — [63] 6 Psyche Table 1: Continued. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as — Pheidole vallifica Forel — [89] Orasema fraudulenta (Reichensperger) Psilogaster fraudulentus Pheidole megacephala (Fabr.) — [90] Orasema minuta Ashmead — Pheidole nr. tetra Creighton — [11,83] — Temnothorax allardycei (Mann) — [11,83] Orasema minutissima Howard — Wasmannia auropunctata (Roger) — [91] — Wasmannia sigmoidea (Mayr) — [92] Orasema monomoria Heraty — Monomorium sp. — [93] Orasema occidentalis Ashmead — Pheidole pilif era (Roger) — [94] Orasema pireta Heraty — Solenopsis sp. — [85] Orasema rapo (Walker) — Eciton quadriglume (Haliday)** — [83] Orasema robertsoni Gahan — Pheidole dentata Mayr — [95] Orasema salebrosa Heraty — Solenopsis invicta Buren — [85] — Solenopsis richteri Forel — [96] Orasema simplex Heraty — Solenopsis invicta Buren — [97] — Solenopsis macdonaghi Santschi — [85] — Solenopsis quinquecuspis Forel — [85] — Solenopsis richteri Forel — [96] Orasema simulatrix Gahan — Pheidole desertorum Wheeler — [98] Orasema sixaolae Wheeler and Wheeler — Solenopsis tenuis Mayr — [63] Orasema sp. B1 nr. bakeri Solenopsis geminata (Fabr.) — [83] B1 nr. bakeri Solenopsis xyloni MacCook — [83] Orasema sp. B2 nr. bakeri Pheidole nr. californica Mayr — [83] B2 nr. bakeri Pheidole nr. clementensis Gregg — [83] B2 nr. bakeri Pheidole sp. — [83] B2 nr. bakeri Tetramorium sp. — [83] Orasema sp. Cl nr. costaricensis Pheidole dentata Mayr — [83] Orasema sp. — Pheidole bilimeki Mayr P anastasii Emery [99] Orasema sp. — Pheidole paiute Gregg — [94] Orasema sp. nr. bouceki Heraty — Pheidole sp. — [83] Orasema sp. uichancoi-group — Pheidole sp. — [93] Orasema susanae Gemignani — Pheidole nr. tetra Creighton — [83] Orasema tolteca Mann — Pheidole hirtula Forel P vasleti var. acohlma [100] Orasema valgius (Walker) 0. pheidolophaga Girault Pheidole sp. — [53] Orasema wheeleri Wheeler 0. wheeleri Ashmead Pheidole ceres Wheeler — [70] 0. viridis Ashmead Pheidole dentata Mayr — [55, 70] 0. viridis Ashmead Pheidole sciophila Wheeler — [55, 70] 0. viridis Ashmead Pheidole tepicana Pergande P kingi subsp. instabilis Emery [55, 70] 0. viridis Ashmead Pheidole tepicana Pergande P carbonaria Pergande [55, 70] Orasema Worcester i (Girault) 0. doello-juradoi Gemignani Pheidole radoszkowskii Mayr P nitidula Emery [71,96] Orasema xanthopus (Cameron) — Solenopsis invicta Buren — [83, 96] — Solenopsis quinquecuspis Forel — [85] — Solenopsis richteri Forel — [101] — Solenopsis saevissima (F. Smith) — [102] Psyche 7 Table 1: Continued. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as Orasemorpha eribotes (Walker) — Pheidole sp. — [54] Orasemorpha myrmicae (Girault) — Pheidole sp. — [83] Orasemorpha tridentata (Girault) Eucaromorpha wheeleri Brues Pheidole proximo Mayr — [103] Orasemorpha xeniades (Walker) — Pheidole tasmaniensis Mayr — [83] Pogonocharis browni Heraty — Gnamptogenys menadensis (Mayr) — [11] Pseudo chalcur a gibbosa (Provancher) — Camponotus herculeanus (L.) — [46] — Camponotus laevigatus (F. Smith) — [104] — Camponotus novaeboracensis (Fitch) C. ligniperdus var. novaeboracensis [70] — Camponotus sp. Ivicinus Mayr — [104] Pseudo chalcur a nigrocyanea Ashmead — Camponotus sp. — [105] Pseudo chalcur a sculpturata Heraty — Camponotus planatus Roger — [11] Pseudometagea schwarzii (Ashmead) — Lasius neoniger Emery — [106] Rhipipalloidea madangensis Maeyama, Machida, and Terayama — Camponotus (Tanaemyrmex) sp. — [107] Rhipipalloidea mira Girault — Polyrhachis femorata F. Smith — [11] Schizaspidia convergens (Walker) — Odontomachus haematodus (F.) 0 . haematodes [55] Schizaspidia nasua (Walker) — Odontomachus rixosus F. Smith — [11] Stilbula arenae Girault — Polyrhachis sp. Cyrtomyrma sp. [54] Stilbula cyniformis (Rossi) S. cynipiformis Camponotus aethiops (Fatr.) C. marginatus Fatr. [68] Schizaspidia tenuicornis Camponotus japonicus Mayr C. herculeanus ssp. japonicus [108] Schizaspidia tenuicornis Camponotus obscuripes Mayr C. herculeanus ssp. ligniperdus v. obscuripes [66, 108] S. cynipiformis Camponotus sanctus Forel C. maculatus r. sanctus [62] Stilbula polyrhachicida (Wheeler and Wheeler) Schizaspidia polyrhachicida Polyrhachis dives F. Smith Polyrhachis (Myrmhopla) dives [109] Stilbuloida calomyrmecis (Brues) Schizaspidia calomyrmecis Calomyrmex purpureus (Mayr) — [103] Stilbuloida doddi (Bingham) Schizaspidia doddi Camponotus sp. — [52] Timioderus acuminatus Heraty — Pheidole capensis Mayr — [93] Tricoryna chalcoponerae Brues — Rhytidoponera metallica (F. Smith) Chalcoponera metallica var. critulata [103] Tricoryna ectatommae Girault — Rhytidoponera sp. Ectatomma sp. [110] Tricoryna iello (Walker) — Rhytidoponera sp. — [11] Tricoryna minor (Girault) — Rhytidoponera metallica (F. Smith) — [11] — Rhytidoponera victoriae (Andre) — [11] Tricoryna sp. nr. alcicornis (Boucek) — Rhytidoponera violacea (Forel) — [11] Zulucharis campbelli Heraty — Camponotus sp. — [11] Chalcidoidea: Eulophidae (5) Horismenus floridensis (Schaulf and Bou&k) Alachua floridensis Camponotus atriceps (F. Smith) C. abdominalis (Fabr.) [111] Alachua floridensis Camponotus floridanus (Buckley) — [111] Horismenus myrmecophagus Hansson, Lachaud, and Perez- Lachaud — Camponotus sp. ca. textor Forel — [112] 8 Psyche Table 1: Continued. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as Myrmokata diparoides Bou&k — Crematogaster sp. — [113] Pediobius marjoriae Kerrich — Lepisotia sp. Acantholepis sp. [114] Unidentified sp. {^.Horismenus) nr. Paracrias Crematogaster acuta (Fabr.) — [109, 112] Chalcidoidea: Eurytomidae (4) Aximopsis ajfinis (Brues) Conoaxima ajfinis Azteca sp. [115] Conoaxima affnis Azteca alfari Emery Azteca alfari subsp. lucidula var. canalis [116] Conoaxima affnis Azteca pittieri Forel — [117] Aximopsis aztecicida (Brues) Conoaxima aztecicida Azteca alfari Emery Azteca alfaroi [115] Conoaxima aztecicida Azteca constructor Emery — [115] Aximopsis sp. Conoaxima sp. Azteca salti Wheeler Azteca xanthochroa (Roger) subsp. salti [116] Aximopsis sp. aztecicida) Conoaxima sp. {1 aztecicida) Azteca alfari Emery — [118] Conoaxima sp. aztecicida) Azteca australis Wheeler — [118] Conoaxima sp. aztecicida) Azteca ovaticeps Forel — [119] Conoaxima sp. aztecicida) Camponotus balzani Emery — [118] Chalcidoidea: Perilampidae (1) Unidentified sp. Pachycondyla luteola (Roger) [119] Ichneumonoidea: Braconidae (11 + 4*) Elasmosoma berolinense Ruthe — Camponotus spp. — [120] — Camponotus vagus (Scopoli) — [121] — Pormica fusca L. — [48] — Eormica japonica Motschoulsky — [122] — Eormica pratensis Retzius — [123] — Eormica rufa L. — [124-126] — Eormica sanguinea Latr. — [48] — Eormica spp. — [120] — Lasius niger (L.) — [48, 56] — Polyergus sp. — [127] Elasmosoma luxemburgense Wasmann — Pormica rufibarbis Fabr. — [128, 129] Elasmosoma michaeli Shaw — Pormica obscuripes Forel — [130] E. sp. nr. pergandei Ashmead Pormica obscuriventris clivia Creighton — [131] Elasmosoma pergandei Ashmead* — Camponotus castaneus (Latr.) C. melleus (Say) [132] — Pormica integra Nylander — [126] — Pormica subsericea Say — [126] Elasmosoma petulans Muesebeck* — Pormica integra Nylander — [133] — Pormica opaciventris Emery — [127, 133, 134] — Pormica pergandei Emery P. rubicunda Emery [133, 134] — Pormica rubicunda Emery*** — [127, 133, 134] — Pormica subintegra Wheeler P. subintegra Emery [133] — Pormica subsericea Say — [133] Psyche 9 Table 1: Continued. Hymenopterous parasitoids Associated ant host References Species Referred to as Species Referred to as Elasmosoma schwarzi Ashmead* — Pormica schaufussi Mayr — [127] — Polyergus lucidus Mayr — [127] Elasmosoma vigilans Cockerell — Pormica perpilosa Wheeler — [94] — Pormica subpolita Mayr — [135] Elasmosomites primordialis Brues — Lasius sp. {hchiejferdeckeri Mayr) — [136] Kollasmosoma marikovskii (Tobias) — Pormica pratensis Retzius — [137] Kollasmosoma platamonense (Huddleston) Elasmosoma platamonense Cataglyphis bicolor (Fabr.) — [127] — Messor semirufus (Andre) — [138] Kollasmosoma sentum van Achterberg and Gomez — Cataglyphis ibericus (Emery) — [129] Neoneurus auctus (Thomson) Euphorus bistigmaticus Morley Pormica pratensis Retzius — [139, 140] Euphorus bistigmaticus Morley Pormica rufa L. — [139, 140] Neoneurus clypeatus (Forster)* Elasmosoma viennense Giraud Pormica rufa L. — [141] Neoneurus mantis Shaw — Pormica podzolica Francoeur — [142, 143] Neoneurus vesculus van Achterberg and Gomez — Pormica cunicularia Latr. — [129] Ichneumonoidea: Ichneumonidae (3 + 2*) Eurypterna cremieri (de Romand) Pachylomma cremieri Pormica rufa L. — [144] Pachylomma cremieri Pasius fuliginosus (Latr.) Pormica fuliginosa [145-148] — Pasius niger (L.) — [123] Pachylomma cremieri Pasius nipponensis Forel — [149] Ghilaromma fuliginosi (Donisthorpe and Wilkinson) * Paxylomma fuliginosi Pasius fuliginosus (Latr.) — [150, 151] Hybrizon buccatus (Brebisson) Pachylomma buccata Pormica rufa L. P. rufa var. rufo -pratensis [152] Pachylomma buccata Pormica rufibarbis Fabr. — [152] Pachylomma buccata Pormica sanguinea Latr. — [152] Pachylomma buccata Nees Pasius alienus (Forster) Donisthorpea aliena [24] Pachylomma buccatum Pasius brunneus (Latr.) — [144] Pachylomma buccata Pasius flavus (Fabr.) — [152] — Pasius grandis Forel — [129] Pachylomma buccata Pasius niger (L.) — [140] Pachylomma buccata Nees Myrmica lobicornis Nylander — [24] Pachylomma buccata Nees Myrmica ruginodis Nylander — [24] Pachylomma buccata Myrmica scabrinodis Nylander — [153] Pachylomma buccata Tapinoma erraticum (Latr.) — [152] Hybrizon rileyi (Ashmead)* — Pasius alienus (Forster) — [154] Unidentified Hybrizontinae (gen. nov. sp. nov.) — Myrmica kotokui Forel — [149] * : attack was not observed, but there is strong evidence that all of the species of this genus reported as associated with an ant species are true primary parasitoids of this host. **: uncertain report of association with the host (e.g., ants of the genera Pogonomyrmex and Messor do not have cocoons — contrary to what is reported in the original reference — and were probably misidentified), uncertain identification of the ant host (ambiguity between 2 or more species), or wasps not found directly within the nest of the presumed host (e.g., found near a nest — perhaps only by chance — or found on refuse deposit — perhaps as a prey — ). *** : erroneous report (misidentification of either the parasitoid or the ant host), or erroneous emendation of the host species. 10 Psyche Figure 1: Winged females of the diapriid wasp, Plagiopria passerai (white pointer) in a nest of the formicine ant Plagiolepis pygmaea, just after emergence from queen pupae. Photos courtesy of L. Passera. search for dipterous hosts, such as Tetramopria aurocincta Wasmann found in nests of Tetramorium caespitum (L.) [128]. This wasp is in fact a parasitoid of the puparia of Compsilura concinnata Meigen (Diptera: Tachinidae), a pri- mary parasite of the lepidopteran Hyphantria cunea (Drury) [160]. Occasionally, diapriids enter ant nests for temporary shelter since some species hibernate in the host nest as do Solenopsia imitatrix Wasmann and Lepidopria pedestris Kieffer in the nests of Solenopsis fugax (Latr.) [37, 164]. Only a few diapriids are true parasitoids of ant brood. Ever since the pioneering work of Wasmann in 1899 [128], most diapriids found in ant nests were assumed either to parasitize insect myrmecophiles (dipteran or coleopteran) inside the host nest or, less frequently, to be primary parasitoids of ant larvae. However, the first record of a diapriid positively reared from ant brood was reported just in 1982 by Lachaud and Passera [37], who reared Plagiopria passerai from cocoons of queens of the formicine Plagiolepis pygmaea (Figures 1(a) and 1(b)). As far as known, diapriid parasitoids attacking ants develop as solitary or gregarious, koinobiont endoparasitoids of the host larvae [34, 36, 38, 169], and worker and/or reproductive immature stages can be parasitized [37, 169, 170]. Ramos-Lacau et al. [35] observed oviposition of Acanthopria sp. in young ant larvae under laboratory conditions. Late parasitized larvae are easily recognized by their dark coloration, compared to nonparasitized larvae, due to the developing wasp visible through the cuticle [35, 36, 38]. Worker ants do not discriminate between parasitized and nonparasitized larvae [35, 38, 169], but adult parasitoids are aggressively attacked by their hosts under laboratory conditions [35, 36]. From the 121 diapriine species in 34 genera that have been collected in association with ants [30], development of immature stages as parasitoids of ant larvae has been demonstrated for only 26 species in 7 genera, most of which are only known at the level of morphospecies (Table 1): 15 species of Acanthopria, 3 of Mimopriella, 1 of Oxypria, 1 of Plagiopria (P. passerai), 4 of Szelenyiopria, and 2 of Trichopria (T. formicans and Trichopria sp.) [34-38, 169, 170]. The ant hosts of these diapriines belong to 8 species in only 4 genera: the myrmicine fungus-growing ants Cyphomyrmex, Trachymyrmex, and Acromyrmex and the formicine Plagi- olepis. Fifteen species of Belytinae belonging to 1 1 genera have also been reported from ant nests [30, 171-173], but none has been reliably reared from the ants, and their actual relationship with their hosts remains unknown. In some cases, the rate of parasitism can reach high levels. Two recent studies have provided important details of the biology of diapriids and have also investigated their impact on ant-host populations. Fernandez-Marin et al. [36] found that between 27 and 70% of the colonies of 2 species of Cyphomyrmex were parasitized by one species in Puerto Rico and by up to 4 concurrent morphospecies of diapriids in Panama. Similarly, the work of Perez-Ortega et al. [34] showed that another fungus-growing ant, Trachymyrmex cf. zeteki, was attacked by a diverse community of diapriids in Panama, with a mean intensity of larval parasitism per ant colony of 33.9%, and a prevalence across all ant populations of 27.2% (global data for all 6 diapriid morphospecies present at the study site). 2.2. Chalcidoidea. The superfamily Chalcidoidea is consider- ed as one of the most abundant, species-rich, and biologically diverse groups of insects with 23,000 species described and a conservative estimation of about 400,000 to 500,000 species in over 2040 genera distributed in 19 families [32, 174-178]. Though some species are phytophagous, most Chalcidoidea are parasitoids of other insects, and numerous species are currently used as biological control agents against insect pests. 2.2.1. Chalcididae. Chalcididae is a moderate-sized family with more than 1450 species and over 85 genera. Chalcids are primary parasitoids of Lepidoptera or, to a much lesser extent, of Coleoptera, Diptera, Hymenoptera, and Neu- roptera, and various species are hyperparasitoids of other hymenopterous parasitoids [179]. Most often they parasitize host larvae or pupae, but a few species can parasitize eggs. Very few species, like Epitranus chilkaensis (Mani) (referred to as Anacryptus chilkaensis) found with the formicine Camponotus compressus (Fabr.) in the Barkuda Island (India) [180], are known to be associated with ants [179, 181], but true parasitoidism has rarely been documented. Only species of the genus Smicromorpha seem to be specialized as parasitoids of the larvae of the green ant, Oecophylla smaragdina. The only unquestionable (see [44]) record of parasitoidism is that of Dodd in the early 20th century, describing Smicromorpha doddi in North Psyche 11 Queensland (Australia) parasitizing larvae of this weaver ant, “depositing eggs upon them when the workers are using their silk-spinning larvae for the purpose of binding the leaves together when building a new nest” [41]. No other example of true parasitoidism has ever been quoted for the genus Smicromorpha but, more recently, adults of another species of this genus, S. masneri, were reported emerging from O. smaragdina nests collected in Vietnam and maintained in controlled green-house conditions in the USA, which strongly suggests that these wasps are also primary para- sitoids of weaver ants [44]. Moreover, two other species, S. keralensis [43] and S. minera [42], have been observed hovering over nests of O. smaragdina in India and Australia, respectively, a behavior likely to be related to parasitism of ants (see below under Braconidae and Ichneumonidae). For such reasons, all these members of the genus Smicromorpha can reasonably be suspected of being true parasitoids of the larvae of this ant host and were included in our list (Table 1). 2.2.2. Encyrtidae. Encyrtidae is a large family of parasitic wasps, currently including more than 460 genera and 3700 species, and is one of the key chalcidoid families for the biological control of insect pests [178, 182, 183]. Most encyrtids are primary endoparasitoids of immatures or, less commonly, adults of Coccidae and Pseudococcidae; others are hyperparasitic through other hymenopterous parasitoids, and some can attack insects in other orders, mites, ticks, or spiders [184, 185]. Some species are polyembryonic, a single egg multiplying clonally in the host, producing large numbers of identical adult wasps. At least 25 species of encyrtid wasps representing 16 genera are known to be indirectly associated with ants through primary parasitism of the trophobionts they exploit and protect [32]; for example, the species Anagyrus ananatis Gahan is indirectly associated with the ant Pheidole mega- cephala through the trophobiotic Pseudococcidae present in their nest [186]. However, very few encyrtids have been reported as directly associated with ants. Apart from Taftia prodeniae Ashmead, which was found to exhibit a phoretic association (wasps were found clinging to the ant’s antennae) with the dolichoderine ant Dolichoderus thoracicus (F. Smith) (referred to as D. bituberculatus (Mayr)) [187], and an unidentified species recently reported from a refuse deposit of the ecitonine ant Eciton burchellii [188], only Holcencyrtus wheeleri (Ashmead) (referred to as Pheidoloxenus wheeleri), found in nests of the myrmicine ants Pheidole tepicana Pergande (referred to as P. instabilis) [70] and P. ceres Wheeler (referred to as P. ceres var. tepaneca Wheeler) [100], has been suspected of being “probably also entoparasitic on these ants or their progeny during its larval stages” [1]. However, the parasitic relationship was never proved. Only very recently a Neotropical, gregarious endoparasitoid species, Blanchardiscus sp. {Ipollux) (determination by J. S. Noyes), was recorded from French Guiana attacking pupae of the ponerine ant Pachycondyla goeldii [45] and thus constitutes the first true case of parasitism on ants for this family. However, no information has yet been published, and the exact identification of the species still needs to be confirmed. 2.2.3. Eucharitidae. This is a small family but the largest and most diverse group of hymenopteran parasitoids attacking ants since all of its members, where the host is known, parasitize ant brood [11, 66, 72, 78, 83, 189-191]. Fifty-three genera and more than 470 species are currently described and distributed in three subfamilies: Oraseminae, Eucharitinae, and Gollumielinae. All of the species have a highly modified life cycle [63, 66, 76, 83, 108]. Like the Perilampidae [191] and the ichneumonid species Euceros frigidus [192], but unlike most parasitic wasp species, eucharitid females deposit their eggs away from the host nest, in or on plant tissue (leaves and buds) [72, 189] (Figures 2(a) and 2(b)), and the very active, minute (less than 0.13 mm), strongly sclerotized first- instar larva is termed a “planidium” (Supplementary material 2 available online at doi: 10. 1155/2012/134746). It is responsible for gaining access to the host ant brood by using various phoretic behaviors including either attachment to an intermediate host (as in some orasemine species [11, 72, 83, 86, 88, 93] and, possibly, in Gollumiella antennata (Gahan) ([190] but see [72]) or, more generally, to foraging ant workers. On occasions (as is apparently the case for Pseu- dochalcura gibbosa and Gollumiella longipetiolata), attractive substances are suspected to be present in or on the eggs [46, 72]. Within the nest, the planidium attaches itself to an ant larva (Figures 2(c) and 2(d)): Eucharitine planidia attach externally to the host larva, whereas orasemine and gollumielline planidia partially burrow into the host larva, in the thoracic region just posterior to the head capsule [11, 70, 72]. All of the Eucharitidae develop as koinobiont, larval-pupal ectoparasitoids. At molting of the host larva, the planidium migrates to the ventral region, just under the legs (Eigure 2(e)), of the newly formed ant pupa for further development which is only completed when the host pupates [76, 83, 93, 189] (Supplementary material 3 available online at doi: 10. 11 55/20 12/ 134746). In general, only one parasitoid develops per host but, occasionally, more than one adult eucharitid can develop in a single host (superparasitism) (Eigure 2(f)) [72, 83], especially when larger brood (sexual brood) is parasitized [193, 194], and one exceptional case of multiparasitism involving two different species from two different eucharitid genera {Dilocantha lachaudii and Isomerala coronata) has even been reported from a single pupa of the ectatommine ant Ectatomma tuberculatum [79]. In almost all of the cases, adults emerge among ant brood (but see [77] ), and, even if in some cases they are well treated within the nest by their hosts (as is the case for Orasema coloradensis which is transported, cared for, and even fed by the workers of Pheidole bicarinata [70]), they have to leave the host nest to reproduce. Ants show only moderate aggression to newly emerged eucharitids [58, 70, 75, 106, 189, 195, 196], suggesting passive or active chemical mimicry of the host ants [58, 75, 195]. If the parasitoid wasps do not exit their host nest by themselves, ant workers transport them outside (Eigure 2(g)) as if they were refuse [58, 77, 196], ultimately enhancing wasp dispersal. Parasitism is very variable and localized in time and space [106, 193, 194]. A very high local prevalence may lead to only a low impact at the regional scale, suggesting that these parasitoids do not 12 Psyche (d) (e) Figure 2: Life cycle of a typical eucharitid wasp, (a) Female Dilocantha lachaudii ovipositing on Lantana camara L. (Verbenaceae). (b) D. lachaudii female with eggs scattered on leaf surface, (c) Planidium (white pointer) attached upon an Ectatomma tuberculatum larva. Insert: SEM picture of a planidium. (d) Two D. lachaudii swollen planidia (white pointers) feeding upon an E. tuberculatum larva, (e) 2nd instar larva (white pointer) relocated after host pupation, (f) Two D. lachaudii pupae from a single host pupa. The host cocoon has been removed, (g) E. tuberculatum worker transporting a recently emerged D. lachaudii female. Photos: J.-P. Lachaud and G. Perez-Lachaud. have a major influence on the dynamics of their ant host population [194]. According to Heraty [11], the hypothesized phylogeny of Eucharitidae is highly correlated with the subfamilies of their ant hosts and responsible for differences in behavior related with egg placement, activity of the planidium, and access to the ant host. Oraseminae {Orasema, Orasemorpha, and Timioderus) primarily attack myrmicine ants (numerous species of Pheidole and Solenopsis, and some species of Diplorhoptrum, Monomorium, Temnothorax, Tetramorium, and Wasmannia, see Table 1), and exceptionally formicines {Formica subnitens and F. areas comptula in the case of Psyche 13 O. coloradensis, [88]) or ecitonines {Eciton quadriglume in the questionable case of O. rapo, [11, 83]). For Eucharitinae, the only two host records for the tribe Psilocharitini {Neol- oshanus) concern the ponerine genus Hypoponera [83], while the numerous members of the tribe Eucharitini are essen- tially parasitic on medium to large ponerines {Pachy- condyla, Odontomachus, and Dinoponera) and ectatommines {Ectatomma, Gnamptogenys, Typhlomyrmex, and Rhytido- ponera), but also on myrmeciines {Myrmecia) and numerous formicines {Anoplolepis, Calomyrmex, Camponotus, Catagl- yphis, Eormica, Easius, and Polyrhachis); without exception, all of the scarce records of associations of eucharitines with myrmicine ants {Messor, Myrmica^ and Pogonomyrmex) are highly doubtful (Table 1). Finally, the only host record for the Gollumiellinae concerns a formicine (Paratrechina) . The hosts of most eucharitid genera seem to be restricted to only one or a few closely related ant genera and, for a long time, all species were considered as host-specific parasitoids, at least at the host genus level [83]. Flowever, recent results [76, 78, 79] raised questions concerning the degree of host specificity in eucharitids and about the factors that determine the association of these parasitoids and their hosts. Results in the guild of eucharitid parasitoids associated with ponerine ant species in southeastern Mexico and French Guiana suggest that some eucharitid wasps tend to be oligophagous in their host choice: some eucharitid species can attack different hosts from different genera and different subfamilies such as Kapala iridicolor, which parasitizes one species of Ectatomma, two of Gnamptogenys, and one of Pachycondyla [76, 78]. Furthermore, concurrent parasitism has been reported for Ectatomma tuberculatum, which is simultaneously parasitized by Dilocantha lachaudii, Isomerala coronata, and Kapala sp. [79], or for E. ruidum parasitized by two Kapala species, K iridicolor, and K izapa [76, 193]. 2.2.4. Eulophidae. The family Eulophidae is the largest of the Ghalcidoidea with up to 4470 species in 297 genera. The majority of the species are primary parasitoids attacking a large variety of insects (mainly Lepidoptera and Goleoptera, but also Diptera, Thysanoptera, and Flymenoptera), and occasionally mites or spiders. Many species are facultative or obligate hyperparasitoids of other Hymenoptera, and some are even phytophagous. Entomophagous larvae can develop as koino- or idiobionts, gregarious or solitary, and ecto- or endoparasitoids, and according to the species, eulophids can attack eggs, larvae, pupae, or even the adults of their hosts [197]. Despite the large number of species in this family, parasitization of ants is uncommon among Eulophidae, and only few associations involving eulophid wasps and ant hosts have been reported to date. Almost all are from genera belonging to the subfamily Entedoninae. Three concern species indirectly associated with ants as they parasitize insects living in ant nests: Pediobius acraconae Kerrich which has been reported [114] from a last instar larva of the pyralid lepidopteran Acracona remipedalis Karsch found in a nest of Grematogaster depressa (Latr.) or C. africana Mayr in Nigeria, and both Micro donophagus woodleyi Schauff in Panama Figure 3: Larva of the neotropical weaver ant Camponotus sp. ca. textor parasitized by the gregarious endoparasitoid Horismenus myrmecophagus (Eulophidae). Several wasp larvae can be observed through the host cuticle. Photo: G. Perez-Lachaud. and Horismenus microdonophagus Hansson et al. in Mexico, which parasitize larvae of Microdon sp. syrphid flies living in nests of the dolichoderine Technomyrmex fulvus (Wheeler) (referred to as Tapinoma fulvum) [198] and of the formicine Gamponotus sp. ca. textor [112], respectively. Three other species (two Entedoninae and a Tetrastichinae) have been reported associated with ant nests, but direct parasitism on the ant brood was not clearly established in any of these cases: Myrmobomyia malayana Gumovsky and Boucek with nests of an ant species of the genus Dolichoderus in Malaysia [ 199] , an unidentified species of Horismenus from the bivouac and refuse deposits of the army ant Eciton burchellii [188], and an unidentified species of Tetrastichus from a nest of the formicine Myrmecocystus mexicanus Wesmael in Nevada [94]. In fact, only five species are known as true primary parasitoids of ants (Table 1). An unidentified gregarious parasitoid, apparently closely related to the genus Paracrias (according to Gahan in [109]), possibly Horismenus sp. [112], was recorded parasitizing larvae of the myrmicine Grematogaster acuta in Guyana, the prepupae of another unidentified species of Grematogaster were parasitized by Myrmokata diparoides [113] in Cameroon, Pediobius marjo- riae was reared from cocoons of the formicine ant Eepisiota sp. in Uganda [114], and two species of Horismenus, H. floridensis and H. myrmecophagus, were found parasitiz- ing the pupae of Gamponotus atriceps and C. floridanus in Florida [111], and of the weaver ant Camponotus sp. ca. textor in Mexico [112], respectively. In the latter two cases, Horismenus larvae develop as gregarious endoparasitoids of the ant larvae (Figure 3), and large numbers of parasitoid individuals can develop from the same host: up to 21 for H. floridensis and between 4 and 12 for H. myrmecophagus. Finally, two other cases deserve to be added to this list since two other ant species have recently been found parasitized by eulophids: the ponerine ant Pachycondyla crenata (Roger) in Mexico and an unidentified species of Camponotus {Den- dromyrmex) in French Guiana [112]; however, the identity of the parasitoids has not been confirmed yet. 14 Psyche 2.2.5. Eurytomidae. Eurytomidae is a moderate-sized family with 90 genera and at least 1400 nominal species [13, 32, 200, 201]. Eurytomid wasps exhibit a wide range of biologies, but most of the larvae are endophytic either as seed or plant stem eaters or as parasitoids of gall formers or other phytophagous insects. Most species are primary or secondary parasitoids, attacking eggs, larvae, or pupae of various arthropods (Diptera, Coleoptera, Elymenoptera, Eepidoptera, Orthoptera, and Araneae). A few species have been reported as indirectly associated with ants, like Eurytoma rosae Nees von Esenbeck found with Easius flavus and Eurytoma sp. found with Eormica (?) rufibarbis (misidentified as Polyergus rufibarbis) [20], but most probably these eurytomids only fed on the gall- forming cynipid larvae and/or on the gall tissue on Rosa spp. which are visited by these ant species, without any direct relationship with the ants. Recently, various adults of a new genus and species, Camponotophilus delvarei Gates, were found within nests of the weaver ant Camponotus sp. ca. textor [202], but the exact nature of their relationship with the ants remains unclear. As a matter of fact, only 3 or 4 species from the single genus Aximopsis (see Table 1) have been reported from Guatemala, Gosta Rica, Guyana, Golombia, and Peru as parasitoids of queens of various species of dolichoderine ants {Azteca alfari, A. australis, A. constructor, A. pitieri, A. ovaticeps, and A. salti) and one formicine {Camponotus balzani), all of which colonize Cecropia spp. internode chambers by chewing a hole through a prostoma and entering the internode. The parasitoids attack only founding queens and feed on their host, while the internode chamber is sealed with parenchyma scraped from the internal stem walls [115, 116, 118]; there is never more than one wasp larva or pupa per foundress ant [117]. Queen parasitization was thought to occur before they entered their dwellings (Bailey, in [115]); however, as suggested by Davidson and Eisher [119], the location of the ant host may occur through searching for host plants since female Aximopsis were observed to visit various seedlings, where they inspected newly sealed prostoma. This fact has been confirmed recently. A picture of an A. affinis female ovipositing through a prostoma into an Azteca queen at La Selva Biological Station, Gosta Rica, was provided by Weng et al. [203] (their Eigure 16). In this site, among the internodes that harbored Azteca ants, 43% contained dead queens, of which 13% contained A. affinis [203]. 2.2.6. Perilampidae. Perilampidae is a small family closely related to the Eucharitidae, composed of up to 270 species from 15 genera. A feature shared with Eucharitidae is that the first-instar larva, the “planidium”, is responsible for gaining access to the host, rather than the egg-laying female [191]. Most species are hyperparasitoids on ichneumonid wasps or tachinid flies which are primary parasitoids of Hymenoptera or Eepidoptera, or parasitoids of wood-boring platypodid and anobiid beetles, and some species can attack Orthoptera, Neuroptera, or Elymenoptera [190, 204]. Association of perilampids with ants seems extremely casual. The only report deals with an unidentified species from Peru found parasitizing cocoons of the ponerine ant Pachycondyla luteola, inhabiting internode chambers of a Cecropia, with as many as nine perilampid wasps emerging from a single pupa of this ant [119]. Eiowever, no other details were ever published, and the species apparently remained undescribed. 2.3. Ichneumonoidea. The superfamily Ichneumonoidea, with only two extant families, accounts for more than 40,000 species around the world, and there are estimated to be approximately 100,000 species [205-207]. Most are primary ecto- or endoparasitoids, idio- or koinobionts, especially attacking immature stages of a wide variety of insects and arachnids, and more occasionally adults. Some members use many different insects as hosts, and others are very specific in host choice. Various ichneumonoids are successfully employed as biological control agents in controlling insect pests such as flies or beetles. 2.3. L Braconidae. This is a very large family with 48 sub- families, more than 1050 genera and about 17,600 described species worldwide and exhibiting a variety of biologies [207-209]. The total number of species is estimated to be 40-50,000. Many braconids parasitize nymphal stages of Hemiptera, Isoptera, and Psocoptera; a few genera also par- asitize adult Coleoptera and EEymenoptera [209]. Two major lineages occur within the Braconidae: (a) the cyclostome braconids, most of which are idiobiont ectoparasitoids of concealed Eepidoptera and Coleoptera larvae although many are koinobiont endoparasitoids of Diptera and Hemiptera, and (b) the noncyclostome braconids which are all endopar- asitoids, and most generally koinobionts, typically attacking an early instar of their hosts (see [210] for a comprehensive overview of their biology). Numerous braconid species have been reported in asso- ciation with ants. Some, such as Compsobraconoides sp. [28] and Trigastrotheca laikipiensis [29], are predatory on several developmental stages of ants. Others, such as Aclitus sappaphis Takada and Shiga found in nests of Pheidole fervida Smith [211, 212], Paralipsis enervis (Nees von Esenbeck) found with Easius niger [213], or P. eikoae (Yasumatsu) found with E. japonicus Santschi (referred to as E. niger (L.)) and E. sakagamii Yamauchi and Hayashida [212, 214], are in fact primary parasitoids of root aphids and can only be considered as indirectly associated with the aphid-attending ants; however, they have developed highly sophisticated rela- tionships with their hosts involving chemical mimicry and chemical and tactile communication to obtain regurgitated food (trophallaxis). Eor several other species, the exact nature of the asso- ciation with the ant host has not been clearly established, but at least 15 euphorine species can be considered as true parasitoids of adult ants even if direct evidence of oviposition has been obtained for only 11 of them (see Table 1). All of these parasitoids are grouped in three extant genera, Elasmosoma, Kollasmosoma, and Neoneurus, and one fossil genus, Elasmosomites, all belonging to the tribe Neoneurini. Evidence from Eocene Baltic amber, as demonstrated from an individual of Elasmosomites primordialis emerging from the abdomen of a Easius worker (Eigure 4(a)), indicates that Psyche 15 the parasitoid association between neoneurine braconids and ants has been in existence for at least 40 million years [136]. Although oviposition into the abdomen of adult worker ants has been reported on several occasions [56, 120, 121, 126, 127, 140], detailed descriptions were rare and, until recently, restricted to only two species. In the case of N. mantis attacking Formica podzolica, Shaw [142, 143] gave interesting information both on the “perching” behavior displayed by the parasitoid females in their ambush strategy to locate their hosts and on the attack sequence which is completed in less than 1 s and is characterized by a reduction of the usual braconid oviposition sequence, the first two steps (antennation of the host and ovipositor probing) being entirely lost in favor of speed. For E. michaeli, Poinar [131] not only described the attack behavior, exclusively focused on major workers of Formica obscuriventris clivia (Figure 4(b)), but also provided invaluable information on the altered behavior of parasitized ants, on the development of the immature stages, and on cocoon formation and adult emergence. Immature stages of Neoneurini parasitoids attacking adult ants develop as koinobiont endoparasitoids in the abdomen of workers, and fully developed larvae leave the host to pupate in the soil [131]. Very recently, slow motion video recordings were used to describe the oviposition behavior in adult ants for 3 other species [129], and we refer the reader to their excellent films, which show the variability in oviposition behavior within the tribe. Neoneurini wasps parasitize worker ants in the vicinity of the nest entrance(s), or while foraging. Females of Elasmosoma luxemburgense hover over the nest entrance of Formica rufibarbis and attack workers from behind, grasping the ant abdomen with the three pairs of legs involved, and probably ovipositing through the anus. The whole behavioral sequence (alighting, grasping, ovipositor insertion, and takeoff) lasted a mean of 0.73 s. The ants were aware of these attacks, turning around and chasing the wasps with open mandibles ([129] doi: 10. 3897/zookeys. 125. 1754.appl). Females of Kollasmosoma sentum attack workers of Cataglyphis iberica in the vicinity of nest entrances, or when carrying prey and walking more slowly than usual. Attacks usually occurred during the brief stops characterizing Cataglyphis workers walks. The wasps were extremely fast and attacked the ants from behind. Oviposition took place in both the dorsal and ventral surfaces of the ant’s gaster, likely through intersegmental membranes. Wasps adjusted their alighting strategies according to the direction of their own approach to the targeted ant, and to the position of the ant’s gaster (horizontal or vertical position, distinctive for the genus Cataglyphis), and accomplished extraordinary pirouettes. The whole oviposition behavior lasted only 0.05 s on average. The ants were often aware of the presence of the para- sitoids, aggressively turning around with open mandibles, or extending their hind or middle legs to hit them ([129] doi: 10. 3897/zookeys. 125. 1754.app2). Finally, N. vesculus females alight and probably oviposit in the mesosoma of Formica cunicularia workers. As for N. mantis [142, 143], they were observed ambushing or hovering over the nest entrance. Females preferentially attacked ants while at a vertical (b) Figure 4: (a) Elasmosomites primordialis larva (white pointer) emerging from the abdomen of a Lasius worker in Baltic amber. Photo courtesy of G. Poinar Jr. (see [136]). (b) Elasmosoma michaeli larva leaving its Eormica obscuriventris clivia host to pupate in the soil. Photo courtesy of G. Poinar Jr. position (going up a tree trunk, e.g.). The wasps approached the ants from behind, alighted, held the ant’s thorax with their raptorial fore legs, bent their abdomen towards the postero-lower part of the ant’s thorax, and oviposited. The ovipositor is thought to be inserted near the posterior coxal cavities. The whole oviposition behavior lasted a mean of 2.02s ([128] doi: 10.3897/zookeys. 125. 1754.app3). With few exceptions, neoneurine wasps have been found in association with formicine ants [129, 207, 215, 216]. It is thought that formic acid used by these ants could serve also as a kairomonal stimulant to host-seeking hymenopterous parasitoids [120, 127, 129]. Far less is known about the fate of parasitized ants. According to Poinar [131], Formica ants parasitized by E. michaeli form an assembly along the edge of their superficial nest when the parasitoid larvae are about to leave the host to pupate. This behavioral modification is thought to increase the survival of adult wasps. Several morphological and behavioral adaptations, apart from rapidity of attack, contribute to the success of these wasps in parasitizing aggressive adult ants: for example, the vestigial tarsal claws and enlarged pulvilli (suction like disks, [130, 131, 217]) of Elasmosoma spp., or the raptorial fore 16 Psyche legs of Neoneurus spp., enable wasps to grasp and hold the ant firmly while ovipositing. Likewise, the peculiar ventral spine of K. sentum females, located on the fifth sternite, could help to fix the wasp’s position during oviposition, when the body of the wasp goes back tending to the vertical position, and fore legs detach from the ant’s cuticle. Finally, the longitudinal disposition of K. sentum females’s tarsi on the ant metasoma, one over the other, enables the necessary rotation of the body to adjust itself to the position of the ant’s gaster, before oviposition. The wasp rotates counterclockwise if the right tarsus is placed over the left one; and if the left tarsus is placed over the right one, the rotation is clockwise. 2.3.2. Ichneumonidae. Ichneumonidae is the largest family in the Hymenoptera with about 23,330 described species worldwide in 46 subfamilies and 1207 genera; the total number of species is estimated to be more than 60,000 [207, 218, 219]. Most of the members of this large family are parasites of holometabolous insects, but a few species para- sitize spiders (egg sacs, spiderlings, or adults) or egg sacs of pseudoscorpions. Many ichneumonids are hyperparasitoids of other ichneumonoids or of tachinid flies, and some species are egg-larval parasitoids, laying an egg in the host egg but consuming the host in its larval stage [218, 219]. Various species of the genus Gelis (all of them initially referred to as Pezomachus) and a few others of the genera Agr other eutes, Aptesis, Pleolophus, and Thaumatogelis have been reported by various authors to be associated with ants of the genera Lasius, Formica, Myrmica, Temnothorax, and Solenopsis [24, 56, 220-222]. However, no information is available on the exact relationship with their ant host, except that in some cases (such as Pleolopus micropterus (Graven- host) (referred to as Pezomachus micropterus) and T. vulpinus (Gravenhorst) (referred to as Pezomachus vulpinus)), they were clearly reported as “found in the nest of Formica rufa, not reared from cocoons"' [220]. Until now, true ichneumonid parasitism on ants has been demonstrated only for 3 species, all belonging to the subfamily Hybrizontinae and very likely to the same tribe Hybrizontini. The most ancient report dates back to 1852 [145] and concerns Eurypterna cremieri described as hovering over a nest of Lasius fuliginosus in Germany. This behavior, suspected to be related to the search of an appropriate host, was later confirmed by different authors not only for the same host species in France and Italy [146-148] but also for three other species of ants in the genera Lasius and Formica in France, England, and Japan [123, 144, 149]. In the early 20th century, Gobeli [148] described how four females of E. cremieri were hovering over trails of L. fuliginosus, while ants were moving their nest to another nest site, inspecting each ant worker that was transporting a larva. The female parasitoids quickly drew closer to the larva, and folding up the abdomen touched it, presumably depositing an egg. Such behavior was only observed with ants transporting a larva and did not trigger any reaction from the workers. In spite of the interesting information supplied, this report passed more or less unnoticed until 2010 when the parasitic nature of this behavior could be confirmed (and even photographed) concerning Lasius nipponensis transporting brood between two nests [149]. Only workers carrying something in their mandibles were tracked by E. cremieri females hovering about 2 cm above them. And only those carrying a larva were attacked after a sudden dive of the wasp which gripped the targeted larva with the tarsi of its fore and middle legs, bent its abdomen down, exerted its ovipositor, and oviposited in the larva before flying away in search of a new host. The complete sequence lasted less than 1 s and elicited some brief excitement from the worker ant. Dissection of a stung ant larva showed that a wasp egg was present in the somatic cavity. Another undescribed Hybrizontinae species (gen. nov. sp. nov.) was similarly reported by the same authors as hovering over workers of the slow moving ant Myrmica kotokui which were holding something in their mandibles. As for E. cremieri, only those carrying a larva were more closely inspected and were attacked in a similar manner as previously described, but in that case, the complete attack sequence lasted longer (3-4 s), and oviposition itself took at least 1 s. A third case of ant larval parasitism has very recently been confirmed and involves Hybrizon buccatus females. This species had been frequently reported in association with (or hovering over) different ant species from various genera {Myrmica, Lasius, Formica, and Tapinoma, see Table 1) [24, 140, 144, 146, 152, 153] and was reared from nests of Lasius alienus where the ichneumonid naked pupae had been found among ant-host cocoons [ 150] . But it was not until 2011 that the oviposition into larvae transported by Lasius grandis workers could be observed and filmed during brood transfer between two nest entrances [ 129] . Only final instar larvae were attacked, in a very similar way to that previously described for E. cremieri, and the complete sequence lasted between 0.40 and 0.58 s. Chemical and/or visual cues are likely to be involved in the location of the ants’ trail since H. buccatus females have been observed continuously hovering over the trail for a period of time, even in the absence of ants. Finally, considering both the hovering behavior as a reliable evidence of parasitism and the fact that all three ichneumonid parasitoids known until now to attack ants are restricted to the Hybrizontinae, two other cases are likely to be added to our list: Ghilaromma fuliginosi and H. rileyi which have been reported swarming and hovering over the nests of Lasius fuliginosus [ 150, 151 ] or attracted to a disturbed nest of L. alienus [154], respectively. However, in both cases, direct oviposition into ant larvae or adults needs to be confirmed. 3. Conclusions Since the last paper on parasites of social insects by Schmid- Hempel [7], the number of reliable records of parasitoid wasps attacking ants and their brood has grown dramatically from about 43 species to at least 138 belonging to 9 hymenopteran families. Furthermore, the knowledge of the biology and behavior of those wasps and the nature of their interactions with ants has significantly progressed, though many gaps still remain. Most likely, hymenopterous parasitoids of ants are more abundant than suggested by our list of reliable records, and future studies focusing on the Psyche 17 immature stages of ants under close scrutiny would certainly increase this list substantially. All castes of ants and all developmental stages, excepting eggs, are the target of parasitoid wasps. For example, neoneurine braconids parasitize adult worker ants while foraging or performing other activities outside the nest [129, 131, 143], while eurytomids of the genus Aximopsis attack adult queens at the very moment of nest foundation [115, 116, 118, 119]. However, in most cases, ant larvae are the target of parasitoid attacks, either inside or outside their nests. Larvae can be parasitized outside the protective walls of the nest during transportation when ants move from one nest to another as for some euphorine braconids and hybrizontine ichneumonids [129, 149], or while being employed to fix or build a new nest as occurred for the green weaver ant larvae attacked by the chalcidid Smicromorpha [41]. Most often, ant larvae are attacked inside the nest, notwithstanding the pugnacious character of ants. For eucharitid and perilampid wasps, planidia are transported by phoresis into the targeted nest where they actively search for a larval host. The extremely small size of the planidia is assumed to facilitate both entrance into the host colony and initial parasitization [195], but in most other parasitoid wasps (diapriids, encyrtids, entedonine eulophids, and some eurytomids), it has been assumed that it is the female that searches for a host nest, enters it, and oviposits on or in the larval host. So far, however, how the females gain access into the ant nest and complete the oviposition process has never been described, and the initial stages of development of these parasitoids are in most cases unknown (but see [35, 131]). Hymenopterous parasitoids attacking ants exhibit a wide array of biologies and developmental strategies: ecto- or endoparasitism, solitary or gregarious, and idio- or koino- biosis. Besides, the behavioral strategies evolved to cope with ant aggression or to exploit the communication system of ants are also impressive. Most of these parasitoids belong to families with species using a wide range of insects or arthropods as primary hosts, and in many cases of recorded associations between parasitic wasps and ants [20, 23, 112, 114, 128, 160, 186, 198], the primary host of the parasitoids is not the ant but another insect species present in the ant nests. Such indirect association through parasitism of trophobionts or other myrmecophiles suggests that a possible path to the parasitization of ants by hymenopterous parasitoids could have evolved as a shift from the initial primary host (Diptera, Coleoptera, or other insect myrmecophiles) to the ant host larvae through a gradual process of association and integration with the ant hosts. Such a hypothesis proposed for diapriids by Huggert and Masner [160] and widened by Hanson et al. [223] to hymenopterous parasitoids in general might apply for numerous families, and a supporting example has recently been suggested among eulophids [112]. However, other evolutionary paths are likely to be involved in the case of eucharitids and perilampids and those species that attack adult ants and deserve further study. Despite a significant increase in our knowledge of hymenopterous parasitoids of ants in the last 15 years, the remark of Schmid-Hempel [7] concerning parasitism in social insects in general: “t/zc existing knowledge is bound to be a massive underestimation, since the true abundance and distribution of parasites remain to be discovered” is still, more than ever, a topical subject. Most hymenopterous parasitoids attacking ants remain to be discovered. Moreover, despite the presumed importance of some of them as natural enemies of ants, few quantitative data are available on the impact of these natural enemies on their hosts (see [224]). Based on their abundance and success in attacking ant hosts [36, 83, 193, 194], some parasitoid wasps like, for example, diapriids and eucharitids, seem excellent potential models to explore how parasitoids impact ant colony demography, population biology, and ant community structure, and further studies focusing on these issues will certainly contribute to deepen- ing our knowledge on this important group of parasites. Acknowledgments The authors thank M. W. Gates, C. Hansson, J. M. Heraty, M. Loiacono, and T. C. Narendran for making available various bibliographic references, and L. Passera and G. Poinar Jr. for kindly supplying their original pictures of ant attack by Plagiopria passerai, Elasmosomites primordialis, and Elasmosoma michaeli. They are also indebted to T. G. Narendran, G. Poinar Jr., and an anonymous reviewer for constructive comments and suggestions on a previous version of this paper, and to Peter Winterton for grammatical improvement. References [1] W. M. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 609572, 4 pages dohlO.l 155/2012/609572 Research Article Age-related and Individual Variation in Male Piezodorus hybneri (Heteroptera: Pentatomidae) Pheromones Nobuyuki Endo,^ Tetsuya Yasuda,^ Takashi Wada,^ Shin-etsu Muto,^ and Rikiya Sasaki^ ^ Agro-Environment Research Division, NARO Kyushu Okinawa Agricultural Research Center (KARC), 2421 Suya, Koshi, Kumamoto 861-1192, Japan ^Division of Plant Protection, NARO Agricultural Research Center (NARC), 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan ^Ecomone Division, Euji Elavor Co., Ltd., 3-5-8 Midorigaoka, Hamura, Tokyo 205-8503, Japan Correspondence should be addressed to Nobuyuki Endo, enobu@alfrc.go.jp Received 15 September 2011; Revised 7 December 2011; Accepted 29 December 2011 Academic Editor: Jeffrey R. Aldrich Copyright © 2012 Nobuyuki Endo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Males of the Piezodorus hybneri stink bug produce a pheromone comprising j5-sesquiphellandrene (Sesq), (R)-15-hexadecanolide (R15), and methyl (Z)-8-hexadecenoate (Z8). We collected airborne volatiles from individual P. hybneri males and analyzed them by GC-MS. Daily analysis from 1 to 16 days after adult emergence showed that pheromone emission started around 3 to 6 days after adult emergence and peaked (~1 |Wg/male/day) on day 11. The proportion of Sesq tended to increase with age to about 80% on days 12 to 16. On the other hand, the proportion of R15 tended to decrease with age. The proportion of Z8 reached a maximum of about 34% on day 9 but otherwise remained below 20%. The total amount of pheromone emitted by individual males varied considerably: three males emitted more than 10 /rg, whereas another three males emitted little or no pheromone and failed to survive by the end of the experiment. These results suggest that the amount of P. hybneri pheromone and its blend ratio could be affected by the male’s physical conditions, such as vitality and age. 1. Introduction The stink bug Piezodorus hybneri (Heteroptera: Pentatomi- dae) is an important soybean pest in southern Japan [1, 2]. Male adults of R hybneri attract conspecific adults of both sexes [3] via a pheromone comprising jS-sesquiphellandrene (Sesq), (R)-15-hexadecanolide (R15), and methyl (Z)-8-hex- adecenoate (Z8) [4]. These synthetic chemical mixtures at- tract conspecific adults, especially females in fields [5]. Our previous study [6] revealed that P. hybneri males produce the pheromone simultaneously with their development to sexual maturity, and that diapausing males produce no pheromone components; thus, the pheromone is likely to play a role in sexual communication. The average amount of Sesq in whole-body extracts increased steadily until day 30 after adult emergence, whereas the other two components peaked at day 10 and then decreased somewhat [6]. Consequently, the proportions of the pheromone components, especially Sesq, changed with age. These findings suggest that the pro- portions of components in emissions also change with age. However, pheromone production might not coincide with emission. In addition, marked variation in the pheromone component ratio among individuals of the same population was found the southern green stink bug, Nezara viridula (Heteroptera: Pentatomidae) [7]. Therefore, it is necessary to monitor the pheromone emission of P. hybneri over time from the same males to examine variation in the pheromone blend. Mating behavior of P hybneri males began on day 4 after adult emergence and showed high mating activity between days 5 and 15 [6]. Development of the ectodermal accessory gland, which is involved in reproduction and an indicator of male sexual maturity, showed that males fully matured by day 10 [6] . Thus, in this study, we collected and analyzed the volatiles from individual P. hybneri males from 1 to 16 days after adult emergence. 2 Psyche Table 1: Individual variation of pheromone titer and extraction from Piezodorus hybneri males. Male Days of first detection Pheromone emission Daily maximum 1 5 - 1 6 d Sum (1-16 d) Amount extracted at 16 d (pg) 1 5-6 0.91 0.09 2.91 12.07 2 4-5 3.20 0.58 14.54 27.49 3 4-5 0.75 0.26 4.22 10.51 4 5-6 1.81 1.51 12.41 48.81 5 5-6 0.31 0.00 0.62 0.00 6 — 0.00 — 0.00 — 7 5-6 0.09 — 0.09 — 8 4-5 0.32 — 1.03 — 9 3-4 1.03 0.29 5.68 5.18 10 3-4 3.47 0.50 18.62 27.02 Average 1.32 0.46 6.01 18.73 2. Materials and Methods 2. 1 . Insects. Adults of P. hybneri were caught in soybean fields of the NARO Kyushu Okinawa Agricultural Research Center (32°52'5"N, 130°44'2"E), Kumamoto, Japan, in 2005. Their progeny were kept in the laboratory (24 ± 1°C, 16L-8D photoregime) and used for experiments in March 2006. The bugs were reared on a diet of soybean (Glycine max) seeds, red clover (Trifolium pratense) seeds, and water. 2.2. Collection of Airborne Volatiles with Glass Beakers. Following the method of Yasuda et al. [8] with some modifications, we collected and analyzed the volatiles from individual P. hybneri males (n = 10) in glass beakers. Collection started 1 day after adult emergence. A single adult male was confined in a 50 mL glass beaker with a few soybean seeds and moist cotton. The beaker was placed upside down and sealed with aluminum foil. The male was kept in the beaker for 24 h under laboratory conditions. After 24 h, the male was removed and the beaker surface was rinsed with 3 mL hexane containing 2 pg octadecane as an internal standard. After this treatment, any pheromone component as well as internal standard was not detected from the hexane rinsing the beaker. The hexane was collected for analysis of volatiles. The male was placed in a new beaker with food and the process was repeated until day 16. After the final collection, the males that survived were extracted with 2 mL hexane containing 2pg octadecane as an internal standard and then rinsed once with 1 mL hexane. All extracts were stored in glass vials with Teflon-lined screw caps at -20° C until analysis. Extracts were concentrated to ca. 100 |WL in an evaporator just before gas chromatography — mass spectrometry (GC-MS) analysis. 2.3. GC-MS Analysis. Quantitative GC-MS analysis was done on an Agilent 6890 N GC with an HP-5 ms column (30 m X 0.25 mm ID X 0.25 pm film thickness; Agilent Technologies) and an Agilent 5975i Network Mass Selec- tive Detector using an internal-standard method. Mass spectrometry data by selected ion monitoring (SIM) and full scan (range: 35-350 m/z) were acquired synchronously. Quantitative (selected) and reference ions for SIM were m/z 254 and 57, respectively, for octadecane, m/z 204 and 69 for (±)-j3-sesquiphellandrene, m/z 210 and 55 for (±)- 15-hexadecanolide, and m/z 268 and 55 for methyl (Z)-8- hexadecenoate. Injection was performed in splitless mode with a split/splitless injector using an Agilent 7683 series automatic liquid sampler at 250° C. Helium was used as the carrier gas at a constant flow of l.OmL/min. The GC oven temperature was an initial 50° C (2-min hold), increased to 240°C at 15°C/min, and then held for 5 min. To determine the quantity of each component, standard curves obtained using known amounts of authentic chemicals with the internal standard (octadecane) were used. 2.4. Chemicals. (±)-j3-Sesquiphellandrene, (±)-15-hexade- canolide, and methyl (Z)-8-hexadecenoate were synthesized according to a previous report [4]. 3. Results The temporal patterns and the amounts of pheromone emis- sion by males varied greatly among individuals (Figure 1). Nine out of the 10 males emitted pheromone (Table 1). Emission started 3 to 6 days after adult emergence, peaked (~1 f/g/male/day) on day 10 or 11, and remained high until day 16. The maximum average emission was 1.32 |Wg/day, and two bugs exceeded 3 ^ug/day. The total amount of pheromone varied more than 30 -fold among surviving individuals (0.62- 18.62 pg). Male No. 6 did not emit any pheromone and died on day 11. Males No. 7 and 8 emitted little pheromone and died on days 7 and 15, respectively. There was a strong correlation between the amount of pheromone collected on day 16 and the amount extracted on day 16 (r^ = 0.885, P = 0.0016). The proportions of the three pheromone components, especially Sesq, showed great variability (Figures 1 and 2). The proportion of Sesq tended to increase with age to about 80% on days 12 to 16. On the other hand, the proportion of R15 tended to decrease with age. The proportion of Z8 reached a maximum of about 34% on day 9, but otherwise remained below 20%. Psyche 3 No. 2 No. 5 100 - 80 - 60 - 40 - 20 Figure 1: Daily changes in pheromone emission and the proportion of /1-sesquiphellandrene from individual males of Piezodorus hyhneri. The bar shows the total amount of pheromone (^wg/male/day). The line chart indicates the proportion of j8-sesquiphellandrene (%) among the total pheromone components. Inverted triangle indicates the day when the bugs died. Because male No. 6 did not emit any pheromone, there was no data available in this figure. 4. Discussion Repeated collection of volatiles with a glass beaker from individual males over a period of 15 consecutive days showed that the pheromone component ratio varied with male age. The proportion of Sesq tended to increase with age and reached about 80% by days 12 to 16. This trend in variable pheromone blend ratios agreed with our previous results obtained from extracts of males [6]. The high correlation between the quantities of pheromone in emissions and those in body extracts, on day 16, indicates that pheromone emission parallels pheromone production in this species. In some heteropteran species, variations of the pherom- one blend ratio among individuals or with physiological condition have been reported. In N. viridula, the pheromone blend ratio varied among individuals within a population [7, 9], although individuals’ ratios remained constant [9]. Large variability of pheromone component ratios in the bean bug Riptortus pedestris (Heteroptera: Alydidae) was also reported [10]. Recently, Moraes et al. [11] reported that food conditions affected the pheromone ratios in the Neotropical brown stink bug Euschistus hews (Heteroptera: Pentatomidae). We report here that large changes in the pheromone blend of R hyhneri males occur during the first 2 weeks of adulthood. This is the first study documenting age- related shifts in the pheromone blend of Heteroptera. The pheromone blend ratio is generally calculated from the amounts of components emitted at a specific age. 4 Psyche ■ Sesq □ R15 ■ Z8 Figure 2; Proportions of each pheromone component ofPiezodorus hybneri males in relation to adult age. Sesq: j^-sesquiphellandrene; R15: (R)-15-hexadecanolide; Z8: methyl (Z)-8-hexadecenoate. Ver- tical lines represent SE. However, this approach is based on the premise that the ratio remains constant throughout adult life. Leal et al. [4] reported that the pheromone blend ratio of R hybneri was Sesq : R15 : Z8 = 10:4:1. However, our results show that the ratio was not constant and was affected by male age. This may be the case in other species, and thus it should be taken into consideration when pheromone blend ratios are determined. Pheromone quantity could be also affected by the male physical conditions. Pheromone production of P. hybneri is paralleled to the development of male sexual maturity [6], and young R hybneri males cannot produce or emit a large amount of pheromone. In addition, our results show a large variation in pheromone quantity among individuals, and the poor pheromone emitters died early. These results suggest that only vital and sexually mature males can produce sufficient pheromone. The biological function or attractiveness of each pherom- one components or its blends for R hybneri is largely un- known. In fields, each R hybneri pheromone component alone lacked the attractiveness, while three-component mix- ture was attractive to R hybneri [5] . Leal et al. [4] showed that the full three-component mixture was attractive more than the any binary mixtures in laboratory conditions, whereas the attractiveness of the different blend ratios has never been investigated. In order to clarify the biological functions of R hybneri pheromone blend or its components, it is necessary to compare the attractiveness among different pheromone blends or male ages. Acknowledgments The authors thank Dr. J. R. Aldrich (USDA ARS) for review- ing the draft. They also thank K. Nagata (NARO/KARC) for her assistance in rearing the bugs. References [1] S. Kono, “Ecological studies of stink bugs injuring soybean seeds for developing effective control measures,” Special Bulletin of the Hyogo Prefectural Agricultural Institute, vol. 16, pp. 32-68, 1991. [2] T. Wada, N. Endo, and M. Takahashi, “Reducing seed damage by soybean bugs by growing small- seeded soybeans and delaying sowing time,” Crop Protection, vol. 25, no. 8, pp. 726- 731,2006. [3] H. Higuchi, “Attraction of conspecific individuals by adults ol Piezo dorus hybneri (Gmelin) (Heteroptera: Pentatoraidae),” Japanese Journal of Applied Entomology and Zoology, vol. 43, no. 4, pp. 205-206, 1999. [4] W. S. Leal, S. Kuwahara, X. Shi et al., “Male- released sex pheromone of the stink bug Piezodorus hybneri,” Journal of Chemical Ecology, vol. 24, no. 11, pp. 1817-1829, 1998. [5] N. Endo, R. Sasaki, and S. Muto, “Pheromonal cross-attraction in true bugs (Heteroptera): attraction of Piezodorus hybneri (Pentatomidae) to its pheromone versus the pheromone of Riptortus pedestris (Alydidae),” Environmental Entomology, vol. 39, no. 6, pp. 1973-1979, 2010. [6] N. Endo, T. Yasuda, K. Matsukura, T. Wada, S. E. Muto, and R. Sasaki, “Possible function of Piezodorus hybneri (Heteroptera: Pentatomidae) male pheromone: effects of adult age and dia- pause on sexual maturity and pheromone production,” Ap- plied Entomology and Zoology, vol. 42, no. 4, pp. 637-641, 2007. [7] M. A. Ryan, C. J. Moore, and G. H. Walter, “Individual var- iation in pheromone composition in Nezara viridula (Heter- optera: Pentatomidae): how valid is the basis for designating “pheromone strains”?” Comparative Biochemistry and Physiol- ogy B, vol. Ill, no. 2, pp. 189-193, 1995. [8] T. Yasuda, N. Mizutani, N. Endo et al., “A new component of attractive aggregation pheromone in the bean bug, Riptortus clavatus (Thunberg) (Heteroptera: Alydidae),” Applied Ento- mology and Zoology, vol. 42, no. 1, pp. 1-7, 2007. [9] N. Miklas, M. Renou, I. Malosse, and G. Malosse, “Repeatabil- ity of pheromone blend composition in individual males of the southern green stink bug, Nezara viridulaf Journal of Chemical Ecology, vol. 26, no. 11, pp. 2473-2485, 2000. [10] N. Mizutani, T. Yasuda, T. Yamaguchi, and S. Moriya, “Indi- vidual variation in the amounts of pheromone components in the male bean bug, Riptortus pedestris (Heteroptera: Alydidae) and its attractiveness to the same species,” Applied Entomology and Zoology, vol. 42, no. 4, pp. 629-636, 2007. [11] M. G. B. Moraes, M. Borges, M. Pareja, H. G. Vieira, F. T. P. de Souza Sereno, and R. A. Laumann, “Food and humidity affect sex pheromone ratios in the stink bug, Euschistus herosf Physiological Entomology, vol. 33, no. 1, pp. 43-50, 2008. Hindawi Publishing Corporation Psyche Volume 2012, Article ID ITbTil-, 11 pages dohlO.l 155/2012/725237 Review Article Myrmica Ants and Their Butterfly Parasites with Special Focus on the Acoustic Communication E Barbero, D. Patricelli, M. Witek, E. Balletto, L. P. Casacci, M. Sala, and S. Bonelli Department of Animal and Human Biology, University of Turin, via Accademia Albertina 13, 10123 Turin, Italy Correspondence should be addressed to S. Bonelli, simona.bonelli@unito.it Received 30 September 2011; Accepted 18 December 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 F. Barbero et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. About 10,000 arthropod species live as ants’ social parasites and have evolved a number of mechanisms allowing them to penetrate and survive inside the ant nests. Myrmica colonies, in particular, are exploited by numerous social parasites, and the presence of their overwintering brood, as well as of their polygyny, contributes to make them more vulnerable to infestation. Butterflies of the genus Maculinea are among the most investigated Myrmica inquilines. These lycaenids are known for their very complex biological cycles. Maculinea species are obligated parasites that depend on a particular food plant and on a specific Myrmica species for their survival. Maculinea larvae are adopted by Myrmica ants, which are induced to take them into their nests by chemical mimicry. Then the parasite spends the following 1 1-23 months inside the ants’ nest. Mimicking the acoustic emission of the queen ants, Maculinea parasites not only manage to become integrated, but attain highest rank within the colony. Here we review the biology of Maculinea/ Myrmica system with a special focus on some recent breakthrough concerning their acoustical patterns. 1. Butterflies and Ants Most myrmecophiles are commensals or mutualists, which live undisturbed or even actively protected within the forag- ing areas or territories of ants [1-3]. Their functional and evolutionary ecology, as well as their truly amazing diversity, have been reviewed by Wasmann [4], Donisthorpe [5], Hinton [6], Malicky [7], Holldobler and Wilson [1], DeVries [8, 9], Fiedler [10, 11], Pierce et al. [12], and others. The interactions that have evolved between insects and ants range from loose facultative associations to obligate de- pendency (as concerns butterflies, see [3, 11, 13, 14]). The nests of eu-social arthropods, including insects such as ants, bees, wasps, or termites, are aggressively defended from predators and intruders alike. As a consequence, these nests provide very safe havens for any roughly ant-sized organism having evolved the necessary adaptations to penetrate them and to become accepted as “self” by the workers’ caste [4, 5, 15]. Around 10,000-15,000 insect morphospecies have evolved as social parasites of ants, thus accounting for a significant proportion of the world’s biodiversity. Yet, despite the many species, most ant social parasites are exceedingly rare or localized, in comparison to the abundance and distribution not only of their ant hosts but also of other symbionts, which loosely interact with ants [1, 16, 17]. Myrmecophily is widespread among Lepidoptera, most particularly as concerns the Riodinidae and Lycaenidae [9, 12], which are often globally referred to as “lycaenoids” [10], and which make up approximately 30% of all known Papil- ionoidea [18]. Their relationships with ants can be mutualis- tic or parasitic and vary from facultative to strictly obligate. In the case of facultative myrmecophiles, the survival of butterfly larvae does not depend on the presence of attendant ants, and associations are unspecific. In other words, these lycaenoids can use ants belonging to several different species, or even subfamilies [11, 12]. On the contrary, in obligate ant associations, butterfly immatures are dependent on ants’ presence, at least in some part of their life cycle and interac- tions are much more species specific [11, 12]. Achieving a myrmecophilous life style requires evolving numerous special adaptations, which are necessary for avoid- ing ant aggression and for communicating with ants. The cuticle of many myrmecophilous butterfly larvae is thicker than in other groups of Papilionoidea and the head can be retracted under a sclerotized plate [7, 19]. Frohawk [20] was the first to observe that most myrmecophilous butterfly 2 Psyche (c) (d) Figure 1: (a) Forager worker of Myrmica ant. (b) Trophallaxis between attendance worker and Maculinea larva, (c) Maculinea rebeli foodplant: Gentiana cruciata. (d) Mating of MacM/fncfl butterflies. larvae have dorsal nectar organs (DNOs), whose “honeydew” secretion attracts and pacifies ants, and plays an essential role in the maintenance of ant attendance [12]. Additionally, many lycaenoid caterpillars possess specialized epidermal glands, pore-cupola organs and tentacle organs, whose secretions are apparently not directly used by ants, but can somehow manipulate their behaviour [21-23]. Moreover, some butterfly species produce cohorts of other chemical and/or acoustical signals, which are involved in their inter- actions with ants [12]. 2. The Parasites: Maculinea Butterflies One of the most intensively studied systems in which both the communication channels are investigated concerns parasitic Maculinea butterfly larvae and their Myrmica host ants (Figures 1(a) and 1(b)) [24-26]. During the past decades butterflies of genus Maculinea (Figure 1(d)) have become “flagships” of European biodiversity conservation [24] and are perceived as umbrella species covering many grassland communities [27-29]. Some recent publications [30-32], based on both molec- ular and morphological data, have shown that species of Maculinea and Phengaris form a monophyletic group, where the three Chinese Phengaris species are basal. According to Fric et al. [32] Maculinea Van Eecke, 1915 should be consid- ered a junior subjective synonym of Phengaris Doherty, 1891. Possible alternatives are that Maculinea is, as subjectively, considered subgenus of Phengaris, or a distinct genus in its own right. On the other end the obligate myrmecophilous life style of Maculinea has attracted a vast number of studies, many of which appeared in leading scientific journals. Maculinea is a model organism for studies on the origin and evolution of parasitic interactions and of host-parasite communication channels [11, 24-26, 30, 33]. Maculinea have also attracted a great deal of attention from a conservationist’s point of view [34-37]. Eor this rea- son some of the authors have asked the International Com- mission on Zoological Nomenclature to conserve the name Maculinea against Phengaris in all cases when the two are considered subjective synonyms. The decision by the ICZN is still pending and we will continue to use Maculinea rather than Phengaris, at least for the moment. Another point is that no molecular evidence is available to distinguish Maculinea rebeli from Maculinea alcon and some authors have argued that the first of them is an ecotype of M. alcon [32]. Also in this case we have decided to stick to the traditional interpretation that M. alcon and M. rebeli represent separate clades (species) and in this paper we Psyche 3 will use the name Maculinea reheli to designate what might represent the xerophilous ecotype of M. alcon. European Maculinea species need urgent conservation actions, indeed four are mentioned in the European Red List of Butterflies and three of them are included in the Annex IV of the Habitats Directive [38, 39]. These lycaenids are known for their very complex biological cycles. Maculinea species are all obligated parasites that depend on a particular food plant and on a specific Myrmica species for their survival. After having spent 10-15 days feeding on a species-specific host plant (Eigure 1(c)), the 4th instar larvae of all Maculinea species drop to the ground and wait until they are found and carried into an ant nest by a Myrmica worker [40-44]. Once in the ant colony, Maculinea species differ in their alimentary strategy: (i) Maculinea alcon and Maculinea reheli utilize a “cuckoo” strategy, and are mostly fed directly by attending workers (trophallaxis) [42] (Eigures 1(a) and 1(b)), they are known for experiencing “contest” competition at high densities [45], (ii) Maculinea arion and Maculinea teleius are “predatory species” and directly prey on ant brood, expe- riencing “scramble” competition when overcrowded in the host colony [46], while (iii) the alimentary strategy of Macu- linea nausithous has not yet been fully clarified, with some authors suggesting the coexistence of both “cuckoo” and “predatory” strategy and others considering it as a “cuckoo” species [24, 47]. Maculinea larvae spend 11 or 23 months inside their host colonies. In many populations two separate cohorts of larvae spending either one or two years inside the ants’ nest are known to exist [33, 48-50]. The polymorphic growth pattern found in Maculinea populations is likely to have evolved for ergonomic, or perhaps hedge-betting reasons. Two are the key moments in the life cycle of these but- terflies: (i) the choice of an optimal food-plant on which to lay eggs and (ii) the first direct interaction with the host ants. The place where females lay their eggs is crucial for a myrmecophilous butterfly, to ensure its brood the chance to be adopted by a specific host ant. Because the worker ants’ foraging range is limited, selecting an “ideal” oviposition site requires that both the phenological stage of the larval food plant (short-term larval fitness) and the presence of suitable host ants (long-term larval fitness) are taken into account. The female’s selection of a valuable oviposition plant is influenced by a variety of factors. Plants are generally selected by females on the basis of their buds’ phenology, while the presence of the host ants in the near surroundings of the plant may be variously insured depending on local situations and perhaps on the species. In some cases the host-plant and the Myrmica ant share a similar ecological niche, so that their overlap ensures population persistence [51-54]. In other cases, however, female butterflies mostly choose those plants which occur in the ants’ foraging range [55-59]. To the best of the authors’ knowledge, nothing is known about the mechanism providing butterfly females with the ability to discriminate among host plants placed inside/outside the foraging range of a Myrmica colony. The other hot point of research on Maculinea butterflies is their host specificity with ants, both for its relevance in coevolutionary dynamics and as a background for conserva- tion strategies. While Maculinea caterpillars induce workers of any Myrmica species to retrieve them by chemical and acoustical deception [26, 60] , their survival till the adult stage will depend largely on which ant-species has found the larva [41-44,61]. Before the 1970s a nonextensive study of Maculinea host specificity led scientists to consider all Myrmica species and, in some cases other ant’s genera (e.g., Lasius), as potential host of these butterflies. In the following decades Thomas et al. [61 ] revealed a clear host specificity pattern involving each of the five European Maculinea species. In their work authors demonstrated that the survival of every Maculinea species was linked to single and different Myrmica ant species, while the adoption by a non-host species caused a large decrease in the survival rate of these butterflies. More recently, the large amount of data collected by many researchers all across Europe, confirmed these general guidelines, but demonstrated that host specificity patterns are much more complex and hosts may vary geographically all along the range of each Maculinea butterfly. The only species that apparently keeps a single host is M. nausithous [34, 62, 63], which shows a clear adaptation to Myrmica rubra all over its distribution [47, 61, 62, 64]. The only known exception to this occurs in Transylvania, where it exploits M. scahrinodis as alternative host [65]. Data on other Maculinea species show a much more complicated pattern, which demonstrates that host specificity occurs at the population or, at least, at the regional scale. Several works have shown that M. teleius, M. arion, M. alcon, and M. reheli may be locally adapted to some Myrmica species previously considered as nonhost [29, 50, 64-72] and in the case of the latter two species have developed the ability to successfully exploit more than one host species in the same site creating real multiple host populations [25, 73]. 3. The Host: Myrmica Ants Myrmica ants are hosts of Maculinea butterflies, but their colonies are infested by numerous other social parasites such as the larvae of the hoverfly Microdon myrmicae (Diptera Syrphidae; see [74, 75]), or by parasitic ant species of the same genus [76]. Reasons for this apparent asymmetry are unclear, but may be related to the biological cycle of these ants. The genus Myrmica has a Holarctic distribution. Most of the species, however, are found in Europe and Asia, while a smaller proportion occurs in North America [77]. Colonies are widespread and can be found in various kinds of habitat, such as meadows, forests, steppes, or mountains [76]. Although the biology of many Myrmica species has not been studied in detail, it seems that a general life style is common to all ants of this genus [76]. Most colonies contain on average 200-500 workers, as well as from one to many functional queens [78, 79] . New nests can be either funded by a single newly mated queen or, more often, by budding pre- existing colonies [45]. Oviposition starts in early spring and lasts throughout the summer, while it stops in autumn when temperature is decreasing [76]. Part of the larvae develop rapidly but others enter diapause and overwinter. The latter 4 Psyche L: SEl EHT: 20 kV HD: 33 mm MAC: x 844 photo: 41 50 fim I 1 E: SEl EHT: 25 kV HD: 12mm MAC: x 318 photo: 46 100 /^m I 1 Figure 2: Morphology (upper part) and sounds (lower part) of the acoustical organs of (a) Maculinea rebeli pupa and (b) Myrmica schencki queen. group includes both workers and all the gyne-potential larvae [80]. Some of these life history traits of Myrmica ants make them more vulnerable to infestations by social parasites. One of the most important is presence of overwintered ant larvae particularly essential for survival of the predatory Maculinea larvae, which start their intensive growth inside host colony at the beginning of spring and use overwintered ant brood as their food resource [49, 81]. Another significant trait that make Myrmica ants a proper host for many social parasites is that many Myrmica species live in polygynous colonies and some of them such as M. rubra, M. ruginodis, or M. rugulosa may contain a relatively high number of workers [76, 77]. This results in lower relatedness among worker nest mates [78, 82]. Many studies [83-85] showed that high genetic variance may be beneficial for social insects colonies, but it can also increase the likelihood of being infested by social parasites, because of the greater variance in nest mate recognition cues. It was indicated that Microdon mutabilis (Linnaeus, 1758) (Diptera: Syrphidae), a social parasite of Formica lemani ants, more often infests host colonies where genetic relatedness is lower [86]. A similar situation was found for colonies of M. rubra infested by M. alcon [87]. Therefore, a cost of polygyny existing in most of Myrmica species is that their colony communication signals (e.g., chemical or acoustical) tend to be broader and more hetero- geneous than in monogynous ant species and their colonies can be more easily invaded by cheats that mimic these sig- nals. 4. Acoustical Pattern in the Maculinea-Myrmica System The more fine-tuned the host-parasite relationship is, the more intriguing studying how the host’s deception can be achieved is. The communication of social insects is mainly based on chemical cues [1], but also the acoustic channel is used, thus it is clear that the parasite has to bypass the host’s chemical and acoustical system to enter and live in its colonies [88]. Cuticular hydrocarbons have long been assumed to play a fundamental role in the nest mate recognition of social insects. All individuals living in the same society share a bouquet of chemicals, which serves as a “colony odour” and enables them to discriminate between nest mates and strangers. Additional variation in hydrocarbon pattern is associated with differences in sex, caste, and developmental stage [89, 90]. The fact that caterpillars of Maculinea butterflies use chemical mimicry to become adopted and to infiltrate colonies of their hosts was first proposed by Elmes et al. [42], while the first experimental evidence was produced by Akino et al. [43], who found that the chemical profile of Maculinea rebeli resembles that of its host more than those of other Myrmica species. Even though sound production is not usually the domi- nant strategy, acoustic communication plays a fundamental role in some groups of insects [91]. Depending on the taxon, sound productions may have a number of functions, ranging Psyche 5 from mate attraction to courtship, aggression, defence, or recruitment of foragers, at least in social insects. Recently, it has been suggested that sounds play a role in the modulation of other signals. This was demonstrated to occur at least in honeybees [92-96]. The role of stridulations in ant communication was un- derestimated for a long time [8, 26] , also because of our scant understanding of the structures involved in the production and the reception of the acoustic signals. Stridulations, however, have long been known to occur in 4 ant subfamilies [97, 98]. In these ants, sounds are produced by a minutely ridged stridulating organ (pars stridens) positioned on the middle-dorsal part of the 4th “abdominal” segment and by a spike (plectrum) jutting from the postpetiole’s rear margin [26, 99-103] (Figure 2(b)). When an ant moves its abdomen, the two parts rub on each other and emit a series of “chirps” [1, 103, 104]. Stridulations are variously defined depend- ing on the transmitting medium. They are sounds, when transmitted by air, or vibrations, if transmitted by substrate. Myrmecologists have long believed that ants cannot “hear” the aerial component of a stridulation but perceive substrate- transmitted vibrations [105]. This notion was based on experience obtained in the early 20th century [106, 107], and has been indirectly confirmed ninety years later by the discovery of a subgenual organ in Camponotus ants [108]. More recently, however, a seminal paper by Hickling and Brown [105] provided fresh impulse to studies on the possible perception of air-transmitted sounds heating the debate on this subject [109, 110]. Flickling and Brown [105] maintain that ants cannot perceive the aerial component of sounds over a long distance (i.e., 1 m), but largely use short range acoustic communication (i.e., 1 cm). Acoustic communication plays a wide range of roles in the ants’ social behaviour, from reciprocal attraction to inter- caste interactions. In most cases, these stimuli are effective only at small range and are mainly used as signals of alarm, for foragers’ recruitment, mating requests, intimidation, and aposematic “threatening”, as well as to modulate other kinds of signals [1,92, 111-118]. Functions of stridulations have been intensively sur- veyed in Atta ants, where foragers’ calls are most frequent when leaves of the highest quality for fungal cultures are found [119]. Myrmica workers frequently stridulate during trophallaxis, particularly the receiving worker, when food decreases [120, 121]. Intercaste acoustical communication has been recorded in only a few instances. Mating queens of Pogonomyrmex hadius stridulate to signal to males when their spermathecae are full [111] whereas, in Atta, leaf- cutting workers stridulate when they are ready to return to the nest. This behaviour induces individuals of the smallest “minim” caste to climb onto the leaf fragment where from there they protect their larger sisters from attack by phorid flies during the journey home [117]. Until recently, there was no direct evidence that different members of an ant society produced distinctive caste-specific sounds to induce appropriate patterns of behaviour either in fellows or in other castes. At least two studies, however, suggested that different castes produce distinctive signals: the major workers of Atta cephalotes make sounds that are more intense and carry further than those of their smaller nest mates [122], while the space between the ridges of the pars stridens of queens exceeds that of workers in four Mcssor species [102]. Our own findings demonstrated that Myrmica schencki queens generate distinctive sounds that elicit increased benevolent responses from workers, thereby reinforcing their supreme social status [26, 123]. These findings demonstrated that acoustical communication within the vast subfamily Myrmicinae (to which Messor spp. and Myrmica spp. belong) is more variable and conveys more social information within ant colonies than was previously recognized. In this group, stridulations also fulfil the strict adaptationist definition of biological communication, in which both the signal and the response are adaptive [26, 124, 125]. Since acoustic signals convey quite complex information, not only between worker ants while outside the colony (e.g., during foraging), but also within the nest and between castes, we started research aimed at understanding whether some social parasites, such as butterfly larvae, could interfere with this communication system. Lycaenid larvae, in fact, have long been known to be able to emit stridulations even if their life cycle is not linked at any degree to the ant presence, but sounds produced by myrmecophilous species are more complex and frequent than those emitted by nonmyrme- cophilous species [22]. More in general, however, studies aiming at clarifying the function of interspecific acoustic communication in myrmecophilous Lepidoptera are scarce. Most of these studies considered butterfly larva stridulations as a merely defensive signals [6, 126] or, more rarely, as aggregation messages [127]. Sounds produced by lycaenid pupae and caterpillars originate from different organs; the former from tooth-and-comb stridulatory organs between the fifth and sixth segments [12, 126, 128, 129] (Figure 2(a)), whereas caterpillar sounds may emanate from muscular contraction and air compression through the tracheae [130]. The acoustics of mutualistic lycaenid species does not ob- viously mimic ant stridulations, and ants attraction has been demonstrated only in the pupae of one extreme mu- tualist species (i.e., Jalmenus evagoras see [12, 131]. On the contrary, the larval calls of four Maculinea species are similar in pulse rate and band width to those of their hosts, although the level of apparent mimicry is to the genus Myrmica rather than to individual host ant species [132]. The same study showed that Myrmica larvae are mute, suggesting that in this trait Maculinea caterpillars are mimicking an adult ant cue, but no direct cause-and- effect relationship was revealed (recordings by DeVries et al. [132] were restricted to distressed worker ants and cater- pillars, and were not played back to the ants). Studying the Maculinea reheli/ Myrmica schencki system, we recently demonstrated the first case of acoustical mimicry in an ant social parasite [26]. In particular we demonstrated that Maculinea rebeli larvae and pupae are able to mimic the sounds produced by Myrmica schencki queens (Figures 2(a) and 2(b)), thus obtaining a high status in the host colony hierarchy. Queens, that never come out of the nest, produce peculiar stridulations, which attract workers. Ethological experiments revealed that the acoustical signals produced by Maculinea rebeli larvae elicit the same benevolent responses 6 Psyche in the worker ants as those emitted by their queen(s). When recordings of unstressed adult M. schencki were played back to laboratory cultures of workers, the sounds of both castes induced benign responses including aggregation and antennation at the speaker. Moreover, when workers were played their queen’s sounds, they stood “on guard” on the speaker to a much greater extent than when worker sounds were played, each holding the characteristic posture adopted by a Myrmica worker when protecting an object of high value to the colony [26]. Maculinea reheli caterpillars are rescued ahead of the ant brood when a colony is disturbed, and are fed in preference to host ant larvae when food is scarce [48]. Neither chemical mimicry nor their begging behaviour explains why M. reheli caterpillars are treated in preference to host ant brood. Instead, we have suggested that acoustical cues are employed [26]. Thus it is possible that acoustical mimicry does not occur in Maculinea reheli only, but rather provides another route for the infiltration of other Maculinea species, as well as for other myrmecophilous insects [26]. Acoustical mimicry can also be related to the level of interaction between host and parasite, or may play a role in host-specificity. In particular, in the Maculinea/Myrmica system the level of host’s integra- tion within the colony results from the two distinct parasites’ foraging strategies. In the so-called “cuckoo” species, Mac- ulinea larvae become perfectly integrated members of the colony, as they need to be tended by worker ants. Larvae of predator species, in contrast, will prey on the ants’ brood and spend much of their life hidden in the remote chambers of the nest. DeVries et al. [132] showed that also the caterpillars of the predatory Maculinea species produce sounds that appear to mimic Myrmica (worker) stridulations, although in nature they are less closely integrated with their host’s society [14], so that they might be less perfect acoustical mimics of their hosts. We tested [124] this hypothesis by comparing the acoustics of unstressed Maculinea arion caterpillars and pupae with those of the queens and workers of its host ant, Myrmica sahuleti, and with data obtained for Maculinea reheli and Myrmica schencki, but found no evidence that M. reheli is a closer mimic of M. schencki than M. arion is to M. sahuleti [26]. We also compared the worker and queen sounds ofM. sahuleti, and those of two other ants, Myrmica scahrinodis and M. schencki, to determine whether the distinctive acoustical communication system occurring in the different castes of M. schencki exists in its congeners. We found that stridulating queens from two additional Myrmica species (i.e., M. sahuleti and M. scahrinodis) make distinctive sounds from those of their workers by using morphologically distinct organs [124]. Interestingly, the calls produced by queen from the three Myrmica species were indistinguishable from each other, as were workers’ stridu- lations even at a less extent. This suggests that acoustics plays little or no part in the cues used by Myrmica to distinguish between kin and nonkin, or other species of ant and mem- bers of their own society. Indeed numerous studies demon- strate the predominant role of chemical cues and the gestalt odour in colony recognition or between physiological states within an ant society [ 1 ] . However, our recent results suggest that acoustical communication, in isolation, is capable of signalling at least the caste and the status of a colony member, as well as of inducing appropriate behaviour towards it by the attending workers [124]. In other words, acoustical mimicry is genus rather than species specific, as DeVries et al. [132] concluded. We have not yet studied whether different castes of Myrmica ants responded differently when played the same sounds, although this seems probable, because Myrmica schencki queen respond aggressively when introduced to Maculinea reheli pupae (which mimic queen sounds) whereas the workers tend them gently [26]. 5. Concluding Remarks To our knowledge, although 10,000 species of ant social parasites may exist [24] particularly among the Coleoptera, Diptera and Lepidoptera [ 1 ] , acoustical mimicry has rarely been examined outside the case of Maculinea. Together with Di Giulio and his collaborators, we recently surveyed the acoustical emissions of Paussus favieri (Coleoptera, Paussinae), a myrmecophilous paussine beetle which lives in the nests of the ant Pheidole pallidula [133]. The presence of stridulatory organs in members of the myrmecophilous ground beetles tribe Paussini has long been known. However, due to the rarity of these beetles and the challenges in rearing them in captivity, sounds emitted by these organs have never been investigated, as well as their biological significance. The complexity of P. favieri s sound repertoire suggests that it has an important role in its interaction with P. pallidula. We strongly believe that the implementation of studies on acoustic communication will bring about significant advances in our understanding of the complex mechanisms underlying the origin, evolution and stabilisation of host- parasite relationships. To improve our understanding of how important and how generalised acoustic mimicry is we also need to clarify which sensory structures are involved in sound perception processes, both in queen and worker ants. Nobody, so far, has ever investigated the possibility that the larvae and pupae of myrmecophilous lycaenids may perceive the sounds emitted by conspecifics, or by their host ants. 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Ingram, kingram@colgate.edu Received 1 October 2011; Revised 7 December 2011; Accepted 12 December 2011 Academic Editor: Alain Lenoir Copyright © 2012 Krista K. Ingram et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study examines the distribution and invasion dynamics of Wolbachia in a recently established Formica fusca population. Pre- liminary data revealed the intermittent infection of Wolbachia across colonies, providing the opportunity to test for ecological factors affecting the acquisition and spread of the parasite. Only 35% of colonies are infected in this population. Both infected and noninfected nests have similar dispersion patterns that approximate a random distribution, suggesting that transmission of Wolbachia between adjacent colonies is not common. There is no difference in the infection rate between workers and brood, indi- cating that workers are not actively eliminating the infection. Our results show no significant association between Wolbachia infec- tion and nest size; however, infected colonies tend to be larger than noninfected colonies. Linally, Wolbachia infection was not asso- ciated with queen number. Overall, our results suggest no large fitness differences between infected and noninfected colonies, although small fitness effects cannot be ruled out for this population. 1. Introduction Wolbachia is common endosymbiotic bacteria of arthropods, crustaceans, mites, and nematodes that induces a variety of effects on their hosts to promote their own spread within the host population [1-6]. It is estimated that Wolbachia is pre- sent in 20 to 75% of all arthropods [2, 7] including more than 90 species of ants [2, 8-14]. Within ant species, high levels of multiple Wolbachia infection are documented, including up to 4 strains of Wolbachia in single individuals [10, 13]. Wolbachia transmission normally occurs through vertical maternal transmission [5, 14, 15]. The parasite has been shown to increase transmission via manipulation of repro- duction and the sex ratio of the host using a number of differ- ent mechanisms (reviewed in [16-19]). Wolbachia infection can benefit host females through positive fitness effects or via cytoplasmic incompatibility (Cl) [4]. Cl prevents infected males from successfully mating with a noninfected female or with a female infected with a different strain of Wolbachia [19]. Other mechanisms by which Wolbachia can bias host sex ratio in favor of infected females include male killing. parthenogenesis, and feminization [19]. In social insects, worker control of sex allocation requires Wolbachia-mediat- ed manipulation of worker’s behavior to result in a favorable sex ratio [20]. To date, studies of sex ratio in ants have pro- vided little evidence for Wolbachia-induced manipulations of sex ratio in ants [14, 15, 20]. Wolbachia can also spread via horizontal transmission of the parasite between species [ 13, 14, 21-23] . Occasional hori- zontal transmission has been documented and occurs most frequently between related species [5, 24], In addition, the presence of multiple Wolbachia strains within a species shows evidence for horizontal gene transfer between host species or recombination events among Wolbachia strains [3, 10]. Less is known about the infection dynamics of Wolbachia within a single host species. Across several ant species, Wolbachia in- fection prevalence appears near fixation within some popula- tions [5, 10, 14] , However, other populations vary in the pre- valence of infection across colonies. Even within infected col- onies, not all workers harbor the infection [14, 22], suggest- ing that Wolbachia is not transferred readily between work- ers. There is also evidence that infection rates of workers are 2 Psyche lower than infection rates of worker brood [14] suggesting a loss of infection with age. We studied the distribution and infection dynamics of the Wolbachia parasite in a recently established Formica fusca population. F. fusca is a pioneering species and rapidly colo- nizes open environments prior to competition from other species [25, 26]. Within a single population, this species can establish both monogynous and polygynous colonies [25, 27]. A preliminary study suggested that Wolbachia is present in this population, but that only a subset of colonies is infect- ed, contrasting previous findings of near fixation prevalence rates in related ants species [5, 14, 15, 20]. We surveyed the population to determine the prevalence of Wolbachia infec- tion across colonies and to test whether infection is associ- ated with nest size, a proxy for colony size [28] , nest location, production of sexuals, and queen number. 2. Methods 2.1. Study Population. The isolated study population of For- mica fusca inhabits a disturbed meadow of grasses and gold- enrod that borders a temperate conifer forest in Hamilton, New York (N 42° 48. 134 W 075° 30.343). While the exact colonization date is not known, estimates of the appearance of nest mounds at the site range from 10 to 15 years ago. Other ant species present at or near the site include Formica species, Leptothorax longispinosus, Tapinoma sessile, Campon- otus americanus, Lasius species, Myrmica punctiventris, and Monomorium minimum. Formica fusca ants were the most common ants found at the site. We did not check for the pre- sence of Wolbachia in other ant species. 2.2. Nest Characteristics. All nests within the study site were mapped with GPS coordinates using Google Earth and ArcView (Figure 1). The Clark- Evans nearest neighbor method was used to infer dispersion of Wolbachia across colonies [29], with R = I indicating random dispersion and R = 0 indicating clumped dispersion. In order to test wheth- er R was significantly different from 1, a critical value, c, was calculated according to Clark and Evans [29] using a f-dis- tribution. Significant differences between infected and non- infected colonies were tested by comparing R-values using the F distribution. The size of the nest mound was measured in two direc- tions across the nest entrance; the longest diameter and the one perpendicular to the longest. Measurement extended to the edge of the raised mound. The area of the nest mound was calculated as the area of an ellipse with the two perpen- dicular measures halved as radii. The average nest mound area was calculated, and mean nest mound area of infected and noninfected nests was compared with a two-tailed t-test. Nests were designated as either “small,” nests smaller than the mean nest area, or “large,” nests larger than the mean. A Fish- er’s Exact Test was used to determine association between in- fection and nest size. Across infected colonies, the proportion of infected individuals was compared to nest area using Ken- dall’s coefficient of rank correlation. Nest size was used as a proxy for colony size following the association described in Tuzzolino [28]. 2.3. Sample Collection. Workers, worker brood, and repro- ductive brood were collected from all 35 colonies within the boundaries of the sampling site from late June to early Au- gust, 2011, during the period when reproductives are most abundant (unpublished data, [28]). Samples were collected from nests in both shady and sunny locations during late morning hours. Temperature during collection averaged bet- ween 24 and 29° C. Nests were watered with approximately 10 liters of water in the afternoon preceding collection to facil- itate the sampling of reproductives [28]. During collection, small areas were probed with trowels to determine location of brood chambers and workers and brood were aspirated into vials with minimum disturbance to the nest. When no brood chambers were found, shovels were used to extract more dirt from the surface to collect workers. The duration of collec- tion was limited to 20 minutes. A Fisher’s Exact Test was used to compare the number of reproductives obtained during this sampling period in infected and noninfected colonies. Worker and brood samples were immediately frozen at -20° C. DNA was extracted from all samples using 100 uE of a 10% Chelex solution (Bio-Rad), and samples were boiled for 15 min and spun for 1 min at 13,000 rpm. The superna- tant from worker samples was placed directly into a PGR re- action; the supernatant from brood samples was diluted 1:10 with water. We sampled 20 colonies for presence of Wolbachia; for one of these colonies, microsatellite data was not available, resulting in a sample size of 19 colonies for comparisons of infection with queen number. 2.4. Population Survey of Wolbachia. All samples were ampli- fied with 18S primers (18SF1 and 18SR1; [30]) to confirm that the DNA extractions were successful. Each sample was then amplified twice with Wolbachia specific primers (wsp 8 IF and wsp 69 IR; [31]) to confirm presence or absence of Wolbachia infection. Detection rate of Wolbachia infection was estimated at greater than 99%. For all reactions, samples were run in 25 uL of the following reaction mixture: IX of lOX PGR buffer, 0.2 mM each of dNTPs, 1.5 mM MgGb, 0.5 uM each of primer, 1 unit of Taq polymerase (5U/uE), and 1 uE DNA. Samples were run on a Bio-Rad DNAengine PTG thermocyder with the following protocol: 94° G for 5 min, 47 cycles of 94°G for 30s, 55°C for 45 s, 72°C for 1:30 min, hold at 72°G for 7 min. Amplified products were run on 1.5% agarose gels and analyzed as presence/absence of a 610 bp band. Samples with no band after two runs were designated as noninfected samples. 2.5. Microsatellite Analysis. We genotyped 20 workers per colony from a total of 34 colonies at 5 microsatellite loci: FE42 [32], FE12, FE29, [33], FY7, FY15 [34]. PGR amplifica- tions were performed in a 25 fiL final volume containing 1 X of lOX PGR buffer, 0.2 mM each of dNTPs, 1.5 mM MgGb, 0.5 uM each of primer, 1 unit of Taq polymerase (5U/uE), and 1 uL DNA. Samples were run on a Bio -Rad DNAengine Psyche 3 12 33 32 »31 30 29 28 Hamilton Road 1 « 13 *14 N 0 S 50 100 150r*« 27 5 22 • 18 34 24 ^ Infected 9 Non-infected • N/A Figure 1; Map of the study site showing all colonies in the population. Infected colonies: red circles; noninfected colonies: blue circles; untested colonies: yellow circles. PTC thermocycler with the following protocol: 94° C for 5 min, 27 cycles of 94° C for 30 s, 48/55° C for 30 s, 72° C for 45 s, hold at 72° C for 3 min. Amplified fragments were anal- yzed on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) and sized using GeneMapper 4.1 and 400ROX size standard from Applied Biosystems. All allele calls were manually verified. Effective queen number was estimated from pairwise worker-worker relatedness values between colonies using Re- latedness 4.2 according to the equation outlined in Krieger and Keller [35]. 3. Results In this population, 35% (7/20) of colonies were infected with Wolbachia. In infected colonies, the average proportions of individuals that were infected per colony (±SD) were workers, 0.62 ± 0.31; worker brood, 0.83 ± 0.32; reproductive brood, 1.00. In infected colonies, there was no difference bet- ween the proportions of infected workers per colony versus the proportions of infected worker brood per colony {t = 0.82, P = 0.44). Noninfected colonies were no more likely to have reproductives than infected colonies (P = 0.53). Only one of the seven infected colonies and three out of 13 non- infected colonies produced sexuals, so it was difficult to test associations between infection and colony sex ratio. The ratio (R) of average distance to nearest neighbor to the expected distance based on density was 1.08 and 0.83, res- pectively, for noninfected and infected colonies, suggesting that both noninfected and infected nests occur in a random distribution. The R-value for infected colonies was not signi- ficantly different from one (R = 0.83, t = 0.43, P = 0.85). The distribution of infected nests was not significantly diffe- rent from noninfected nests {F = 0.24, P = 0.70). The average nest mound size was 2203 cm^. Prevalence of infection is not associated with large nest size when compar- ing small and large nests (R = 0. 12). Mean nest size of infect- ed colonies was nearly double the size of noninfected colonies, but this difference was not significant (infected = 3101 cm^; noninfected = 1632cm^; / = 1.16, P = 0.26). Among infected colonies, nest area was not related to the proportion of infected individuals within a colony (t = 0.41, P = 0.249). 4 Psyche The population has a high level of genetic diversity with the number of alleles per locus ranging from 6 to 14 and ex- pected heterozygosities for each locus ranging from Hg = 0.44 to 0.83. There is significant genetic structure between nests in the population (Fst = 0.20 ± 0.13), and there is no evidence for isolation by distance across nests {y = 9E~^^x+0.28;R^ = 0.014), suggesting that dispersal occurs primarily via mat- ing flights and not by budding of queens and workers to adja- cent nest sites (unpublished data). Of the 19 colonies for which both Wolbachia infection and queen number were tested, 40% of infected colonies were monogynous and only 9% of noninfected colonies were monogynous, but this difference was not significant (P = 0.30). The average queen number in infected colonies was 2.37 ± 1.06 and was not significantly different from the aver- age queen number in noninfected colonies, 2.17 ± 0.86 (t = 0.46, P = 0.65). 4. Discussion The results from this study reveal a snapshot of early Wol- bachia infection in a recently established Formica fusca pop- ulation. The recent introduction of this population offers the unique opportunity to test for ecological correlates of Wol- bachia infection and spread. Infected nests in the population were broadly scattered throughout the study site, and the probability of infection was not predicted by closest neigh- bors, indicating little or no transmission of Wolbachia bet- ween colonies. In one area, three infected nests are closely clumped together, but these nests are likely satellite nests of the same colony due to their close proximity. This result con- trasts previous studies of other Formica species where Wol- bachia seems to infect a high proportion of colonies within populations [5, 10, 14]. In addition, a survey of 32 species of Formica found multiple strains of Wolbachia infection in all species and sharing of parasite haplotypes across distant host mtDNA haplotypes, suggesting historical horizontal trans- mission between species [5]. One possible explanation for these differences is that many Formica species tend to form long-term, stable populations and these species may have enhanced opportunities for hori- zontal transmission. Formica fusca is an ephemeral species that invades relatively open spaces and establishes colonies that later may be outcompeted by more aggressive species [25]. These short-lived populations may not persist long enough to permit extensive horizontal transmission. Alter- natively, the difference in prevalence rate among populations may be due to the fact that a population that spreads from an initial infected foundress or group of infected queens may also have complete transmission of infection to all nests via vertical transmission. For example, the fact that three close- ly spaced nests are infected likely resulted from vertical trans- mission of Wolbachia preceding the split into satellite colon- ies. Our results suggest that this newly established popula- tion was founded by multiple introductions of Formica fusca, some of which were infected with Wolbachia and others that were not. If Wolbachia is transmitted between colonies in this host population, it has not had time to spread. Determining whether the current infections across this population repre- sent the same Wolbachia strain or represent introductions of separate strains of the parasite will help disentangle the history of Wolbachia infection in this population. An addi- tional possibility is that newly established colonies in this population were infected with Wolbachia via horizontal transmission of neighboring ant species. A parallel study of Wolbachia infection in other ant species within the popula- tion would provide the data necessary to test this hypothesis. Although no significant association between nest size and infection prevalence was found, 9 out of 12 “small” nests do not harbor infections and mean nest size of infected nests is nearly double that of noninfected nests. The trends suggest that nest size may be positively associated with the prevalence of Wolbachia given a larger sample size. Such a finding would contradict the argument, as proposed and rejected by Wense- leers et al. [14], that Wolbachia may have deleterious effects on the colony via reducing worker biomass. Alternatively, because nest size is correlated generally with colony age, older colonies may have been established from foundress queens that emigrated from an infected population and the newly established, smaller colonies represent a recent introduction from a noninfected population. Multiple introductions and/ or recent acquisition of the Wolbachia parasite might also ex- plain the low prevalence rate of Wolbachia in this population (34%), which contrasts previous findings of near fixation of the parasite [5, 10, 14]. Interestingly, in other species, Wolbachia infections are not common across introduced populations [ 1 1, 36] . In Argentine ants and fire ants, selective pressures of colonization may impede the ability of an in- fected population to successfully colonize [ 1 1, 36] . This effect is not seen in this Formica population. Within colonies, the infection rate can vary between work- ers, brood, and reproductives. In our study population, 100% of reproductives, 87% of worker brood were infected and 63% of workers were infected. This pattern resembles that seen in Formica truncorum species (95% in sex M, 94% sex F, 87% worker brood, and 45% workers) [14], but the dif- ferences in infection rate in this study were not significant. Thus, our results provide little support to the hypothesis that adult workers may be able to rid themselves of infection. There is no strong selective pressure inhibiting the loss of infection in workers because workers produce only males and are essentially an evolutionary dead-end for this parasite. Overall, the results of this study show no large deleterious fitness effects of infection on colony size or longevity; infec- tion does not appear to decrease longevity of the colony, at least within the time scale of this population expansion. Wol- bachia infection could also cause a decrease in the number of reproductives produced, limiting the reproductive success of colonies. In this population, few colonies produce sexuals, but the production of sexuals does not appear to be linked to the presence of infection as both infected and noninfected colonies produce sexuals. It is important to note that the infected colony that produced sexuals produced only 4 males, compared to the greater than 16 reproductives produced in noninfected colonies. This result does support the finding of reduced biomass of sexual brood in infected Formica trun- corum colonies [14]. Differences in sex ratios of infected Psyche 5 colonies could also result from infections [14, 15, 20, 37], but the small numbers of reproductives produced in this popula- tion make it difficult to test sex ratio predictions. Finally, Wolbachia infection is not correlated with queen number in this population of Formica fusca. In native popu- lations of fire ants, monogyne colonies harbor a higher fre- quency of Wolbachia infection than polygyne colonies. The difference in prevalence rate may be due to a reproductive advantage to monogyne colonies because these queens are less likely to produce diploid males when founding new colo- nies [11]. This pattern was not seen in our study or in stud- ies of related species of Formica ants [8, 20]. Both studies show no difference in the infection rate of monogyne ver- sus polygyne colonies. Thus, Wolbachia infection does not appear to be associated with queen number in Formica ants and is not likely to affect the genetic diversity of Formica fusca colonies. Acknowledgments Special thanks to the Meyer family for access to their pro- perty. This study was funded by the Division of Natural Sciences Undergraduate Summer Research Program and the Department of Biology at Colgate University. References [1] K. Hilgenboecker, P. Hammerstein, P. Schlattmann, A. 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Pamilo, “Evolution of social insect colo- nies,” in Sex Allocation and Kin-Selection, Oxford University Press, Oxford, UK, 1996. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 169564, 6 pages doi:10.1155/2012/169564 Research Article Cytogenetics of Oryctes nasicornis L. (Coleoptera: Scarabaeidae: Dynastinae) with Emphasis on Its Neochromosomes and Asynapsis Inducing Premature Bivalent and Chromosome Splits at Meiosis B. Dutrillaux and A. M. Dutrillaux UMR 7205, OSEB, CNRS/Museum National d’Histoire Naturelle, 16, rueBujfon, CP 32, 75005 Paris, France Correspondence should be addressed to B. Dutrillaux, bdutrill@mnhn.fr Received 14 September 201 1; Accepted 13 December 2011 Academic Editor: Howard Ginsberg Copyright © 2012 B. Dutrillaux and A. M. Dutrillaux. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The chromosomes of specimens of Oryctes nasicornis from three locations in France and two locations in Greece were studied. All karyotypes have an X-Y-autosome translocation: 18, neoXY. Two male specimens from France (subspecies nasicornis) displayed an unusual behaviour of their meiotic chromosomes in 30-50% of spermatocytes, with asynapsis at pachynema, premature bivalent and chromosome split at metaphases I and IF The karyotypes remained balanced at metaphase I, but not at metaphase II. These particularities mimic the meiotic behaviour of B chromosomes and question about their existence, reported earlier in Spanish spec- imens. Due to the variable character of B chromosomes, complementary analyses are needed. To our knowledge, such meiotic par- ticularities have not been described, beside cases of infertility. In specimens from Corsica (subspecies laevigatas) and Greece (sub- species kuntzeni), all spermatocytes I and II had a normal appearance. The meiotic particularity may thus be limited to male speci- mens from subspecies nasicornis. 1. Introduction Beside pathological conditions such as malignancies or chro- mosome-instability syndromes, intraindividual variations of chromosomes are rare. Because of its usual stability, the kar- yotype of a limited number of cells is thought to represent that of a whole individual. This stability prevails for germ cells, so that parental and descendant karyotypes are similar. Consequently, the chromosome analysis of a limited number of cells from a limited number of individuals most frequently gives valuable information about the karyotype of their spe- cies. Exceptions exist, however, among which the presence of B chromosomes represents a major cause of numerical varia- tion and polymorphism. B chromosomes have been describ- ed in plants and animals. They are characterized by a number of criteria among which is their particular meiotic behaviour: they do not pair like autosomes and tend to undergo prema- ture centromere cleavage and non-disjunction at anaphase. This leads to variations of their number from cell to cell and descendant to descendant [ 1 ] . Insect cytogenetics has essentially been developed through spontaneously dividing germ cells at diakinesis/metaphase I and metaphase II. At these stages, chromosome morpho- logy is not optimal for analysis. Among several thousand of chromosome formulas reported in coleopterans, the pres- ence of B chromosomes was noticed in about 40 instan- ces [1-5]. Oryctes nasicornis L. 1758 (Coleoptera: Scarabaei- dae: Dynastinae) is one of the very first insects in which dis- pensable supernumerary chromosomes were described [6] and later on considered as B chromosomes. This observation was quoted in reviews on both insect cytogenetics [5, 6] and B chromosomes [ 1 ] . Having analysed the mitotic chromosomes of a male specimen of O. nasicornis L. 1758, we were surprised to ob- serve a karyotype different from its earlier descriptions. It had neither a Xyp (p for parachute, [6]) sex formula nor 2 Psyche supernumerary B chromosomes, but neoXY as a conseq- uence of an X-Y-autosome translocation. B chromosomes being dispensable, we studied specimens from other localities and performed meiotic analyses to understand the causes of these discrepancies. We did not find B chromosomes, but, in two out of seven specimens, there were quite unexpected meiotic particularities. From pachytene to spermatocyte II stages, recurrent asynapsis, nonpairing, and premature cen- tromeric cleavages mimic the behaviour of B chromosomes. Checkpoints controlling meiotic chromosome behaviour have been identified, from yeast to mammals [7, 8]. They monitor elimination of spermatocytes with abnormal chro- mosome synapsis [9]. In some Oryctes nasicornis specimens, the anomalies at metaphase I and II, as consequences of pachytene asynapsis, suggest the low stringency of these checkpoints. 2. Material and Methods Two male specimens (number 1 and 2) of O. nasicornis were obtained from the breeding developed at the Museum of Besan^on (France). They were captured as larvae in the Besa- n^on area and are assumed to correspond to the nasicornis subspecies. They metamorphosed in June 2006. Two adult male specimens were captured in April 2007 (specimen num- ber 3) and September 2010 (specimen number 4), at Bois-le- Roi, at the Fontainebleau forest border (48° 27' N, 2° 42' E). They are assumed to belong also to the nasicornis subspecies. Another male (specimen number 5) was captured near Porto Vecchio, Corsica (41° 36' N, 9° IT E), in June 2007. It is as- sumed to belong to the laevigatas Heer 1841 subspecies. Einally, two males were captured in Greece, one (specimen number 6) near Oros Kallidromo (38° 44' N, 22° 39' E) in may 2010 and one (specimen number 7) near Kalambaka (39° 47' N, 21° 55' E) in June 2011. They are assumed to be- long to the kuntzeni Minck subspecies. Pachytene bivalent chromosome preparations were obtained following a long hypotonic shock and meiotic and mitotic metaphases after treatment with 0.88 M KCL for 15 min. and another 15 min. in diluted calf serum (1vol.) in distilled water (2 vol.) [10, 11]. Chromosomes at various mitotic and meiotic stages were studied after Giemsa and silver stainings and Q- and C-banding. Image capture was performed on a Zeiss Phomi 3 equipped with a high-resolution camera JAI M4-f- and IKAROS (Metasystems) device or a Eeica Aristoplan equip- ped with a JAI M300 camera and ISIS (Metasystems) device. 3. Results Mitotic Karyotype (Figure 1 ). It is composed of 18 chromo- somes, including three sub-metacentric (number 1, 2 and 8) and five acrocentric (number 3-7) autosomal pairs. All of them carry large and variable heterochromatic segments around the centromeric region. The X chromosome is sub- metacentric and the Y acrocentric. Their size is much larger than that of gonosomes of most other Scarabaeid beetles. All heterochromatin is positively stained after C-banding and heterogeneously stained after Q-banding (not shown) which 6 7 8 Figure 1: Mitotic karyotype of number 1 from Besan^on) after %f 3 ' ' 4 ' ' 5 ' X Y Oryctes nasicornis male (specimen 2-banding. f / K t. / { . i * * ■ Vn.. W ? ft 3 4 Hi L . : Vi V ** h 8 X 5 Figure 2: Karyotype from a spermatocyte at pachynema after the Giemsa staining (left), NOR staining displaying nucleoli (N) (cen- tre), and C-banding treatment (right). Acrocentric bivalents 5 and 6 are not synapsed, but associated by their heterochromatic short arms (arrows). Heterochromatin is more compact than on mitotic chromosomes. Specimen number 3 from Bois-le-Roi, as is the case in the next figures. indicates its heterogeneous composition. Beside the varia- tions of the amounts of heterochromatin, all specimens had the same chromosome complement, as reported [4, 12]. Pachytene Chromosomes (Figures 2 and 3). As expected from the mitotic karyotype, nine bivalents were generally observ- ed. They could be identified by the amount and position of their heterochromatin, although heterochromatin was glob- ally more compact than in mitotic cells. The sex bivalent was quite characteristic. It had a large synapsed segment, similar- ly to autosomes, followed by juxtacentromeric heterochro- matin, and a compact segment. This was interpreted as the result of an X-Y-autosome translocation, the autosomal por- tion forming the long arm and the sex chromosomes forming the short arm. This translocation explains the low number of chromosomes (18 instead of 20 in most Scarabaeidae) and the large size of the sex chromosomes (the short arm relative length matches that of the X of other Dynastinae with a free X). Thus, the mitotic karyotype formula is 18, neoXY. Silver staining displayed a strong staining of all heterochromatin, as in most coleopterans. In addition, round nucleolar-like stru- ctures were recurrently associated with the short arm of a small acrocentric bivalent that we defined as number 6. Thus, according to previous studies [13], the Nucleolar Or- ganizer Region (NOR) is located on chromosome 6 short arm (Figure 2). The above description refers to observed spermatocytes. However, one or several bivalents displayed Psyche 3 Figure 3: Spermatocytes at pachynema after the Giemsa staining (left) and C-banding (right) displaying asynapsis of chromosomes 8 (a) and sex chromosomes (b). either asynapsis or incomplete synapsis in 29% and 41% of the spermatocytes from specimen number 2 and 3 from Besan^on and Bois-le-Roi, respectively (Table 1). Smaller acrocentrics (numbers 6 and 7) were the most frequently in- volved, but all bivalents, including the sex bivalent (Figures 2, 3(a) and 3(b)), could be occasionally affected. In all instan- ces, the non-synapsed autosomes were lying close to each other, suggesting either their premature desynapsis or defi- cient synapsis. The two homologues remained frequently at contact by their heterochromatic regions (Figure 2). Conver- sly the neoX and neoY chromosomes could be completely separated (Figure 3(b)). Specimen number 1 was immature and spermatocytes at pachynema of specimen number 5 could not accurately be studied and could not be considered as control. In specimen number 4 from Bois-le-Roi and spec- imens number 6 and 7 from Greece, the synapsis was strictly normal. We applied the same cytological techniques to speci- mens from more than other 100 species and observed such pachytene asynapsis only once and at a low frequency. Diakinesis/Metaphase I (Figure 4). This stage was the most frequent in all the specimens studied: a total of 696 cells could be examined. Most of them displayed nine bivalents (biv), among which the sex bivalent could be identified by its asymmetrical constitution, as in other species with translocation-derived neoXY. No particularities were noticed in specimens number 4 to 7, whereas 43% and 34% of cells from the specimens number 2 and 3 (Table 1) displayed uni- Table 1: Numbers and percentages of mitotic and meiotic cells analysed in specimen number 2 from Besan^on and number 3 from Bois le Roi. Cells were scored as abnormal (abnl) when they display- ed asynapsis (pachynema), univalents (diakinesis/metaphase I) or monochromatidic chromosomes (metaphase II), and normal (nl), when all chromosomes were in correct phase. Besan^on- image no. 2 Bois-le-Roi-image no. 3 nl abnl % abnl nl abnl % abnl Mitotic 32 0 0 0 0 Metaphase Pachynema 34 10 29 36 25 41 Diakinesis/ Metaphase I 41 31 43 195 99 34 Diakinesis/ Metaphase II 25 11 31 40 58 59 valents (univ), respectively. Their number was inversely pro- portional to that of bivalents: 9 biv + 0 univ; 8 biv + 2 univ; 7 biv + 4 univ; 6 biv + 6 univ, demonstrating that two uni- valents replaced one bivalent. The univalent occurrence, ob- served at both early diakinesis and late metaphase I, did not seem to depend on the progression towards anaphase. It preferentially involved smaller and sex chromosomes. Metaphase II (Figures 5 and 6). No particularities were noticed among the 56, 48, 50 and 27 metaphases II analyzed 4 Psyche % Figure 4: Spermatocytes at metaphase I after the Giemsa staining (top) and C-banding (bottom) with eight bivalents and two univalents (arrows). ■ © ^ @ ® © Figure 5: Spermatocyte at metaphase II displaying eight bi-chro- matidic and two single-chromatid chromosomes, presumably num- ber 6 (arrows). from specimens 4, 5, 6, and 7, respectively. All were com- posed of 9 double-chromatid chromosomes. In specimens 2 and 3, 31% and 59% of metaphases II, respectively, compris- ed more than 9 chromosomes. C-banding allowed us to dif- ferentiate mono-chromatidic (monoc) and bi-chromatidic (bic) chromosomes. The number of monoc was roughly in- versely proportional to that of bic: 9 bic + 0 monoc, 8 bic +2 monoc (Figure 5), 7 bic -1- 4 monoc, and 6 bic + 6 monoc. In a proportion of metaphases II, however, the ratio bic/ monoc was different, indicating that aneuploidies occurred, as consequence of segregation errors at anaphase I (Figure 6). The premature centromeric split preferentially involved the small acrocentrics, the metacentric 8, and the sex chromo- somes. 4. Discussion The karyotypes of the specimens of O. nasicornis studied here obviously do not contain B chromosomes. O. nasicornis is a widespread species in Western Europe, with eleven sub- species identified. The first mention of its karyotypic partic- ularities was reported on Spanish specimens, which belong to the grypus Illiger 1803 subspecies [4] . The specimens from Besan^on and Bois-le-Roi belong to the subspecies nasicor- nis. These two locations cover only a small part of the whole distribution area of the subspecies, but they are sufficiently distant (about 300 km) to assume that they do not constitute an isolate with abnormal gametogenesis. The specimens from Corsica and Greece, in which we failed to detect any meiotic particularity, belong to the subspecies laevigatas and kuntzeni, respectively, and there are no available data on the chromosomes of other specimens from this subspecies. Thus, the question of both the presence of B chromosomes and/or atypical meiosis, in relation with subspecies, remains open and needs further investigations. The high recurrence of asynapsis and premature centro- meric cleavage may be an artifact induced by hypotonic shock and spreading. However, the techniques used for pach- ynema and other meiotic stages were different, and we found fairly similar rates of aberrations at all stages. Furthermore, technical artifacts can hardly explain aneuploidies at meta- phase II. We applied these techniques on meiotic chromo- somes from many species of coleopterans without B chromo- somes and observed such particularities only once at a low rate. Conversly, when B chromosomes were duly identified, they had a particular pairing leading to non-disjunctions at anaphase I, hence duplications and losses in spermatocytes II and variable numbers in descendants. It has no effect upon the phenotype, which indicates they carry no genes with major effect on the phenotype [I]. Here, all chromosomes can be involved in abnormal meiotic pairing. At metaphase II, 30-50% of spermatocytes displayed premature chro- mosome cleavage, which should induce a high rate of Psyche 5 Figure 6: Unbalanced sister metaphases II after the Giemsa staining (top) and C-banding (bottom). One acrocentric (presumably number 7) is single-chromatid on the left, while the complementary mono-chromatidic chromosome is in excess on the right (arrows). unbalanced gametes. Indeed, aneuploid spermatocytes II were observed and a reduction of reproductive fitness should be expected, but we have no indication that it is the case. Furthermore, it is noteworthy that the rates of asynapsis at pachynema, premature bivalent cleavage at metaphase I, and premature centromere split at metaphase II are roughly simi- lar at both intra- and interindividual levels. This suggests that metaphase I and II anomalies are direct consequences of pachytene asynapsis, and that there is both synapsis and checkpoint flaws at pachynema [8, 9] . It will be interesting to establish karyotypes of a series of eggs laid by parents with these meiotic particularities to know whether or not they induce a high rate of aneuploidies at early stages of develop- ment. Another point of interest, in the karyotype of O.nasicornis, is the presence of neo-sex chromosomes. As described in the Scarabaeid beetles Dynastes Hercules and Jumnos ruckeri, their meiotic behaviour, with an autosome-like synapsis of a long portion, indicates they originated from an X-Y-autosome translocation [13, 14]. As in these species too, the autosomal portion is separated from the original X component by the centromere, that is, constitutive heterochromatin. The insu- lating role of heterochromatin has been discussed for long in mammals, where it prevents inactivation spreading from the late replicating X to the attached autosome in female somatic cells [15]. In meiotic prophase of the male, heterochromatin also isolates euchromatin from the inactivated sex chromo- somes [16]. In Drosophila, the gene dosage compensation between males and females somatic cells is achieved by the overexpression of genes from the single X of the males [17]. This may also be the case of the beetle Dynastes Hercules, but this was shown only for NOR expression [ 13] . In Gryllotalpa fossor (Orthoptera), the dosage compensation is of the mam- malian type [18]. Finally, in Musca domestica (Diptera), no dosage compensation seems to exist [19]. These different sit- uations demonstrate the existence of several regulatory mechanisms for X-linked gene expression in insect somatic cells. Whatever this mechanism, that is, over- or underex- pression, there is an important character which is the exis- tence of an epigenetic control spreading over large chromo- some segments, if not whole chromosomes. We proposed that, in insects with overexpression of the X-linked genes in the male, as Drosophila, heterochromatin might play this insulating role [ 13] . This fits with the observation that in the few instances where an X-autosome translocation carrier Drosophila is fertile, the break point originating the translo- cation occurred within heterochromatin of the X ([20] and references herein). The presence of heterochromatin between gonosomal and autosomal components in the neo-sex chromosomes of O. nasicornis provides another example 6 Psyche suggesting the role of heterochromatin to avoid spreading of cis-acting epigenetic control elements. In conclusion, this study shows that two chromosomal particularities exist in O. nasicornis. One is an X-Y-autosome translocation, frequently deleterious for reproduction, unless specific conditions prevent position effect, due to the dif- ferent regulation of sex chromosomes and autosomes. Such translocations are not exceptional in Coleoptera, compared to other animals such as mammals. The other particularity is much more exceptional: two male specimens of O. nasicornis nasicornis display meiotic alterations usually considered as deleterious for fertility. These specimens were caught at two distant localities, which suggests these alterations are spread in the population and do not drastically prevent reproduc- tion. Progress in the molecular biology of meiosis has shown the multiplicity of genes involved in synaptonemal com- plex formation and recombination [21, 22]. One of them may be altered in some specimens of O. n. nasicornis and maintained if associated with some hypothetical advantage. A third particularity, that is, the presence of B chromosomes, reported in specimens from Spain, may be an incorrect interpretation of the meiotic particularity described here and warrants further studies. Acknowledgments The authors are indebted to Jean- Yves Robert and Frederic Maillot, Museum de Besan^on, France, and Laurent Dutril- laux, who provided us with the specimens from Besan^on area and Corsica, respectively. References [1] R. N. Jones and H. Rees, B Chromosomes, Academic Press, London, UK, 1982. [2] C. Juan and E. Petitpierre, “Chromosome numbers and sex determining systems in Tenebrionidae (Coleoptera),” in Ad- vances in Coleopterology, M. Zunino, X. Belles, and M. Bias, Eds., pp. 167-176, AEG Press, Barcelona, Spain, 1991. [3] E. Petitpierre, C. Segara, J. S. Yadav, and N. Virkki, “Chromo- some numbers and meioformulae of chrysomelidae,” in Bio- logy of Chrysomelidae, P. Jolivet, E. Petitpierre, and T. H. Hsiao, Eds., pp. 161-186, Kluwer Academic Publishers, Dodrecht, The Netherlands, 1988. [4] N. Virkki, “Akzessorische chromosomen bei zwei kafern, Epi- cometis hirta und Oryctes nasicornis L. (Scarabaeidae),” An- nales Academice Scientiarum Fennicce, vol. 26, pp. 1-19, 1954. [5] J. S. Yadav, R. K. Pillai, and Kaaramjeet, “Chromosome num- bers of Scarabaeidae (Polyphaga: Coleoptera),” The Coleopter- ist Bulletin, vol. 33, pp. 309-318, 1979. [6] S. G. Smith and N. Virkki, Insecta 5: Coleoptera, vol. 3 of Ani- mal Cytogenetics, Gebriider Borntraeger, Berlin, Germany, 1978. [7] N. Bhalla and A. 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DutriUaux, “Synaptonemal complexes in Gerbillidae: probable role of intercalated heterochromatin in gonosome-autosome translo- cations,” Cytogenetics and Cell Genetics, vol. 43, no. 3-4, pp. 161-167, 1986. [17] V. Gupta, M. Parisi, D. Sturgill et al., “Global analysis of X- chromosome dosage compensation,” Journal of Biology, vol. 5, article 3, 2006. [18] S. R. V. Rao and M. Padmaja, “Mammalian-type dosage com- pensation mechanism in an insect -Gryllotalpa fossor (Scud- der)- Orthoptera,” Journal of Biosciences, vol. 17, no. 3, pp. 253-273, 1992. [19] A. Diibendorfer, M. Hediger, G. Burghardt, and D. Bopp, “Musca domestica, a window on the evolution of sex-deter- mining mechanisms in insects,” International Journal of Devel- opmental Biology, vol. 46, no. 1, pp. 75-79, 2002. [20] M. Ashburner, Drosophila: A Laboratory Handbook, Gold Spring Harbor Laboratory Press, Gold Spring Harbor, NY, USA, 1989. [21] M. D. Ghampion and R. S. Hawley, “Playing for half the deck: the molecular biology of meiosis,” Nature Cell Biology, vol. 4, pp. 50-56, 2002. [22] P. E. Gohen, S. E. Pollack, and J. W. Pollard, “Genetic analy- sis of chromosome pairing, recombination, and cell cycle con- trol during first meiotic prophase in mammals,” Endocrine Reviews, vol. 27, no. 4, pp. 398-426, 2006. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 312054, 12 pages doi:10.1155/2012/312054 Research Article Associations of Two Ecologically Significant Social Insect Taxa in the Litter of an Amazonian Rainforest: Is There a Relationship between Ant and Termite Species Richness? Amy L. Mertl,^ James F. A. Traniello,^ Kari Ryder Wilkie,' and Reginaldo Constantino^ ^ Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA ^ Departamento de Zoologia, Universidade de Brasilia, 70910-900 Brasilia, DF, Brazil Correspondence should be addressed to Amy L. Mertl, amymertl@gmail.com Received 1 October 2011; Accepted 29 December 2011 Academic Editor: David E. Bignell Copyright © 2012 Amy L. Mertl et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In spite of the ecological dominance of Neotropical ants and termites, little is understood about how their interactions influence their species richness and distribution. We surveyed ground-dwelling termite and ant species in a primary rainforest in Ecuador and analyzed ecological correlates of diversity. Termite richness was positively correlated with ant richness and abundance of twig- nesting ants. We found no evidence of competition for twigs between termites and ants. No ecological factors were correlated with termite diversity although elevation and twig and log abundance influenced ant diversity. When ant richness was compared to the richness of termites employing different predator defenses, a positive correlation was found with soldierless termites, but not genera employing chemical or mechanical defense. Our results suggest that multiple ecological factors influence ant and termite diversity, and that ant predation on termites may have a greater effect than competition between ant and termites for nest sites and food sources. 1. Introduction Ants (Order Hymenoptera, Family Formicidae) and termites (Order Isoptera) are among the most prevalent and abun- dant animals in terrestrial ecosystems [1-3]. Worldwide, 14,550 ant species and subspecies in 283 genera have been de- scribed to date [4], and termites are comprised of more than 2866 species in 287 genera [5]. Ants and termites are both entirely eusocial and often form large colonies, contributing to their ecological dominance. In a central Amazonian terra firme rainforest in Brazil, ants and termites were found to constitute approximately 75% of soil fauna biomass [6] and 30% of total animal biomass [2]. In this region. Beck [6] estimated that there were 8.6 million ants and 1.3 million termites in a hectare. Ants and termites are most diverse in New and Old World lowland tropical forests, respectively. Termites reach their peak in the Afrotropics, whereas ant diversity is greatest in the Neotropics [7, 8]. Humivo- rous termites can be exceptionally diverse and abundant in the Afrotropics [7]. In some areas, termites may constitute up to 95% of the soil insect biomass [9] and local abundance may reach 10,000 termites per square meter [10]. The extraordinary abundance and diversity of ants and termites underscores their importance to tropical terrestrial ecosystems [1, 11-15]. Both ants and termites provide sig- nificant ecosystem services, their roles varying in accordance with their very different feeding biology. Termites are decom- posers [9] and humivores [16], whereas ants are predators, herbivores, scavengers, and fungivores [17]. In the tropics, leaf-litter and soil-dwelling ants turn over more earth than earthworms [18]. Carnivorous ants are among the dominant predators of invertebrates [1]. In contrast, termites are among the most important decomposers of cellulosic materi- als. Termites consume as much as 30% of leaf litter in tropical rainforests [19, 20] and can remove 12-57% of available dung in one month [21]. Plant growth tends to be improved on or near termite mounds due to soil modification [22] and higher nutrient content and water availability [23, 24]. 2 Psyche Termite food sources — litter, twigs, and decayed wood — are all typical nest sites of ants. Termite feeding may thus po- tentially limit ant nest site availability and hence diversity. However, ant/termite mutualisms have been reported [1, 25- 32], and by sharing nest sites, the presence of termites may increase ant diversity [33]. Dejean et al. [25] found 151 ant species in 725 termitaries and postulated that the availability of suitable nesting sites may impact ant diversity. Termites may also influence ant distribution and diversity because they can represent a significant component of ant diets [34]. Ants in genera such as Centromyrmex, Leptogenys, Metapone, Opthalmopone, Pachycondyla, and Paltothyreus may feed exclusively on termites [1, 27, 28, 35-38]. While most ant species seem to prey on termites without having a major impact on the survival of established termite colonies [39-41], some ant species may significantly reduce termite populations [37, 42, 43]. Indeed, ant predation is considered to be a major selective force in the evolution of termite defensive strategies [38, 44, 45]. The above-cited studies indicate that Neotropical leaf- litter ants and termites interact primarily as predators and prey and nest mutuals, and termites potentially are indirect competitors with ants for nest sites. The consequences of these interactions could influence ant and/or termite diver- sity and distribution. Ants have been shown to influence termite abundance, at least in the case of inquilines [46]. Ant and termite diversity in lowland tropical rainforests appears to be especially high [47-49], but there are few comprehensive species inventories. Regional surveys of ants and termites tend to focus on one clade or the other, but rarely both, and their ecological relationships thus remain poorly understood. Here we report on the results of a survey of the diversity and distribution of ants and termites in the litter layer of a Neotropical rainforest in western Ecuador. Given their varied associations, the relationship between ant and termite diversity and distribution is difficult to predict a priori. However, based on the common occurrence of ant predation on termites and potential competition between ants and termites for wood as a nest site or food source, respectively, we hypothesize that (1) the diversity/abundance of twig- and wood-nesting ants will decrease with increasing termite diversity/abundance in the litter due to reduced nest site availability; (2) the diversity of ant genera known to contain termitophagous species will increase with termite diversity; (3) the diversity of ants will increase with the diversity of termites employing antipredatory strategies potentially less efficacious than chemical defense [45, 50-52]. 2. Site Description and Sampling Methods Ants and termites were collected at the Tiputini Biodiversity Station (TBS) in the western Amazonian rainforest of Ecuador (Orellana Province, 0°37'55"S and 76°08'39"W, altitude 230 m, annual rainfall ~3000 mm), bordering Yasuni National Park. The study site is predominantly primary lowland rainforest with a diverse tree community dominated by the palm Iriartea deltoidea [53]. This region of western Amazonia has been identified as a major tropical wilderness of exceptional richness [54] and holds the global record for regional ant richness [55]. Assessments of the richness of ground-dwelling ants and termites were carried out along three 200 m transects (A, B, and C), with collection points every 10 m. At each sampling point, ants and termites were collected with a pitfall trap (diameter 9 cm, volume 400 mL, collected after 48 hours), a 1 m^ Mini- Winkler sample, a bait station (including tuna, peanut butter, cookie, and quinoa collected after 30 min- utes), and hand-collecting via careful examination the litter and understory for 15 minutes. The following environmen- tal variables were measured at each of the 60 collection sites (instrumentation/methodology noted in parentheses): microelevation (altimeter), slope (clinometer), canopy cover [56, crown illumination ellipses index], leaf litter depth (average of 10 measurements at each sampling point), bare ground percentage, number of plants/ m^, number of twigs and logs/m^, and volume of twigs and logs/m^. Elevation var- ied only 18 m over our transects (from 206-224 m), though this variation is enough to affect ant diversity [55]. We use the term microelevation because any effects of this variable are likely related to changes in slope and/or water table level rather than climatic variability. A detailed description of the site and methods can be found in Ryder Wilkie et al. [57] and Ryder Wilkie et al. [55]. Means and ranges for all measured environmental variables can also be found in Ryder Wilkie et al. [55]. Ants and termites were collected and identified to species or morphospecies. We noted when ants and termites were found utilizing the same twig or decayed log. Termites were identified at the University of Brasilia, where vouchers have been deposited. Ant vouchers have been deposited in the collection of the Harvard Museum of Comparative Zoology. To evaluate the potential for competition between ants and termites for twigs in the litter layer, we exhaustively searched leaf litter in 84 three X three meter plots (756 m^) and examined all twigs for nesting ants or feeding termites. We measured the volume of all twigs utilized by each group and rated decay on a scale of 1 (no decay, audible snap when broken) to 5 (highly decayed, crumble at touch) [58] . We also chose one meter at random in each plot and recorded the volume and decay of all twigs within the meter in order to estimate twig availability at TBS. Details of this methodology can be found in Mertl et al. [59]. These data were used to estimate the abundance of ants and termites in twigs but not species richness, as samples of termite species were not collected in these plots. We define “ground-dwelling” ants and termites as those nesting or active within the litter layer and therefore found using any of the above collection methodologies. Though we have previously collected ants in the canopy and subter- ranean strata at TBS [55], here we focus only on the litter strata where our methodologies sampled both ants and ter- mites effectively. Our collecting methods were adapted from the ALL protocol, which targets ground-dwelling ants [8]. These methods, which include hand collecting, litter-search- ing, the use of Winkler devices, and pitfall traps, strongly overlap with those used to sample termites in the litter layer Psyche 3 [60-62] . Our survey does not reflect total termite diversity at TBS, as both canopy and subterranean strata are important to consider when sampling total termite diversity [63]. 3. Data Analysis To assess sampling coverage, species accumulation curves were determined for both ants and termites using Estimates V 8.2.0 [64]. Multiple regression was used to determine the effects of environmental variation on ground-dwelling ant and termite species richness at the 60 principal sites (20 sites each along transects A, B, and C). At each site, measures of species richness of ground-dwelling ants and termites were based on the incidence of species in pitfall traps, baiting stations, hand collecting, and Winkler samples. Richness was regressed against the eight environmental variables noted previously. An exploratory correlation coefflcient matrix of these variables showed that all were at most weakly correlated {R^ < 0.41), supporting their independence and allowing for the use of multiple regression (least squares method). Slope, number of twigs and logs, and total volume of twigs and logs were log-transformed prior to regression to improve normality. Canopy cover, percent bare ground, and number of plants could not be normalized through transformation; however, a linear model provided a good fit to the data based on plots showing no correlation between residuals and fitted values (F < 0.001, P > 0.05 for all regressions) and normally distributed residuals. Separate regressions were run for total species richness of ants and termites. Regression was performed using JMP version 5.0.1 (SAS Institute Inc., Cary, NC, 1989-2002). To explore competition between ants and termites for wood, we correlated the diversity of twig- and wood-nesting ants and termite diversity based on nesting habits of ants [55], as well as the abundance of twig-nesting ants versus the abundance of twig-foraging termites in the 84 3 X 3 m plots. We also analyzed the volume and decay distributions of twigs used by twig-nesting ants and twig-foraging termites compared to the available distribution of twigs using ANOVA and Tukey’s HSD with JMP version 5.0.1. To examine the role of predation and antipredator de- fenses as determinants of ant and termite species richness, we categorized termite genera according to strategies of soldier defensive behavior (chemical and mechanical defense), sep- arating soldierless termite species in the analysis [44, Table 1]. Although termite soldiers equipped with mandibles to attack predators may possess cephalic glands producing a variety of secretions that could play a role in defense, we classified these species as having mechanical defenses because it is their first line of action and rapid mandibular strike can immediately dispatch an opponent [65]. Nasutitermitinae species, in contrast, rely on terpenoid glues produced by the frontal gland and discharged through the nasus to deter predators [51, 66, 67]. We recognize that soldierless apicotermitine termite species are not defenseless due to their lack of a soldier caste because workers are equipped with a frontal gland and/or paired dehiscence glands that rupture and burst the body wall to discharge defensive compounds Table 1: List of termite species collected at TBS, and their category of soldier defensive strategy. Termite species Defense Anoplotermes sp. 1 Soldierless Anoplotermes sp. 2 Soldierless Anoplotermes sp. 3 Soldierless Anoplotermes sp. 4 Soldierless Armitermes minutus Mechanical Atlantitermes sp. Chemical Coptotermes cf testaceus Mechanical Cornicapritermes sp. Mechanical Cornitermes sp. Mechanical Crepititermes verruculosus Mechanical Cylindrotermes cf. nordenskioeldi Mechanical Cylindrotermes flangiatus Mechanical Cylindrotermes parvignathus Mechanical Heterotermes tenuis Mechanical Nasutitermes callimorphus Chemical Nasutitermes ephratae Chemical Nasutitermes guayanae Chemical Nasutitermes intermedius Chemical Nasutitermes llinquipatensis Chemical Nasutitermes longirostratus Chemical Nasutitermes sp. Chemical Nasutitermes surinamensis Chemical Neocapritermes pumilis Mechanical Neocapritermes villosus Mechanical Rotunditermes bragantinus Chemical Ruptitermes sp. Soldierless Triangularitermes triangulariceps Chemical Velocitermes beebei Chemical [68, 69]. We correlated ant diversity with the diversity of termite species in each category of defense. In addition, we examined the species richness of termites in each of the three termite defense categories in samples in which army ants were present or absent using a Mann-Whitney (7-test. We also examined the correlation between the species richness of ant genera known to contain a high proportion of ter- mite predators {Acanthostichus, Basiceros, Centromyrmex, Leptogenys, Pachycondyla, Strumigenys, and Tranopelta) and total termite richness. 4. Results Twenty-eight termite species in 15 genera (Table 1) and 257 ant species in 56 genera were identified (Table 2, see also [55]). Termites were found at 50 of 60 sample sites (83%). Termites were encountered in 5/60 baiting stations (8.3%), 7/60 pitfall traps (11.7%), 31/60 Winkler (51.7%), and 43/60 hand-collection samples (71.7%). Ants were present in all collections. Species accumulation curves (Figure 1) indicate that additional sampling is required to inventory total species 4 Table 2: List of ant species collected from ground samples at TBS. Psyche Acromyrmex coronatus Hylomyrma sagax Pheidole exigua Acropyga decedens Hypoponera c.f creola Pheidole fimbriata Acropyga donisthorpei Hypoponera e.f distinguenda Pheidole fracticeps Acropyga fuhrmanni Hypoponera c.f. inexorata Pheidole horribilis Acropyga guianensis Hypoponera e.f parva Pheidole lemnisca Amblyopone cf. cleae Hypoponera perplexa Pheidole metana Anochetus diegensis Hypoponera STD 10 Pheidole midas Anochetus mayri Hypoponera STD 1 1 Pheidole sp. nr. nitella Apterostigma auriculatum Hypoponera STD 12 Pheidole peruviana Apterostigma sp. 2 Hypoponera STD 13 Pheidole pholeops Apterostigma sp. 3 Hypoponera STD 14 Pheidole sabella Apterostigma sp. 4 Hypoponera STD 15 Pheidole sagax Apterostigma sp. 5 Hypoponera STD 16 Pheidole sarpedon Apterostigma sp. 6 Hypoponera STD 17 Pheidole scalaris Apterostigma sp. 7 Hypoponera STD 20 Pheidole scolioceps Azteca SJ-AA Hypoponera STD 21 Pheidole tobini Azteca SJ-I Hypoponera STD 22 Pheidole triplex Azteca SJ-M Labidus eoecus Pheidole tristicula Azteca SJ-P Labidus punctatieeps Prionopelta amabilis Basiceros conjugans Lachnomyrmex scrobiculatus Probolomyrmex petiolatus Basiceros manni Leptogenys gaigei Pseudomyrmex tenuis Basiceros militaris Leptogenys imperatrix Pyramica beebei Brachymyrmex cavernicola Leptogenys nigricans n. sp. Pyramica decipula Brachymyrmex KTRW-001 Leptogenys ritae Pyramica denticulata Brachymyrmex KTRW-005 Megalomyrmex balzani Pyramica depressiceps Brachymyrmex KTRW-007 Megalomyrmex cuatiara Pyramica eggersi Brachymyrmex KTRW-014 Megalo myrmex fo reli Pyramica glenognatha Brachymyrmex KTRW-015 Megalomyrmex incisus Pyramica gundlachi Brachymyrmex KTRW-016 Megalomyrmex mondabora Pyramica metopia Camponotus atriceps Megalomyrmex n. sp. near drifti Pyramica schulzi Camponotus claviscapus Megalomyrmex silvestrii Pyramica subedentata Camponotus femoratus Megalomyrmex timbira Pyramica urrhobia Camponotus integellus Mycetarotes acutus Pyramica villiersi Camponotus planatus Mycetarotes unknown Pyramica zeteki Camponotus rapax Mycocepurus smithii Rhopalothrix n. sp. 1 Camponotus senex Myrmelachista KTRW-001 Rogeria blanda Camponotus WM-009 Myrmelachista KTRW-003 Rogeria ciliosa Camponotus WM-01 0 Myrmicoerypta longinoda Rogeria JSC-001 Carebara ungulate Neivamyrmex pseudops Rogeria JSC-002 Carebara panamensis Nomamyrmex esenbecki Rogeria lirata Carebara paya Ochetomyrmex neopolitus Rogeria micromma Carebara urichi Oehetomyrmex semipolitus Rogeria scobinata Carebarella KTRW-001 Octostruma iheringi Rogeria tonduzi Cephalotes minutus Octostruma KTRW-002 Rogeria unguispina Crematogaster carinata Octostruma KTRW-003 Sericomyrmex sp. 1 Crematogaster erecta Octostruma KTRW-004 Sericomyrmex sp. 2 Crematogaster flavomicrops Octostruma KTRW-005 Solenopsis SC-02 Crematogaster levior Octostruma KTRW-006 Solenopsis SC-03 Crematogaster limata Octostruma KTRW-007 Solenopsis SC-05 Crematogaster nigropilosa Octostruma KTRW-008 Solenopsis SC-06 Psyche 5 Table 2: Continued. Crematogaster sotobosque Odontomachus biumbonatus Solenopsis SG-08 Crematogaster stollii Odontomachus haematodus Solenopsis SG-09 Crematogaster tenuicula Odontomachus meinerti Solenopsis SG-11 Cyphomyrmex cf. minutus sp. 1 Odontomachus panamensis Solenopsis SG-12 Cyphomyrmex cf minutus sp. 2 Odontomachus yucatecus Solenopsis SG-14 Cyphomyrmex cf rimosus Oxyepoecus ephippiatus Solenopsis SG-16 Cyphomyrmex costatus Pachycondyla apicalis Solenopsis SG-17 Cyphomyrmex laevigatus Pachycondyla arhuaca Solenopsis virulens Cyphomyrmex sp. 2 Pachycondyla constricta Stegomyrmex connectens Cyphomyrmex sp. 3 Pachycondyla crassinoda Stegomyrmex manni Cyphomyrmex vorticis Pachycondyla gilberti Strumigenys cosmostela Discothyrea denticulata Pachycondyla harpax Strumigenys elongata Discothyrea horni Pachycondyla inversa Strumigenys perparva Discothyrea JSC-001 Pachycondyla laevigata Strumigenys precava Discothyrea sexarticulata Pachycondyla lunaris Strumigenys smithii Dolichoderus imitator Pachycondyla verenae Strumigenys trinidadensis Dolichoderus rugosus Paraponera clavata Strumigenys trudifera Eciton hamatum Paratrechina cf fulva Tapinoma sp. Eciton vagans Paratrechina cf steinheili Thaumatomyrmex sp. Ectatomma edentatum Paratrechina KTRWOOl Trachymyrmex cf. bugnioni Ectatomma lugens Paratrechina KTRW003 Trachymyrmex diversus Ectatomma tuberculatum Paratrechina KTRW004 Trachymyrmex farinosus Gigantiops destructor Paratrechina KTRW005 Trachymyrmex ruthae Gnamptogenys haenschi Paratrechina KTRW008 Tranopelta subterranea Gnamptogenys horni Pheidole ademonia Typhlomyrmex pusillus Gnamptogenys kempfi Pheidole allarmata Typhlomyrmex rogenhoferi Gnamptogenys KTRW-001 Pheidole ALM-006 Wasmannia auropunctata Gnamptogenys mediatrix Pheidole ALM-01 3 Wasmannia cf lutzi Gnamptogenys mina Pheidole ALM-025 Wasmannia scrobifera Gnamptogenys minuta Pheidole ALM-026 Gnamptogenys moelleri Pheidole ALM-028 Gnamptogenys pleurodon Pheidole ALM-03 1 Gnamptogenys simulans Pheidole ALM-032 Gnamptogenys striatula Pheidole amazonica Gnamptogenys sulcata Pheidole astur Hylomyrma blandiens Pheidole biconstricta Hylomyrma dolichops Pheidole cephalica Hylomyrma immanis Pheidole cramptoni Hylomyrma praepotens Pheidole deima richness of both ants and termites at TBS. Nonparametric estimators ICE, Jackknife 1, Jackknife2, and Chao2 (classic) suggest the actual richness of ground-dwelling termites is between 40 and 87 species, and actual ground-dwelling ant richness is between 341 and 383 species. Nine occurrences of termite presence in ant nests were recorded, including ter- mites from four genera {Cylindrotermes, Nasutitermes, Rup- titermes, and Triangularitermes) found with ants in the gen- era Acropy^a, Gnamptogenys, Hylomyrma, Hypoponera, Pach- ycondyla, Pheidole, Strumigenys, and Tranopelta (Table 3). Ground-dwelling ant species richness increased with mi- croelevation (t = 5.83, P < 0.0001), as well as the number of logs and twigs/m^ in sample plots (f = 4.70, P = 0.035). The remaining variables were not significant predictors of ant richness across all species, although some variables were significantly correlated in specific genera [55]. A linear multiple regression model showed no significant relationship between the environmental variables measured and termite species richness (Fj , 52 = 0.80, P = 0.614, adjusted = -0.03). 6 Psyche Chao2 Jackknife2 ICE Species observed -A- Jackknife 1 Chao2 Jackknife2 ICE Species observed Jackknife 1 (a) (b) Figure 1: Species accumulation curves for (a) ants and (b) termites. Each sample consists of the total species collected via Winkler device, pitfall trap, baiting, and hand collecting at each sampling point (n = 60). Values for nonparametric estimators ICE, Jack 1, Jack 2, and Chao 2 (classic) are also shown. Table 3: Ant and termite species collected in the same nest/ food source. Termite species were found inside the same twig as the ant species listed next to them, though in separate chambers. Ants and termites were not found to occupy the same galleries within a twig. Termite Ant Cylindrotermes flangiatus Strumigenys precava Cylindrotermes parvignathus Pachycondyla constricta Nasutitermes ephratae Gnamptogenys pleurodon Nasutitermes intermedins Hylomyrma dolichops Nasutitermes intermedins Pheidole amazonica Nasutitermes llinquipatensis Tranopelta subterranea Ruptitermes sp. Hypoponera c.f distinguenda Ruptitermes sp. Acropyga decedens Triangularitermes triangulariceps Pheidole biconstricta Species richness of termites was positively and signifi- cantly correlated with ground-dwelling ant species richness (Figure 2, Spearman’s Rho = 0.353, P = 0.006, n = 60). When the relationship between species richness in ant gen- era known to include termite predators {Acanthostichus, Basiceros, Centromyrmex, Leptogenys, Pachycondyla, Strumi- genys, and Tranopelta) and termite species richness was analyzed separately, no significant correlation was found (Figure 3, Spearman’s Rho = 0.239, P = 0.068, n = 60). A significant positive correlation was found between termite species richness and the species richness of twig- and log- nesting ants (Figure 3, Spearman’s Rho = 0.482, P = 0.007, n = 60). There was a significant positive correlation between ant nest abundance and termite forager occurrences in twigs (Spearman’s Rho = 0.49, P < 0.0001). In respect to the vol- ume of twigs used, ants (mean 108 ± 461 cm^, n = 342) Figure 2: Relationship between the species richness of ground- dwelling ants and termites (Spearman’s Rho = 0.353, P = 0.006, n = 60). showed no difference from the mean available twig size (52 ± 993 cm^, n = 4509, Tukey’s HSD P = 0.554), while termites fed on twigs that were larger than those used for nests by ants (365 ± 915 cm^, n = 138, P = 0.022) and larger than twigs of average available size (P = 0.001). Conversely, mean decay of twigs utilized by termites (3.47 ± 0.852) did not vary from the overall mean decay of twigs (3.57 ± 0.89, P = 0.368), whereas ants nested in twigs that were significantly less decayed than the overall mean decay value (3.35 ± 0.880, P < 0.0001). When the richness of termites in each of the three termite defense categories was compared between samples in respect to the presence or absence of army ants, no significant differences were found (mechanical: Z = 0.01, P = 0.990; chemical: Z = -1.71, P = 0.088; soldierless: Z = 0.23, P = 0.82). When ant richness was compared to the diversity Psyche 7 Figure 3: Correlation between termite species richness and (a) the species richness of ant genera known to contain termite predators (Spearman’s Rho = 0.239, P = 0.068, n = 60) and (b) the species richness of twig and log nesting ant species (Spearman’s Rho = 0.482, P = 0.007, n = 60). Ant species richness (c) Figure 4: Correlation between ant species richness and (a) the species richness of termites with chemical defense (Spearman’s Rho = 0.195, P = 0. 136, n = 60), (b) the species richness of termites with mechanical (mandibular) defense (Spearman’s Rho = 0.159, P = 0.225, n = 60), and (c) the species richness of soldierless termites {Anoplotermes and Ruptitermes) (Spearman’s Rho = 0.379, P = 0.003, n = 60). of termites employing these different predator defenses, a sig- nificant positive correlation was found for the soldierless termite genera Ruptitermes and Anoplotermes (Figure 4, Spearman’s Rho = 0.379, P = 0.003, n = 60), but not for termites with chemical (Figure 4, Spearman’s Rho = 0.195, P = 0.136, n = 60) or mechanical (mandibular) defense (Figure 4, Spearman’s Rho = 0.159, P = 0.225, n = 60). 5. Discussion We found 265 ant species and 28 termite species in the litter layer of a lowland tropical rainforest and estimated actual species richness at 346-390 for ground-dwelling ant species and 40-87 for ground-dwelling termite species. Ant species diversity at other Neotropical localities ranges from 74 to 520 8 Psyche species [70-79], while Neotropical termite species diversity, including localities in western Amazonian Brazil, ranges from 26 to 100 species [7, 19, 80-85]. These inventories differ greatly in area surveyed, time frame, collection methods, and goals. Our inventory results fall within the lower end of this range. This is not surprising, because we did not sample subterranean or canopy strata. Accumulation curves indicate that both ant and termite faunas were undersampled [55]. Some of our sampling methods (particularly baiting, in which termites were found in less than 10% of samples) favored the collection of ants. Our Winkler collections as well as hand- collecting and litter-sampling nevertheless yield- ed termites, including soil-feeding species, in over 50% of samples. Our results suggest that termite and ant diversity are significantly correlated, perhaps reflecting the nature of their ecological interactions. Termite diversity was not correlated with any of the environmental variables we measured, while ant diversity was correlated with microelevation and the number of twigs and logs in a plot. The positive correlation between termite and ground- dwelling ant species richness suggests that both groups respond similarly to the same, as yet unidentified, environmental factor. We cannot eliminate the possibility that soil humus depth and soil pH [83], clay content and moisture retention [86, 87], dominant tree species [88], total litter weight [89], specific mineral content [87, 90], or other factors influence the diversity and distri- bution of both groups. However, the relationship between termite and ant diversity may also result from their mutual interactions rather than abiotic preferences and tolerances. Termite distribution can be influenced by intra- and inter- specific competition [91, 92], but we are unable to assess the influence of such interactions in our study or separate it from those of ant/termite interaction. We hypothesized that diversity and abundance of twig- and wood-nesting ants will decrease with termite diversity and abundance, due to the potential for competition with termites for wood resources in the litter. However, we found the opposite pattern: diversity of twig- and wood-nesting ants was positively correlated with termite diversity and the abundance of ants and termites in twigs was also positively correlated. We also found no clear evidence of competition for twigs based on the size or decay of twigs utilized by each group; termites were found foraging in larger twigs than those used for nests by ants, and ants nested in twigs that had less-than-average decay, while termites showed no apparent preference. Termites may select larger twigs for more efficient feeding, while ant species having different body sizes and colony sizes may use a wider variety of nest sizes. Indeed, litter- nesting ants in terra firme forest at TBS utilized only 2% of available twigs for nesting, suggesting that competition for nest sites maybe negligible [59]. Termite feeding may also be less influenced by decay level than ant presence. Termites and ants may therefore not compete for twig resources. Contrary to our second hypothesis, the diversity of ant genera known to include termite predators was not signif- icantly correlated with termite diversity. It may be that in- creased termite diversity does not generate diverse, spe- cific trophic niches for ants, because ants do not appear to specialize solely on any given termite species. More detailed information on the diets of Neotropical ant species could offer important insights (i.e., [93]). Army ants can be key- stone predators in invertebrate communities [94] and army ant raids have been shown to increase with the density of potential prey [95], but we did not find any effect of the presence or absence of army ants on the species richness of termites. The lack of a detectable effect could be due to the nomadic habits of army ants and the relatively sedentary nature of termites and their potentially great abundance. Our results may reflect the importance of opportunistic predation on termites by twig- and wood-nesting species. Predatory ants can negatively influence termite populations [96, 97]. In African grassland, however, overdispersed Odontotermes mounds create patches of productivity that positively influ- ence the biomass and fitness of aerial and arboreal arthro- pods as well as some vertebrates [22]. Predators (spiders and geckos) are more abundant near termite mounds. In our system, predator/prey interactions between ants and termites in the litter may be more significant than competition for wood as a nest site for ants or food source for termites, and ant species density might therefore increase as a function of the diversity and abundance of termite prey species. Our third hypothesis was supported: we found a positive correlation between ant species richness and soldierless termite genera. However, the association did not hold for termites with chemical or mechanical defense. The autothytic defenses of soldierless termite species may have relatively low efficacy, compared to nasutitermitinae species and other species groups with mobile chemical defenses and secretions that can be repeatedly discharged without loss of soldiers. Chemical defenses are considered to be highly effective in deterring ant predation [98, 99], and Nasutitermes, a genus characterized by its prominent terpenoid secretion defenses, is the most species-rich termite genus. Clearly, experimental research is required on the efficacy of termite defenses in the field to fully test this hypothesis. Our work represents one of the few attempts to survey ants and termites simultaneously and examine how their ecological associations could influence their species richness. As the two most prevalent invertebrates in tropical forests, the dynamics between ants and termites may have strong implications for other invertebrate groups. Ants are sym- bionts with many species of plants and insects [1, 100]. If the distribution of termites strongly affects ant distributions, termites may also indirectly affect the distribution of mutual- istic species. The decomposition and nutrient cycling services provided by termites may also be indirectly controlled by ant predation. Additional studies are needed to better under- stand mutual influences in these two diverse and abundant groups in the Neotropics as well as how secondary effects of their relationship may impact the functioning of tropical ecosystems. Acknowledgments This work developed from a collaboration between J. F. A. Traniello and the late Sally Levings. The authors fondly Psyche 9 remember Sally, and thank her for past intellectual contri- butions and enduring inspiration. They are very grateful to Stefan Cover for training in ant taxonomy and assistance with identifications. They thank Shawn Dash, David Donoso, Brian Fisher, Stephanie Johnson, John Lattke, John Longino, Bill Mackay, Scott Powell, Ted Schultz, Gordon Snelling, Jeffrey Sosa-Calvo, James Trager, Minsheng Wang, and Phil Ward for assistance in ant species identifications. They thank Kelly Swing and the other directors and staff of Tiputini Bio- diversity Station for assistance in the field. Megan Johnson and Clifton Meek assisted with ant and termite sampling. David Donoso, Pablo Araujo, Paulina Rosero, Van Le, Helen Mary Sheridan, Stephanie Chu, and Winston McDonald pro- vided valuable assistance in the laboratory. Piotr Naskrecki assisted with the MANTIS software package for collection data management. They especially thank Yves Roisin for detailed and constructive comments on a prior draft of the manuscript. This paper was supported in part by an NSF Graduate Research Fellowship to A, L. Mertl and indirectly supported by NSF Grant lOB 0725013 to J. F. A. Traniello. 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Ghua, “Explaining the abundance of ants in lowland tropical rain- forest canopies,” Seienee, vol. 300, no. 5621, pp. 969-972, 2003. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 239392, 10 pages dohlO.l 155/2012/239392 Review Article Towards a Better Understanding of the Evolution of Specialized Parasites of Fungus-Growing Ant Crops Sze Huei Yek, Jacobus J. Boomsma, and Michael Poulsen Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark Correspondence should be addressed to Sze Huei Yek, syek@bio.ku.dk Received 5 October 2011; Accepted 12 December 2011 Academic Editor: Alain Lenoir Copyright © 2012 Sze Huei Yek et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lungus- growing ants have interacted and partly coevolved with specialised micro fungal parasites of the genus Escovopsis since the origin of ant fungiculture about 50 million years ago. Here, we review the recent progress in understanding the patterns of specificity of this ant-parasite association, covering both the colony/population level and comparisons between phylogenetic clades. We use a modified version of Tinbergen’s four categories of evolutionary questions to structure our review in complementary approaches addressing both proximate questions of development and mechanism, and ultimate questions of (co) adaptation and evolutionary history. Using the same scheme, we identify future research questions that are likely to be particularly illuminating for understanding the ecology and evolution of Escovopsis parasitism of the cultivar maintained by fungus-growing ants. 1. Introduction 1.1. The Attine Fungus-Growing Ants. Fungus -growing ants (Hymenoptera: Formicidae: Attini) form a monophyletic tribe of primarily tropical ants that obligately depend on fungal cultivars (Agaricales: mostly Lepiotacea: Leucoco- prineae). The ants provide the fungus with optimal growth conditions, and in return, the fungus serves as the main food source for the ants [1, 2]. The symbiosis between fungus -growing ants and their fungi originated about 50 million years ago [3-6] from a single ancestor that was most likely a generalist forager [3]. Subsequently, the Attini have diversified to encompass approximately 230 described species, distributed across 14 ant genera [4, 7]. Colonies of fungus-growing ants are typically founded by a single queen, who carries a piece of the fungus cultivar from her natal colony in the infrabuccal pocket [8] during her nuptial flight [9-11]. The Attini are divided into the “higher attine” and the phylogenetically basal “lower attine” genera based on their cultivars [5]. Lower attine cultivars are largely unmodified and resemble free-living Leucocoprini, whereas higher attine cultivars (including those of leaf-cutting ants) are highly derived [ 12] . The clonal propagation of cultivars through vertical transmission [2, 13] predicts ancient association and con- gruence between the ant and fungal cultivar phylogenies. High degrees of congruence have indeed been found at the deep phylogenetic levels in both higher [14, 15] and lower attines [12, 14]. However, the phylogenetic inter- action specificity breaks down within, and occasionally between, ant genera and their cultivar strains, indicating that switches and/or reacquisitions of new garden cultivars have occurred (e.g., [12, 16-19]). While the higher-attine fungi no longer persist outside the symbiosis, lower attine fungi have free-living close relatives, which is likely to facilitate gene flow and reacquisitions of symbionts [12]. Cultivar switches can be induced in the laboratory, including the formation of chimeric gardens [20-22]. However, consistent with predictions from host-symbiont conflict theory [23], mature individual colonies appear to consistently maintain a single fungus clone, at least in leaf-cutting ants {Atta and Acromyrmex) where this has been best studied [24, 25]. Clonally propagated monoculture crops are expected to be particularly prone to infection with parasites and pathogens [26], because they represent an attractive resource that should be easy to exploit. This “Red Queen” logic 2 Psyche [27, 28] assumes that parasites and hosts are involved in evolutionary arms races, in which unpredictable genetic heterogeneity, due to sexual recombination, is the most powerful defence against parasites that have short generation times relative to their hosts [28-30]. Single asexual cultivar clones thus seem to represent a liability for the farming symbiosis [25] that needs to be overcome by active protection by the host ants (see below). Colony-level monoculture does not imply population- wide monoculture, as is often the case in modern human crops that are vulnerable to disease. With the possible exception of some species, there is likely to be considerable strain diversity across neighbouring colonies [16, 18] that should discourage the spread of infections between colonies. 1.2. Specialised Coevolved Parasites. Microfungal parasites in the genus Escovopsis (anamorphic Hypocreales) have been known for more than a century to overgrow fungus gardens of laboratory colonies [1, 13, 31], but the formal status of Escovopsis as a disease was confirmed only just over a decade ago when Currie et al. [32] showed that Escovopsis fulfils Koch’s four defining postulates [33] for causative disease agents. This included evidence that Escovopsis (i) is found in abundance in diseased but infrequently in apparently healthy colonies, (ii) can be isolated from diseased colonies, (iii) can cause disease when colonies are artificially infected, and (iv) can be reisolated from diseased experimental colonies [32] . It was also shown that Escovopsis has a directly negative impact on the ant cultivar [32, 34, 35] through the secretion of compounds that break down the cultivar mycelium [36]. As fungus-growing ants rely on healthy fungus gardens for growth and reproduction, this implies that Escovopsis is a potentially serious threat to ant fitness [35]. Deep -level phylogenetic congruence has been found between the fungus-growing ants, their cultivars, and Escov- opsis parasites, suggesting a long history of codivergence within the attine agricultural systems [37]. However, cophy- logenies at lower levels appear to be punctuated with occa- sional host switching of the parasites [38], consistent with ongoing arms races [37], although null hypotheses of genetic drift in isolated parasite populations can usually not be dismissed. Even within ant genera, there is some evidence for ant- cultivar- Escovopsis pairing specificity. Four morphologically and genetically distinct Escovopsis types parasitize the cul- tivars maintained by Aptero stigma, a basal fungus -growing ant genus [39]. These have so far been categorised as “brown,” “yellow,” “pink,” and “white,” but are genetically distinct and likely different subspecies or species (cf. [39]). Even within these groupings, there is evidence for speci- ficity: “pink” Escovopsis appears to infect only G3 cultivars and (rare) “white” Escovopsis only G2 cultivars, whereas “brown” and “yellow” Escovopsis commonly coinfect G2 cultivars (cf. Table 1 in [39]). Current evidence suggests that these pathogen lineages display patterns of phyloge- netic congruence with their fungal host [39], maintained by chemotaxis and host resistance in nonnative (i.e., not naturally occurring) combinations [40]. A similar scenario of association specificity is apparent for the lower attine genus Cyphomyrmex, where subclades of a single Escovopsis morphotype (pink) are phylogenetic congruent with corre- sponding clades of cultivar host genotypes [41] . In the higher attine ants, Trachymyrmex and Sericomyrmex are infected by specific Escovopsis parasites that are phylogenetically distinct from the two clades that parasitize Atta and Acromyrmex leaf-cutting ants [37, 38]. Within the leaf-cutting ant genera, Escovopsis infections are nonspecific [38], confirming the high degree of ant-cultivar specificity of all extant leaf-cut- ting ants to a single species of Attamyces symbiont [42]. 1.3. Defence Strategies against Escovopsis . Fungus -growing ants, especially the leaf-cutting ants, have elaborate pro- phylactic fungus grooming and weeding behaviours to keep their cultivar free from parasites [44, 45]. In Acromyrmex, minor workers are particularly efficient at restricting spore germination [45] , and major workers appear to recruit minor workers to infected sites, thereby potentially increasing the efficiency of disease suppression [46]. If spores manage to escape the attention of minor workers and germinate, major workers appear to perform the task of removing infected garden pieces (weeding) [45]. Task specialization between castes thus appears to make hygienic policing more efficient in general, which has been proposed to be normally sufficient for eliminating generalist fungal parasites, but not for completely eradicating Escovopsis infections [44] . To control Escovopsis infections, fungus -growing ants may also use metapleural gland secretions, which contain an array of compounds with antibiotic properties [48, 69]. In a seminal study, Fernandez-Marin et al. [47] described highly coordinated and challenge-specific foreleg movements along the metapleural gland opening (metapleural gland grooming), which allowed Atta and Acromyrmex ants to precisely target the application of antibiotic secretion to their gardens. In combination with metapleural gland grooming, fungus -growing ants utilize their infrabuccal pocket (located in the oral cavity) as a further filtering and sterilising device. After grooming, the ants collect Escovopsis spores in this pocket, where they are sterilised by an as-of-yet unknown mechanism (potentially metapleural gland compounds), after which the infrabuccal pellet is expelled on the colony refuse pile [47, 49, 70]. The cuticle of major garden workers is often covered with a thick white growth of Actinobacteria [50, 51], which produce antimicrobial compounds that aid in the protection of the fungal cultivar from Escovopsis [49-51, 64, 71] and possibly other parasites [65] . These beneficial Actinobacteria are reared by the ants and housed in cuticular crypts, tuber- cles, or other modifications associated with subcuticular exocrine glands [52]. Most work on the Actinobacteria has focused on specifically associated lineages of Pseudonocardia [51, 55]. Pseudonocardia appears to be vertically transmitted by default [50], but phylogenetic evidence indicates that events of horizontal transfer and incorporation of free-living Pseudonocardia to the symbiosis have occurred [55-57]. Recent studies have further shown that other Actinobacteria Psyche 3 genera (mainly Streptomyces) are often also present [57- 61], but their degree of specificity with the symbiosis is less clear. There is little doubt that cuticular Actinobacteria cultures serve active defence functions in the symbiosis, but clarifying the relative importance of predominantly vertically transmitted Pseudonocardia and horizontally transmitted other defensive microbes will need much further work. Ant cultivars, the hosts of Escovopsis parasitism, are able to launch defences themselves by secreting chemical compounds that suppress Escovopsis growth. This has been tested in the Apterostigma and Cyphomyrmex [41, 43], where antifungal compounds secreted by the cultivar appeared to be more effective in suppressing the growth of Escovopsis strains that are unknown to infect them in nature, but less effective against their native Escovopsis strains [41, 43]. Such cultivar responses towards novel Escovopsis strains might result in limitations for Escovopsis host switching outside the agricultural system that they are adapted to. Overall, therefore, the defences of the ants, the Actinobacteria, and the cultivar appear to reinforce each other in suppressing Escovopsis infection and proliferation within attine ant fungus gardens (see e.g.. Figure 10.1 in [22]). L4. Trade-Offs between Alternative Defence Eunctions. Over the course of millions of years of selection on the interaction between fungus -growing ants and Escovopsis, different ant genera have diversified in their specific utilization and combination of alternative defence mechanism to reduce the impact of Escovopsis. This has been best studied in species of the leaf-cutting ant genera Atta and Acromyrmex. Escovopsis infections appear to be more prevalent in Acromyrmex than Atta colonies [35], possibly due to differences in the efficiency of alternative defensive strategies. First, the chem- ical compounds in the metapleural glands differ between Acromyrmex than Atfa, reviewed in [53], making it inevitable that compounds with different antimicrobial properties are produced (cf. [48]). Second, Actinobacteria are abundant in Acromyrmex and essentially absent in Atta [54]. Third, the rate of metapleural gland grooming differs in a contrasting manner, with Atta increasing grooming rates after Escovopsis infection and Acromyrmex maintaining a constantly low rate of metapleural gland grooming [54]. Differences in metapleural gland chemistry, grooming rate, and Actinobacteria coverage indicate that trade-offs between these alternative defensive strategies are likely, conceivably because these defences are known to be costly [72, 73]. Different defences may target the same parasite, but with different modes of action. For example, in Acromyrmex, metapleural gland secretions kill Escovopsis spores but show limited effect on hyphae [48] , while Actinobacteria secretions suppress hyphal growth but do not kill spores [64] . A similar scenario has been proposed for two other genera of higher attine ants, Trachymyrmex and Sericomyrmex, as certain species from the former genus have abundant Actinobacteria cover and low frequencies of metapleural gland grooming, while Sericomyrmex has very few Actinobacteria and a higher frequency of metapleural gland grooming [72]. 2. Using Tinbergen’s Four Quadrats to Structure Attine-Escovopsis Research Nikolaas Tinbergen was a Dutch ethologist and ornithologist who received a Nobel Prize in Physiology or Medicine in 1973 together with Karl von Frisch and Konrad Lorenz for their joint work on the organization and elicitation of individual and social behaviour in animals [74]. Tinbergen’s four categories of evolutionary questions were originally developed to obtain an integrated explanation for animal behaviour, based on complementary understanding of prox- imate mechanisms (1) and ontogenetic developments (2), as well as ultimate selection forces resulting in adaptive evolution of individuals (3) and long-term evolutionary change of populations or higher-level clades (phylogenetic history) (4) [75]. Tinbergen’s framework has since been used in many research programs throughout the life sciences [76-78] but has, to our knowledge, not been applied to host-parasite interactions. For the purpose of the present paper, we modify Tinbergen’s framework to encompass a classification of questions that have been (Table 1), or could be (Figure 1), addressed to better understand the evolutionary ecology of attine ant-Escovopsis interactions. Table 1 summarizes how studies available so far can be grouped into Tinbergen four quadrats framework. This was relatively straightforward for the ultimate questions of adaptive evolution and phylogenetic history, but not always for the proximate ontogeny and mechanism categories, be- cause available research tools have so far not allowed much understanding of the (epi)genetics behind developmental pathways and phenotypic plasticity. It is, therefore, also ar- guable that the questions addressed in our ontogeny and mechanism categories are rather ambiguous, in being both technologically challenging and relatively imprecise in their fit to a single Tinbergen quadrat. We nonetheless felt that making a first attempt to structure a research agenda was worthwhile and have chosen to group questions of Escovopsis specialization in the ontogeny quadrat and questions of cultivar utilization and defences by the ants and fungal sym- bionts in the mechanism quadrat. In the sections below, we utilise these groupings to formulate how new experimental work, combined with the increasing availability of genome sequences, may allow novel insights in Escovopsis parasitism. 3. Tinbergen’s Ontogeny Quadrat 3.L Escovopsis Recognition of Cultivars. In vitro assays have shown that Escovopsis can recognize native cultivar hosts through chemotaxis, followed by directed growth of the par- asite towards the cultivar, the secretion of parasite enzymes breaking down cultivar cells, and absorption of cultivar cell contents [36]. In contrast, Escovopsis is not able to utilize nonnative cultivar strains and can even be inhibited by them [41, 43]. The mechanisms and genes underlying parasite differentiation between native and nonnative host cultivars remain unknown, that is, we neither know the identity or the evolution of the chemicals (what does Escovopsis recognize?) nor the genes coding for the chemicals produced and their 4 Psyche Table 1: Overview of available studies on Escovopsis virulence in gardens of fungus -growing ants, and our assortment of these studies into the four Tinbergen quadrats. Quadrat Study focus References Ontogeny Pathology, impact, and prevalence [32, 34, 35] Genetic and chemical basis of Escovopsis recognition of cultivars [36, 38-41, 43] Mechanism Ant behavioural defences [44-47] Chemical defences [47, 48] Actinobacteria defences [49-52] Cultivar defences [40,41,43] Phylogeny Population-level specificity [38-41, 43] Cross-phylogeny specificity [38,51] Adaptation Susceptibility/ resistance to metapleural gland compounds ([48], reviewed in [53, 54]) Degree of Actinobacteria specificity [55-63] Susceptibility/resistance to Actinobacteria secretions [50, 58, 60, 61, 64, 65] Host cultivar use [38-41, 43] Objects of explanation Development/Historical Progression in current form Single form What organisms need to function and why those functions arose Questions Proximate How organisms work by describing their developmental and functional traits Ontogeny (i) Escovopsis recognition of cultivars (ii) Genetic basis for Escovopsis recognition by the ants (iii) Trade-offs between alternative defences Mechanism (i) Escovopsis transmission between colonies (ii) Colony-level virulence Evolutionary How evolution has shaped organisms to acquire their extant forms Phylogeny (i) Origin and diversification of the association (ii) Phylo-geographic patterns, coevolutionary interactions, and dispersal Adaptation (i) Evolutionary potential of Escovopsis as a parasite (ii) Evolutionary consequences of host-parasite interactions Figure 1: Tinbergen’s four quadrat framework applied to evolutionary questions about Escovopsis parasitism of fungus- farming ant crops. Ontogeny refers to the description of development, from DNA to progressive phenotype, mechanism refers to the physiological and cellular processes that organisms have available, phylogeny refers to the idiosyncratic evolutionary history of a lineage, and adaptation refers to traits that acquired their extant function because of specific selective advantages, modified from [66-68]. evolutionary history. Ongoing genome sequencing of culti- vars and Escovopsis, as well as efforts to isolate the chemicals involved, will thus allow considerable progress to be made. Two evolutionary explanations for the maintenance of Escovopsis-cultiyar utilization patterns seem possible. The nonadaptive explanation would hold that Escovopsis strains (or species) would be subject to consistent genetic drift in isolated populations, so that they would lose adaptations to allopatric hosts by chance. The alternative adaptive expla- nation would hold that populations are mostly panmictic, so that genes coding for innovative pathogen traits and defensive recognition and resistance traits of cultivars would tend to coevolve. If so, Escovopsis would track cultivar evolution in continuous, but variable, arms races reminiscent of a geographic mosaic of coevolution [79] . If the latter is the case, expectations are that positive selection on specific gene complexes (e.g., recognition or resistance genes) will likely have left signatures of enhanced dN/dS ratios compared to housekeeping and neutral genes, while nonsignificant dN/dS ratios would make the nonadaptive null hypothesis more likely. In general, it seems unlikely that Escovopsis popu- lations are highly structured (see also below), but solid empirical evidence on this is lacking. 3.2. Genetic Basis for Escovopsis Recognition by the Ants. Ants are able to discriminate between Escovopsis and other fungi and behave accordingly [44, 45]. Natural selection in the ant host is expected to select for genes involved in the recognition and removal of Escovopsis from the fungus garden, as this is predicted to provide a selective advantage. Further, Escovopsis has the potential to be much more virulent than any general fungal weeds of attine ant colonies, at least in the higher attine system where virulence has been studied, implying stronger selection on Escovopsis recognition pathways in the Psyche 5 ants compared to pathways mediating the recognition of weed fungi. The genetic basis of Escovopsis recognition has not been explored, but genomic tools will make this possible in the years to come [80, 81]. For example, two leaf-cutting ant genomes are now published [82, 83] and a sequenced Escovopsis genome will soon follow (anonymous reviewer, personal communication), providing the tools necessary for such new approaches to studying behavioural recognition mechanisms. Recognition of, and concomitant behavioural responses to, Escovopsis infection are faster and last longer than the response to general fungal pathogens [44, 47], leading to the prediction of higher levels of recognition gene expression in the presence of Escovopsis. Flowever, it is conceivable that the mechanism of recognition of Escovopsis and other fungi by the ants does not differ but that responses do, so that it is rather genes underlying behavioural removal responses that are differentially expressed. 3.3. Trade-Ojfs between Alternative Defences. Defences against Escovopsis include behavioural removal (including self- and allo-grooming), glandular secretions, cultivar de- fensive compounds against nonnative Escovopsis, and com- pounds with antibiotic properties derived from Actinobacte- ria. These defences all involve interactions on the ant cuticle and are expected to require coordinated interactions to avoid negative interference. In Acromyrmex octospinosus, the metapleural gland secretions do not appear to harm the Actinobacteria, so that both defences can be freely expressed [54] . Further, complementarity is expected to maximize cost- benefit ratios of defences as well as to avoid redundancies. It is conceivable that differences in Actinobacteria cover between closely related ant species, such as A. octospinosus and A. echinatior [72], reflect more recent adjustments (trade-offs) in the relative importance of defences between the species. Explorations of defence trade-offs have only been done in some higher attines, leaving questions of this kind unexplored in most of the fourteen extant fungus -growing ant genera. We propose that utilizing the phylogenetic framework of structural modifications over the course of the association between fungus -growing ants and Actinobacteria [52] would offer a good basis for future work to understand the dynamics of defence components across the attine tribe. The relative usage of metapleural gland grooming and the chemistry of glandular and bacterial secretions in Acromyrmex/ Trachymyrmex versus Atta! Sericomyrmex exemplify how such comparative approaches can be insight- ful [54]. However, considering the vast diversity of cultivar usage, Actinobacteria communities, substrate choice, and ant life-history traits, it is conceivable that defence strategies and trade-offs in unstudied attine ants might be different from those found in the higher attines. 4. Tinbergen’s Mechanism Quadrat 4.1. Escovopsis Transmission between Colonies. The success of parasitism is tightly linked to the transmission frequency between host colonies [84]. The most common transmission for fungal spores is passive dispersal through the air, but this is unlikely to be the case for Escovopsis because it sporulates inside colonies and has wet spores [35]. The mechanism of Escovopsis transmission, therefore, continues to be enigmatic, with untested hypotheses of commensal garden arthropods vectoring spores between colonies, or foraging ants picking up spores via encounters outside the nest as reasonable leads [41, 84]. Both mechanisms could be further facilitated by attine colonies nesting in each others close proximity. Culture-based attempts to isolate Escovopsis from potential vectors are, therefore, needed for a better understanding of transmission modes. Expectations are that Escovopsis is more likely to be transmitted between colonies by commensal arthropods. This is so, because foragers presumably rarely, if ever, enter other colonies, and are therefore unlikely to pick up Escovopsis spores from nonnative infected colonies, and because workers are efficient at recognizing and removing Escovopsis spores from their cuticle (e.g., [85, 86]). In contrast, commensal arthropods moving between colonies are not expected to have evolved such avoidance behaviours towards Escovopsis. 4.2. Colony-Eevel Virulence. The within-nest dynamics of Escovopsis infections remain a frontier awaiting exploration. Escovopsis can coexist with other nonmutualistic filamentous fungi within colonies without colonies displaying signs of infection [62, 87-89]. However, it is not known if infection sets out shortly after Escovopsis introduction, or if Escovopsis spores remain dormant in the colonies until an outbreak of mycelial growth is triggered by external factors. To begin to understand these dynamics, two essential questions need to be addressed. Firstly, we need a better understanding of the level of metabolically active spores and hyphae of Escovopsis in normally functioning and apparently healthy colonies. This could be obtained through quantitative PCR approaches, so that amounts of Escovopsis biomass and levels of metabolic activity, measured as gene expression, can be estimated. Ideally, this should be explored over time to also determine temporal variation. Only when we have a better idea of such dynamics, we can begin to explore the role of the ants in mediating these threats. Secondly, if spore-dormancy is the rule, work should address what factors trigger within-colony outbreaks. One approach that could potentially address this is long-term field surveys of natural colonies to better understand the interplay between ecological fluctuations, (e.g., temperature, rainfall, and food availability), intrinsic factors (e.g., loss of queen, imbalance of worker to garden ratio, and emergence of reproductives), and infection dynamics. 5. Tinbergen’s Phylogeny Quadrat 5.1. Origin and Diversification of the Association. The appar- ent presence of Escovopsis throughout the fungus -growing ants suggests that an ancestral Escovopsis was present as a parasite in the first ant cultivars that were domesticated ca. 50 million years ago (cf. [37, 90]). However, an alternative scenario is that Escovopsis parasitism originated shortly after the early attine ants had become irreversibly committed to farming. The latter would indicate that Escovopsis parasitism 6 Psyche was not merely a passive carry-over process, but that the highly peculiar garden phenotype of domesticated fungi created a novel niche to parasites like Escovopsis. Finding that Escovopsis parasitism would also occur in free-living relatives of lower attine garden symbionts would make an origin predating ant fungiculture more likely, but several lines of indirect evidence suggest that the “new garden niche” model is more likely to apply. First, Actinobacteria cultures on the cuticle of attine ants arose also shortly after these ants became farmers [52], and it would be hard to imagine that the origin of this costly biocontrol habit was not somehow related to Escovopsis infections. Second, the impact of Escovopsis on fungus -growing ant cultivars is likely to be particularly high because colonies keep a high density of cultivar mycelium without sufficient own defences. Third, it is striking that the only clade of attine ants that secondary developed a radically different and much less conspicuous garden phenotype, the yeast-rearing Cyphomyrmex, have secondary lost Escovopsis as a parasite [4]. To date, there are two described species of Escovopsis, with E. weberi from a Brazilian Atta species thought to be the monotypic species of the genus [91]. Later, a morphological- ly distinct E. aspergilloides was isolated from Trachymyrmex ruthae in Trinidad [92]. Both large scale (cf. [37, 38]) and lower-level lineage diversity [39-41] are considerable, suggesting that there are more Escovopsis species associated with fungus -growing ants. Molecular species delineation based on conserved genes such as EF-la and 18S rRNA is unlikely to distinguish lineages that diverged recently, so that more sensitive marker studies are needed. Recent multilocus sequence analyses (MLSAs) have provided the opportunity to estimate divergence dates for crucial nodes in phylogenetic trees [4, 19] and would thus also offer novel insights when applied to an Escovopsis phylogeny [37]. Approaches of this kind will ultimately allow conclusions about the origin of Escovopsis parasitism (before or after attine ants became farmers) and the rates of Escovopsis evolution in different host clades. 5.2. Phylo -Geographic Patterns, Co evolutionary Interactions, and Dispersal. Coevolutionary theory predicts that geno- typic and phenotypic variation across the geographic range of a host-parasite association can lead to parasite adapta- tions to locally available host genotypes, while becoming maladapted to nonnative genotypes [93]. A prerequisite for such coevolutionary interactions is that host populations are genetically structured, so that gene flow between populations remains limited [93]. In fungus-growing ants, only a single study has attempted to explore such coevolutionary dynam- ics (in the ant species Apterostigma dentigerum [43]). This showed the presence of six distinct host genotype clusters across Central America, while structuring was essentially absent in the parasite, indicating that Escovopsis genotypes are not tightly tracking those of the host [43] . We would expect that other fungus -growing ant- cultivar-Tscovopsfs interactions will mirror the findings in Apterostigma, since cultivars are vertically transmitted by default while Escovopsis is horizontally transmitted. There- fore, the population structure in Escovopsis could be explainable if their sticky spores would use vectors for long distance dispersal that are not available to dispersing ants. It would be tempting to speculate that other arthropods living in attine nests might have this vector function, but examples of such long distance flyers vectoring spores are presently lacking. Alternatively, wind dispersal of small leaf fragments with Escovopsis spores would also seem a realistic mechanism for parasite populations to become less viscous than host populations. Future studies addressing relative dispersal efficiencies of partners in the attine ant symbiosis would seem most informative if they could span geographic areas that would be large enough to include natural barriers that would differentially affect Escovopsis spores and dispersing ant queens transmitting fungus-garden symbionts. 6. Tinbergen’s Adaptation Quadrat 6.L Evolutionary Potential of Escovopsis as a Parasite. As already mentioned, Escovopsis has probably persisted as a parasite of fungus -growing ant gardens since the origin of ant fungiculture 50 million years ago [4, 37]. If that is so, “Red Queen” like arms races with the ant and fungal hosts may have at least periodically occurred, so that genetic diversity of the parasite is likely to be substantial [26-29]. However, the sexual “teleomorph” of Escovopsis has never been observed so that Escovopsis may not have sexual re- production, similar to many other Ascomycetes [94]. Lack of sex would not necessarily preclude the integration and exchange of genetic material between different anamorphous mycelia within nests, provided that coinfections occur with some frequency. This is because asexual Ascomycetes can undergo genetic exchange between strains after hyphal merging (anastomosis) and parasexual heterokaryosis (the exchange of cell nuclei) [95]. If such exchanges lead to mitotic crossovers, then there is potential for recombination between genetically different strains [95]. It will be very interesting to investigate whether the Escovopsis genome still shows signs of such genetic recombination. The presence of coinfections within individual nests is a prerequisite for such genetic exchanges. Both Atta and Acromyrmex leaf-cutting ants appear to frequently harbour genetically distinct Escovopsis strains, including ones appear- ing in two separate phylogenetic clades [63]. Similarly, in the paleoattine genus Apterostigma, fungus gardens are infected by four distinct Escovopsis morphotypes “brown,” “yellow,” “pink,” and “white” [39]. This implies the potential for ex- change of genetic material between coinfecting strains within colonies. By explicitly addressing this question, we could gain insight both in the dynamics of coinfections (e.g., facilitation, inhibition, the role of the order of infection precedence) within colonies and in the putative species status of different Escovopsis morphotypes. 6.2. Evolutionary Consequences of Host- Parasite Interactions. A common question in the evolutionary study of host-patho- gen interactions is whether coevolutionary arms races are al- most continuous or relatively rare. This is partly because of the difficulty of testing such dynamics when exploring Psyche 7 biological systems in real time. Fungus-growing ants have evolved extensive complementary defences to deal with Esco- vopsis, but the parasite nevertheless prevails at relatively high population-level frequencies, ranging from 27-75%, de- pending on the ant genus and geographic location (e.g., [32, 88]). This finding suggests that Escovopsis continues to exert selection pressure on the ant hosts, potentially leading to concomitant changes in ant defences. All this is suggestive of, but not decisive evidence for, antagonistic coevolution (cf. [96]). The efficiency of behavioural defences (grooming/weed- ing) in attine ants is known to have an impact on the virulence of Escovopsis [44, 45]. Under a coevolutionary sce- nario, expectations are that Escovopsis has exerted selection pressures on the ants to optimize their behavioural response towards native parasite strains. Such a scenario would predict that infections with (avirulent) nonnative strains of the parasite would not elicit the same efficient response from the ants. Similarly, if metapleural gland grooming behaviour and chemistry have been shaped by coevolutionary interactions with Escovopsis, then we would expect that the grooming rate and the chemical secretion cocktail would be adapted to inhibit native parasite strains more than nonnative strains. The coevolutionary patterns arising from interactions between Escovopsis and the Actinobacteria are inevitably different from those between Escovopsis and direct defences by the ants. Two, perhaps nonmutually exclusive, scenarios derived from Red- Queen dynamics in relation to Acti- nobacteria defence have been proposed. The first scenario suggests that Actinobacteria in the genus Pseudonocardia evolve in response to antibiotic resistance evolving in Esco- vopsis. Evidence supporting the potential for this to be the case comes mainly from observations of variation in the propensities of different Pseudonocardia-derived antibiotics, including the presence of Escovopsis strains that are resistant [55, 64]. Phenotypic variation is a prerequisite for such dynamics to be maintained, as this is what natural selection can act on. However, no studies have as yet shown that changes in Pseudonocardia genes for antibiotic production do indeed change in response to Escovopsis susceptibility. A second possible scenario is that attine ants frequently acquire strains of bacteria from the environment that have novel antibiotic properties against Escovopsis, be it either Pseudonocardia [55] or other Actinobacteria [57, 61, 65]. Evidence for such acquisitions comes from survey data showing that free-living Pseudonocardia are phylogeneti- cally interspersed with ant-associated clades [55], and that additional Actinobacteria with antibiotic properties (mainly Streptomyces) can be obtained from the ant cuticles or gardens of colonies. We expect that characterizations of the antibiotic profiles produced by the major clades of Pseudono- cardia that associate with fungus -growing ants will clarify the role that these alternative acquisition mechanisms have played in maintaining a successful Pseudonocardia-defence against Escovopsis. Further studies will also benefit from a more explicit emphasis on exploring how and to what extent such horizontal acquisitions of novel Actinobacteria occur, and whether they have a selective advantage for ant colony fitness. 7. Conclusions Since the discovery of Escovopsis parasitism of fungus - growing ants less than 15 years ago, we have obtained a broad understanding of prevalence, impact, role, and coevo- lution of the parasite with the attine ant-fungus symbiosis. Nevertheless, many fundamental questions remain unan- swered, including the origin of the host-parasite association, its presence and potential role outside attine ant nests, parasite transmission between colonies, and within-colony disease dynamics. We know that Escovopsis is attracted to specific ant cultivars in some cases, but the generality of this phenomenon and the underlying recognition mech- anisms are unknown. Several defences against Escovopsis are known, including prophylactic behaviours, metapleural gland grooming and compounds, and Actinobacteria sym- bionts, which all contribute to reducing the impact of Escov- opsis. However, we know little about the context-specific efficiency of these alternative and complementary defences, and only in some cases do we have a crude understanding of the potential trade-offs involved. More detailed phylogenetic studies of the association specificity of ants, fungal cultivars, Escovopsis, and Actinobacteria are needed to improve our interpretations of reciprocal interactions observed. Although the Tinbergen framework did not allow us to do full justice to the complexity of this host-parasite interaction, we feel that it does provide a useful structuring device for the research agenda that will be required to make further progress in understanding this unique genus of crop -pests of fungus - growing ants. Acknowledgments The authors thank Panagiotis Sapountzis, Jelle van Zweden, Eric Caldera, and Jeremy Thomas Poulsen for comments on an early draft of this manuscript, and three anonymous reviewers for valuable comments and suggestions. S. H. Yek and J. J. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 273613, 9 pages doi:10.1155/2012/273613 Research Article Interactions among Carbon Dioxide, Heat, and Chemical Lures in Attracting the Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae) Narinderpal Singh, ^ Changlu Wang,^ Richard Cooper,^ and Chaofeng Liu^ ^ Department of Entomology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA ^ Department of Statistics, Purdue University, West Lafayette, IN 47907, USA Correspondence should be addressed to Narinderpal Singh, nsingh@aesop.rutgers.edu and Changlu Wang, cwang@aesop.rutgers.edu Received 3 October 2011; Revised 19 December 2011; Accepted 21 December 2011 Academic Editor: Mark M. Feldlaufer Copyright © 2012 Narinderpal Singh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Commercial bed bug {Cimex lectularius L.) monitors incorporating carbon dioxide (CO 2 ), heat, and chemical lures are being used for detecting bed bugs; however, there are few reported studies on the effectiveness of chemical lures in bed bug monitors and the interactions among chemical lure, CO 2 , and heat. We screened 12 chemicals for their attraction to bed bugs and evaluated interactions among chemical lures, CO 2 , and heat. The chemical lure mixture consisting of nonanal, l-octen-3-ol, spearmint oil, and coriander Egyptian oil was found to be most attractive to bed bugs and significantly increased the trap catches in laboratory bioassays. Adding this chemical lure mixture when CO 2 was present increased the trap catches compared with traps baited with CO 2 alone, whereas adding heat did not significantly increase trap catches when CO 2 was present. Results suggest a combination of chemical lure and CO 2 is essential for designing effective bed bug monitors. 1. Introduction Hematophagous arthropods use a variety of visual, mechan- ical, chemical, and thermal cues to detect vertebrate hosts [1]. Host searching behavior in unfed bont tick, Atnbly- omma hehraeum Koch [2, 3], and Glossina spp. (Diptera: Glossinidae) [4] is stimulated by carbon dioxide (CO 2 ) emitted by mammalian hosts. Odors from human skin [5], sweat, breath and body odors from cattle, birds, and mice [6] , bird feathers or skin [7], and bird uropygial glands [8] play a major role in attracting different families of hematophagous mosquitoes. R-(-)-l-octen-3-ol, an enantiomer of 1-octen- 3-ol, was found attractive to field populations of adult mosquitoes [9]. Geranyl acetone (E and Z enantiomers), a component of human sweat, elicited strong electroantenno- gram responses in female Anopheles gambiae Giles [10] . The resurgence of bed bugs {Cimex lectularius L.) in recent years stimulated research on bed bug behavior [11,12] with the goal of developing effective bed bug monitoring tools. It is known that bed bugs use GO 2 [11-13], heat, and chemical odors to locate their hosts [11, 12, 14, 15]. Among the chemical lures, geranyl acetone, l-octen-3-ol, and L-lactic acid have been reported to be attractive to bed bugs [16, 17]. Bed bug airborne aggregation pheromones including (E)-2-hexenal, (E)-2-octenal, (2E, 4E)-octadienal, benzaldehyde, nonanal, decanal, sulcatone, (+)-limonene, (-)-limonene, and benzyl alcohol were attractive to bed bug nymphs in olfactometer bioassays [18]. These chemicals could potentially be used for monitoring bed bugs; however their effectiveness has not been tested yet in arenas or under conditions that simulate field conditions. Anderson et al. [11] demonstrated the effectiveness of a trap baited with CO 2 (50-400 mE/min), heat (37.2-42.2°G), and a chemical lure comprised of 33.0 /rg propionic acid, 0.33 jWg butyric acid, 0.33 pg valeric acid, 100 pg l-octen-3-ol (octenol), and 100 pg E-lactic acid. In a separate study, Wang et al. [12] confirmed the effectiveness of GO 2 (169 mE/min) and heat (43.3-48.8° G) in their attraction to bed bugs. Until 2 Psyche / 18 cm ^ ) (a) (b) Hand warmer (c) Figure 1: Experimental setup for determining bed bug attraction to nonchemical and chemical lures: (a) pitfall trap used in all bioassays; (b) a plastic tray arena with a baited and an unbaited trap; (c) a wooden door arena with a baited trap and an unbaited trap. present, there are no studies investigating the interactions among chemical lures, heat, and CO 2 . Bed bugs hide during the day and are difficult to locate as they are small and elusive. Therefore, developing effective monitoring tools has been recognized as a critical component in the current campaign for fighting the bed bug resurgence [19]. Most of the available monitors incorporate one or several nonchemical and chemical lures to attract and capture hungry bed bugs foraging for blood meals. However, the data on the role of various lures in the effectiveness of monitors are very limited. Studying the interactions among nonchemical and chemical lures has immediate practical significance in designing more effective monitors which can be used to detect the presence of small numbers of bed bugs or as an alternative control method. The objectives of this study were (1) screening for chemical lures that are attractive to bed bugs, (2) testing the effect of CO 2 release rate and heat source on trap catches and (3) determining the interactions among chemical lures, CO 2 , and heat in attracting bed bugs. 2. Material and Methods 2.1. Insects. Bed bugs were collected from an infested house in Lakewood, NJ. They were maintained in plastic containers (4.7 cm height and 5 cm diameter) with folded paper as harborages at 26° C ± 1°C, 40 ± 10% relative humidity, a 12 : 12-hour (L : D) photoperiod, and were deprived of food for the entire duration of the study. There was a great variation in their hunger levels ranging from very hungry to very well fed at the time of collection. We immediately started the experiments after collection using hungry bugs based on color of the insect abdomen. Only males and large bed bug nymphs were used in this study. Females were not tested to avoid mating and laying eggs in the arenas. All bioassays were conducted within 3 months after bed bugs were collected. 2.2. Pitfall Trap and Experimental Arenas. Pitfall traps were used to evaluate the attractiveness of various lures. The pitfall trap was an inverted plastic dog bowl (600 mL volume, 18 cm diameter, 6.4 cm depth, and 1 mm thickness) (IKEA, Baltimore, MD, USA) (Figure 1(a)). The outer wall of the trap was covered with a layer of paper surgical tape (Caring International, Mundelein, IL, USA), which was painted black with ColorPlace spray paint (WalMart Stores Inc., Bentonville, USA). Bed bugs preferred black color to white color in our preliminary bioassays. Two types of experimental arenas were used: (a) wooden door arenas (200 by 76 cm by 6.4 cm) (length by width by height) with wooden floor and (b) plastic tray arenas (80 by 75 by 5 cm) (length by width by height) with bottom lined with brown paper (Figure 1(b)). The brown paper was never changed during the entire study. A layer of fluoropolymer resin (DuPont Polymers, Wilmington, DE, USA) was applied to inner walls of the experimental arenas to prevent the bugs from escaping. A layer of this resin was also applied to inner walls of the pitfall traps in a similar fashion to confine the bed bugs that fell into the traps. A filter paper (15 cm diameter) was placed on the floor in the center of each arena, and then a plastic ring (13.3 cm diameter and 6.4 cm height) was placed on the filter paper for confining the bed bugs. A piece of folded cardboard and folded fabric was placed on the filter paper inside the ring to provide harborages for bed bugs. Six and four additional paper harborages measuring 5.1 cm long and 3.3 cm wide were placed along the edges of the floor of each wooden and tray arena, respectively. Two wooden door arenas were located at least 6 m away from each other in a 15 m long and 9 m wide room at 23-25°C. Two additional wooden door arenas were located in two 4 m long and 2.3 m wide rooms at 24-25° C. These rooms had air current through vents on the ceilings or through the open door. In experiments using plastic tray arenas, four arenas Psyche 3 were placed simultaneously in a nonventilated, closed room measuring 4 m long and 2.3 m wide at 24-25°C. A 12 : 12- hour (L : D) cycle was maintained in all the rooms that were used for bioassays. 2.3. Ejfect of CO 2 Release Rate on Bed Bug Trap Efficacy. Four door arenas were used and each arena had an unbaited control pitfall trap and a pitfall trap baited with CO 2 . The two traps were placed at opposite ends equidistant (85 cm) from the center. The experiment was tested over 4 consecutive days. On each day, a different CO 2 release rate was used in each arena following a Latin square design. The CO 2 source was 5 lb cylinders (Airgas East Inc., Piscataway, NJ, USA). The tested release rates were 200, 300, 400, and 500mL/min. The rate was determined as mL of bubble fluid displaced by CO 2 per unit of time using a Bubble- O-Meter (Bubble-O-Meter, Dublin, Ohio, USA). The CO 2 was introduced into 240 mL plastic cups that were placed on the pitfall traps (Figure 1(c)). Two holes were made on the lid of each plastic cup for CO 2 to escape. Fifty bed bug nymphs and adult males were released into the center of each arena and confined with a plastic ring. The bugs were acclimated for approximately 15 hours prior to the start of the experiment. At 1 hour after dark cycle, CO 2 was released and the plastic ring confining the bugs was removed. The numbers of bed bugs trapped in the pitfall traps and those in the arenas were collected and counted only after 8 hours with the aid of a flashlight. An 8-hour period has been observed to be sufficient for observing the effect of lures on bed bug behavior in preliminary bioassays. After counting, dead and moribund bugs were replaced with healthy bugs in each arena. All bugs were placed back to the center of the arenas and confined with plastic rings for 15 hours before starting the next bioassay. 2.4. Effect of Heat on Bed Bug Trap Efficacy. This experiment was conducted in four plastic tray arenas. Mini hand warmers were used as the heat source (Grabber, Grand Rapids, MI, USA). Two pitfall traps were placed at opposite corners of each arena equidistant (25 cm) from the center. One trap received either two or four mini hand warmers, and the other trap was used as an unbaited control. The surface temperature of the hand warmer was 40-48° G during the first 6 hours. The air temperatures on the floor of arenas 1 cm away from the pitfall trap baited with 2 and 4 hand warmers were 0.2-0. 3° G and 0.5-0.6°G, above the ambient temperature, respectively. The air temperatures at the lip of pitfall trap baited with 2 and 4 hand warmers were 0.8-0.9°G and 1.3-1.6°G, above the ambient temperature, respectively. These temperatures were based on hourly recordings of one monitor during the first 6 hours after trap placement using a thermocouple thermometer (Gole- Parmer Instrument Gompany, Vernon Hills, IL, USA). The ambient temperature was recorded in the center of each arena equidistant (25 cm) from all traps and 3 cm above arena floor. Each treatment was replicated 6 times over 3 consecutive days. Fifty bed bugs were released into each arena and the testing procedure was the same as that in Section 2.3. 2.5. Effect of Heat on Bed Bug Trap Efficacy When CO 2 is Present. CO 2 at 200 mL/min was selected based on results from Section 2.3. This rate is similar to the respiration rate of an adult human at rest (250 mL/min) [20]. GO 2 alone or in combination with 2, 3, or 4 mini hand warmers was tested in four wooden door arenas on the same day under similar conditions to those in Section 2.3. Each treatment was assigned to a different arena, and the experiment was repeated four times over four consecutive days following a Latin square design. Each arena had an unbaited control trap and a baited pitfall trap placed on opposite ends of the test arena. Fifty bed bugs were released into each arena and the testing procedure was the same as that in Section 2.3. 2.6. Screening of Chemical Eures for Attraction to Bed Bugs in Eour-Choice Bioassays. Twelve known or potential bed bug chemical lures (Table 1) were evaluated for their attrac- tiveness to bed bugs in plastic tray arenas. Most of them were provided by Bedoukian Research Inc. (Danbury, GT, USA). Three chemicals were purchased from Sigma- Aldrich Go. (St. Louis, MO, USA). One chemical was purchased from New Directions Aromatic (Ontario, Ganada). Among them, styralol, benzyl alcohol, 6-methyl-5-hepten-2-one, and Insect Biting Lure, were potentially attractive to bed bugs (Robert Bedoukian, personal communication). The chemicals were randomly divided into 4 groups. Each group was tested in the same arenas to evaluate the attractiveness of the chemicals. A 50 pL aliquot of each chemical was dispensed on cotton within a 0.7 mL microcentrifuge tube. The lid of each tube had a 2 mm diameter opening to allow for slow release of the chemical. Eour pitfall traps were placed at four corners equidistant (25 cm) from the center. Three traps in each arena were baited with three different chemical lures belonging to the same group listed in Table 1 and the fourth trap was an unbaited control. Each group of chemical lures was tested 8 times over two consecutive days. Eifty bed bugs were released into each arena and the testing procedure was the same as that in Section 2.3. 2.7. Attractiveness of Chemical Eures to Bed Bugs in Two- Choice Bioassays. Nonanal, l-octen-3-ol, spearmint oil, coriander Egyptian oil, L-lactic acid, and L-carvone exhibited significant attraction to bed bugs in Section 2.6. These chem- icals were further evaluated to confirm their attractiveness to bed bugs using two-choice bioassays. The experimental setup and testing procedure were similar to Section 2.6. The difference was that only two traps were placed at opposite corners of each arena (Eigure 1(b)). One trap was used as an unbaited control and the other trap received a chemical lure. Each chemical lure was evaluated 8 times over two consecutive days. The baited and unbaited trap positions in each arena were switched on the second day to eliminate any positional effect that could influence the trap catch. 2.8. Relative Attractiveness of Chemical Eures to Bed Bugs in Eour-Choice Bioassays. The relative attractiveness of four most effective chemicals, nonanal, l-octen-3-ol, spearmint oil, and coriander Egyptian oil identified from Section 2.7, 4 Psyche Table 1: Percent bed bugs in pitfall traps baited with three chemical lures and an unbaited control in each arena. Group Chemical lure N Mean (%) ± SE F P value Source of material l-Octen-3-ol 8 28.3 ±2.5* 8.60 0.0001 Bedoukian Research Inc. L-Lactic acid 8 25.7 ± 2.7* Bedoukian Research Inc. I Coriander Egyptian oil 8 24.2 ± 4.8* New Directions Aromatic Control 8 12.0 ± 1.3 Arena 8 10.0 ± 3.9 L-carvone 8 27.5 ±3.5* 6.90 0.0001 Bedoukian Research Inc. Spearmint oil 8 25.0 ±2.2* Bedoukian Research Inc. II Styralol 8 16.4 ± 2.4 Bedoukian Research Inc. Control 8 14.6 ± 1.1 Arena 16.4 ± 2.2 Nonanal 8 27.7 ±3.2* 4.84 0.002 Sigma-Aldrich Co. III Benzyl alcohol 8 25.1 ± 3.4* Sigma-Aldrich Co. 6-Methyl-5-Hepten-2-one 8 20.9 ± 2.5 Sigma-Aldrich Co. Control 8 15.1 ± 1.8 Arena 11.2 ±2.6 Insect Biting Lure 4 16.8 ± 1.6 0.57 0.63 Bedoukian Research Inc. IV R-Octenol + NH 3 HCO 3 4 15.3 ± 3.3 Bedoukian Research Inc. Z-Geranyl Acetone 4 13.0 ± 2.4 Bedoukian Research Inc. Control 4 12.0 ±3.7 Arena 42.7 ± 3.6 * Indicates significantly different from the unbaited control within each group (P < 0.05). was evaluated using the same method as that in Section 2.6. Each of the four traps in each arena was baited with one of these chemicals. Four arenas were used to obtain four replicates. 2.9. Attractiveness of a Chemical Lure Mixture to Bed Bugs. Nonanal, l-octen-3-ol, spearmint oil, and coriander Egyp- tian oil were confirmed with significant attraction to bed bugs from Section 2.7. We examined the attractiveness of a mixture of these four chemical lures. Ten microliter of each chemical was dispensed onto cotton within a 0.7 mL microcentrifuge tube. The experimental setup was similar to Section 2.7 (Figure 1(b)). Each plastic tray arena had two traps: one trap was used as an unbaited control and the other trap received the chemical lure mixture. Four tray arenas were used. The experiment was repeated the next day. The baited and unbaited trap positions in each arena were switched on the second day. Other procedures were the same as those in Section 2.3. The attractiveness of the four-chemical lure mixture was also compared with each individual lure component. A 40 fiL of individual chemical lure was dispensed on cotton within a 0.7 mL microcentrifuge tube. Two traps were placed at opposite corners. One trap received one of the four chemicals and the other trap received the four- chemical lure mixture. Four arenas were used. On each day, a different chemical was tested in each arena. The experiment was repeated four times over four consecutive days following a Latin square design. Other procedures were the same as those in Section 2.3. 2.10. Attractiveness of a Chemical Lure Mixture When CO 2 and Heat Are Present 2.10.1. Comparison between CO 2 Alone and CO 2 + Chemical Lure + Heat. Two door arenas were baited with CO 2 (200 mL/min) and two arenas were baited with a combina- tion of CO 2 (200 mL/min), heat (4 mini hand warmers), and the chemical lure mixture as discussed in Section 2.9 (Figure 1(c)). The experiment was repeated four times over four consecutive days to obtain 8 replicates. The baited and unbaited trap positions in each arena were switched after two days. 2.10.2. Comparison between CO 2 Alone and CO 2 + Chemical Lure. Two door arenas were baited with CO 2 (200 mL/min) and two arenas were baited with a combination of CO 2 (200 mL/min) and the chemical lure mixture. The exper- iment was repeated three times over three consecutive days to obtain 6 replicates. The baited and unbaited trap positions in each arena were switched after the second day. The experimental procedures were the same as those in Section 2.3. 2.11. Statistical Analyses. Bed bug distribution among traps in each arena was summarized as percentage of bed bugs in traps and percentage of bugs that remained in the arena. Generalized mixed linear models (PROG GLIMMIX) were used to analyze the count data [21]. The model accommodates random effects (cohort), repeated measures, and overdispersion. In all experiments, only those bed bugs Psyche 5 100 1 +1 80 ■ ^ 60 ■ — I — ““ — I — ^ — I — 200 300 400 500 CO2 release rate (mL/min) 100 1 C/D +1 80 - c ^ 60 - Number of hand warmers ■ Unbaited trap H Baited trap 0 Arena Figure 2: Effect of CO 2 release rate on bed bug trap efficacy. ■ Unbaited trap Q Baited trap 0 Arena Figure 3: Effect of heat on bed bug trap efficacy. that appeared in the traps were analyzed. Those bugs that remained in the arenas at the end of the experiments were weak, inactive, or behaviorally different from those actively seeking for a host. Previous observations indicate that the presence of bed bugs in a trap had no significant effect on the probability of trapping additional bed bugs. Therefore, the bed bugs in the traps were considered independent events and were not related to gregarious behavior. The data for Sections 2.10.1 and 2.10.2 were pooled for analyzing differences among treatments. 3. Results The different CO 2 release rates had no significant effect on trap catches (F = 2.23, df = 3, P = 0.08) (Figure 2). In each test arena, the probability (mean ± 95% confidence interval) of bed bugs being caught in a trap baited with 200, 300, 400, and 500 mL/min CO 2 was 94.6 ± 2.6, 97.4 ± 1.8, 91.7 ± 3.2, and 85.9 ± 4.1%, respectively. Fleat (two or four mini hand warmers) significantly increased trap catches (P < 0.05) although there were no significantly differences between the two heat sources (P = 0.08, df = 1, P = 0.77) (Figure 3). The probability of bed bugs being caught in traps baited with two and four hand warmers was 64.7 ± 4.3 and 66.4 ± 3.9%, respectively. There were no significant differences among pitfall traps baited with CO 2 alone or in combination with 2, 3, or 4 hand warmers in door arenas (Figure 4) (P = 0. 61, df = 3, P = 0.60). The probability of bed bugs being caught in traps baited with 200 mL/min alone and in combination with 2, 3, and 4 hand warmers was 93.2 ± 2.6, 95.8 ± 2.0, 92.2 ± 2.8, and 91.0 ± 2.8%, respectively. Out of the twelve bed bug attractants evaluated in four-choice bioassays, nonanal, l-octen-3-ol, spearmint oil, coriander Egyptian oil, L-lactic acid, L-carvone, and benzyl alcohol baited traps caught a significantly higher number of bugs than their corresponding controls (P < 0.05) (Table 1). In two-choice bioassays, nonanal, spearmint oil, l-octen-3- 01, and coriander Egyptian oil baited traps caught signifi- cantly more bugs than L-lactic acid and L-carvone baited 100 n w c/D +1 C oc c 0^ 80 - Treatment ■ Unbaited trap Q Baited trap 0 Arena Figure 4: Effect of heat on bed bug trap efficacy when CO 2 is present. traps (P = 10.02, df = 5, P = 0.0001) (Figure 5). Nonanal, spearmint oil, l-octen-3-ol, and coriander Egyptian oil were not significantly different from each other (P > 0.05). The probability of bed bugs caught in traps baited with nonanal, spearmint oil, l-octen-3-ol, coriander Egyptian oil, L-lactic acid, and L-carvone was 75.1 ±3.3, 73.9 ±3.0, 69.0 ± 3.7, 67.3 ± 4.0, 55.2 ± 3.8, and 51.9 ± 4.3%, respectively. Eurther analysis in four-choice experiments showed that pitfall traps baited with nonanal captured a significantly higher number of bed bugs than spearmint oil, l-octen-3- ol, and coriander Egyptian oil (P = 6.43, df = 3, P = 0.0002). In each arena, the probability of bed bugs being trapped in nonanal, coriander Egyptian oil, l-octen-3-ol, and spearmint oil baited traps was 41.5 ± 4.0, 19.6 ± 3.0, 18.3 ± 4.0, and 20.6 ± 4.0%, respectively. 6 Psyche Treatment ■ Unbaited trap H Baited trap Q Arena Figure 5: Attractiveness of chemical lures to bed bugs in two-choice bioassays. Treatment ■ Chemical lure mixture H Lure components 0 Arena Figure 6: Attractiveness of a lure mixture compared with single lures. The traps baited with a chemical lure mixture com- prising nonanal, spearmint oil, l-octen-3-ol, and coriander Egyptian oil captured significantly higher numbers of bed bugs than the unbaited control traps (P < 0.05). The probability of bed bugs trapped in chemical lure mixture baited traps was 71.0 ± 2.8%. These chemical lure mixture baited traps were significantly more attractive to bed bugs than any of the four individual lure components (P < 0.05) (Figure 6). The probability of bed bugs trapped in chemical lure mixture baited traps when compared with nonanal, coriander Egyptian oil, l-octen-3-ol, or spearmint oil baited traps was 66.9 ± 3.6, 70.4 ± 3.5, 71.1 ± 3.6, and 72.6 ± 3.4%, respectively. Traps with a combination of either chemical lure mixture + CO 2 , or chemical lure mixture + CO 2 + heat captured significantly more bed bugs when compared to the traps baited with CO 2 only (P = 24.81, df = 2, P = 0.0001). However, bed bug counts in traps baited with chemical lure mixture + CO 2 were not significantly different than those in traps baited with chemical lure mixture + CO 2 + heat (P >0.05). The probability of bed bugs being caught in traps baited with CO 2 , chemical lure mixture -t- CO 2 , and chemical lure mixture + CO 2 + heat was 71.7 ± 1.9, 87.5 ± 2.0, and 88.8 ± 1.7%, respectively (Figure 7). 4. Discussion Our experiments demonstrated the attractiveness of four chemical lures to bed bugs: nonanal, l-octen-3-ol, spearmint oil, and coriander Egyptian oil. Among these, nonanal was the most attractive chemical lure. Nonanal has been reported to play a major role in the chemical ecology of triatomine bugs [22], Aedes aegypti L. [23], and Anopheles gambiae [24]. Nonanal was also the major compound found in odorant profiles of humans, chicken, and pigeon and elicited strong response in antenna of southern house mosquito, Culex pipiens quinquefasciatus Say [25]. Traps baited with 100 n ■ Unbaited trap H Baited trap 0 Arena Figure 7: Effect of chemical lure mixture and heat + lure mixture when CO 2 is present. nonanal and CO 2 caught higher number of southern house mosquitoes than traps baited with CO 2 alone [25]. 1-Octen- 3-ol has been reported to attract different blood sucking insects including bed bugs [11, 12], Triatoma infestans Klug [26], Glossina spp. [27], and Aedes and Culex spp. mosquitoes [28, 29]. Spearmint oil and coriander Egyptian oil are plant derived. L-carvone is the major component (51%) present in spearmint oil [30]. However, L-carvone did not significantly increase trap catch in two-choice bioassays. Its enantiomer. Psyche 7 D-carvone, has been patented as an attractant for Culicidae mosquitoes [31]. Spearmint oil and carvone (L and D enantiomers) were found very attractive to both nymphs and adults of spot clothing wax cicada, Lycorma delicatula White [32]. Coriander Egyptian oil has the aroma similar to odors emitted by bed bugs [33] . CO 2 was very attractive to bed bugs regardless of the CO 2 release rates being used when tested in door arenas, indicating that 200mL/min rate is sufficient for attracting bed bugs in a room that is 2 m in length. Marx [13] and Anderson et al. [11] reported that bed bugs can locate a host that is 150 cm and 86 cm away. The 200mL/min rate seems to have exceeded the bed bug response threshold and any higher concentrations above that were not helpful in enhancing their responses in door arenas. Measuring the CO 2 gradient at various locations of the arenas might be helpful to establish the relationship between CO 2 release rate and bed bug responses. Under field conditions where a typical room is much larger, the minimum effective CO 2 release rate might be larger. Moreover, bed bug hunger levels, air current, and presence of a human host will affect the minimum effective CO 2 rate. Adding a mixture of four attractants (nonanal, 1-octen- 3-ol, spearmint oil, and coriander Egyptian oil) increased bed bug trap catches when CO 2 was present, indicating the additive effect of chemical lures and CO 2 on bed bug host searching behavior. Similarly, Allan et al. [7] found greater attraction in Culex spp. by the combined use of feathers and CO 2 than by using each component alone. Mixture of 1- octen-3-ol with CO 2 was reported to be more attractive than CO 2 alone in Culex salinarius [34, 35]. Tropical bont ticks, Amblyomma variegatum E, were found to be more attracted to pheromone + CO 2 than CO 2 alone [36]. Host seeking in A. variegatum involves activation and a nondirectional searching activity by CO 2 and a directional movement to pheromone and to other host emanating odors [36]. Hematophagous hemipteran, Triatoma infestans Klug, which is closely related to C. lectularius, also uses a combination of host cues to locate a host. CO 2 served as a long range cue in its nonoriented searching behavior and when a bug arrives in close proximity of its host, then radiant heat and chemical odors from the host oriented it to the exact host location [37]. It is possible that bed bugs host searching behavior follows a similar sequence to that of T. infestans or A. variegatum. The presence of either two or four hand warmers (or a 0.8-1.6°C difference in temperature between the lip of the trap and the ambient air) attracted bed bugs from a distance of 25 cm. The role of heat became insignificant when used in combination with CO 2 in wooden door arenas, indicating adding heat when a gradient of CO 2 concentration was present in the environment was not helpful in increasing trap catches. In contrast, the role of chemical lure mixture was significant even when CO 2 was present. The arena substrates were never cleaned or changed during the study period. They could retain natural attrac- tant/chemical cues, which also persist in natural infestations. We wanted to mimic field conditions and determine if the traps can attract the bugs that were already acclimated to the arenas with feces and their associated pheromones present. Results from such experimental conditions would more likely correspond well to those obtained under field conditions. Wang et al. [38] showed the effectiveness of pitfall traps baited with CO 2 alone for detecting very low level bed bug populations. But none of the bed bug monitors provide 100% assurance of the presence/absence of bed bugs in field environments. Results from this study suggest adding an inexpensive chemical lure to a trap may significantly improve the trap efficacy and provide more accurate monitoring of bed bug infestations. Wang et al. [38] suggested that an effective monitor can be used in unoccupied infested rooms to trap the hungry bed bugs and for reducing the probability of bed bugs dispersing into adjacent uninfested rooms. An effective monitoring/trapping system for bed bugs could also be combined with insecticides to kill bed bugs that are attracted to lures or baited traps. It is noteworthy to mention that the bed bug strain, hunger level, arena size, and test room conditions had signif- icant impacts on test results in our preliminary experiments. Even within a test arena, there could be a location effect. In a ventilated room, bed bugs that are downwind of the baited trap are more likely to be exposed to the plume of CO 2 , chemical lure, or heat. When testing the effect of chemical lures or heat alone, we used small plastic arenas and a room with still air. Using a door-sized arena or in a ventilated room could not detect the attractiveness of chemical lures or heat. 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Kenneth Grace Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Mama, 3050 Made Way, Gilmore 310, Honolulu, HI 96822, USA Correspondence should be addressed to J. Kenneth Grace, kennethg@hawaii.edu Received 30 September 2011; Revised 21 December 2011; Accepted 3 January 2012 Academic Editor: David E. Bignell Copyright © 2012 N. K. Hapukotuwa and J. K. Grace. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tunneling behavior and the spatial dispersion of tunnels constructed by the subterranean termites Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann) (formerly known as C. vastator Light) (Blattodea; Rhinotermitidae) were examined in foraging arenas. The results indicated that these two termite species construct quantitatively different tunnel systems, supporting visual observations made in earlier studies. Coptotermes gestroi constructed thin, highly branched tunnels, while C. formosanus tended to construct wider and less branched tunnels. Tunnels of C. gestroi showed more spatial dispersion than those of C. formosanus, and this species constructed a larger number of tunnels compared to C. formosanus. The presence or absence of food (wood) within the arena did not influence the tunneling pattern of either species. Although previous observations have suggested that these two termite species exhibit different tunneling behaviors; this is the first quantification of the differences. Comparative studies of the foraging behavior of subterranean termite species contribute to our understanding of their distribution and ecology and may help to improve pest management programs, particularly those based on placement of toxic baits. Moreover, differences in tunneling patterns may reflect different foraging strategies optimized for either tropical (C. gestroi) or sub tropical/ temperate (C. formosanus) environments. 1. Introduction Subterranean termites (Family Rhinotermitidae) create ram- ified tunnel systems above or beneath the soil to locate their cellulosic food [1]. Sometimes these tunnels, which range from tens to hundreds meters in length, connect multiple feeding sites [2] . The morphology and architecture of termite tunnels are highly diverse, with the spatial distribution, tunnel length, width, and volume differing according to the species [3]. Lee et al. [4] observed that the tunnel geo- metry results from concerted work by a group of termite in- dividuals. Campora and Grace [5] found some evidence that average worker size has an effect on group tunneling efficiency. They hypothesized that physically larger workers may have advantages in the mechanics of tunneling. Also, tunneling behavior may be induced by both internal and external factors such as physiological, genetic, and environ- mental conditions [6]. Campora and Grace [7] reported that when termites were introduced into the center of a two- dimensional foraging arena filled with sand, tunnels were uniformly distributed around the perimeter of the initiation site. Su et al. [8] observed that none of these tunnels looped back towards the origin. It is difficult to identify which spe- cific factor is responsible for observed tunnel characteristics, since tunneling behavior is the outcome of complex interac- tions among multiple physiological functions [6]. For exam- ple, Arab et al. [9] reported that termite trail pheromones elicit both orientation and recruitment behaviors. The relationships among observed tunneling behaviors and external (environmental) factors are easier to study since 2 Psyche these factors can be more objectively defined [6]. Houseman and Gold [1] observed that tunneling rates increased in response to the concentration of sand in the soil. Su and Puche [10] and Green et al. [11] found that soil moisture positively affected tunneling rates; and Arab and Gosta- Leonardo [12] reported that increased temperature increased tunneling rates. Cornelius and Osbrink [13] concluded that physical properties of soil affected both tunneling through the soil and above-ground construction of shelter tubes (pro- tected runways between the soil and wood), and that termites tunnel through sand faster than through top soil or clay. The tunneling behavior of subterranean termites is difficult to study in the field due to their cryptic habitats [14]. As a result, detailed analyses of foraging and tunneling behavior are largely limited to laboratory bioassays. Those experiments may help to illuminate the consequences of tunneling behavior for termite foraging strategies, and the link between the individual and population levels in tun- neling dynamics [15]. In a previous study, Grace et al. [16] observed differences in the tunneling patterns of the two subterranean ter- mites found in Hawaii, Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann). Coptotermes gestroi was for- mally known as Coptotermes vastator Light in the Pacific region, until synonymized by Yeap et al. [17], and Hawaii is one of the few places in the world where C. gestroi and C. formosanus cooccur [18-20]. Coptotermes gestroi ap- peared to construct thin and highly branched tunnels, while tunnels of C. formosanus were wider and less branched. However, these visual observations were made in the course of feeding experiments, and the bioassay design did not permit any quantification or analyses of these apparent differences. In a comparative study of feeding and tunneling activity with Malaysian termites, Yeoh and Lee [21] depicted tunnel patterns for C. gestroi similar to those reported by Grace et al. [16], but did not include C. formosanus nor ana- lyze the patterns. Coptotermes gestroi primarily occurs in the equatorial zone, while C. formosanus is subtropical in dis- tribution, and Grace et al. [16] suggested that the apparent differences in tunneling might reflect different foraging strategies on the part of these two termite species optimized to fit the distribution of cellulosic resources in their respec- tive environments. Visual observations alone of biological or behavioral phenomena, without analysis, are of limited value in testing hypotheses. Thus, the present study was initiated in order to quantify these apparent differences, using two-dimensional foraging arenas (Figure 1) already proven useful for analysis of C. formosanus tunneling behavior. Coptotermes gestroi cur- rently has a very limited distribution in Hawaii, and were limited to a single-field collection site for this species. How- ever, we compensated for this very limited colony replication (atypical of termite behavioral studies) with appropriate sta- tistical analyses. To mimic the situations normally encoun- tered by tunneling termites, we also included arenas with two different susceptible woods as well as arenas with no wood present in the study, in order to determine if these different conditions resulted in different tunnel patterns. 2. Materials and Methods 2.1. Foraging Arenas. We used six test arenas (three for C. formosanus and three for C. gestroi) with each arena consisting of two sheets of transparent acrylic, separated by a third sheet with the interior cut out to form a 10 cm rim around the perimeter as described by Gampora and Grace [7]. The upper sheet was 85 x 85 x 0.25 cm and the lower 85 X 85 X 0.50 cm; the two sheets were fixed together by peripheral fastening screws through the 10 X 0.25 cm rim in order to form an inner experimental space of dimensions 75 X 75 X 0.25 cm (Figure 1). Eight small square spacers were distributed on the upper surface of the lower sheet to maintain a uniform separation of the upper and lower sheets, and the space between them filled with ca. 8 kg silica sand (40-100 mesh, 150-425 pm sieve, Fisher Scientific, Fair Lawn, NJ) and moistened with deionized water to approx. 18% water content by weight [5]. To present an array of baits, 16 holes were drilled in four rows across the upper sheet (3.1 cm diameter, 20 cm separation, see Figure 1), each able to accommodate a plastic snap-cap vial (48 mL capacity) from which the bottom had been removed. For baited treat- ments, a preweighed wooden block (Douglas Fir, Pseudotsuga menziessii, and Southern Yellow Pine, Pinus spp., separate arenas for each wood) was placed in each. At the center of the upper sheet, a larger hole of 8.2 cm was drilled to accom- modate a capped plastic jar of 500 mL capacity, again with the bottom removed: this served as the termite release cha- mber and contained two wet filter papers to provide food and moisture for the termites at the outset of the experiment. During filling, the sand was spread manually across the arena before closure, allowing no sand-free spaces to be formed under the upper sheet [23]. Finally, all arenas were stood on a metal grid supported by four glass jars and kept in a dark room. Three fluorescent lights were spaced evenly under each arena for observation of the developing tunnelling patterns. 2.2. Termites and Bioassay Procedures. Coptotermes for- mosanus individuals were collected from a field site on the Manoa campus of the University of Hawaii, adjacent to Miller Hall (21°17'N; 157°99'W; 23.1m above sea level) (Figure 2). Coptotermes gestroi individuals were collected from a field site on the west side of Oahu, approximately 40 k from the campus (Barber’s Point riding stables, Kalaeloa; 21° 19'N; 158°02'W; 9 m above sea level) (Figure 3). Termites were collected from each site in wood placed beneath plastic buckets on the soil surface and were brought to the laboratory. They were removed from the wood and counted using techniques modified from those of Tamashiro et al. [24], Su and La Fage [25], and Grace et al. [26]. Approx. 1500 termites comprising 90% workers and 10% soldiers were introduced into each arena via the release chamber. Six arenas were employed simultaneously, with three allocated to each termite species, one of each set of three allocated to each type of timber (Douglas Fir or Southern Yellow Pine) and one of the three containing no wood and designated as a nonfeeding control. The arenas were arrayed in a rectangular fashion (2 X 3) in a dark room lit only by Psyche 3 Figure 1: Schematic drawing of a subterranean termite foraging arena, after Campora and Grace [22]. Inner space between top and bottom halves of the arena is filled with moist silica sand. Figure 3: Coptotermes formosanus field site near Miller Hall, Figure 2: Coptotermes gestroi field site at Kalaeloa, Oahu, Hawaii. University of Hawaii at Manoa, Oahu, Hawaii. the lights beneath each arena and were allocated randomly to each species and wood treatment. Arenas were observed daily over 22 days. Temperature and humidity were recorded each day using an indoor data logger (HOBO UlO Temp/RH Data Logger). For the first five days, observations and photographs were made every six hours. From the sixth to 22nd day, observations were made every 12 hours. To monitor tunnel construction, digital photographs were taken using a Nikon D40 digital camera. 2.3. Data Analyses. Digital images of the arenas were ana- lyzed using Adobe Acrobat Professional 8 software [27]. The parameters of interest were length of tunnels (primary, secondary, tertiary, quaternary, and other), width of tunnels (mean width), and angles formed between the primary and secondary tunnels of the two species. Tunnels were classified as primary if they originated from the release site, secondary if they branched from a primary tunnel or originated at a feeding portal intercepted by a primary tunnel, and tertiary 4 Psyche if they branched from a secondary tunnel or originated at a feeding portal intercepted by a secondary tunnel [7] . These parameters were used to calculate the following variables for each arena: total distance (TD: sum of all tunnel lengths in each arena), area occupied (A: TD X mean tunnel width), numerical density within the tunnel (D: number of termites released/A), and mean speed of tunnel growth (S: TD/time) [28]. Statistical analyses were carried out using Minitab 15 software [29]. Analysis of variance (ANOVA) was used to test for significant differences in the total tunnel length (L), total mean width of tunnels {W), mean speed of tunnel construc- tion (S), area occupied (A), and numerical density (D). ANOVA was also used to test for differences between species and the effect of presence of wood in the experiment. Tukey HSD assessed differences between colonies in pairwise comparisons. A t-test (two sample test) was used to compare results between arenas without wood and with wood. To compare different parameters measured in tunnel construc- tion trials with C. gestroi and C. formosanus, the data were grouped as discussed below. 3. Results After placement in each release chamber (central jar), the termites remained largely clustered in the central jar for a few hours (1-6), while some termites of both species started to move outward into the arenas. For the first six to seven days, most of the termites in all arenas were very active and made tunnels over ca. 75% of each arena. They appeared to engage in several activities throughout the test period: mov- ing back and forth within tunnels, actively excavating sand during tunnel formation, and gathering in either small (20- 40) or large groups (more than 100) at apparently random locations. From days 7-22, some wood blocks were encoun- tered (relatively very few), and fed upon and in some cases covered with silica sand. As previously noted with C. formosanus [7], empty bait ports in the arenas without wood were also intercepted by termite tunnels. Arenas with C. gestroi showed a low rate of food location ( 1-4 wood blocks out of 16) and a low feeding rate (by visual estimation). However, C. formosanus demonstrated a relatively high rate of wood block location (3-10 blocks out of 16) and a higher feeding rate. During the last few days of the test, termite activity was low, with many termites dead and living termites gathered in groups. Tunnel network systems differed between the two species. As previously noted by Grace et al. [16], C. gestroi con- structed a large number of highly branched, narrow tunnels, while C. formosanus made fewer, wider, and less branched tunnels (Figures 4 and 5, Table 1). There were some differences in the parameters measured in tunnel construction between the two species (Table 2). Coptotermes gestroi built longer (F = 3.85, df = 5, P = 0.012) and narrower (P = 10.62, df = 5, P = 0.031) tunnels, at a faster rate (P = 3.84, df = 5, P = 0.012), and occupied a smaller total area (P = 27.30, df = 5, P = 0.006) than C. formosanus. The lowest termite mortality was record- ed in the sixth arena with C. formosanus, and the highest Figure 4: Coptotermes formosanus tunnels in foraging arena. Figure 5: Coptotermes gestroi tunnels in foraging arena. mortality was also recorded with C. formosanus in the fourth arena. Mean mortality values were high for both termite species, indicating that the prolonged laboratory exposure was stressful to the termites. In addition, differences in tunnel density and angles between primary and secondary tunnels were also noted, as indicated in Table 2. There were no differences in the number of tunnels in arenas with and without susceptible wood with either termite species (Table 1 and Figure 6). Thus, tunneling patterns for the two species could be directly compared without the con- founding variable of food source. 4. Discussion The results of this study indicate that C. gestroi and C. formosanus construct quantitatively different tunnel systems. Psyche 5 Table 1: Number of tunnels in each foraging arena (C: control arenas without wood; CG: Coptotermes gestroi, CF: Coptotermes formosanus) . Arena no. Species Primary Secondary Tertiary Quaternary Other Total 1 CG 5 17 23 18 10 73 2 CG 12 13 28 6 16 75 3(C) CG 11 18 28 11 28 96 4 CF 7 14 12 16 10 59 5(C) CF 4 7 7 10 11 39 6 CF 3 7 10 7 4 31 Table 2; Comparison of parameters measured in tunnel construction with C. formosanus (CF) and C. gestroi (CG). (TD: total length of tunnels constructed; W: mean width of tunnels; A: area occupied; D: numerical density within tunnels; S: speed of tunnel growth; p: primary tunnels; s; secondary tunnels; wo; workers; so: soldiers). Arena No Species TD (cm) W (cm) A (cm^) D (no/cm^) S (cm/days) Angles-p and s Survival No. wo : so Mortality % 1 CG 1043.56 0.08 87.66 15.40 47.43 52.13 663 : 20 54.47 2 CG 715.85 0.07 50.83 26.56 32.53 53.21 913:29 37.2 3(C) CG 1294.45 0.06 81.55 16.55 58.84 54.22 924 : 36 36 4 CF 706.87 0.20 166.64 8.10 37.13 38.81 485:35 65.33 5(C) CF 611.37 0.35 212.76 6.35 27.78 38.55 658 ; 66 51.73 6 CF 496.0 0.50 247.01 5.47 22.55 41.10 1040:133 21.8 P value 0.012 0.031 0.006 0.024 0.012 0.805 (C: Controls without wood, Termite species comparison by ANOVA and Tukey, P < 0.05). Species ■ Primary ■ Secondary Figure 6: Number of primary and secondary tunnels in foraging arenas with (Douglas Fir and Southern Yellow Pine) and without (controls) food present. CG: C. gestroi; CF; C. formosanus. Within each species, tunnel numbers with and without food present were not significantly different (t-test: P = 0.118). substantiating the qualitative visual observations of Grace et al. [16]. Coptotermes gestroi makes a large number of narrow tunnels with many branches, whereas C. formosanus makes a smaller number of wider and less -branched tunnels (Tables 1 and 2). Coptotermes formosanus occupies a larger tunneling area than C. gestroi and thus may locate a greater number of individual wood resources. These results support the hypothesis suggested by Grace et al. [16] that C. gestroi employs a foraging strategy of intensive local search, while C. formosanus constructs a network of longer exploratory tunnels extending over a greater area in order to locate more spatially disparate resources. Grace et al. [16] also suggested that these differences in tunneling behavior may reflect differences in the spatial distribution of cellulosic resources in the tropical (C. gestroi) and subtropical (C. formosanus) environments favored by each of these termite species. Although not addressed in the current study, the interac- tion between the trail pheromone of termite species and their food search patterns might lead to more efficient location of food [12]. Trail pheromone plays a fundamental role in the orientation and recruitment of termites [30] . Minimizing the energy used in search tunnel formation would certainly be advantageous to a termite colony. An efficient search system should optimize total tunnel length and mean tunnel length within a given search area, resulting in fairly direct foraging routes to food sources [3]. Although C. formosanus appeared to be more energy efficient by constructing a few, wider tunnels compared to the network of small, branched tunnels created by C. gestroi, the relative efficiency of these tunnel patterns in food location and accumulation would depend upon the distribution of resources in the surrounding en- vironment. We also examined whether different tunneling patterns resulted when there was no wood to locate within the arena. However, there were no statistically significant differences in tunnel numbers with or without the presence of wood. Gampora and Grace [7] also found that the presence or absence of wood did not affect the basic tunneling pattern of C. formosanus. In additional work, we intend to examine whether the different tunnel patterns of these two species result in more or less rapid food discovery when resources are clumped, as might be found more often in the subtropical regions home to C. formosanus. Grace et al. [16] suggested that tunneling patterns may reflect such regional variation in the distribution of woody resources. 6 Psyche We conclude that tunneling patterns, rates, the numbers of individual tunnels produced by C. formosanus differ from those of C. gestroi. The presence or absence of wood did not influence each species’ innate tunneling patterns. Based upon the results of this study, we are currently designing a foraging arena appropriate to test the hypothesis of Grace et al. [16] that the different tunneling patterns of C. gestroi and C. formosanus may reflect regional variation in the dis- tribution of woody resources upon which these termites depend. The highly branched network of tunnels charac- teristic of the equatorial C. gestroi would appear more effi- cient for locating more uniformly distributed cellulosic re- sources, while the longer and unbranched tunnels of C. formosanus would provide a more efficient route to the widely separated woody resources found in subtropical or temperate environments. Thus, in a foraging arena contain- ing widely separated (clumped) wood, one would expect that C. formosanus would locate these resources more quickly than C. gestroi. Whether or not this is the case remains to be seen, but correlation between observable termite behaviors, such as tunneling patterns, and particular characteristics of the species’ environments would provide a useful tool for studying the ecology of these cryptic insects, and for under- standing the current and future distributions of invasive ter- mite species. Acknowledgments The authors are grateful to Robert Oshiro and Maria Aihara- Sasaki, for technical assistance and to Julian R. Yates, III, and Mark Wright for reviewing drafts of this paper. Thanks are also due to Cory Campora for providing them with arena construction details and to NH’s husband, Chaminda Wijesundara, for his help in various ways. Funding for this paper was partially provided by USDA-ARS Specific Co- operative Agreements 58-6615-9-200 and 58-6435-8-294 and Mclntire-Stennis and Flatch funds administered by the College of Tropical Agriculture and Human Resources, Uni- versity of Hawaii at Manoa. 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Chaves, “Foraging activity and demographic patterns of two termite species (Isoptera: Rhinotermitidae) living in urban landscapes in southeastern Brazil,” European Journal of Entomology, vol. 102, no. 4, pp. 691-697, 2005. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 178930, 10 pages doi:10.1155/2012/178930 Review Article A Paratransgenic Strategy for the Control of Chagas Disease Ivy Hurwitz/ Annabeth Fieck,^ Nichole Klein, ^ Christo Jose,^ Angray Kang,^ and Ravi Durvasula' ^ Department of Internal Medicine, Center for Global Health, Health Science Center, University of New Mexico and New Mexico VA Health Care System, Albuquerque, NM 87131, USA ^ Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London El 4AT, UK Correspondence should be addressed to Ravi Durvasula, ravi.durvasula@va.gov Received 25 October 2011; Accepted 9 December 2011 Academic Editor: Jocelyn G. Millar Copyright © 2012 Ivy Hurwitz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chagas disease results from infection with the parasite Trypanosoma cruzi. This disease remains a significant cause of morbidity and mortality in central and south America. Chagas disease now exists and is detected worldwide because of human migration. Control of Chagas disease has relied mainly on vector eradication however, the development of insect resistance to pesticides, coupled with cost and adverse health effects of insecticide treatments, has prompted our group to investigate novel methods of transmission control. Our laboratory has been instrumental in the development of the paratransgenic strategy to control vectorial transmission of T. cruzi. In this paper, we discuss various components of the paratransgenic approach. Specifically, we describe classes of molecules that can serve as effectors, including antimicrobial peptides, endoglucanases, and highly specific single chain antibodies that target surface glycoprotein tags on the surface of T cruzi. Furthermore, we address evolving concepts related to field dispersal of engineered bacteria as part of the paratransgenic control strategy and attendant risk assessment evaluation. 1. Chagas Disease American trypanosomiasis, or Chagas disease, is caused by the protozoan Trypanosoma cruzi. Between 8-11 million people worldwide are infected, and, of these, approximately 50,000 will die annually [1]. In 2000, the annual cost of morbidity and mortality attributed to Chagas disease in en- demic countries was estimated to be close to US$8 billion [2]. Two years later, the WHO estimated the burden of Chagas disease to be as high as 2.7 times the combined burden of malaria, schistosomiasis, leishmaniasis, and lep- rosy [3]. Though traditionally a disease endemic to Mexico, central, and south America, human migration has resulted in reported cases of T. cruzi infection worldwide [4]. For example, cases of Chagas disease have been reported in Portugal [5], Spain [6, 7], France [8, 9], and Switzerland [10], countries that are favored for immigration from Latin America. Reports from Australia estimate T. cruzi infection in 16 of every 1,000 Latin American immigrants [1, 5], and Chagasic heart disease was reported in Brazilian immigrants of Japanese origin in Japan [6]. There have been numerous reported cases of Chagas disease resulting from unscreened blood transfusions and organ donation. Further, the parasite can be congenitally passed from mother to child. However, this disease is most often transmitted to humans by T. crwzi-infected blood- sucking triatomine bugs. These insects are members of the heteropteran family Reduviidae. The major vectors for Chagas disease in central and south America are Rhodnius prolixus (Figure 1(a)) and Triatoma infestans (Figure 1(c)), respectively. These bugs thrive in thatch and adobe of poorly constructed homes during the heat of the day, coming out in the cooler hours of the night to feed. Carbon dioxide emanating from the breath of the sleeping vertebrate victims as well as ammonia, short chain amines, and carboxylic acids from skin, hair, and exocrine glands are among the volatiles that attract triatomines. These insects are often dubbed “kissing bugs,” from their common habit of biting the face, which is often exposed during sleep. The bite of triatomine bugs is painless, allowing the insect to feed without interruption. As the insect engorges, it defecates. If the insect is infected with T. cruzi, the parasite will be in the 2 Psyche Figure 1: Triatomine bugs, (a) R. prolixus, picture adapted from http://www.jyi.org/articleimages/185/originals/img0.jpg; (b) T. gerstaeckeri, picture adapted from http://theearlybirder.com/insects/hemiptera/reduviidae/index.htm; (c) T. infestans, picture adapted from http://www. k-state.edu/parasitology/546tutorials/ARTHFlG01.JPG; (d) T. sanguisuga, picture adapted from http://bugguide.net/node/view/5164; (e) T. rubida, picture adapted from http://bugguide.net/node/view/185220. fecal material and will then enter the bloodstream when the victim scratches the irritated bite wound. There are currently more than 300,000 people infected with T. cruzi living in the United States. Most acquired the disease while residing in endemic areas [11]. Although T. crwzi- infected vectors and animals are found in many parts of this country [12], there have only been 5 documented cases of autochthonous (indigenous) transmission in the US [13]. Surveys of Reduviid bugs in the American Southwest have shown high rates of T. cruzi infection in T. rubida (Figure 1(e)), T. protracta, T. sanguisuga (Figure 1(d)), and T. gerstaeckeri (Figure 1(b)) [12, 14-16]. Species-specific behavior of the north America triatomines may explain why more autochthonous transmission is not observed. In a study by Klotz et al. [17], field caught T. protracta and T. rubida were allowed to feed on live immobilized white mice and the defecation pattern was observed for one hour. Of the 71 triatomines observed, only 30 (42 percent) produced a fecal droplet within one hour after feeding. Sixty-seven percent of these defecated 1.5-6 cm from the mouse, whereas 33 percent defecated 7-10 cm away from the mouse. None of the bugs defecated on the mouse. The transmission cycle of T. cruzi is complex. Reduviid vectors become infected with T. cruzi when they feed on vertebrate blood containing bloodstream trypomastigotes (Figure 2). The trypomastigotes differentiate into epimastig- otes in the midgut and then to infective metacyclic trypo- mastigotes as they move further into the hindgut of the insect. When the triatomine bug takes its next blood meal, the trypomastigotes are defecated onto the bite wound of its victim. Transmission of the parasite therefore occurs through contamination of the bite site. Upon entry into the blood stream of the vertebrate host, the trypomastigotes colonize muscle and neuronal tissue where they form intracellular amastigotes. These proliferating cells will form pseudocysts, which after several successive cell divisions, will asynchronously transform into trypomastigotes. The trypo- mastigotes then escape from the pseudocysts into the blood Psyche 3 Triatomine bug stages Triatomine bug takes a blood meal ^ (passes metacyclic trypomastigotes in feces, ^ trypomastigotes enter bite wound or mucosal membranes, such as the conjunctiva) Metacyclic trypomastigotes in hindgut Multiply in midgut ^ Triatomine bug takes O Epimastigotes in midgut a blood meal ^^(trypomastigotes ingested) http /Amwm dpd cdc gov/dpdx A infective stage diagnostic stage Human stages Metacyclic trypomastigotes penetrate various cells at bite wound site. Inside cells they transform into amastigotes. Trypomastigotes ^ can infect other cells and transform into intracellular amastigotes in new infection sites. Clinical manifestations can .result from this infective cycle. Amastigotes multiply by binary fission in cells of infected tissues. A Intracellular amastigotes transform into trypomastigotes, then burst out of the cell and enter the bloodstream. Figure 2: An infected triatomine bug takes a blood meal and releases trypomastigotes in its feces near the site of the bite wound. Trypomastigotes enter the host through the wound (1). Inside the host, the trypomastigotes invade cells near the site of inoculation, where they differentiate into intracellular amastigotes (2). The amastigotes multiply by binary fission and form pseudocysts (3). Following several cycles of division, these cells will asynchronously differentiate into trypomastigotes, which are then released into the circulation as bloodstream trypomastigotes where they can start infecting cells from other tissues (4) . The cycle of infection continues as another triatomine bug becomes infected by feeding on human blood containing the circulating parasites (5). The ingested trypomastigotes transform into epimastigotes in the vector’s midgut (6). The parasites multiply in the midgut (7) and differentiate into infective metacyclic trypomastigotes in the hindgut (8). This figure is adapted from http://www.dpd.cdc.gov/dpdx. and lymph to invade new cells. The cycle of transmission continues when another triatomine bug takes a blood meal from the infected vertebrate. Chagas disease is characterized by three successive stages. The acute stage, often characterized by generalized malaise, fever, swelling of the lymph nodes, and enlargement of the liver and spleen, is minimally symptomatic and lasts from 4 to 8 weeks. The hallmark of acute infection is the chagoma, an inflammatory skin lesion that develops at the site of a triatomine bug bite. The lesion is a result of lymphocytic infiltrate, intracellular edema, and adjacent reactive lym- phadenopathy due to the intramuscular presence of T. cruzi at the site of inoculation. When the bite is on the face near the eye, the characteristic Romana’s sign occurs (Figure 3). The acute phase is then followed by an indeterminate phase that can last between 10 to 20 years. During this period, there are few clinical manifestations but active replication of T. cruzi and periodic release of bloodstream forms of the parasite into the circulation occurs. Approximately, 30 percent of these patients progress to symptomatic chronic Chagas disease, which often manifests in the 4th or 5th decade of life [18]. Hallmarks of chronic infection include inflammation of the heart muscles, enlargement of the esophagus, and enlargement of the colon. For the most part. Figure 3: Romana’s sign is characterized by unilateral palpe- bral edema, conjunctivitis, and lymphadenopathy. Photograph is adapted from WHO/TDR 2011. the gastrointestinal manifestations of chronic Chagas disease are geographically restricted and play a lesser role in the over- all disease burden of Chagas disease. However, progressive heart disease is a leading public health concern throughout much of central and south America. The chronic phase of Chagas disease is incurable and on average is associated with a ten-year shortening of life span. Whereas acute Chagas disease is treatable with appropri- ate and timely antiparasitic medication, the chronic phase of 4 Psyche this debilitating disease often goes undiagnosed. Antipara- sitic treatment of chronic disease is of questionable clinical benefit and is often limited by adverse drug reactions and side effects. In the absence of vaccines and effective drug therapies, control of Chagas disease had relied largely on measures aimed at vector eradication. To date, several large- scale insecticide-based efforts have been undertaken with considerable success. In 1991, an international coalition of governmental agencies from Argentina, Bolivia, Brazil, Chile, Paraguay, Uruguay, and Peru started The Southern Cone Initiative to minimize transmission of T. cruzi by T. infestans. This coalition developed educational programs aimed at reducing human contact with T. infestans, as well as blood bank screening programs to eliminate transmission of T. cruzi. Successes of this program include a Pan-American Health Organization awarded certificate for the disruption of Chagas disease transmission by T. infestans to Uruguay, Chile, and Brazil [19]. However, sustainability of this pro- gram has been called into question due to the need for con- tinued application of pyrethroid insecticides and possibility of reinfestation of domestic structures [19]. The poor effects of pyrethroid insecticides are mainly caused by their short- lasting residual effects in outdoor sites exposed to sunlight, high temperatures, rain, and dust [20, 21]. The reduced effectiveness of pyrethroids was further compounded by the development of triatomine populations that were resistant to a variety of insecticides in Chagas disease endemic areas [22]. This, coupled with recent surveillance data indicating resurgence of human infections, particularly in the Argentinan Gran Chaco [23], would suggest that current insecticidal programs for control of vectorial transmission of Chagas disease are failing, and that novel, effective, and sustainable supplementary tactics are critically needed to maintain suppression of Chagas disease transmission. 2. The Paratransgenic Approach, an Alternative Strategy to Control Vectorial Transmission of T, cruzi Triatomine bugs subsist on a blood-restricted diet. To sup- plement their basic nutritional and developmental needs, these insects have developed a symbiotic relationship with nocardioform actinomycetes [24]. These soil-associated bac- teria are thought to aid in the processing of B complex vitamins and are essential to the survival of the bug. The sym- biosis of these bacteria with several triatomine species and the amenability of these cells to genetic manipulation are the cornerstones of the paratransgenic strategies aimed at interrupting vectorial transmission of T. cruzi. Rhodococcus rhodnii is a soil-associated nocardioform actinomycete. It also lives extracellularly in the gut lumen of R. prolixus in close proximity to T. cruzi. This microbe is transmitted from adult triatomine bugs to their progeny through coprophagy, the ingestion of fecal material from other bugs. Rhodococcus rhodnii is critical to the growth and development of R. prolixus [24]. Rhodnius prolixus nymphs that lack gut-associated symbionts (aposymbiotic) do not reach sexual maturity and most die after the second developmental molt. Introduction of the bacteria to the first or second instar nymphs permits normal growth and maturation. In 1992, we transformed R. rhodnii with pRrI.I, a shuttle plasmid containing a gene encoding resistance to the antibiotic thiostrepton, to support the hypothesis that a transgene-carrying symbiont could be introduced into R. prolixus [25]. These transformants were introduced into aposymbiotic first instar R. prolixus nymphs via artificial membrane feeding and reared in the presence and absence of thiostrepton. Examination of bacteria from the gut of insects carrying the transformed symbionts demonstrated that thiostrepton resistant colonies could be recovered from these vectors, regardless of antibiotic presence, for up to 6.5 months after infection. These studies demonstrated that genetically modified R. rhodnii symbionts expressing a selectable gene product could be stably maintained in R. prolixus without adverse effects on insect survival and fitness, thereby substantiating the paratransgenic approach. For the paratransgenic strategy to work, it is imperative that a population of symbiotic bacteria, that is, amenable to culture and receptive to genetic manipulation, be identified within a given disease -transmitting vector. The fitness of these symbionts should not be compromised nor should their normal functions within the vector be affected fol- lowing genetic manipulation. The transgene products, when expressed in proximity to the target pathogen, should inter- fere with pathogen development in the vector, and should not be detrimental in any way to the vector. Finally, the dispersal technique used to spread the genetically modified symbiont/commensal to naturally occurring vector popu- lations should minimize the spread of the transgene to other organisms in the vector’s environment, which include both the nontarget microbiota inside the vector and other organisms that live in the same ecological niche. Since our initial experiments, we have adapted the paratransgenic strategy to numerous other vector-borne disease systems, including sand-fly-mediated leishmaniasis [26, 27] and sharpshooter- mediated Pierce’s disease [28]. The strategy is also being developed and extrapolated into shrimp mariculture [29] . In this paper, we will focus on work relating to the paratransgenic control of Chagas disease with a number of effector molecules, specifically, antimicrobial peptides (AMPs), recombinant endoglucanases that disrupt the surface of the parasite, and functional transmission- blocking single-chain antibodies. 2.1. Antimicrobial Peptide Genes. Cecropin A is an AMP that was isolated from the giant silk-worm moth Hyalophora cecropia [30]. This AMP is 38 amino acids in length. Cecropin A lyses cells by binding to and covering the parasite membrane surface, effectively dissipating transmembrane electrochemical gradients [31]. We cloned the DNA sequence for cecropin A into the pRrl.l shuttle vector to produce pRrThioCec. This plasmid was then used to transform R. rhodnii [32]. Paratransgenic R. prolixus were generated with the cecropin A-expressing symbionts and allowed to engorge on T. cruzi-laden human blood until they reached sexual maturity. Hindgut contents from paratransgenic insects carrying pRrThioCec-transformed R. rhodnii were Psyche 5 either devoid of T. cruzi trypomastigotes (65 percent) or maintained markedly reduced titers of the parasite (35 percent) [32]. In contrast, all control insects harboring untransformed R. rhodnii or R. rhodnii transformed with pRrl.l (original shuttle vector without cecropin gene) that were infected with T. cruzi in the same manner carried mature trypomastigotes. This study provided proof of concept for the paratransgenic strategy, and suggested that other AMPs might be employed singly or in concert to elicit complete elimination of infective parasites in the hindgut of T. cruzi vectors. A large number of AMPs have been and are being discovered that function in a variety of ways, including dis- ruption of cell membranes similar to cecropin A, interference with host metabolism, and inactivation of cytoplasmic com- ponents [31]. Many AMPs are also capable of discriminating between host and invading organisms, thereby permitting the expression of recombinant AMP’s from certain cell lines without deleterious effects to a host insect. In vitro studies were carried out with six AMPs selected from different insect sources to determine their differential toxicity profiles against host bacterial strains and T. cruzi parasites [33]. AMP’s were identified that displayed high toxicity against T. cruzi (LCioo < lO^wM) compared to R. rhodnii (MBC > 100 jUM) in single synthetic peptide treatment regimens. These peptides; apidaecin, cecropin A, magainin II, and melittin, were employed in pairwise treatment protocols against T. cruzi. Dual peptide treatments of T. cruzi showed synergistic or additive effects between different AMP’s result- ing in increased toxicity over any single AMP treatment. The best example for this was observed with apidaecin. When administered alone to T. cruzi, apidaecin killed the parasite at the 10 [iM dose, but when used in combination with melittin, magainin II, or cecropin A, complete lethality to T. cruzi was seen at 1.0 /^M — a tenfold decrease in the necessary lethal concentration. While all combinations exhibited additive activity compared to single AMP treatments, synergistic activity was observed when magainin II was applied in combination with apidaecin or melittin (Figure 4). It has been inferred from the pair-wise treatment data that the additive and synergistic effects observed could improve the 65 percent rate of T. cruzi elimination seen in the initial in vivo studies with cecropin A. Furthermore, the use of peptides in combination could reduce the development of peptide resistance in target T. cruzi populations. Rhodococcus rhodnii has been transformed with expres- sion plasmids for the four peptides (melittin, magainin II, apidaecin, and cecropin A) and expression of these molecules was confirmed by ELISA and western blot (Fieck et al., in prep.). The shuttle vector employed for these studies expressed AMP gene sequences from the Mycobac- terium kansasii-a antigen promoter and export signal sequence. Selection of positive transformants was achieved for both the E. coli cloning host and R. rhodnii symbiont by growth on carbenicillin and confirmed by colony PCR. Cell lysates from AMP-transformed R. rhodnii have been shown to be toxic to T. cruzi in single- and pairwise in vitro toxicity assays. In vivo experiments are currently underway at the Centers for Disease Control (CDC) to test the toxicity of products from single and dual peptide-carrying symbionts to T. cruzi in aposymbiotic R. prolixus nymphs. 2.2. Single-Chain Antibodies. Antimicrobial peptides act as direct effectors in the paratransgenic model by physically damaging cell structure or metabolic function, resulting in parasite death. Single-chain antibodies (scFv) comprise a second class of effector molecules selected to negatively impact T. cruzi development and transmission by acting through an indirect mechanism. In this design, scFvs with binding specificity to T. cruzi surface proteins interfere with the physical contact between trypanosomes and the vector that is essential for parasite development. This interference model predicts that the activity of the effector scFv molecules would be specific to parasite development and elicit fewer negative effects on the vector or transformed symbiont. Parasite maturation, which involves metacyclogenesis of T. cruzi from noninfective epimastigotes to infective trypo- mastigotes, occurs in the gut of the triatomine bugs and is an important step in the transmission of Chagas disease [34]. This maturation process is dependent on interactions between the surface epitopes of T. cruzi and the gut lumen of the insect vector [35] and would be the target of scFv effector activity. Single-chain antibodies usually consist of variable re- gions of heavy and light chains of immunoglobulins connected by a flexible linker, (Gly4Ser) n = 3-5, that permits the two protein domains to interact effectively with their corresponding antigen [36]. The fact that DNA sequences for scFvs can be cloned into expression plasmids and expressed from bacterial transformants renders these molecules uniquely suited to this system of insect paratrans- genesis. To test the ability of a scFv to be expressed and functional within the gut of the Reduviid vector, the pRrMDWK6 expression shuttle plasmid was constructed with a marker gene coding for a murine antiprogesterone antibody frag- ment, rDB3 [37]. Constitutive expression of rDB3 from pRrMDWK6-harboring symbionts was under control of the M. kansasii-a antigen promoter/signal sequence and could be quantified by ELISA for progesterone binding activity. Aposymbiotic R. prolixus nymphs were exposed to DB3- expressing R. rhodnii symbionts and allowed to develop on blood meals. Subsequent examination revealed that the rDB3 antibody fragment was synthesized by the transformed R. rhodnii and secreted into the gut lumen throughout the development of the nymphs to the adult stage (6 months). Protein extracts from the gut of paratransgenic R. prolixus bound progesterone suggesting that the presence and activity of scEvs could be maintained in the environment of the insect gut [37]. Eurther evidence for this was provided by similar experiments carried out with T. infestans, another major vector of Chagas disease. A Cory neb acterium sp., a bacterium closely related to R. rhodnii, was identified as a symbiont in this vector. We successfully transformed this bacterium to express rDB3 from the pRrMDWK6 shuttle vector, and generated paratransgenic T. infestans lines [38]. ELISA analysis of gut extracts from these paratransgenic bugs 6 Psyche o >0 On u C o o w o C r^ tn O c/2 r^ CT3 1/ DC c CD X O Melittin alone Melittin + cecropin A Melittin + apidaecin Melittin + magainin Apidaecin alone Apidaecin + melittin Apidaecin + cecropin Apidaecin + magainin (a) Antimicrobial peptide kill curves cecropin combinations (b) Antimicrobial peptide kill curves magainin combinations Cecropin alone Cecropin and melittin Cecropin and apidaecin Cecropin and magainin Magainin alone Magainin and cecropin Cecropin and apidaecin Cecropin and melittin (c) (d) Figure 4: Antimicrobial peptide kill curves for dual-combination treatments of T. cruzi cultures. Results averaged from triplicate samples in three separate experiments and displayed as the percent change in absorbance at 600 nm compared to untreated controls. Figure is adapted from Fieck et al. Trypanosoma cruzi: synergistic cytotoxicity of multiple amphipathic antimicrobial peptides to T. cruzi and potential bacterial hosts [33]. showed the presence of rDB3 antibodies capable of binding to progesterone [38]. Progression from the paratransgenic system employing a marker scFv to one utilizing effector scFv’s required the development of antibodies with strong binding affinities to parasite surface coat proteins. Trypanosoma cruzi does not synthesise or catabolise free sialic acid, but expresses a developmentally regulated sialidase which is used for surface sialylation by a trans-sialidase mechanism [39]. If the appropriate galactosyl acceptor is available the sialyl- transferase activity of the T. cruzi sialidase is greater than its hydrolytic activity. This implies a mechanism that is capable of remodelling the T. cruzi glycan surface using host glycoconjugates as the sialyl donor. Such sialylation might Psyche 7 Figure 5: Molecular assembly of an affinity fluorescent protein. The REDantibody shown here has mRFP engineered between the VH and VL domains of the scFV, resulting in a highly stable red fluorescent targeted affinity probe. Figure adapted from Markiv et al. Module based antibody engineering: a novel synthetic REDantibody [36]. provide protection for T. cruzi from the innate immune responses. This large family of cell surface sialyated mucin- like glycoproteins plays an essential role in the parasite’s life cycle [35], and, therefore is excellent targets for scFv binding. Two well characterised murine monoclonal antibodies, B72.3 [40] and CA19.9 [41], that bind sialyl-Tn and sialyl-(le)a surface glycans, respectively, were selected for application to the paratransgenic system incorporating scFv’s for control of Chagas disease transmission [36]. Synthetic DNA sequences encoding the variable regions of the heavy and light chains of these monoclonal antibodies were used to assemble the complete coding sequences for the scFvs. In place of the standard 15 amino acid linker between the heavy and light chain fragments, a monomeric red fluorescent protein (mRFP) derived from the red fluorescent protein cloned from the Discosoma coral, DsRed [42], was inserted as a rigid linker that conferred extra stability and fluorescence to the scFvs (Figure 5). Binding to fixed T. cruzi epimastigotes and fluorescent optical properties of these scFvs were demon- strated using confocal microscopy (Figure 6). The gene sequences for these scFvs are currently being inserted into the E. coli/R. rhodnii shuttle vector for the generation of traceable scFv-expressing symbionts and subsequent generation of effector scFv containing paratransgenic R. prolixus. 2.3. ^-1,3-Glucanase. Endoglucanases comprise a third class of trypanocidal molecules expected to function as effector molecules in the paratransgenic system for Chagas disease control. j8-l,3-glucanase is part of an endoglucanase com- plex isolated from Arthrobacter luteus called lyticase. This molecule functions by breaking the 1-3 and 1-6 glycosidic linkages of surface glycoproteins [43]. The surface of T. cruzi is covered by a thick coat of glycoproteins proposed to play a role in the binding of the cell body and flagellum Figure 6: Confocal image of anti-sialyl-Tn REDantibody targeting glycan structures on the surface of T. cruzi epimastigotes. Figure adapted from Markiv et al. Module based antibody engineering: a novel synthetic REDantibody [36]. to membranes in the vector gut [44]. This binding is necessary for T. cruzi to complete its development and, as a consequence, is essential for its transmission. In unpublished work, we showed that A. luteus lyticase was efficient in lysing T. cruzi while being nontoxic to R. rhodnii and R. prolixus. We inserted the cDNA for j5-l,3-glucanase into our shuttle plasmid and isolated extracts from the transformed R. rhodnii for use in toxicity assays against T. cruzi. Although j5-l,3-glucanase was originally described as being effective only as a part of the lyticase complex in the presence of a complementary alkaline protease [43], we have demonstrated that recombinant j5-l,3-glucanase is biologically active and clears T. cruzi at low concentrations even in the absence of the protease (Jose et al., in prep.). Toxicity of the recombinant j5-l,3-glucanase against T. cruzi is comparable to A. luteus lyticase complex. These results emphasize the potential for use of j5-l,3-glucanase as another effector molecule in a paratransgenic strategy to control Chagas disease transmission. The in vivo toxic effects of recombinant j5-l,3-glucanase expressed from symbiotic R. rhodnii transformants in R. prolixus will be determined using the previously described experimental approach. The three classes of molecules described above target T. cruzi differentially. An effective paratransgenic strategy for field application would involve the delivery of these effector molecules in combination, for example: AMPs with scFvs or AMP’s with endoglucanases. This strategy should reduce not only transmission of T. cruzi, but also the development of resistance resulting from prolonged treatment with a single effector. 3. Preparations for Field Trials In anticipation of field trials, we have tested the efficacy of a simulated triatomine-fecal preparation, CRUZIGARD, consisting of an inert guar gum matrix dyed with India ink, as a method for delivery of engineered R. rhodnii to closed colonies of R. prolixus [45]. The CRUZIGARD preparation was mixed with 10^ colony forming units (CFU)/mL of genetically modified R. rhodnii and used to impregnate cages constructed of thatch and adobe building materials 8 Psyche from Chagas -endemic regions of Guatemala (Olopa) [45]. In these experiments, field caught adult R. prolixus from the Olopa district were placed in the cages and removed after eggs were laid. Nymphs were allowed to mature in the CRUZIGARD-treated cages. Nine months later, genetically altered R. rhodnii were detected in approximately 50 percent of Fi adults and comprised nearly 95 percent of total CPUs in these bugs, demonstrating that CRUZIGARD may be useful as a gene dispersal strategy even in environments where competing microbes are present. To increase the volume and duration of CRUZIGARD ingestion, and conse- quently increase rates of vector inoculation with transformed symbiont, on-going collaborations to develop triatomine attractants and semiochemicals to supplement the current CRUZIGARD formulation are underway. We realize that deployment of genetically altered R. rhodnii into the field may have profound environmental consequences. In a recent publication, we evaluated the risks of horizontal gene transfer (HGT) between R. rhodnii and Gordona rubropertinctus, a closely related nontarget Gram- positive actinomycete [46] . We developed a model that treats HGT as a composite event whose probability is determined by the joint probability of three independent events: gene transfer through the modalities of transformation, transduc- tion, and conjugation. Genes are represented in matrices, with Monte Carlo method and Markov chain analysis used to simulate and evaluate environmental conditions. The model is intended as a risk assessment instrument and predicts an HGT frequency of less than 1.14 X 10 per 100.000 generations at the 99 percent certainty level [46]. This predicted transfer frequency is less than the estimated average mutation frequency in bacteria, 10"^ per gene per 1.000 generations. These predictions were further substan- tiated when laboratory studies that involved coincubation of R. rhodnii and G. rubropertinctus in conditions highly conducive to HGT resulted in no detectable HGT. These results would suggest that even if HGT were to occur between R. rhodnii and G. rubropertinctus, the transgene would likely not persist in the recipient organism, and that the likelihood of these unwanted events is vanishingly small [46] . To further minimize gene spread to nontarget arthro- pods, we are engaged in the development of a strategy to determine the minimum amount of transformed symbionts necessary to prevent T. cruzi transmission to humans, and an effective method of CRUZIGARD application suitable for at- risk domiciles in the endemic region. We are also engaged in the development of second-generation paratransgenic delivery systems that utilize microparticle encapsulated, genetically altered R. rhodnii for targeted release in the gut of the triatomine bug. Finally, we realize that the field release of engineered bacteria cannot occur until a risk assessment framework is in place. Because such information will not be readily available through field release trials, we are working with collaborators to develop a framework involving rigorous mathematical modeling and simulations. Outputs of these models will be integral to informing risk assessment and regulatory oversight of the paratransgenic program, and, ultimately, to permit field trials of the paratransgenic strategy. 4. Conclusion Chagas disease affects the lives of millions of people world- wide and remains a major cause of mortality and morbidity, as well as economic loss [47]. Increased attention from the World Health Organization and interest from governments of endemic regions have yielded desirable results for control of Chagas disease transmission. However, success of disease control with large-scale insecticide-based approaches, as demonstrated through the Southern Cone [48], central American [49], Andean Pact [50], and Amazonian Initiatives [49], has been dimmed by the looming possibilities of envi- ronmental toxicity, human health impacts, cost of repeated applications, and development of vector resistance. We describe a novel and potentially environmentally friendly method to control vectorial transmission of Chagas disease. This paratransgenic approach is based upon genet- ically manipulating symbionts of the triatomine vectors to express effector molecules that would kill or prevent the development of the parasite within the gut of the insect. We have demonstrated the feasibility of this approach in a number of laboratory-based experiments, using effector molecules such as AMPs, endoglucanases and highly specific scFvs. A number of collaborations are underway to evaluate environmental risk related to field release of the geneti- cally modified symbionts. We believe that eventual field application of the paratransgenic approach could provide a more effective and feasible alternative to current strategies of Chagas disease control in endemic regions of the world. Conflict of Interests The authors declare that they have no competing interests. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 437589, 6 pages dohlO.l 155/2012/437589 Research Article Dung Beetles (Coleoptera: Scarabaeinae) Attracted to Lagothrixlagotricha (Humboldt) and Alouatta seniculus (Linnaeus) (Primates: Atelidae) Dung in a Colombian Amazon Forest Jorge Ari Noriega Laboratorio de Zoologia y Ecologia Acudtica (LAZOEA), Universidad de Los Andes, Bogota, Colombia Correspondence should be addressed to Jorge Ari Noriega, jnorieg@hotmail.com Received 5 October 2011; Accepted 20 December 2011 Academic Editor: D. Bruce Conn Copyright © 2012 Jorge Ari Noriega. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dung beetles are among the most important insects in the Neotropics. Some species use a wide range of food sources, whereas other species are highly specialized. This study compares the use of two-primate excrement by an assemblage of dung beetles in a tropical forest in Colombia. Dung of Lagothrix lagotricha and Alouatta seniculus was used to attract beetles. A total of 32 species (47.7% of the species recorded for the area) were found on the two types of excrement studied, demonstrating that primate excrement is an important resource. The niche overlap between both feces is 27.03%, which indicates a high degree of resource specialization. Although these two primate species are found in the same areas, their diets vary greatly to permit a high degree of differentiation in beetle species. A study that includes dung of others primates would create a more complete panorama of resource overlap in the assemblage. 1. Introduction Dung beetles are among the most important insect assem- blages in the Neotropics, due to their important role in nutrient recycling, seed dispersal, and helminthes control, as they use omnivorous and herbivorous mammals dung as their food resource [1, 2]. Some species of dung beetles can use a wide range of food sources, from carrion to dung or more specific resources such as mushrooms, fruits, diplopods, decomposing vegetation, detritus, and eggs [1, 3-10]. Other species are highly specialized, using certain mammal dung [4, 1 1-15] or a more specific resource like primate dung of Alouatta spp. [3, 16- 24] as well as Lagothrix spp. [25, 26]. Although, diverse aspects of the natural history and ecology of dung beetles have been widely studied [2, 27-30], little is known about their specialized use of some resources, especially primate dung. For this reason, this paper presents a study comparing the use of two primate excrement types by an assemblage of dung beetles in a tropical forest in Colombian Amazon region. Up to the present, no study has compared the preferences between two different primates excrement in the same locality. 2. Materials and Methods 2.1. Study Area. This study was carried out at the Center for Ecological Research of the Macarena (CIEM), located at 2° 40' N and 74° 10' W at an altitude of 350 m. The area is in a lowland tropical wet forest, located on the right bank of the Duda River, at the eastern border of Tinigua National Natural Park (Meta Department, Colombia). The predominant vegetation is mature forest [31] with a single rain cycle (dry period between December and March), The annual precipitation average is 2400 mm, with the least rainfall recorded in January (0 mm) and the greatest amounts in May-July (530 mm) [32], The study was conducted in the primary habitat of the area, mature land forest, characterized by a continual tree canopy with a height of 25 to 30 m and emergent trees that 2 Psyche reach up to 35 m [33]. Seven species of primates coexist in the area and of these, Lagothrix lagotricha (Humboldt, 1812) (Woolly Monkeys) and Alouatta seniculus (Linnaeus, 1766) (Red Howler Monkeys) are the most abundant [34]. 2.2. Field Sampling 2.2.1. Lagothrix lagotricha Dung (see [25]). From january to July 1997 (including part of dry and rainy season), a group of woolly monkeys was followed for 60 h each month, and dung from a single focal individual was collected during the day. Dung beetles attracted to the dung were collected in plastic bags with the monkeys dung sample {n = 520). Dung was collected between 5 minutes after defecation. Additionally, five pitfall traps on soil surface were used during 72 hours in mature forest (replaced the dung daily), in order to complement the sample for nocturnal and delayed visitors. 2.2.2. Alouatta seniculus Dung. During January 1998 (dry season), a group of howler monkeys was followed and in the morning, when the monkeys defecated all the species attracted to the dung for 10 minutes were collected. After- wards, the largest possible quantity of dung was collected in a hermetic container. The collected excrement was placed the same day in 25 mL cups along a 300 m transect in 10 pitfall traps. The traps were placed 30 m apart for a period of 24 hours. This methodology was carried out on three occasions with two days between each sampling effort. The specimens collected were preserved in a 70% alcohol and taken to the Museum of Natural History at the University of Los Andes (ANDES-E), where they were deposited. They were identified to species level using keys, in comparison with the other specimens and the assistance of specialists. 2.3. Data Analysis. Percentage overlap between the dung types was determined using the Jaccard similarity analysis by PAST software. In addition, to compare the composition of species recorded on the two types of dung, an analysis was carried out using the Levin’s breadth of niche index with the standardization proposed by Hurlbert [35]. The MacArthur and Levin’s index of niche overlap was calculated with the modification proposed by Pianka [36], using the species as resources and the types of dung as the species [37]. 3. Results A total of 32 species were found on the two types of excre- ment studied, seven of those found on L. lagotricha were only identifiable to the genus level and thus appear as morph species in Table 1. It is probable that these morph species are contained in the species already identified for A. seniculus, but the material obtained for the two excrement types could not be compared. The analysis comparing the two excrements was thus conducted for two possible extreme scenarios: (a) where the seven morph species not identified by L. lagotricha are contained within the species identified for A. seniculus and (b) where these species are not contained and are different species. The species found on the primate dung sample account for 47.7% of the 67 total species recorded for the area [15], demonstrating that the excrement of both primate species is without doubt an important resource in the area. 18 species were found on the excrement of L. lagotricha, of which seven were exclusive to this type of excrement (or 13 given scenario (a)). Likewise, 19 species were found on the excrement of A. seniculus of which 14 are exclusive (or 8 in scenario (a)). Five species were recorded on both types of excrement (1 1 in scenario (a)). The similarity between both dung types is higher in scenario (a) (Jaccard’s mean value = 42.5%, SD = 9.04%) than in scenario (b) (Jaccard’s mean value = 18.5%, SD = 6.85%). In addition, the structure of the assemblage in each scenario fluctuates in the species in common related with the dung type they use (Figure 1). The Levin’s niche breadth index does not present significant differences between both species (L. lagotricha = 0.016, A. seniculus = 0.017, mean value IL = 1.958). The niche overlap index presented a mean value of 0.2703 (72.97%), with no significant differences between both species (L. lagotricha over A. seniculus = 0.277, A. seniculus over L. lagotricha = 0.2631), while the Renkonen overlap percentage was 15%. In addition, it is interesting to note that on both dung types, it is possible to found rolling species (telecoprids) and tunnelers (paracoprids), while the dwellers (endocoprids) were only collected on the dung of A. seniculus (Figure 2). 4. Discussion Estrada et al. [14] registered an overlap greater than 80% for the dung beetles assemblage on the excrement of A. seniculus and Nasua narica (L., 1776), presenting a high number of common species with some variations in abundance, as a few were recorded exclusively on a single bait. In this study, the relation found between the overlap percentages in the two excrements was the opposite, a range between 15.6% (scenario (b), n = 5 spp.) and 34.3% (scenario (a), n = ll spp.), which indicates that, for this locality, few species are generally associated with both types of dung. These results indicate that there is a high degree of food resource specialization, despite both excrements com- ing from primates, there are sufficient differences in diet, microhabitat, and behavior of each species to permit a degree of differentiation between the species that make use of each dung [38-42]. Although these two species are found in the same areas, their diets vary greatly, as L. lagotricha consumes insects, fruits, and leaves, while the diet of A. seniculus is primarily foliage [25], and these differences affect the consistency, nutritional composition and smell of their respective excre- ments. Additionally, these species use different forest strata, produce excrement at different places, and times and the mobility and number of individuals per group are different [41]. It is probable that the use of different forest strata [41] affects the spatial disposition of the dung and distribution of the species that use these food resources. In addition, it is possible that seasonal difference in diet proportion affects the number of shared species between primates dung because Psyche 3 a Ph b/D p a c/5 > a u p a < o u G u CL| G cb G O Ph ^G n X > X) cb u Dh c/5 cb cb G cb 3 3 G cb w 1 n 0.9 - 0.8 - 0.7 - 0.6 - 0.5 - I ^ 0.4 H 0.3 - 0.2 - 0.1 - CPQU o<1W B ^ p p CLi Q-i ^ ^ CLi (1> P-i ^ U U C3 OCCUOOQ^^OU a cb c o y G u b/D G C3 4-» cb X cb u X cb C3 G Q o u u O o “T" 10 ~r“ 20 ~r“ 30 (a) o ..Q C O s '3 9" c i ^ 8 ^ i X ^ •-H c IS IS p B Q O O O w w a is o X > t3 . 2 .. 3 u ^ G O Dh 3 D b/D P-l G cb G cb IS X u a c a g IS 4-» cb G X cb s .w u < U U o u o U O < o G O u o *G I c75 1 - 0.9 - 0.8 - 0.7 - 0.6 - 0.5 - 0.4 - 0.3 - 0.2 - 0.1 - “I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 (b) Figure 1; Dendrogram cluster analysis of similarity (Jaccard’s index) among species and the dung type they use in scenarios (a) and (b), CIEM-Meta, Colombia. 4 Psyche Table 1: List of the species associated with each of the primate’s dung, with their relocation habits (T: tunneler, R: roller, and D; dweller), in the CIEM station, Meta, Colombia. Tribe Species rh L. lagotricha A. seniculus Ateuchini Ateuchus murrayi (Harold, 1868) T X — Ateuchus pygidialis (Balthasar, 1939) T — X Ateuchus sp. T X — Coprini Canthidium aurifex (Bates, 1887) T — X Canthidium cupreum (Blanchard, 1843) T — X Canthidium funebre (Balthasar, 1939) T X — Canthidium onitoides (Perty, 1830) T — X Canthidium ruficolle (Germar, 1824) T X X Canthidium sp. A T X — Canthidium sp. B T X — Canthidium sp. C T X — Dichotomius compressicollis (Luederwaldt, 1929) T — X Dichotomius problematicus (Luederwaldt, 1922) T X — Ontherus pubens (Genier, 1996) T — X Coptodactylini Uroxys bidentis (Howden and Young, 1981) T — X Uroxys sp. A T X — Uroxys sp. B T X — Deltochilini Canthon aequinoctialis (Harold, 1868) R — X Canthon angustatus (Harold, 1867) R X X Canthon femoralis (Chevrolat, 1834) R X — Canthon fulgidus (Redtenbacher, 1867) R — X Canthon juvencus (Harold, 1868) R — X Canthon luteicollis (Erichson, 1847) R X X Canthon sp. R X — Deltochilum amazonicum (Bates, 1887) R X — Oniticellini Eurysternus hamaticollis (Balthasar, 1939) D — X Eurysternus velutinus (Bates, 1887) D — X Onthophagini Onthophagus buculus (Mannerheim, 1829) T — X Onthophagus haematopus (Harold, 1875) T X X Phanaeini Oxysternon conspicillatum (Weber, 1801) T X — Phanaeus cambeforti (Arnaud, 1982) T — X Phanaeus chalcomelas (Perty, 1830) T X X L. lagotricha A. seniculus Figure 2; Richness of the three relocating guilds (R: rollers, T: tunnelers, D: dwellers) in the two dung types (I. lagotricha and A. seniculus) in the area, CIEM-Meta, Colombia. the diet of these species is more similar in the rain season, but at dry time it is very different (P. Stevenson pers. comm.). The rolling species can use the dung that remains on leaves, while tunnelers and dwellers do not, but even so the dominant group on both excrement types is the tunnelers. It is interesting to note that many of the species recorded on the two excrements were frequently found perching in the study area (Noriega, unpublished data). The amount of dung that remains on leaves is very small compared to what reaches the ground, but some of the species that utilize the dung that remains on leaves did not fall into the traps were placed at ground level [43]. A study that includes other possible resources, such as the dung of others primates species in the area and that of other mammals, would create a more complete panorama of Psyche 5 resource overlap in the beetle assemblage, clarifying which species have generalist habits and which really are specialists, allowing to approach the quantification of the interspecific competition for this locality. Acknowledgments The author would like to thank Carlos Arturo Mejia, Rami- ro Montealegre, Juan Cristobal Calle, and Alexandra Vega for their help during the fieldwork; Emilio Realpe for the support during the laboratory work; Fernando Vaz-de-Mello for taxonomic support; Carolina Rudas and Pablo Steven- son for valuable information; Castor Guisande and Daniel Monroy for statistic collaboration; Pablo Stevenson, K. Isawa, Julio Louzada, Ricardo Botero, Carolina Vizcaino, and Nora Martinez for helpful comments, and suggestions which improved the paper; To David Morris for kindly checking the English version; two anonymous reviewers for the critical reading of this paper. References [ 1 ] B. D. Gill, “Dung beetles in tropical american forests,” in Dung Beetle Ecology, I. Hanski and Y. Camberfort, Eds., chapter 12, pp. 211-230, Princeton University Press, Princeton, NJ, USA, 1991. [2] E. Nichols, S. Spector, J. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 840860, 12 pages dohlO.l 155/2012/840860 Research Article Chemical Integration of Myrmecophilous Guests in Apbaenogaster Ant Nests Alain Lenoir,' Quentin Chalon,' Ana Carvajal,^ Camille Ruel,^ Angel Barroso,^ Tomas Lackner,^ and Raphael Boulay^’^ ^ Institut de Recherche sur la Biologic de VInsecte, IRBI, UMR CNRS 7261, Universite Francois Rabelais, 37200 Tours, France ^Estacion Bioldgica de Dohana, CSIC, 41092 Seville, Spain ^ Department of Forest Protection and Game Management, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Praha, Czech Republic Departamento de Biologia Animal, Universidad de Granada, 18071 Granada, Spain Correspondence should be addressed to Alain Lenoir, alain.lenoir@univ-tours.fr Received 13 October 2011; Accepted 7 December 2011 Academic Editor: Jean Paul Lachaud Copyright © 2012 Alain Lenoir et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Social insect nests provide a safe and favourable shelter to many guests and parasites. In Apbaenogaster senilis nests many guests are tolerated. Among them we studied the chemical integration of two myrmecophile beetles, Sternocoelis hispanus (Coleoptera: Histeridae) and Chitosa nigrita (Coleoptera: Staphylinidae), and a silverfish. Silverfishes bear low quantities of the host hydrocarbons (chemical insignificance), acquired probably passively, and they do not match the colony odour. Both beetle species use chemical mimicry to be accepted; they have the same specific cuticular hydrocarbon profile as their host. They also match the ant colony odour, but they keep some specificity and can be recognised by the ants as a different element. Sternocoelis are always adopted in other conspecific colonies of A. senilis with different delays. They are adopted in the twin species A. iberica but never in A. simonellii or A. subterranea. They are readopted easily into their mother colony after an isolation of different durations until one month. After isolation they keep their hydrocarbons quantity, showing that they are able to synthesize them. Nevertheless, their profile diverges from the host colony, indicating that they adjust it in contact with the hosts. This had never been demonstrated before in myrmecophile beetles. We suggest that the chemical mimicry of Sternocoelis is the result of a coevolution with A. senilis with a possible cleaning symbiosis. 1. Introduction Ant colonies often host microcosms of myrmecophile guests, mostly arthropods that take advantage of ant nest favourable environment and food resources [1-3]. The largest known association is the army ant Eciton burchellii with more than 300 guest species [4]. Interactions with ants range from true predators, commensals that live on ant food remains, mutualists, and parasites [3, 5, 6]. In order to get accepted they must break the ant colony “fortress” which is based on a chemical recognition system by which ant workers are able to recognize and exclude aliens. More precisely, a colony- specific mixture of cuticular hydrocarbons has been shown to constitute the recognition pheromone of most ant species [7, 8] . Several strategies have been described for the chemical integration of myrmecophiles into ant colonies. Chemical mimicry is achieved either in a few cases by biosynthesising the same hydrocarbons as their host (but this is rare) or more generally by acquiring them through cuticular contacts and/or grooming (e.g., the guest actively licks the host’s cuticle; see reviews by [9-11]). Myrmecophiles like woodlice, mites, phorid flies, and snails can also be “chemically insignificant,” that is, their cuticle bears very small amounts of hydrocarbons as it has been shown in Leptogenys [6]. Similarly, callow ants are chemically insignificant which allows them to get accepted in alien colonies during the first hours after emergence (see [12]). Another possibility of integration has been discovered recently in social insects: guests and parasites can be chemically “transparent” if they have only saturated hydrocarbons, which are not involved 2 Psyche in recognition [13]. Nevertheless, some myrmecophiles like Pella in Lasius fuliginosus colonies do not present chemical mimicry, simply escaping from the ants or using appease- ment or repelling behaviour [14]. In the present study we conducted a survey of all arthro- pods living in the nest of the gipsy ant Aphaenogaster senilis in southern Spain. Then, we compared the chemical integration of two myrmecophiles beetles {Sternocoelis hispanus and Chitosa nigrita) with that of an undetermined silverfish. We hypothesized that guests specialized with only one host (like Sternocoelis) have coevolved with it and biosynthesize the hydrocarbons while host-generalists like silverfish would mimic passively their hosts and can shift easily to different host species. To test for host specificity and relate it to chemical distance, we designed adoption experiments with Sternocoelis in conspecific colonies and congeneric species. We then analysed the mechanisms of chemical mimicry looking at the effects of separation of the beetles from their host. After two weeks, the exogenous hydrocarbons of the myrmecophile beetle Myrmecaphodius begin to disappear [15]. Therefore, after one-month isolation, we supposed that all exogenous hydrocarbons acquired by contact with ants had disappeared. As Sternocoelis was frequently observed licking the ant larvae, we investigated possible roles of these beetles in larval predation or prophylaxis. If the beetles fed on larvae by piercing the cuticle (haemolymph feeding on larvae by ant workers is known in Amhlyopone [16]), larvae were supposed to decline. On the contrary, if the beetles fed only by licking the cuticle, larvae will maintain their wellbeing. 2. Material and Methods 2.1. Inventory of Guests in A. senilis Colonies. We completely excavated 57 nests between February 2008 and December 2009 on the banks of Guadalquivir near Sanliicar de Barrameda to list and count all the guests, mites, silverfish, sowbugs, staphylinids, and histerid beetles. 2.2. The Guest Studied 2.2.1. Sternocoelis (Coleoptera: Histeridae). This genus regroups myrmecophile beetles that live in ant nests of several species within the genera Aphaenogaster, Cataglyphis, Formica and Messor [17]. They are frequently found on the brood pile on which they were thought to feed (Figure 1(h)). According to Lewis [18] Sternocoelis feed on larvae and dead adult ants. Otherwise, little is known on their biology and reproduction. Larvae and pupae are unknown [17]. Sternocoelis hispanus (Figures 1(a) and 1(b)) occurs in central and southern Portugal and Spain, as well as in northern and central Morocco (see [19] for details). In the Iberian Peninsula it has been found living only in A. senilis colonies [17, 19]. On the other hand, in Morocco it was found in at least four different species of Aphaenogaster with more than 30 specimens in some nests (Lackner, unpublished). After exposing the colony by turning the stone under which they live, some S. hispanus immediately headed for the security of the nest searching for the nearest gallery, whereas the other attempted to “hitch a ride” by clinging onto the ants (Figures 1(c) and 1(e)). The histerids, rather than the ants (as is the case in Chennium bituberculatum observed in eastern Slovakia; Lackner, unpublished), always actively seek out the ants in order to be transported into the nest. This phenomenon of Sternocoelis riding the ants has so far been observed only in four Sternocoelis species: S. hispanus, S. slaoui, S. arachnoids, and S. espadaler (Lackner, unpublished). As very few is known on S. hispanus biology, we measured the length, width, and weight of the Sternocoelis to search for sex differences. 2.2.2. Chitosa (Coleoptera: Staphylinidae) and Silverfish (Thysanura). They are very active insects moving rapidly into the nest. Very little is known on their biology. Since they are associated with various ant species, they are apparently host generalists. Chitosa nigrita is a rare myrmecophilous species known only from Spain and Morocco [20] (Figure 2). We collected C. nigrita in two colonies of A. senilis (1 and 4). From colony 4 we also collected two silverfish. Silverfish are known to move freely within the entire nest [6] . Chitosa and silverfish were only used for chemical analyses. 2.3. Ant Colonies. In November 2008, 43 S. hispanus beetles were discovered in an A. senilis colony (hereafter, colony 1) in Andalusia, Donana National Park (Las Beles, 36°58.53'N, 6°29.lLW, sea level). Three other colonies were collected, colony 2 and 4, just a few meters from colony 1, and a fourth one (colony 3) collected 60 km apart, near Aznalcazar in a pine forest (37°14.77'N, 6°12.17'W, 36 m). For adoption experiments we used four colonies of di&erent Aphaenogaster species: one colony of A. simonellii (Egine’s island, Greece; 37°45.22'N, 23° 31. 46' E, 580 m), one colony of A. subterranea (Gevennes, France; 44°02.57'N, 3°49.68'E, 370 m) and two colonies of A. iberica (Sierra Nevada, Spain; 37°08.42'N, 3°28.34'E, 1370 m). Like A. senilis and A. simonellii, A. iberica belongs to the subgenus Aphaenogaster, while A. subterranea belongs to a different subgenus (Attomyrma), suggesting it is phylogenetically more distant from A. senilis than the other two. Golonies were maintained in the laboratory in large plastic boxes and fed at libitum with live maggots, pieces of orange, sliced Tenebrio larvae, and a commercial solution for bumblebees (Beehappy). 2.4. Behaviour of Sternocoelis. We performed a behavioural repertoire of the beetles using scan sampling method: during 3 days, we recorded during 50 sequences the behaviour of all beetles that were visible in colony 1 (total number of observations 741). Behaviours were the followings: isolated in the colony (either immobile or moving), on larvae, on prey, on a worker, licked by a worker (see Eigure 1). As the Sternocoelis were observed frequently on the ant larvae, we made small nests with 6 A. senilis nurse worker ants, 6 beetles, and 6 larvae of different developmental stages. The behaviour of the beetles and the number and aspect of larvae were observed during 30 days. 2.5. Adoption Experiments. We observed the behaviour of the Sternocoelis beetles and examined whether the beetles can be Psyche 3 Figure 1; Sternocoelis beetles (Coleoptera: Histeridae). (a, b): Sternocoelis hispanus morphology, sex unknown (photo (b) by Martin Svarc and Peter Koniar); (c): two Sternocoelis slaoui riding on an Aphaenogaster worker (Photo Martin Svarc and Peter Koniar, Larache, Morocco, February 2010); (d): Sternocoelis hispanus beetles feeding on mealworm larvae; (e): S. hispanus jumping on an ant worker; (f, g): aggressive behaviour against allocolonial S. hispanus in the foraging arena-transport by an Aphaenogaster worker (f): aggression; (g): S. hispanus cleaning ant larvae. All photos unless (b) and (c) by Alain Lenoir. Figure 2: Chitosa nigrita (Coleoptera: Staphylinidae) (photo by Alain Lenoir). Determination by Munetoshi Maruyama. 4 Psyche adopted by another A. senilis colony or a colony of another species. Adoption tests were conducted on small colony fragments {n = 6 for A. senilis) containing 120 workers and a brood kept in small flat plaster nests covered with a glass. Observations were realized through a red plastic sheet. The nest communicated with a foraging arena, made of a plastic box where the beetles were introduced. These experimental colonies have been acclimated in the experimental setup for at least 24 h before the adoption experiments were conducted. Consecutive experiments were separated by at least one week. One or two beetles were introduced in the foraging arena of each Aphaenogaster experimental colony. We then measured the following variables: (i) Latency to the first Contact with the beetle (LC); (ii) Total time of Contact between the beetle and ants in the External area (TCE); (iii) total time of TRansport of the beetle by ants into the nest (TR); (iv) total time of Contact between the beetle and ants In the Nest (CIN); (v) the sum of these four durations, the Total Time until Adoption (TTA). In some cases the beetle was again aggressed inside the nest and we added this duration to the first TTA. If the beetle was left in the foraging arena during one week and always neglected, it was considered not adopted and returned to its original nest. Sternocoelis beetles were introduced either into a fragment of their own colony (con- trols, colony 1; n = 10 beetles), a different colony of A. senilis (colony 2 and 3; u = 10 beetles per colony), a colony of A. simonellii {n = A beetles), a colony of A. iherica {n = 12 beetles), or a colony of A. suhterranea (n = 4 beetles). In order to evaluate the chemical integration of Ster- nocoelis into colonies of A. iherica, we made 5 more trials: 3 were adopted and used for chemical analysis, 2 disappeared, probably killed by foragers. 2. 6 . Isolation Experiments. To observe the effect of separation from the Aphaenogaster hosts, groups of 5 Sternocoelis were isolated in a small glass tube with water and food. Individual isolation was not possible as the beetles died rapidly. They were reintroduced into their original nest after 1, 3-4, 5-6, 7-8, and 30 days of isolation {n = 4 for each and n = 6 for 30 days) and we measured the readoption time. These data were compared to controls retrieved directly into the host nest {n = 8). We performed chemical analysis of the hydrocarbons on the 8 controls, 5 beetles isolated for 4 and 8 days, and 6 beetles isolated 30 days (see Section 2.7). 2.7. Chemical Analyses. In a first step we used the whole ants and the whole myrmecophiles. The animals were frozen at - 18°C and immersed in 200 pL of pentane during one hour, and the extract stored at -18°C until analysis. Substances were identified by combined gas chromatography/mass spec- trometry (Turbomass system, Perkin-Elmer, Norwalk, CT, USA, operating at 70 eV) using a nonpolar DB-5HT apolar fused silica capillary column (length: 25 m; ID: 0.25 mm; film thickness: 0.25 /^m). Samples injections were performed in splitless mode for 1 minute, a temperature program from 100° C (2 min initial hold) to 320° C at 6°C min ^ with 5 min of final hold. A mixture of 10 linear hydrocarbon standards (from C20 to C40) was injected at regular time intervals in order to recalibrate retention times. To analyze the effects of social isolation, as we had only a few beetles, we used SPME: the live beetle was held in forceps and rubbed gently on the dorsal and lateral surfaces with a polydimethylsiloxane (PDMS, 7 p fused silica/SS, Supelco, color code green) fiber for 3 minutes. The fiber was immediately desorbed in the GC-MS in the same conditions of pentane extracts. It has been shown that the profiles obtained with SPME and classical solvent extraction are qualitatively identical [21, 22], but a precise quantitative analysis showed that the propor- tions of compounds are slightly different [23], so we made also SPME controls for ants. SPME were made on 4, 8, and 30 days of isolation. An internal standard (eicosane) was added to the extract or deposited on the fiber to measure the hydrocarbon quantities. Hydrocarbons of A. senilis were previously identified [24, 25] and we added some new compounds present in very small quantities. 2.8. Statistics. AN OVA was performed on behavioural data for adoption experiments, Kruskall- Wallis on hydrocarbon quantities of isolated beetles. Statistical analysis of the chemical profiles was done using all peaks that were identified. To determine the level of similarity of the CHC profile of the beetles and their hosts, isolated beetles and between species we used hierarchical cluster analysis (Euclidean distances. Ward’s method) to construct a single-linkage dendrogram (see, e.g., [26, 27]). The Nei index of similarity was used to compare the chemical profiles of the species when large qualitative differences are observed (see, e.g., [27-29]). 3. Results 3.1. Inventory of Guests in Aphaenogaster Nests. Eigure 3 shows the frequency of nests in relation to the number of guests. Many nests did not contain any guest; for example, 51% of nest were free of mites, 98% of histerids beetles. Out of the 57 nests excavated, 23 contained at least one individual of the staphylinid Chitosa nigrita (mean ± SE: 1.1 ±0.3; range: 0-9). Silverfish were present in 14 colonies (mean ± SE: 0.9 ± 0.3; range: 0-14) while sowbugs were found in 15 colonies (mean ± SE: 0.8 ± 0.3; range: 0-13). In Dohana National Park Sternocoelis beetles were rare. With the exception of two colonies that contained 43 and 30 individuals (resp., in November 2008 and July 2011), only five Sternocoelis beetles were found in four different colonies among more than 300 colonies. The mean length of Sternocoelis was 2.01 mm (SE = 0.01; min 1.79, max 2.24, n = 42) and the width 1.29 mm (SE = 0.01, min 1.06, max 1.39, n = 42). The weigh was 2.25 mg (SE = 0.18, min 1.5, Psyche 5 Number of guests □ Mites CH Staphylinid beetles □ Silverfishes □ Histerid beetles CH Sowbugs Figure 3: Frequency of colonies containing 0, 1 to 10, 1 1-20, 21-30, and > 40 guests in A. senilis colonies. max 2.80, n = 8). The distributions were unimodal, and therefore no sexual dimorphism appeared. 3.2. Behaviour and Longevity. Beetles stayed isolated or moved freely inside the host nest (28% of observations; it is a raw indication of the time budget of the beetles). From time to time they clutched to a worker’s leg, jump on it, and stayed there (34.55% of observations) (Beetle on the ant body: Figures 1(c) and 1(e)). As observed by Yelamos [17], they were frequently found near or on the larvae (31.6%) (Figure 1(h)) and fed directly on pieces of Tenehrio (4.3%) (Figure 1(d)). They were occasionally licked by a worker (1.35%). It is possible that in the nests there is competition for food. Beetles had a rather long life, since one year later 18 of them were still alive. We never observed any sexual behaviour nor found any Sternocoelis larvae, so we do not know how these beetles reproduce. In the small nest experiments with larvae we always observed at least 3 beetles on the ant larvae while the others were moving around searching for food. On the larvae they were either immobile or licking the cuticle. After one month, the observations were stopped because we did not observe any larval mortality and the larvae appeared to maintain normally. We never observed any brown spot, which would indicate a piercing of the cuticle. We did not quantify the behaviour of the other guests, but observations indicated that Chitosa beetles and silverfish had a very different behaviour compared to Sternocoelis: they had very few interactions with the host, moving frequently in the nest. Silverfish were very fragile and died in less than 24 hours in the laboratory nests. 3.3. Adoption Tests in Alien Colonies Behaviour (i) when deposited into the foraging arena of the alien colony, the beetle spent some time without contact with ants either because they did not meet it or because they did not perceive their presence. They also simply stopped and inspected the ants, and continued thier way; (ii) at their first contact with the beetle, ants behaved aggressively. They seized them in their mandibles, maintained them on the ground and inspected them with their antennae. They made short attacks with their mandibles (Figure 1(g)). The beetles are difficult to seize with the mandibles in account of their hard, smooth and rounded surface [17, 18] (Figure 1(f)). Some ants stopped after this initial inspection and continued their way; (hi) thence, the ants grasped the beetles by their legs. They were transported either inside the nest, or, on the contrary, farther from the nest. Sometimes the beetle held a prey and it was therefore more difficult to seize. It could also cling to the legs and thus be transported passively. Alternatively, it could cling to the antennae of the ant, which would try to shake it off; (iv) when the beetle reached the nest, it was maintained by ants and received a mixture of aggression and grooming. Sometimes it was transported again into the foraging arena, which indicates a rejection, at least provisory. The adoption was considered success- ful when the beetle was neglected and moved freely. Once adopted inside the nest, it searched rapidly for the chambers with larvae. In A. senilis alien colonies adoption was almost systemat- ically a success except for colony 2 which rejected one beetle (=5% total rejection). All but 2 beetles introduced in A. iber- ica were adopted, 2 were rejected and died {n = 17, = 11%). The duration of the adoption phases is given on Table 1. The latency without contact (LC) and duration of contacts (TCE) in the foraging arena, the transport times (TR) are longer in colony 2. The total adoption times (TTA) is 20 minutes in controls, more than one hour in colony 3 and very long in colony 2 where it attains 38 hours as the beetle is seized and aggressed many times. In A. iberica the duration of contacts in the nest is longer, the total adoption time is also longer (3 hours) but the difference is not significant (Table 1). When the adopted beetles were reintroduced into their A. senilis mother colony, they were aggressed but readopted rapidly. In A. simonellii {n = 4) and A. subterranea {n = 4), the beetles were maintained always in the foraging arena and no adoptions occurred. 3.4. Adoption of Isolated Beetles. When reintroduced into their mother colony, isolated beetles were rapidly readopted in half an hour versus 22 minutes in controls. There is a small tendency to increase the adoption time with the isolation duration but none of the measures were significant (ANOVA, n = 30, Lambda de Wilk = 0.242, ^( 25 , 86 . 943 ) = 1.615, P = 0.054 — details not shown). At 30 days, 2 about 10 (i.e., 20%) died during the isolation. 3.5. Chemical Profile of the Beetles and Hosts. Sternocoelis, Chitosa and silverfish had the same hydrocarbons as their host Aphaenogaster senilis (Figured and Table 2). 6 Psyche Table 1: Duration of different behavioural phases of adoption of Sternocoelis beetles (mean ± SE). LC: latency of the first contact with ants in the external area; TCE: time contacts in the external area; TR: time of transport of the beetle into the nest; CIN: time of contact with ants in the nest; TTA: total time of the adoption. A. iber: A. iberica. LC Mean SE TCE Mean SE TR Mean SE CIN Mean SE TTA Mean SE Col 1 (control) 6.5 2.0 2.0 0.8 2.2 0.3 12.3 3.4 22.9 3.1 Col 2 73.3 42.5 154.9 33 44.7 16.3 73.4 17.3 2491.1 479.1 Col 3 20.6 8 23.7 14.6 7 3.7 19.6 3.5 70.8 22.6 A. iber 6 2 12.7 2.6 2.2 0.5 158.2 21.3 179.3 20.5 ANOVA, Wilk = 0.006, F = 28.45, df = 15, P < 0.00001; in bold significant differences for each column with post hoc Neumann-Keuls (P < 0.001, all other withP > 0.15). Eigure 4; Chromatograms of Aphaenogaster senilis and Sternocoelis hispanus. Numbers refer to hydrocarbons in Table 2. P: phthalate and Sq: squalene pollutants. Aphaenogaster iberica and A. simonellii also had the same hydrocarbons as A. senilis (Nei indexes were close: A. senilis! A. iberica = 0.75; A. senilis! A. simonellii = 0.65; A. iberica! A. simonellii = 0.88). A. subterranea had a very different profile with very small quantities of hydrocarbons (using total peak areas) and 20% of unsaturated alkanes which were absent in all other species. Surprisingly, it has also a lot of heavy hydrocarbons (25.8% had more than 32 carbons) that were not found in other species. This species is mostly subterranean and lacks saturated hydrocarbons protecting against desiccation. The Nei index between A. subterranea and the other Aphaenogaster species is very low (0.211), indicating a high chemical disparity. Therefore, this ant species has not been included in the following analyses. In the first analysis we constructed a dendrogram of chemical distances between the guests and their Apha- enogaster host. It appeared clearly that the four A, senilis colonies had different profiles (Figure 5), confirming pre- vious analyses [30]. All the beetles, both Sternocoelis and Chitosa, were grouped with their host colony, indicating a chemical mimicry fitting the colonial signature. Neverthe- less, beetles aggregated distinctly from their host. The chem- ical distance between colonies did not depend on their geo- graphical distance, and was not linked to the beetle adoption time. Colonies 2 and 3 were equally chemically distant to colony 1 but accepted the beetles more or less rapidly. Apha- enogaster iberica and A. simonellii were close to A. senilis colonies 2 and 3 (data not shown) but the first species accepted the beetles whereas the second did not (but only 4 adoption trials). On the contrary, the silverfish did not match the host colony. Interestingly, Sternocoelis adopted in A, iberica were close to their new host but did not match completely to the new colony. A. subterranea is very different and as expected never adopted the beetles. Psyche 7 Table 2: Hydrocarbon quantities (mean ± SE) in Aphaenogaster simonellii, A. subterranea, A. senilis, A. iberica, Sternocoelis hispanus, Chitosa nigrita, and a silverfish. Blanks indicate the absence of the substance or that it is present only as not quantifiable traces. Peakno. Name Ap/j simonelli Mean SE Aph subterranea Mean SE Aph senilis Mean SE Aph iberica Mean SE Sternocoelis Mean SE Chitosa Mean SE Silverfish Mean SE 1 C25:l 0.19 0.07 0.33 0.08 0.07 0.04 0.77 0.32 1.10 0.52 1.08 0.20 2 C25 1.43 0.20 19.86 3.47 0.29 0.04 1.38 0.31 0.81 0.13 0.67 0.19 4.26 0.37 3 11+13C25 (+7C25) 1.38 0.34 1.04 0.40 1.08 0.17 6.51 1.26 4.70 1.21 0.57 0.16 0.98 0.02 4 5C25 0.33 0.06 0.15 0.15 0.18 0.06 1.41 0.84 0.46 0.08 0.20 0.07 0.83 0.05 5 9,15C25 0.04 0.02 1.21 0.58 1.39 0.52 6 3C25 0.92 0.11 2.26 0.40 1.01 0.18 1.30 0.21 5.05 1.63 1.22 0.38 3.72 0.25 7 5,9C25 0.14 0.02 0.82 0.23 0.49 0.07 0.17 0.07 8 C26 4.31 0.91 2.45 0.49 0.16 0.04 5.04 0.62 0.24 0.07 0.10 0.03 0.75 0.01 9 4,6C25 0.12 0.05 0.85 0.33 0.08 0.02 10 10+12C26 10.67 2.03 0.49 0.13 2.21 0.20 6.48 0.71 2.39 0.27 1.34 0.34 4.91 0.78 11 6+8C26 1.93 0.35 1.76 0.19 0.41 0.12 1.65 0.21 1.06 0.11 0.45 0.17 12 4C26 0.41 0.07 0.29 0.19 1.06 0.18 0.59 0.11 0.36 0.05 0.31 0.07 2.82 0.22 13 10,14C26 2.14 0.28 0.11 0.11 4.49 1.19 1.91 0.30 2.44 0.35 3.23 0.98 5.36 0.10 14 8,12C26 2.07 0.57 0.42 0.09 4.17 0.52 3.23 0.72 15 C27:l 1.46 0.45 16 6,10C26 0.65 0.14 0.05 0.05 1.03 0.19 0.21 0.07 17 4,8C26 0.22 0.12 1.11 0.19 0.94 0.31 18 C27 15.59 3.19 11.27 2.19 3.82 0.37 13.05 1.38 2.83 0.16 0.76 0.04 6.47 0.19 19 4,8,12C26 5.77 0.79 0.20 0.09 2.97 0.30 6.98 0.60 20 9+11+13C27 8.36 1.78 1.43 0.21 18.84 2.08 17.04 1.08 11.01 0.49 9.00 1.84 5.84 0.14 21 7C27 1.15 0.46 0.07 0.07 3.65 0.49 1.03 0.19 3.64 1.02 4.64 1.37 2.12 0.06 22 5C27 2.29 0.39 0.18 0.08 1.50 0.34 0.87 0.22 1.48 0.39 1.83 0.58 1.10 0.05 23 9,13C27 0.43 0.24 3.12 0.20 2.43 0.80 24 3C27 24.55 2.82 1.81 0.54 15.64 1.50 17.53 2.05 7.21 1.15 10.02 0.90 38.77 0.51 25 5,9C27 1.07 0.16 1.96 0.41 0.87 0.22 26 C28 6.17 1.37 2.48 0.21 1.00 0.32 0.67 0.24 0.11 0.04 1.07 0.03 27 3,7+3,9+3,11027 6.09 0.97 1.32 0.47 7.30 0.65 2.47 0.83 5.35 0.28 3.53 0.04 28 10+12028 5.70 1.56 1.29 0.74 7.32 0.70 3.40 0.22 10.32 1.57 8.56 0.56 3.33 0.09 29 6028 1.21 0.29 0.61 0.05 1.02 0.28 0.75 0.06 0.57 0.08 30 4028+10,14028 1.08 0.12 0.44 0.21 6.11 0.96 2.32 0.31 8.41 0.50 10.31 0.65 4.79 0.15 31 6,10028 0.64 0.17 2.01 0.16 0.52 0.24 2.67 0.47 3.40 0.18 1.16 0.07 32 029:1 1.06 0.40 33 4,8+4,10028 0.11 0.08 1.89 0.15 0.27 0.06 2.76 0.26 6.01 0.68 0.82 0.05 34 029 1.14 0.21 9.43 1.09 2.39 0.35 0.48 0.08 3.22 0.51 1.27 0.06 1.78 0.05 35 TM 028 1.38 0.08 1.33 0.04 3.32 0.36 36 11029 0.79 0.23 2.86 0.65 3.90 0.49 4.12 0.53 2.24 0.30 1.95 0.14 0.49 0.03 37 7029 0.19 0.05 2.04 0.49 1.65 0.16 0.45 0.12 0.62 0.20 1.00 0.15 38 5029 0.86 0.12 1.79 0.34 0.32 0.08 0.48 0.24 1.55 0.35 2.03 0.01 39 11,15029 0.14 0.06 0.52 0.20 0.26 0.06 40 7,x029 0.18 0.10 0.76 0.13 1.80 0.64 41 3029 2.36 0.25 42 5,9029 0.26 0.02 0.00 0.00 0.17 0.02 0.72 0.24 0.16 0.06 0.42 0.08 43 030 0.13 0.05 1.14 0.13 0.08 0.03 0.00 0.00 0.24 0.09 1.07 0.47 44 10+12030 0.11 0.02 1.14 0.42 0.17 0.07 0.43 0.24 45 10,14+10,16+12,14030 0.17 0.04 0.86 0.16 0.17 0.08 0.25 0.08 46 031:1 3.32 0.58 47 4,8+4,10+4,12030 0.01 0.01 0.03 0.02 0.01 0.00 48 031 1.15 0.06 49 ? 0.00 0.00 0.05 0.04 0.01 0.01 50 11+13031 2.54 0.46 0.20 0.07 0.19 0.09 0.10 0.09 8 Psyche Table 2: Continued. Peakno. Name Aph simonelli Aph suhterranea Aph senilis Aph iherica Sternocoelis Chitosa Silverfish Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE 51 ll,15±13,xC31 3.23 0.63 0.58 0.20 0.31 0.07 0.08 0.05 52 C32 53 10C32 2.19 0.44 0.26 0.06 54 11C33 l.Yl 0.54 55 11,15C33 3.23 0.75 56 12C34 3.16 0.72 57 11±12C35:1 9.08 2.87 58 C35:l 6.01 1.68 Total 100 100 100 100 100 100 100 n = 5 5 16 11 4 9 2 11+13C25: ll-MethylC25 + 13-MethylC25; 9,15C25: 9,15-DiMethylC25; TMC28: TriMethylC28. In the second analysis, we constructed the dendrogram of isolated Sternocoelis beetles (Figure 6). It revealed that they did not match completely the colony odour in a few days compared to controls maintained in their host colony. Some beetles after 4 or 8 days had always their colony profile — in a red ellipse on Figure 6 — indicating a progressive change. Nevertheless, these changes were not sufficient to induce the rejection of the beetle. We also measured the quantities of hydrocarbons on the cuticles. In the pentane rinses, the hydrocarbon quantities of Aphaenogaster senilis workers were 1099 ng/worker (±860, n = 5), 446 ng (±542, n = 5) for Sternocoelis, and 1567 ng (±1270, n = 5) for Chitosa. These beetles were not chemically insignificant. On the contrary, silverfish had only 34 ng (30.1 and 37.7; n = 2) indicating that these insects are insignificant and not protected against desiccation and explains why they die very rapidly after collection. For isolated Sternocoelis we retrieved by SPME only a very small quantity of hydrocarbons (1 to 5 ng/beetle, see medians in Figure 7), but the profile was comparable to liquid extracts. There were no differences between 4, 8, and 30 days isolated beetles compared to the con- trols (Kruskal-Wallis Chi-square = 3.91, df = 3, P = 0.27) . It shows that the beetles maintained their hydrocarbons quan- tities independently of their host. 4. Discussion The three guest species mimic chemically their host: they have the same hydrocarbons (chemical mimicry sensu lato). This explains why they are tolerated inside the nest without being aggressed the ants and they have the host colony odour. This was predictable for Sternocoelis, which lives intimately with brood in the colony, but it was more surprising for Chitosa which has very few interactions with the host workers. Nevertheless, both species maintain some chemical specificity into the host colony (Figure 5), they are probably recognised as a different though tolerated element. This can be compared to social parasites that also keep their own identity into the host colony [ 1 1, 3 1 ] . It indicates that chemi- cal mimicry is not sensu stricto, it means that ant workers have a double template: they must know and recognize both their nestmates and their guests. The queen also has a slightly different chemical profile and is recognized by workers (see reviews [7, 21, 27]). Recently, Vantaux et al. [27] described chemical mimicry between predatory larvae of a Diomus coccinellid and the little fire ant Wasmannia auropunctata. The myrmecophile larvae of Diomus also segregate separately in the clusters made on hydrocarbons, indicating that it may be general [27]. On the contrary, silverfish while matching the host hydrocarbons also, do not have an A. senilis colony- specific odour. They escape host aggression by avoiding contacts. They probably get their hydrocarbons directly by transfer from the host ant as it has been demonstrated in Malayatelura silverfish and their host Leptogenys using radioisotopes [32]. Adoptions of Sternocoelis are possible in all colonies of A. senilis with different delays. Aphaenogaster colonies are not completely closed [30] , favouring the adoption of beetles that bear the same hydrocarbons in different proportions. The differences in adoption times can probably be explained by the fact that A. senilis colonies are very different in aggression levels [30]. Differences between A. iherica (acceptance) and A. simonellii (rejection) are difficult to explain. They may be due at least partly to the chemical distances with A. senilis being more important to A. simonellii (0.65) compared to A. iherica (0.75). We can also hypothesize that A. iherica are less aggressive, they do not have alkaloids in the venom gland, which may repel the beetles [33]. It may also simply not representative as we had only one small colony of A. simonellii and 4 adoption trials. We discovered that Sternocoelis beetles kept their hydro- carbon quantities even after one-month isolation. Therefore, chemical mimicry by biosynthesis, rather than camouflage, may explain the host tolerance. If the hydrocarbons were transferred from the host passively, they should have disap- peared in a few days because of the rapid turnover of these substances on the cuticle. For example, Myrmecaphodius isolated from their Solenopsis host colony lose their hydro- carbon profile in two weeks [15] and silverfish Malayatelura Psyche 9 Figure 5: Dendrogram of chemical distances (Ward method, Euclidian distances). As: A. senilis colonies (colony 1 Asll to Asl5; col. 2 As21 to 24; col. 3 As31 to 35; col. 4 As 41 to 43). Ch: Chitosa nigrita (colony 1 Chi to 4; col. 4 Ch41 to 44). St: Sternocoelis hispanus (col 1 Stl to St4). Silverfish form col. 4 (mean of 2 individuals). Ward method Euclidian distances Figure 6: Dendrogram of the isolated Sternocoelis beetles (Ward method, Euclidian distances). Controls: Cl (n = 8); 14 (n = 5), 18 (n = 5), 130 (n = 6) Isolated during 4, 8, and 30 days. In the red ellipse, isolated individuals having kept completely their colony odour. 10 Psyche HCs quantities (ng) C Is 4d Is 8d Is 30d □ Median I I 25%-75% Min-max Figure 7: Hydrocarbon quantities (ng/individual; median, quar- tiles, Min-Max) retrieved by SPME on Sternocoelis beetles. C = Controls (beetles in the host nest, n = 8), I4(n = 5), I8(n = 7), and 130 (n = 17): beetles isolated from the hosts during 4, 8, and 30 days. after six or nine days separation from their host Leptogenys showed reduced chemical host resemblance and received more aggression [32], This suggests that Sternocoelis beetles are able to biosynthesize the host hydrocarbons and adjust their profile to the host colony by contacts. It explains why they change a little their profile after isolation, but are always accepted. This is an indication of a coevolution with the host, with a species-specificity of the association. We could not determine whether the chemical mimicry of the Chitosa staphylinid is an active or passive camouflage, but it is probably the latter as the beetle has very few direct interac- tions with the host and these beetles are not species-specific (Maruyama, pers. comm.). The silverfish have very few hydrocarbon quantities and are chemically insignificant, as observed in a species living in Aenictus colonies (but no details are given in the paper, [34]), Nevertheless they also have the host odour, probably acquired simply by contact with the nest material (see above V. Witte pers. comm.), but it is not colonyspecific. It is interesting to note that the inside nest material odour is not colony specific as shown in Lasius niger [23] , This may explain why silverfish are killed in Leptogenys experimental colonies [6]. Chemical mimicry has been studied only in a few beetles in social insects and all the situations are possible. Biosynthesis has been demonstrated using radio-labelling 14C-acetate. It was shown to occur in two species of thermi- tophile Staphylinidae with their host Reticulitermes [35, 36]. Hydrocarbons are also biosynthesized by the larvae of the fly Microdon that are transported in the ant nest [37, 38]. The larvae of the butterfly Maculinea rebeli use a double mecha- nism: they first synthesize the hydrocarbons of the ant brood and later acquire additional hydrocarbons from the ants enhancing the mimicry [39]. Concerning the association of larvae of Diomus coccinellid and the little fire ant the authors suggested mimicry by biosynthesis, but they do not prove it [27] . In all the other cases studied, the myrmecophile mimics passively its host (see [9, 10]). Is the presence of Sternocoelis beetles costly for the ant colony? In the army ant Leptogenys distinguenda workers are able to recognize and kill the intruders (and possibly eat them) to various degrees, which is the mark of a counterstrat- egy of the ant [6]. Nevertheless, Leptogenys are nomadic ants without a permanent nest, and the situation is different in ants that build a nest and mark it with the colony odour. Inside the nest, all individuals including guests are consid- ered as friends as it was first hypothesized by Jaisson [40] and chemically explained in Lasius niger [23]. Apparently, the cost of Sternocoelis is insignificant for the host, but some competition for food is possible as beetles and ant larvae feed in the same chambers and beetles can be very numerous. On the opposite, the beetles licking the ant larvae may benefit if they protect them against parasites and infection. We suggest that this may mean a cleaning symbiosis as known in vertebrates, for example, between a cleaner fish and a client [41]. 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Jones, and J. J. Boomsma, “A mosaic of chemical coevolution in a large blue butterfly,” Science, vol. 319, no. 5859, pp. 88-90, 2008. Hindawi Publishing Corporation Psyche Volume 2012, Article ID 153975, 10 pages doi:10.1155/2012/153975 Review Article The Host Genera of Ant-Parasitic Lycaenidae Butterflies: A Review Konrad Fiedler Department of Tropical Ecology and Animal Biodiversity, Faculty of Life Sciences, University of Vienna, Rennweg 14, 1030 Vienna, Austria Correspondence should be addressed to Konrad Fiedler, konrad.fiedler@univie.ac.at Received 10 October 2011; Accepted 3 January 2012 Academic Editor: Volker Witte Copyright © 2012 Konrad Fiedler. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Numerous butterfly species in the family Lycaenidae maintain myrmecophilous associations with trophobiotic ants, but only a minority of ant-associated butterflies are parasites of ants. Camponotus, Crematogaster, Myrmica, and Oecophylla are the most frequently parasitized ant genera. The distribution of ant-parasitic representatives of the Lycaenidae suggests that only Camponotus and Crematogaster have multiply been invaded as hosts by different independent butterfly lineages. A general linear model reveals that the number of associated nonparasitic lycaenid butterfly species is the single best predictor of the frequency of parasitic interactions to occur within an ant genus. Neither species richness of invaded ant genera nor their ecological prevalence or geographical distribution contributed significantly to that model. Some large and dominant ant genera, which comprise important visitors of ant-mutualistic lycaenids, have no {Formica, Dolichoderus) or very few ant-parasitic butterflies {Lasius, Polyrhachis) associated with them. 1. Introduction Associations between ants and butterfly species in the fam- ilies Lycaenidae and Riodinidae have attracted the interest of naturalists since more than 200 years. Building upon an ever-increasing number of field records and case studies (summarized in [1]) these interactions with their manifold variations and intricacies have developed into a paradigmatic example of the evolutionary ecology and dynamics of interspecific associations [2]. Interactions with ants are most well developed during the larval stages of myrmecophilous butterflies. To communicate with ants, myrmecophilous caterpillars possess a variety of glandular organs and often also use vibrational signals that may modulate ant behaviour [3, 4]. Essentially, interactions between myrmecophilous caterpillars and visiting ants comprise a trade of two com- modities. The caterpillars produce secretions that contain carbohydrates and amino acids [5]. In turn, the ants harvest these secretions, do not attack myrmecophilous caterpillars and the presence of ant guards confers, at least in a statistical sense, protection against predators or parasitoids (reviewed in [2]). Thus, such interactions are basically mutualistic in nature, even though the extent of benefits accruing to both partners may be asymmetric and manipulatory communica- tion (by means of mimicking chemical or vibrational signals of ants) is not uncommon. In certain cases, especially if butterfly-ant associations are obligatory (from the butterfly’s perspective) and involve specific host ants, interactions may extend into other life-cycle stages of the butterflies, such as pupae (if pupation occurs in ant nests or pavilions built by ants to protect their trophobiotic partners), adults (if egg- laying or nutrient acquisition occurs in company with ants), or eggs. The vast majority of known butterfly- ant interactions are mutualistic or commensalic in nature. In the latter case the butterfly larvae benefit from their association with ants, while no costs accrue to the ants. Some few butterflies, however, have evolved into parasites of ants [6]. These unusual associations have served as models for host- parasite coevolution [7] . Ant parasitism requires very precise tailoring of the chemical and mechanical signals employed to achieve social integration into ant colonies. Accordingly, ant- parasitic lycaenid butterflies are highly specific with regard to their host ant use, which also renders them extraordinarily 2 Psyche susceptible to the risk of coextinction [8]. Indeed, many ant- parasitic lycaenids are highly endangered species [9], and the well-studied Palaearctic genus Maculinea is now regarded as a prime example of insect conservation biology [10]. In this essay, I will focus on the ant genera that serve as hosts of parasitic butterflies. First, I summarize which ant genera in the world are known to be parasitized by butterflies. I then discuss whether this host ant use reflects the macroeco- logical patterns seen in mutualistic butterfly-ant associations. Finally, I will explore if the observed host use patterns allow for generalizations and testable predictions, for example, with regard to expected host ant affiliations in underexplored faunas. Specifically, I expected that the number of associated parasitic lycaenids per host ant genera increases with their ecological prevalence, geographical distribution, and species richness. 2. What Constitutes an Ant-Parasitic Butterfly Species? I here use a rather restrictive definition of ant parasitism. I regard a butterfly species as a parasite of its host ants only if (a) the butterfly caterpillars (at least from some develop- mental stage onwards) feed on ant brood inside ant nests (“predators”) or (b) the caterpillars are being fed through trophallaxis by their host ants (“cuckoo-type” parasitism). Both these types of parasitism occur in Maculinea [11], but the extent of the nutrient flow from the ant colony to the caterpillars may vary across species. For example, in some lycaenid species feeding through trophallaxis apparently occurs only as a supplementary mode of nutrient acquisition. Yet include such cases here as parasites of ants, since the re- spective behavioural and communicative strategies are in place. In contrast, I exclude two types of “indirect” parasitism. First, there are a few myrmecophilous lycaenid species that feed obligately on myrmecophytic ant plants. The best doc- umented examples are certain SE Asian Arhopala species on ant-trees of the genus Macaranga [12, 13]. These caterpillars cause substantial feeding damage to the ant-trees and thereby likely inflict costs to the Crematogaster ants that inhibit these trees. Arhopala caterpillars on Macaranga, however, possess a nectar gland and secrete nectar at rates typical for ant- mutualistic lycaenids (K. Fiedler, unpublished observations). They are also not known to elicit trophallaxis or even to prey on ant brood. Accordingly, I did not score these associations as parasitic, but rather as competitors of ants for the same resource (namely, the ant-tree). Analogous cases are known, or suspected, to occur in other tropical lycaenid butterflies whose larvae feed on obligate myrmecophytes, such as various Hypochrysops species in Australia and New Guinea on Myrmecodia ant plants [14, 15]. Similarly, I do not include those lycaenid species (notably in the subfamily Miletinae) whose larvae prey upon ant- attended honeydew-producing homopterans and often also feed on homopteran honeydew [16-20]. In analogy to the case of myrmecophytes, these butterflies compete with ants for the same resources (here: trophobiotic homopterans), but as a rule the caterpillars neither prey on ant brood nor elicit trophallaxis. Some species of the Miletinae, however, are known to supplement their diet through ant regurgitations, and these are included below since they show the behavioural traits considered here as essential for parasitism with ants. Two further restrictions are (1) cases where trophallaxis or predation on ant brood have so far only been indirectly inferred, but not be confirmed through direct observational evidence, are largely excluded. This relates to a couple of tropical lycaenid species for which only old, or very incom- plete or vague, information on their life cycles is available. In these cases, new data are needed, before any conclusions become feasible. (2) The butterfly family Riodinidae is also excluded. Ant-associations occur in at least two clades of Neotropical Riodinidae (tribes Eurybiini and Nymphidiini, see [21, 22] for many case studies and [23] for a tentative phylogeny). Circumstantial evidence exists that in at least one genus within the Nymphidiini (Aricoris) the larvae may feed on trophallaxis received from Camponotus host ants [21], but otherwise the existence of ant-parasitic life habits in the Riodinidae (though not unlikely to exist amongst Neotropical riodinids) must await confirmation. 3. Data Sources Butterfly life-history data were compiled from a large variety of sources, ranging from faunal monographic treatments across hundreds of journal papers to databases in the Internet. The data tables in [1] formed the initial basis, and they have been continually extended and updated ever since [24, 25] . Flere, I focus on that subset of sources where (a) the butterfly species qualifies as a parasite of ants according to the restrictions stated above and (b) the host ant has been reported at least at genus level. Three reasons justify the choice of the ant genus level for the subsequent comparisons. (1) For most ant genera, no modern revisions are available. Thus, proper species identifications are often impossible, especially in tropical realms. (2) Ant genus delimitations are quite stable and recognizable on a worldwide basis ([26], see also http://www.antweb.org). Accordingly, records (often reported by lepidopterists and not myrmecologists) should usually be reliable on this level. (3) Most ant-parasitic lycaenids are not bound to one single ant species, but are affiliated with a couple of congeneric ant species. For example novel Myrmica host ant species continue to be discovered in Eastern Europe for butterflies in the genus Maculinea [27, 28] . Therefore, I performed all analyses on the taxonomic level where the highest reliability can be achieved. Data on species richness of ant genera was extracted from the website antweb.org (as of 9 October 2011). A complete bibliography of the evaluated literature would extend beyond the scope of this essay. Eor ant-parasitic Lycaenidae, many sources have been detailed in [6]. Full information on data sources is available upon request from the author. Psyche 3 4. Summary of Ant Genera That Are Confirmed as Hosts of Parasitic Lycaenid Butterflies Of the 54 ant genera known to attend lycaenid larvae on a worldwide basis ([24], only Liometopum has been added to this list since) just 1 1 genera are for certain recorded as hosts of parasitic butterflies. 4.1. Subfamily Formicinae 4.1.1. Camponotus. This is one of the globally most prevalent ant genera in terms of species richness (>1050 described species) as well as ecological significance. It is also the numer- ically leading ant genus with regard to the number of as- sociated parasitic lycaenid species. At least 9 species of the large Afrotropical genus Lepidochrysops have been recorded from nests of either Camponotus niveosetosus or C. maculatus. Lepidochrysops larvae have a life cycle similar to the Maculinea-Phengaris clade. They initially feed on flowers of plants (mostly in the families Lamiaceae, but also Verbenaceae and Scrophulariaceae). At the onset of their third instar they are adopted by Camponotus workers into the ant colonies where they turn into predators of ant brood. There are more than 125 described Lepidochrysops species [29]. Many of them are microendemics of high conservation concern [30]. Presumably all Lepidochrysops species are parasites of Camponotus ants. The small South African genus Orachrysops is the closest relative of Lepidochrysops. Orachrysops larvae are not parasites of ants, but live in close association with Camponotus ants as leaf, and later root, herbivores of various Fabaceae plants [31]. Orachrysops spe- cies may therefore be seen as models for the evolutionary transition between “normal” phytophagous ant-mutualistic lycaenids and species that are parasites of ants. The East Asian Niphanda fusca is an obligate cuckoo - type parasite of various Camponotus ants [32]. Unusual for ant-parasitic lycaenids, larvae of this species retain a fully functional nectar gland whose secretions are tuned towards the gustatory preferences of their host ants [33]. Life histories of other Niphanda species, that all occur in East and South-East Asia, are unknown. Within the genus Ogyris (13 species in New Guinea and Australia) most species maintain obligate mutualistic associations with ants, but two are reported to occur inside nests of Camponotus species, namely, O. idmo and O. suhterrestris [15, 34, 35]. Finally, for at least two representatives of the aphytophagous African genus Lachnocnema (L. bibulus, L. magna) there is evidence that caterpillars supplement their diet by eliciting trophallaxis from Camponotus ants (in L. bibulus reportedly also from Crematogaster ants). The major nutrient source of Lachnocnema larvae, however, is preying on homopterans and drinking their honeydew excretions. 4.1.2. Oecophylla. The two species of weaver ants in the genus Oecophylla are extremely dominant insects in their habitats in tropical Africa, southern and south-eastern Asia, Australia, and New Guinea. Two lycaenid genera are special- ist parasites of weaver ants. Liphyra (L. brassolis, L. grandis) are predators of the brood of Oecophylla smaragdina in the Oriental region [15, 25], while African Euliphyra {Eu. miri- fica, Eu. leucyania) are cuckoo-type parasites of Oe. longinoda by means of trophallaxis and also steal prey items of their host ants [36]. Many more lycaenid species are associated with weaver ants, including striking examples of obligate and specific interactions, but these all appear to be mutualistic associations. 4.1.3. Polyrhachis. Even though this large ant genus (>600 described species) ranks rather high in the visitors list of lycaenid caterpillars, only one of its reported associated 27 myrmecophilous butterfly species is a parasite. The rare Arhopala wildei in Australia and New Guinea preys on brood in nests of Polyrhachis queenslandica [37, 38]. 4.1.4. Lasius. Ant species of this moderately rich genus (>100 species) are frequent visitors of lycaenid caterpillars, especially in the Palaearctic realm [25]. Shirozua jonasi from East Asia is the only ant-parasitic butterfly known to be affiliated with Lasius ants (L. spathepus, L. fuliginosus, and L. morisitai). The caterpillars apparently receive occasional trophallactic regurgitations, but their principle mode of feeding is to prey on a variety of homopterans and to drink their honeydew excretions [39]. 4.1.5. Lepisiota. Butterflies of the South African genus Aloei- des all have an obligate relationship to ants. Lepisiota capensis is their major host ant [40]. As far as known, most Aloeides species are phytophagous ant mutualists (host plants in the Fabaceae and more rarely the Malvaceae, Zygophyllaceae and Thymelaeaceae), but older larvae of A. pallida have been observed to feed on ant eggs and appear to be completely aphytophagous [40]. 4.1.6. Anoplolepis. Another endemic South African butterfly genus is Thestor, with about 27 recognized species [41]. The life histories of these butterflies are still very incompletely known, but for sure they are essentially aphytophagous, as is the rule in the Miletinae to which this genus belongs. Younger larvae prey on various homopterans, and in at least 3 species {Th. yildizae, rileyi, and basutus) older larvae live inside ant nests where they feed on brood of the ant Anoplolepis custodiens. It is suspected that all Thestor species share this habit [41]. 4.2. Subfamily Dolichoderinae 4.2.1. Papyrius. The small endemic Australian butterfly genus Acrodipsas can be divided into two clades [42]. Larvae of one of these, comprising the species A. brisbanensis and A. myrmecophila, are obligate parasites of Papyrius nitidus [35] from their first instar onwards, that is, without a phytophagous phase as in Lepidochrysops or the Maculinea/ Phengaris clade. Papyrius species are highly dominant components of Australian ant assemblages and serve as mutualistic partners for some additional Australian lycaenids [34]. 4 Psyche 4.3. Subfamily Myrmicinae 4.3.1. Crematogaster. This diverse ant genus (>450 described species) ranks second in terms of associated ant-parasitic lycaenid butterflies. In the lycaenid tribe Aphnaeini (about 260 species, of which >90% occur in Africa) caterpillar- ant associations are nearly always obligatory, and the pre- dominant host ant genus is Crematogaster. Few Aphnaeini species, however, are well established to be parasites of Crematogaster ants. Only one of these is a brood predator {Cigaritis acamas [43]), whereas in other cases trophallactic feeding has been reported (e.g., Aphnaeus adamsi, Chrysoritis (Oxychaeta) dicksoni, Spindasis takanonis, and also S. syama; [40, 44]). Beyond the tribe Aphnaeini, parasitic relationships occur in the Australian Acrodipsas of which three species (A. cuprea, illidgei, and aurata) are predators of Crematogaster ants [35, 42, 45]. According to one old account caterpillars of the aphytophagous African Lachnocnema hihulus (which essentially prey on homopterans and drink their honeydew exudates, see above) also supplement their diet by trophal- laxis obtained from Crematogaster ants [46]. 4.3.2. Myrmica. This genus is famous as being the host of the ant-parasitic Maculinea butterflies in temperate regions of Eurasia. Maculinea comprises about 10-15 species, depending on the status allocated to local forms and cryptic lineages detected through resent sequence analyses [47]. All Maculinea species are either brood predators or cuckoo-type parasites [11] of Myrmica ants. Host specificity was initially thought to be generally high [48], but research over the past two decades has revealed more complex, locally to regionally variable patterns of host specificity [27]. Especially in previ- ously underexplored regions of central and east Europe many new local host associations have been elucidated through thorough field work [28]. Caterpillars of the closely related East Asian butterfly genus Phengaris also parasitize Myrmica species [44, 47]. 4.3.3. Aphaenogaster. There are two Maculinea species from East Asia (M. arionides, M. teleius) for which the use of Aphaenogaster ant species as hosts has been recorded. Both these butterfly species are known to parasitize mainly Myrmica host ants. It remains to be shown to what degree Aphaenogaster ants really qualify as valid hosts. Alternatively, these records might be based on misidentifications or rep- resent rare affiliations that only occur under exceptional cir- cumstances (see the discussion about primary and secondary hosts in [27]). 4.3.4. Rhoptromyrmex. Representatives of this small Oriental ant genus have been observed to attend a range of lycaenid caterpillars in a mutualistic manner. Besides, trophallactic feeding does occur in one unusual case, the Miletinae species Logania malayica. L. malayica larvae prey essentially on homopterans and drink their honeydew exudates, but young larvae also elicit regurgitations from Rh. wroughtonii ants, with which the butterflies are closely and specifically asso- ciated over their entire life cycle [17, 49]. 5. Macroecological Patterns of Host Ant Use among Ant-Parasitic Lycaenidae Butterflies Myrmecophilous associations between lycaenid butterflies and ants are confined to that subset of ant genera which maintain trophobiotic interactions [24]. Trophobiotic ants form a highly significant fraction in terms of their ecological prevalence as well as species diversity. They essentially derive liquid nutrients from extrafloral plant nectar [50, 51] and from the excretions (“honeydew”) of sap -sucking homopter- ans [52, 53]. Lycaenid and riodinid butterfly species that offer nectar-like secretions in exchange for protection largely “hitch-hike” on the behavioural and ecological syndromes which are associated with ant trophobiosis. Harvesting nutrient-rich liquids requires specialized anatomy [54] and behaviour in ants (e.g., trophallactic exchange of liquid food within the colony), with trophobiosis demanding a more complex suite of morphological and behavioural traits than licking-up plant nectar [55]. Ant-parasitic lycaenids form a very small subset of myrmecophilous ant-attended species in that butterfly fam- ily. Not surprisingly, the host ants parasitized by them constitute a small subset of ant genera known to visit and attend caterpillars in mutualistic associations. In two earlier studies the ecological prevalence and geographical distribu- tion of ant genera were shown to be the best predictors for their representation in mutualistic lycaenid-ant associations [24, 25]. Eor parasitic interactions, this pattern changes according to a similar analysis. In analogy to [24], I con- structed a multiple linear regression model, with the number of recorded ant-parasitic lycaenids as response variable and the species richness (log-transformed), representation in lycaenid-ant interactions (log-transformed), ecological prevalence, and geographical distribution of ant genera as predictors. Geographical distribution was scored on a rank scale (from 1 to 10) as the number of faunal regions from which an ant genus is known, using the following 10 regions: West Palaearctic region (Europe eastwards up to the Ural mountains, including Africa north of the Sahara, Asia Minor, and the Near East); East Palaearctic region (Asia east of the Ural mountains, including Japan and Taiwan); India; South East Asia (comprising Thailand, the Malay Peninsula, and the large islands of the Sunda shelf like Sumatra, Borneo, and Java); New Guinea; Australia; Gentral Africa (south of the Sahara to approx. 15° southern latitude); Southern Africa (mainly comprising South Africa, Namibia, Botswana, and Zimbabwe); North America (north of Mexico); Central and South America. Ecological prevalence (sensu [56]) was scored on a rank scale from 1 to 5 (Table 1). The linear model revealed that only the number of associated lycaenid species had a significant and positive relationship with the number of recorded cases of lycaenid- ant parasitism in an ant genus (see Table 2 for full doc- umentation). All three other potential predictors were far from having any significant effect. Inspection of residuals confirmed that the model assumptions were met with reasonable accuracy. Moreover, application of a Ridge cor- rection (with A = 0.1) to account for collinearity among predictors did not change the overall model outcome (data Psyche 5 Table 1: Classification of ant genera known to associate with Lycaenidae caterpillars into prevalence groups. Ant genera are classified into that group which corresponds to the dominance status of its most dominant component species involved in butterfly- ant associations. For example, Formica is scored as “top dominant” since many (but not all) Formica species are territorial key-stone ant species in their respective habitats and communities, adapted from [24]. Class Score Criteria Genera Dominant ants in habitat; defend Myrmicinae: Pheidole; Formicinae: Formica, Oecophylla; Dolichoderinae: Anonychomyrma, Azteca, Forelius, Froggattella, Iridomyrmex, Papyrius Top dominant 5 territories and resources intra- as well as interspecifically; monopolize resources against all heterospecific competitors Myrmicinae: Crematogaster, Meranoplus, Monomorium, Myrmicaria, Solenopsis, Tetramorium; Second- order dominant 4 Subordinate relative to top dominants, but may become dominant in the absence Formicinae: Anoplolepis, Camponotus, Polyrhachis, Lasius, Lepisiota, of these; monopolize resources^ Myrmecocystus; Dolichoderinae: Dolichoderus, Linepithema, Liometopum, Ochetellus, Philidris; Submissive 3 Subordinate to both classes of dominants; usually opportunistic species with generalized feeding habits; rarely defend and monopolize resources against heterospecific ants Myrmicinae: Acanthomyrmex, Aphaenogaster, Myrmica, Rhoptromyrmex; Formicinae: Echinopla, Notoncus, Paratrechina, Prolasius; Dolichoderinae: Dorymyrmex, Tapinoma, Technomyrmex; Ponerinae: Ectatomma Solitary 2 Foraging individually; rarely monopolize resources Myrmeciinae: Myrmecia; Myrmicinae: Cataulacus; Ponerinae: Gnamptogenys, Odontomachus, Rhytidoponera Pseudomyrmecinae: Tetraponera, Pseudomyrmex Cryptic 1 Minute species foraging on the ground or in leaf litter; inferior to all other ants in direct confrontation Myrmicinae: Leptothorax; Formicinae: Brachymyrmex, Plagiolepis; Dolichoderinae: Bothriomyrmex ^ Includes many species that become dominant in disturbed habitats or when introduced as alien species into non-adapted ant communities. not shown). In a stepwise forward model selection, again only the frequency of nonparasitic associations remained as significant predictor. Likewise, using Poisson-type (instead of Gaussian) error distributions did not affect the outcome of this analysis (data not shown). Hence, it is not the ecological or geographical prevalence that is decisive for the establishment of parasitic relationships between lycaenid butterflies and ants. Rather, the more butterfly species do interact with a given ant clade, the more likely it is that some of these interactions may turn, in evolutionary time, into parasitic relationships. This also becomes evident when the incidence of ant- parasitism is plotted against the rank the ant genera have in interactions with lycaenid caterpillar species (Figure 1). Instances of social parasitism are more likely amongst those ant genera that are numerically more important in lycaenid- ant associations in general, whereas again species richness of the respective ant genera had no significant influence (Table 3). A number of ant genera (e.g., Pheidole, Dolichoderus, Formica, and Iridomyrmex) that are ecologically dominant in Table 2: Results of general linear model relating the number of parasitic lycaenid species associated with an ant genus to its species richness (log-transformed), ecological prevalence, geographical dis- tribution, and importance in nonparasitic lycaenid-ant associations (log-transformed). Given are standardized regression coefficients and the F and p scores for each variable. SS; sum of squares; MS: mean of squares. Overall model fit: R = 0.5394, = 0.2332, F4;49 = 5.0288; P = 0.0018. SS df MS F P constant 12.78 1 12.78 2.607 0.113 dominance 0.35 1 0.35 -0.0370 0.072 0.789 associated lycaenid spp. 47.54 1 47.54 0.5051 9.698 0.003 species richness 3.44 1 3.44 0.1201 0.702 0.406 geographic regions 0.018 1 0.018 -0.0097 0.004 0.952 error 240.21 49 4.90217 their habitats and serve as hosts for many well-integrated myrmecophilous ant parasites from other insect groups (e.g., [55]) are thus far completely missing in the host list of 6 Psyche 200 -!□ Crematogaster (9) Dh Dh c U o a 80 60 40 H 20 - 8 - 6 - 4 - 2 - □ Camponotus (14) Lasius(l) Polyrhachis (1) Anoplolepis ■ Oecophylla (4) (3) Lepisiota (1) Rhoptromyrmex (1) Aphaenogaster (2) ^^***^_^Papyrius ( 2 ) ~i 1 1 1 1 1 10 20 30 40 50 60 Rank of ant genus Figure 1; Rank-frequency plot of ant genera of the world involved in myrmecophilous associations of Lycaenidae butterflies, based on 927 record pairs of 497 butterfly species with 54 ant genera. Rank 1: ant genus with largest number of associated lycaenid species reported. Ranks 47 to 54: ant genera with only one associated lycaenid species known thus far. Filled diamonds: ant genera only known to be involved in mutualistic interactions with butterflies; open squares: ant genera that also serve as hosts for ant-parasitic Lycaenidae larvae (with genus name included; figure in parentheses: number of confirmed ant-parasitic lycaenid species). Note log-scale of y-axis. Table 3: Results of a bivariate logistic regression, modelling the incidence of ant-parasitic associations within an ant genus {N = 54 ant genera), in relation to species richness and number of nonparasitic associations with lycaenid immatures (both log- transformed) per ant genus. Given are the regression coefficients bi, their standard errors and corresponding t and p values. Overall model score: Xidf = 6.577; P = 0.0373. bi ± 1 SE t P Constant -3.0864 ± 1.1160 2.766 0.008 Number of associated lycaenid species 0.9105 ± 0.3936 2.313 0.025 Species richness of genus -0.1799 ± 0.2327 0.773 0.443 to ant-parasitism suggested earlier [6]. Likewise, parasitism of Crematogaster ants by Acrodipsas is certainly unrelated in phylogenetic terms to the multiple (and probably again: independent) occurrences amongst single species of Aph- naeini that all belong to larger genera where the majority of species is nonparasitic {Cigaritis, Spindasis, and Aphnaeus) . Overall, the scattered occurrence of ant- parasitism amongst the Lycaenidae gives evidence that such interactions have evolved multiple times, rather independently from another, and under quite different circumstances [6]. Only few such cases have given rise to moderate or even substantial radiations, most notably in the African genus Lepidochrysops (over 120 species) and in the Eurasiatic Phengaris-Maculinea clade (some 10-20 species). The host ant use of the latter remains a mystery in terms of its evolutionary and eco- logical roots. My r mica ants are visitors of only a moderate number of ant-mutualistic lycaenids in the Holarctic region (recorded with 22 species thus far). Moreover, Myrmica ants usually neither form very large colonies nor are they territorial and ecologically dominant in most habitats where they occur today. Hence, they lack typical characters of other host ants of parasitic myrmecophiles. On the other hand recent phylogenetic evidence [47] strengthens the notion that evolution of parasitic associations with Myrmica ants occurred just once, at the base of the Phengaris-Maculinea clade. Similarly, the affiliation with Camponotus ants in parasitic Lepidochrysops as well as mutualistic Orachrysops suggests that specialization on Camponotus hosts predated the evolution of parasitism in that butterfly lineage. Host shifts among ant-parasitic butterflies from one ant host genus to another have apparently rarely occurred in the Lycaenidae. One well- documented case is the Australian genus Acrodipsas, where some species parasitize Papyrius ants, but one clade subsequently shifted to Crematogaster hosts [42]. This rare case even implies a switching of hosts across ant subfamily boundaries. In contrast, the significance of Aphaenogaster recorded as host ants of some East Asiatic Maculinea needs to be rigorously addressed. In all likelihood, these are stray (or even erroneous) records rather than an indication of host shifts beyond ant genus boundaries. ant-parasitic lycaenids. Even considering that trophobiosis is an important evolutionary prerequisite for the estab- lishment of lycaenid- ant interactions (thereby excluding nontrophobiotic ants such as army ants, leaf-cutter ants or harvester ants as potential hosts), the discrepancy in host use between ant-parasitic lycaenids, and other well- integrated myrmecophilous parasites remains striking. Only two ant genera, Camponotus and Crematogaster, have been the target of multiple evolutionary trajectories towards parasitic life habits amongst the Eycaenidae. Even though complete phylogenetic analyses are still lacking for the family Eycaenidae, there can be no doubt that para- sitism of Camponotus through the butterfly genera Lepi- dochrysops, Niphanda, Ogyris, and Lachnocnema has evolved independently — these four butterfly genera are far apart from each other in all systematic accounts of the family Eycaenidae, and they represent all three potential pathways 6. Which Cases of Ant-Parasitism Might Await Detection amongst the Lycaenidae? Starting from the patterns of host-ant use among ant- parasitic Eycaenidae, and in combination with other infor- mation on life-history traits of lycaenid butterflies, I here finally outline a few expectations in which butterfly clades and biomes further instances of parasitic interactions might most likely be uncovered. These expectations are amenable to testing by systematic assembly of further life history data or by evaluating earlier inconclusive reports. One major group of lycaenid butterflies where a larger number of instances of trophallactic feeding by ants can be expected is the subfamily Miletinae. Miletinae larvae are essentially predators of homopterans. Since many homopter- ans are attended by ants and since quite a number of Miletinae larvae also drink honeydew, it would not come as Psyche 7 a surprise to see more cases of trophallaxis with ants being documented in the future. Particularly likely candidates are those Miletinae species that are specifically adapted to spend their entire life cycle (including adult feeding on homopteran honeydew) with individual ant species. This is the case for Logania malayica with Rhoptromyrmex wroughtonii, and analogous candidate species occur in tropical SE Asia {Miletus spp. with Dolichoderus spp.; Allotinus unicolor with Anoplolepis longipes; [18, 20]). In two cases {Allotinus apries with Myrmicaria lutea [17]; Logania hampsoni with Iridomyrmex [15]) parasitic interactions have explicitly been suspected to exist, but until now these cases remain unsup- ported by direct observations of parasitic behaviours of the lycaenid caterpillars (A. Weissflog, personal communication for A. apries). As stated above, it is also quite likely that most, if not all Thestor species in South Africa will turn out to maintain parasitic relationships to Anoplolepis custodiens and allied ants [41]. Such cases of ant-parasitic relationships may also occasionally shift from the lower trophic level of cuckoo -feeding to the higher trophic level of brood predation (as in the genera Liphyra and Euliphyra). However, certain Miletinae do not interact intensively with ants that attend their homopteran prey [17, 57-59]. It is unlikely that traits required to entering into host-specific parasitic butterfly-ant interactions have evolved here. All further examples of ant- parasitism derived from predation on homopterans would obviously fall into the “Miletinae type” [6]. Another lycaenid clade where further cases of ant- parasitism can surely be expected to occur is the tribe Aph- naeini. Even though the few confirmed cases of ant-para- sitism are rather isolated incidences nested within larger clades of ant-mutualists (e.g., Chrysoritis dicksoni in the genus Chrysoritis [60]), further species may show up to depend on nutrients derived from their close association with ants, as has been speculated many times in the literature (for critical reviews see [40, 61, 62]). Most additional instances of ant-parasitism in the Aphnaeini are expected to involve Crematogaster ants (the prevalent ant partner in mutualistic Aphnaeini species), but in Aloeides also further incidences of Lcpisiota-parasitism may be found. Other obvious candidates to furnish more ant-parasitic lycaenids are the genera Lepidochrysops (with Campono- tus), Maculinea, and Phengaris (hitherto undescribed host associations in East Asia expected to refer to Myrmica), and Niphanda (probably with Camponotus). Beyond that, no valid extrapolations seem feasible at present. For exam- ple, the parasitic association between Arhopala wildei and Polyrhachis queenslandica does not seem “predictable” in a phylogenetic framework [13]. The most likely candidates for the discovery of novel ant-parasitic lycaenids of the “Aphnaeini type” are clades where a number of butterfly species show intimate host-specific mutualistic relationships towards specific host ants. From the ant perspective, two genera which account for a very substantial fraction of records with lycaenids (namely, Lasius and Formica) score strikingly low as hosts of ant- parasitic butterflies. The only confirmed case with Lasius involves a species {Shirozua jonasi) whose larvae obtain most of their nutrient income from preying on homopterans and drinking their honeydew. This hairstreak species is ecologi- cally similar to Miletinae butterflies and does not enter into Lasius nests to prey on ant brood. Possibly, the lack of brood being present in Lasius nests over winter poses a constraint in the evolution of ant-parasitism in temperate-zone climates. This would also explain why so far no case of ant-parasitism has been confirmed from the genus Formica. In East Asia, larvae of Orthomiella rantaizana have been found in Formica nests (Shen-Horn Yen, personal communication), but whether these are parasites, commensales, or mutualists of ants remains to be uncovered. Clearly, Lasius as well as Formica species serve as hosts for a large range of well- integrated myrmecophiles [55], but the majority of these parasites have evolved from detritivorous or predacious ancestors, and not from herbivores. Two other sociobiological traits of ant colonies that have been suggested to be related to the evolution of parasitic myrmecophily are the level of polygyny or polyandry, and the brood cycle. With regard to the latter, as already noted above the absence of winter brood may have prevented the intrusion of Holarctic lycaenids as parasites into Lasius and Formica colonies. With regard to ants from the humid tropics, however, seasonal fluctuations in brood availability are less likely to constrain the evolution of lycaenid butterflies into parasites of ants, so that this factor (if valid at all) would have to be restricted to seasonal climates. Genetic intracolo- nial heterogeneity, which can result from the presence of multiple queens and/or the occurrence of multiple matings during their nuptial flight, may facilitate the intrusion of social parasites as well as of parasitic myrmecophiles [63]. It is presently impossible to rigorously test these two hypothe- ses, since data on the colony structure and population demography of many tropical and subtropical ants that are parasitized by lycaenids are too scant. Polygyny seems to be common among ants that serve as hosts [64], but in at least one instance {Camponotus japonicus, the host ant of Niphanda fusca) monogyny and claustral colony foundation have been confirmed [65]. 7. Perspective Ant-parasitic lycaenid butterflies are a bewildering evo- lutionary outcome: carnivores or cuckoo-type feeders in an otherwise phytophagous clade of insects. The commu- nication modes required for integration into their host colonies, the phylogenetic roots, and population genetic consequences of their unusual interactions with ants, and their repercussions into conservation biology [66, 67] will continue to attract the interest of scientists. However, these parasitic interactions encompass only a small minority of myrmecophilous Lycaenidae butterfly species. Also the ant genera involved comprise but a small minority as compared to the range of trophobiotic ants that could potentially be parasitized. For sure, some further extensions can be expected, especially in hitherto underexplored tropical regions or in butterfly clades whose life histories are thus far very poorly documented. Most known ant-parasitic lycaen- ids occur in seasonally cold and/or dry regions [6], where 8 Psyche both the butterfly and the ant faunas are comparatively well covered. It has even been suggested, though not yet rigorously tested, that avoidance of unfavourable seasons might have promoted the entering of ant nests as safe places for lycaenid caterpillars. The detection of additional cases of butterfly-ant parasitism in these regions in all likelihood will not radically turn the robust patterns described here upside down. For tropical faunas, some more unexpected incidences of ant-parasitism may await discovery, yet it does not seem likely that many instances of butterfly caterpillars living in brood chambers of ant nests would have gone undetected thus far. Rather, future progress will be made in uncovering the microevolutionary steps that drive host-parasite co -evo- lution [7]. It will also be rewarding to rigorously assess the macroevolutionary pathways leading to ant-parasitism in a phylogenetically controlled manner. To achieve this goal, besides elucidating the phylogenetic relationships of ly- caenids and their ant hosts, more bionomic data on both of these players, but especially a better documentation of the sociobiology and ecology of the host ants (beyond the well- studied Myrmica case) will be essential. Acknowledgments The author is grateful to Jean-Paul Lachaud for the invitation to write this contribution. Volker Witte and an anonymous reviewer provided helpful comments that served to improve this paper. Shen-Horn Yen, Andreas Weissflog, and Alain Dejean contributed some records of ant hosts of parasitic lycaenid larvae. Alain Heath generously sent him copies of papers that are otherwise difflcult to obtain. 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Hindawi Publishing Corporation Psyche Volume 2012, Article ID 363767, 6 pages dohlO.l 155/2012/363767 Research Article Performance of Tomicus yunnanensis and Tomicus minor (Col., Scolytinae) on Pinus yunnanensis and Pinus armandii in Yunnan, Southwestern China Tao Zhao^’^ and Bo L^gstrom^ ^ Division of Forest Protection, Yunnan Academy of Forestry, Kunming 650224, China ^ Department of Chemistry, Royal Institute of Technology, 10044 Stockholm, Sweden ^ Department of Ecology, Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden Correspondence should be addressed to Tao Zhao, taozhao@kth.se Received 30 September 2011; Revised 16 December 2011; Accepted 14 January 2012 Academic Editor: John A. Byers Copyright © 2012 T. Zhao and B. Langstrom. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pine shoot beetles, Tomicus yunnanensis Kirkendall and Faccoli and Tomicus minor Hartig (Col., Scolytinae), have been causing substantial mortality to Yunnan pine (Pinus yunnanensis Franch) in Yunnan, southwestern China, whereas only a few Armand pine (Pinus armandii Franch) were attacked by the beetles. In order to evaluate the suitability of P armandii as host material for the two Tomicus, adults of both Tomicus were caged on living branches and felled logs of the two pines during shoot feeding and trunk attack phase, respectively. More beetles survived on the living branches of P. yunnanensis than on P. armandii. Tomicus yunnanensis and T minor produced similar progeny in the logs of the two pines. The sex ratio and developmental period were not affected by host species, but the brood beetles emerging from Armand pine weighed less than those from Yunnan pine, suggesting that P. armandii are less suitable to be host of T yunnanensis and T minor. 1. Introduction As the most important forest pests in southwestern China, pine shoot beetles, Tomicus yunnanensis Kirkendall and Fac- coli and Tomicus minor Hartig (Col., Scolytinae), have killed more than 200 000 ha of Yunnan pine (Pinus yunnanensis Franch) in Yunnan province since early 1980s [1], of which T. yunnanensis is considered as a more serious species [2-4]. Since the morphology and gallery system of T. yunnanensis are very similar to Tomicus piniperda L., T. yunnanensis had long been confused with T. piniperda. Molecular and taxonomic studies have, however, demonstrated that T. piniperda is absent in Yunnan [3, 4]. Thus, the aggressive Tomicus species in Yunnan was a new undescribed species, and consequently T. yunnanensis was finally described and named in 2008 [3]. Like the pine shoot beetles in Europe, the life cycle of T. yunnanensis and T. minor is univoltine and contains two phases, a reproduction phase and a maturation feeding phase [5-7]. In Yunnan, adults of the two Tomicus species mate and lay eggs in the inner bark of trunks and large branches of living trees from November to May [6-8] . Larvae and pupae subsequently complete their development there. After emergence, the young adults fly to the crowns of host pine trees where they feed in the shoots and become sexually mature [2, 5, 9]. The main shoot-feeding phase lasts from May to November in Yunnan [5-7]. However, T. minor usually initiates its flight one or two weeks later than T. yunnanensis [1, 2]. Since T. minor usually attack trees that previously have been attacked by T. yunnanensis, it is regarded as a more secondary species in Yunnan [ 1-3] . P. yunnanensis and Armand pine (Pinus armandii Franch) are distributed at similar elevations. Armand pine often grows together with P. yunnanensis in Yunnan prov- ince, but P. armandii has rarely been attacked by T. piniperda and T. minor [7]. The reason for this is poorly understood, but it would be valuable to know if P. armandii really is more resistant to pine shoot beetle damage. In this study, we 2 Psyche compared the suitability of R armandii and P. yunnanensis for maturation feeding and breeding of the two Tomicus species. 2. Materials and Methods 2.1. Origins of Pine Shoot Beetles. The pine shoot beetles were collected from Yunnan pine forests in Yiliang or Shilin County. We recognized the shoots containing maturation feeding beetles by their yellowish needles and then cut off the shoots 5-10 cm below the entrance holes by scissors. The shoots with beetles were collected from the forest a few days before the experiment started, and the beetles were peeled out of the shoots and identified to species under a stereoscope (Nikon SMZ). The Tomicus population in Shilin consisted of mostly T. yunnanensis, but a relatively high proportion of T. minor was found in Yiliang. 2.2. Shoot Cage Experiment. In order to assess the effect of tree species on the maturation feeding of pine shoot beetles, the shoot cage experiment was carried out in a mixed stand of P. yunnanensis and P. armandii in Kunming Tree Garden (25°07" N, 103°00" E, 1953 m above sea level). Most of the trees there were 4-5 m high and exhibited a healthy appearance. No damage history had been recorded. A total of 18 similar trees (nine P. yunnanensis and nine P. armandii) were selected. For each tree, a mid-crown branch with 20-30 current shoots was enclosed within a 2.0 m X 1.5 m net cage. On luly 28, 2000, T. yunnanensis adults (10 beetles/cage) were released in six cages for each host species. T. minor adults were released in three cages for each pine species in the same way on October 24, 2000. The ends of all the cages were closed and fastened by string. On December 25, 2000, all the cages were cut off the trees and taken to the laboratory. The number of attacked shoots in each cage was counted. The numbers of beetles alive, killed by resin, dead from other reasons or missing were recorded. For each attacked shoot, the shoot diameter at entrance hole, the distance from entrance hole to the shoot tip, as well as the length of feeding tunnel (defined as the length from entrance hole to the end of the tunnel) were measured by ruler. 2.3. Breeding Experiments. On December 26, 2000, three P. yunnanensis and three P. armandii trees of similar size were felled in a naturally generated mixed stand in Yiliang County. The lower stem section of each tree was cut into four 50 cm- long logs and placed in 80 X 70 cm net cages. A total of 12 cages were used, six of these each contained two logs of P. yunnanensis or P. armandii, and were divided into two groups, each group containing three P. yunnanensis cages and three P. armandii cages. On December 27, twenty pairs of unsexed T. yunnanensis adults were released to each cage in the first group, and an unsexed mixture of the two Tomicus - species ( 1 1 pairs of T. minor and nine pairs of T. yunnanensis) was released to the second group of cages. After beetles were released, all the logs were checked during the entire experimental period. The new entrance holes were marked and counted twice a week. On Mar. 30, 2001, the new brood adults started emerging. Then the emerging beetles were collected at different intervals ranging from one day to several days. We measured the diameter of each log on April 20. After measurement, we carefully peeled off the outer bark of each log and counted the number of egg galleries constructed by T. yunnanensis and T. minor, respectively. For each egg gallery, we measured gallery length and counted the number of larvae, pupae, and adults remaining in the phloem. The brood production was obtained by summing up the number of emerged adults and brood remaining in the galleries. The brood adults from the second group were identified to species under stereoscope. On April 28, the collected beetles of T. yunnanensis or T. minor were separately dried for each sampling occasion at 60° C for 8 hours, and the dry body weight of the beetles collected at different dates was weighed in 10 -beetle samples using an electronic balance. 2.4. Data Analysis. Data were analyzed using the statistics program “StatPac for windows” (StatPac Inc., 1999). All the means were given as mean ± SD (standard deviation) and compared by Student’s f-test. The percentage data was compared by Chi-square test. 3. Results 3. 1 . Maturation Reeding. The status of pine shoot beetles that were caged in the branches of two pine species was identified as alive, killed by resin, dead from other reasons, or missing at the inspection. Although a few beetles of both species survived on both host species (Table 1), the Tomicus species performed differently on the two hosts. The survival rate of both Tomicus species on P. yunnanensis was two-fold higher than on P. armandii. Correspondingly, the mortality of T. yunnanensis and T. minor on P. armandii (63% and 73% resp.) was somewhat higher than on P. yunnanensis (42% and 50% resp.). Significantly more beetles feeding on P. armandii were killed by resin than that feeding on P. yunnanensis {X^ = 5.44-9.31, P < 0.05). In addition, T. yunnanensis excavated twofold more tunnels on P. yunnanensis than on P. armandii {t = 4.45, P < 0.01), and the tunnels by both Tomicus species on P. yunnanensis were longer than on P. armandii {t = 4.28- 5.61, P < 0.01) (Table 2), indicating that the beetles were more adapted to their principal host than to P. armandii in the shoot feeding phase. 3.2. Oviposition and Brood Production. During reproduction phase, the female adults of either T. yunnanensis or T. minor excavated similar numbers of egg galleries in the logs of P. yunnanensis and P. armandii (Table 3). The brood production of the two beetles was also similar in the two hosts. These results suggest that the oviposition and brood production of T. yunnanensis and T. minor do not differ that much in the logs of the two hosts. Tomicus minor seemed to be in an inferior position in the competition with T. yunnanensis. After the mixture of two beetle species, composed of 55% T. minor and 45% T. yunnanensis, was released in the cages, the resulting egg galleries of T. minor occupied only 24.4% and 19.0% of the Psyche 3 Table 1: The performance of Tomicus yunnanensis and T. minor in the caged shoots of Pinus armandii and P. yunnanensis in Kunming. 60 T. yunnanensis adults were released into the 6 shoot cages on July 28, and 30 T. minor adults were released into 3 cages on October 24, for each tree species. The performances of the beetles were checked on December 25, 2000. Tomicus species Pinus species Total T. yunnanensis P yunnanensis 60 P armandii 60 Chi-square analysis of 2 x 4 contingency table T. minor P. yunnanensis 30 P armandii 30 Chi-square analysis of 2 x 4 contingency table Alive Number of beetles (percentage) Killed by Dead from resin other reasons 13 (21.7) 1 (1.7) 24 (40.0) 7 (11.7) 12 (20.0) 26 (43.3) = 12.5, d.f. = 3,P = 0.006 8 (26.7) 1 (3.3) 14 (46.7) 4 (13.3) 8 (26.7) 14 (46.7) = 7.60, d.f. = 3, P = 0.055 Missing 22 (36.6) 15 (25.0) 7 (23.3) 4 (13.3) Table 2: Feeding tunnels by Tomicus yunnanensis and T minor in the caged shoots of Pinus armandii and P. yunnanensis in Kunming. Ten T yunnanensis adults were released into each cage on July 28, and 10 T minor adults were released into each cage on October 24. The data were collected on December 25, 2000 and are expressed as means ± ISD. Means followed by the different letters in a column are significantly different at P < 0.05 by t-test. Tomicus species Cages Pinus species Tunnel no cage“^ Tunnel length (mm) cage“^ T. yunnanensis 6 P. yunnanensis 16.5 ± 3.7 a 24.5 ± 19.5 a 6 P. armandii 7.5 ± 3.3 b 7.9 ± 5.2 b T. minor 3 P. yunnanensis 12.0 ±2.0 a 16.1 ± 8.7 a 3 P. armandii 11.0 ±6.4 a 9.3 ± 3.3 b total attack density in R yunnanensis and P. armandii, respec- tively. Correspondingly, the brood production of T minor was only 18.9% and 13.7% of the total brood production in P. yunnanensis and P. armandii logs, respectively (Table 3). 3.3. Developmental Period. To investigate the influence of host species on the developmental period of the Tomicus, we estimated the speed of brood development of T. yunnanensis under laboratory conditions by counting the days from median attacking date to the median date of emergence (Table 4). The developmental period of T. yunnanensis was 89 days on P. yunnanensis, and 93 days on P. armandii, demonstrating that the developmental period of T. yunna- nensis was nearly similar in the logs of two host species. 3.4. Size of Emerging Beetles. In addition to developmental period, we also investigated the effect of host species on the size of emerging beetles, by comparing the dry weight of T. yunnanensis emerging from the logs of the two hosts. The result indicated that T. yunnanensis adults reared on P. yunnanensis were heavier than those reared on P. armandii (data not shown). In addition, the dry weight of T. yunnanensis brood adults was strongly related to the date of emergence. The weights of brood adults bred on both P. yunnanensis and P. armandii decreased with time after the initial brood emergence date, indicating an effect of intraspecific competition or deteriorating food quality (Figure 1). 4. Discussion The pine shoot beetles T. piniperda and T. minor have been reported from a large number of pine species and other conifers as well [10], but the principal host for them in Europe is Scots pine {Pinus sylvestris L.). Since the accidental introduction into North America, T. piniperda has been reported from a number of North American pines since the 1990s [11-13]. Experiments in Sweden and France have shown successful development in several exotic pine hosts [14]. Although both T. piniperda and T. minor occur on the exotic host lodgepole pine {Pinus contorta Douglas ex Loudon) in Sweden [15], they perform less well in this host [16]. There is another pine shoot beetle species, Tomicus destruens Wolk, in the Mediterranean area which biologically is more similar to T. yunnanensis than T. piniperda [3], and this species did better on local maritime than on boreal pine species in northern Italy [17]. In Portugal, Vasconcelos et al. found different host preferences between local populations of T. piniperda and T. destruens, that is, that northern populations preferred Aleppo pine {Pinus halepensis Miller) whereas southern populations preferred Italian stone pine {Pinus pineaL.) [18]. The shoot cage experiments showed that T. yunnanensis and T. minor are capable of feeding in the shoots of P. armandii, but more beetles died due to resin and other reasons on P. armandii than on P. yunnanensis. The resistance of conifers against invaders is mainly based on their ability to produce resin [19-22]. The resin of P. armandii was more abundant, and its concretionary speed was slower than P. yunnanensis [23]. In addition, the terpene compositions of the two pine species were also different. The shoot piths of P. armandii trees contain a lower proportion of a-pinene but a higher proportion of j3-pinene than P. yunnanensis (Borg-Karlson, A.-K., unpublished data). The observation that more beetles were killed by resin on P. armandii might be due to the stronger physical repellency and sticky property of its resin and reflected a higher resistance of P. armandii to pine shoot beetles. In addition, the small shoot diameter of P. 4 Psyche Table 3: Oviposition and brood development of Tomicus yunnanensis and T. minor in the logs of Pinus armandii and P. yunnanensis in laboratory condition, after 1 1 pairs of T. minor and 9 pairs of T. yunnanensis were introduced to each cage with two logs of P yunnanensis or P armandii. Gallery and brood production data were collected from three replicates. Larval tunnel gallery”^ and gallery length were the mean from all the egg galleries appeared in the logs (number in the bracket). Data are expressed as means ± ISD. Means followed by the different letters in a column are significantly different at P < 0.05 by t-test. Pinus species Tomicus species Galleries m ^ Brood production m“^ Larval tunnel gallery”^ Gallery length cm P yunnanensis T yunnanensis T minor 83.4 ± 12.4 a 36.80 ± 14.6 b 1449.9 ± 96.3 a 338.6 ± 36.2 b 29.1 ± 6.9 a 9.2 ± 3.1b 6.75 ± 4.2 a (68) 6.18 ± 3.59 a (30) P armandii T yunnanensis T minor 86.8 ± 9.1 a 20.43 ± 7.6 b 1693.2 ± 166.7 a 268.2 ± 23.2 b 22.4 ± 4.5 a 13.1 ± 2.2 b 5.02 ± 2.50 a (68) 4.08 ± 1.85 a (16) Table 4: Developmental periods of Tomicus yunnanensis in logs of Pinus armandii and P. yunnanensis in laboratory condition. The developmental periods were estimated from the median date of entering (50% entrance holes existed) to the median date of emergence (50% of new generation emerging from brood logs). Median date of Median date of Developmental Host species attack emerging period, days P yunnanensis Jan. 4 Apr. 3 89 P armandii Jan. 8 Apr. 12 93 armandii might also contribute to high mortality of Tomicus in this pine species during maturation feeding. The females of the two Tomicus-species accepted P. armandii as brood material, and the brood production of the two species was also similar in the two hosts, indicating that T. yunnanensis and T. minor could reproduce in the logs of P. armandii as well. However, T yunnanensis oviposited later, and the brood development was somewhat slower on P. armandii than on P. yunnanensis, suggesting that this beetle preferred the last host. Similarly, Langstrom and Hellqvist found no variation on brood production and adult weight between T. piniperda beetles reared on lodgepole pine and those reared on Scots pine, but the development time of this beetle was longer on P. contorta than on P. sylvestris [16]. Fiihrer and Miihlenbrock demonstrated that six-toothed spruce bark beetle {Pityogenes chalcographus L.) had similar brood production on its principal and secondary conifer hosts [24]. Differently, Cerezke showed that mountain pine beetle {Dendroctonus ponderosae Hopkins) was able to reproduce successfully in some pine species, but with a considerable variation in the brood production [25]. In our experiments the dry weight of T. yunnanensis brood adults emerged from Yunnan pine was higher than of those beetles that emerged from Armand pine. This observation might be due to qualitative differences in nutritional value and/or secondary metabolisms in the two hosts or just simply have resulted from the difference in phloem thickness of the two tree species. A similar pattern was found for the spruce bark beetle {Ips typographus L.) developing on its native host, Norway spruce [Picea abies (L.) Karsten], as compared to beetles emerging from an exotic host, sitka spruce [Picea sitchensis (Bong) Carriere] [26]. Since heavier bark beetles survive better than the lighter ones (a) Figure 1: Mean dry weight of emerging Tomicus yunnanensis reared on Pinus yunnanensis (a) andP. armandii (b) related to days post the initiation of brood adult emergence. Each dot represents the average adult weight for a 10-beetle sample. [27], and the fecundity of female bark beetles is related to the fat reserves available [28, 29], the lower body weight for T. yunnanensis bred from P armandii could reduce survival of the beetles when they feed in the shoot and lead to less brood production later on, Langstrom and Hellqvist found that the weights of callow adults bred on both P contorta and P. sylvestris decreased over the days following the initiation Psyche 5 of brood emergence [16]. We found the same pattern in the present experiment, both on P. yunnanensis and P. armandii. This pattern indicates an intraspecific competition and/or a deteriorating food quality. 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