Psyche: A Journal of Entomology Psyche: A Journal of Entomology Volume 2014 ISSN: 0033-2615 (Print), ISSN: 1687-7438 (Online), DOI: 10.1155/6152 Copyright © 2014 Hindawi Publishing Corporation. All rights reserved. This is volume 2014 of “Psyche: A Journal of Entomology.” All articles are open access articles distributed under the Creative Commons At- tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents A Modular Cage System Design for Continuous Medium to Large Scale In Vivo Rearing of Predatory Mites (Acari: Phytoseiidae) Juan Alfredo Morales-Ramos and Maria Guadalupe Rojas Volume 2014, Article ID 596768, 8 pages Ovicidal Activity of Couroupita guianensis (Aubl.) against Spodoptera litura (Fab.) Kathirvelu Baskar, Chelliah Muthu, and Savarimuthu Ignacimuthu Volume 2014, Article ID 783803, 5 pages Sex-Pheromone-Mediated Mating Disruption Technology for the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae): Overview and Prospects Wei N. Kong, J. Li, Ren J. Fan, Sheng C. Li, and Rui Y. Ma Volume 2014, Article ID 253924, 8 pages Retracted: Climatic, Regional Land-Use Intensity, Landscape, and Local Variables Predicting Best the Occurrence and Distribution of Bee Community Diversity in Various Farmland Habitats in Uganda Psyche Volume 2014, Article ID 546862, 1 page The Effect of Conspecific Density on Emergence of Lestes bipupillatus Calvert, 1909 (Odonata: Lestidae) Ricardo Cardoso-Leite, Gabriel G. Vilardi, Rhainer Guillermo -Ferreira, and Pitagoras G. Bispo Volume 2014, Article ID 650427, 3 pages Diversity and Composition of Beetles (Order: Coleoptera) of Durgapur, West Bengal, India Moitreyee Banerjee Volume 2014, Article ID 792746, 6 pages Notes on the Biology of the Cixiid Planthopper Cixius meridionalis (Hemiptera: Eulgoroidea) M. L. Bowser Volume 2014, Article ID 769021, 4 pages An Ultrastructural and Fluorescent Study of the Teratocytes of Microctonus aethiopoides Loan (Hymenoptera: Braconidae) from the Hemocoel of Host Alfalfa Weevil, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) Kent S. Shelby, Javad Habibi, and Benjamin Puttier Volume 2014, Article ID 652518, 9 pages Inflorescences of the Bromeliad Vriesea friburgensis as Nest Sites and Food Resources for Ants and Other Arthropods in Brazil Volker S. Schmid, Simone Langner, Josefina Steiner, and Anne Zillikens Volume 2014, Article ID 396095, 9 pages Volatile Organic Compounds from the Clone Populus x canadensis “Conti” Associated with Megaplatypus mutatus Attack Alejandro Lucia, Paola Gonzalez -Audino, and Hector Masuh Volume 2014, Article ID 793298, 6 pages Efficacy of Neem Oil on Cardamom Thrips, Sciothrips cardamomi Ramk., and Organoleptic Studies Johnson Stanley, G. Preetha, S. Chandrasekaran, K. Gunasekaran, and S. Kuttalam Volume 2014, Article ID 930584, 7 pages Preemptive Circular Defence of Immature Insects: Definition and Occurrences of Cycloalexy Revisited Guillaume J. Dury, Jacqueline G. Bede, and Donald M. Windsor Volume 2014, Article ID 642908, 13 pages Prospects for the Use of Pongamia pinnata Oil-Based Products against the Green Peach Aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) Elena A. Stepanycheva, Maria O. Petrova, Taisiya D. Ghermenskaya, and Roman Pavela Volume 2014, Article ID 705397, 5 pages A New Species of Dikrella Oman, 1949 (Hemiptera: Cicadellidae: Typhlocybinae) Found on Caryocar brasiliense Cambess. (Caryocaraceae) in Minas Gerais State, Brazil Luci Boa Nova Goelho, Germano Leao Demolin Leite, and Elidiomar Ribeiro Da- Silva Volume 2014, Article ID 871605, 5 pages Age Stage Two-Sex Life Table Reveals Sublethal Effects of Some Herbal and Chemical Insecticides on Adults of Bemisia tabaci (Hem.: Aleyrodidae) Eatemeh Jafarbeigi, Mohammad Amin Samih, Mehdi Zarabi, and Saeideh Esmaeily Volume 2014, Article ID 164271, 9 pages A Survey of Bedbug {Cimex lectularius) Infestation in Some Homes and Hostels in Gboko, Benue State, Nigeria Onah Isegbe Emmanuel, Alu Cyprian, and Omudu Edward Agbo Volume 2014, Article ID 762704, 5 pages Nothochrysinae (Neuroptera: Chrysopidae): New Larval Description and Generic Synonymy, with a Consideration of Generic Relationships Gatherine A. Tauber Volume 2014, Article ID 839261, 10 pages Evidence for the Absence of Worker Behavioral Subcastes in the Sociobiologically Primitive Australian Ant Nothomyrmecia macrops Clark (Hymenoptera: Formicidae: Myrmeciinae) Robert W Taylor Volume 2014, Article ID 232057, 7 pages Immature Stages and Life Cycle of the Wasp Moth, Cosmosoma auge (Lepidoptera: Erebidae: Arctiinae) under Laboratory Conditions Gunnary Leon-Einale and Alejandro Barro Volume 2014, Article ID 328030, 6 pages Report on a Large Collection oiMerope tuber Newman, 1838 (Mecoptera: Meropeidae), from Arkansas, with Notes on Collection Technique, Sex Ratio, and Male Clasper Size Michael J. Skvarla, Jessica A. Hartshorn, and Ashley P. G. Dowling Volume 2014, Article ID 530757, 6 pages Molecular Population Structure of Junonia Butterflies from French Guiana, Guadeloupe, and Martinique Amber P. Gemmell, Tanja E. Borchers, and Jeffrey M. 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(Apiales: Apiaceae) and Lavandula angustifolia Miller (Lamiales: Lamiaceae) against Tetranychus urticae Koch (Acari: Tetranychidae) Asgar Ebadollahi, Jalal Jalali Sendi, Alireza Aliakbar, and Jabraeil Razmjou Volume 2014, Article ID 424078, 6 pages Application of Asiatic Honey Bees {Apis cerana) and Stingless Bees {Trigona laeviceps) as Pollinator Agents of Hot Pepper {Capsicum annuum L.) at Local Indonesia Earm System Ramadhani Eka Putra, Agus Dana Permana, and Ida Kinasih Volume 2014, Article ID 687979, 5 pages Cryptocephaline Egg Case Provides Incomplete Protection from Generalist Predators (Coleoptera: Chrysomelidae) Matthias Scholler Volume 2014, Article ID 176539, 4 pages Hindawi Publishing Corporation Psyche Volume 2014, Article ID 596768, 8 pages http://dx.doi.org/ 10 . 1 155/2014/596768 Research Article A Modular Cage System Design for Continuous Medium to Large Scale In Vivo Rearing of Predatory Mites (Acari: Phytoseiidae) Juan Alfredo Morales-Ramos and Maria Guadalupe Rojas USDA-ARS National Biological Control Laboratory, Biological Control of Pests Research Unit, 59 Lee Road, Stoneville, MS 38776, USA Correspondence should be addressed to Juan Alfredo Morales-Ramos; juan.moralesramos@ars.usda.gov Received 26 September 2013; Accepted 20 October 2013; Published 9 January 2014 Academic Editor: Cleber Galvao Copyright © 2014 J. A. Morales-Ramos and M. G. Rojas. 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. A new stackable modular system was developed for continuous in vivo production of phytoseiid mites. The system consists of cage units that are filled with lima beans, Phaseolus lunatus, or red beans, P. vulgaris, leaves infested with high levels of the two- spotted spider mites, Tetranychus urticae. The cage units connect with each other through a connection cup, which also serves for monitoring and collection. Predatory mites migrate upwards to new cage units as prey is depleted. The system was evaluated for production of Phytoseiulus persimilis. During a 6-month experimental period, 20,894.9 ± 10,482.5 (mean ± standard deviation) predators were produced per week. The production consisted of 4.1 ± 4.6% nymphs and 95.9 ± 4.6% adults. A mean of 554.5 ± 59.8 predatory mites were collected per harvested cage and the mean interval length between harvests was 6.57 ± 6.76 days. The potential for commercial and experimental applications is discussed. 1. Introduction Phytoseiid mites are very effective predators used mainly in biological control of spider mites, Tetranychus urticae (Koch); however, phytoseiids are known to provide effective control of other mite species and some insects like thrips and white flies [1]. Zhang [2] reported that at least 20 species of phytoseiids have been made commercially available and have been applied mainly on greenhouse plants. The phytoseiid that has been most widely mass-produced and sold commer- cially is Phytoseiulus persimilis Athias-Henriot. Phytoseiulus persimilis is an effective biological control agent of spider mites on vegetables in glasshouses [3-5] and growers around the world use P. persimilis to control T. urticae and other tetranychid mites on crops grown in greenhouses and in the field [6, 7]. Other phytoseiid species produced commercially and used in augmentative biological control of greenhouse pests include Neoseiulus cucumeris (Oudemans), N. barkeri Hughes, N. californicus (McGregor), N. fallacis (German), Iphiseius degenerans (Berlese), and Galendromus occidentalis (Nesbitt) [2]. Gurrent methods of mass production of phytoseiid mites such as P. persimilis rely on greenhouse growth of bean plants for spider mite production and later inoculation with the predatory mite. A pure spider mite culture, free of predators, is also required for rearing. Infested leaves from the pure cul- ture are used to infest bean plants in a different greenhouse. A series of greenhouse benches are inoculated at weekly intervals to provide continuous supply of prey. Predators are later introduced to bean plants heavily infested with spider mites and grown for 2-3 weeks. A section of the bench is harvested when it has reached the maximum predator density [8]. Introduction of P. persimilis into the infested beans requires perfect timing to allow maximum spider mite reproduction without losing the plants to the mite infestation [8]. Predator harvesting often exposes the predators to stress- ful conditions of starvation and many are lost to inefficient collection methods. Enclosed rearing systems offer the potential of greater control of environmental conditions and better containment preventing excessive losses. Several methods for rearing phytoseiid mites in enclosed systems or cages by introducing 2 Psyche prey have been proposed consisting of dishes with a central area limited by a channel filled with machine oil or other liquids [9-11]. Theaker and Tonks [12] reared P. persimilis in floating plastic leads positioned by magnets in the middle of a water- filled container to prevent mites from escaping. A similar method based on a plastic foam block or sponge positioned in the middle of a tray filled with water was described by Overmeer [13] to rear several species including P. persimilis, P macropilis (Banks), Typhlodromus occidentalis Nesbitt, T. pyri Scheuten, Amblyseius (Neoseiulus) fallacis (German), A. potentillae (German), and N. cucumeris (Oudemans). A barrier is formed by placing wet tissue paper around the block with one side touching the water to maintain a continuous saturation [13]. McMurtry et al. [14] describe a method of mass rearing of phytoseiid mites by washing eggs and other spider mite stages from infested leaves. The washed spider mites are then fed to predatory mites reared using the paper- lined block in a water tray method as described by Overmeer [13]. Series of these trays are stacked inside shelved wood boxes. Shih [15] developed a method to separate the prey mites {T. urticae) from plant leaves and an apparatus which used pneumatic pressure to dispense a mix of the prey and corn pollen to rear Amblyseius womersleyi Schicha using the same lined semisubmerged block method. Fournier et al. [16] proposed a cage system for rearing P. persimilis consisting of series of superimposing cylinders filled with bean leaves heavily infested by spider mites. New cylinders with infested leaves are added to the top of the series to supply new prey. The cylinders at the bottom are retired as predators move into cylinders with fresh prey [16]. Another cage system was described by Overmeer [13] consisting of two cardboard ice cream containers glued together and separated by a screen. Infested leaves are placed in the lower side and predators are introduced. New leaves are placed in the upper side where predators tend to move to find new prey. The whole system is flipped over to place new leaves while removing the old material [13]. While many enclosed rearing systems have been effective to mass-produce phytoseiid mites commercially, none of the existing enclosed systems can match the production capabil- ities of the open greenhouse rearing methods. An increas- ing level of sophistication will be required to reach a compara- ble level of production using enclosed systems. The objective of this study was to develop and test a refined enclosed rearing system based on the Fournier et al. [16] cage series. 2. Materials and Methods 2.1. Rearing of the Prey. Spider mites, Tetranychus urticae Koch, were used as prey to feed the phytoseiid mites. Golonies of T. urticae were established from commercial stocks pro- vided by Syngenta Bioline, Oxnard, GA, and were reared on red kidney beans, Phaseolus vulgaris L., and lima beans, Phaseolus lunatus L., cultivars Fordhook 242 and Henderson in a greenhouse. The greenhouse was divided into two areas by using a clear polyethylene curtain. Lima beans were grown in one-half of the greenhouse using 60 x 20 x 20 cm Figure 1: Materials used to construct the cage system. (A) Polypropylene 50 mL centrifuge tube with screwed cover, (B) polypropylene 250 mL lab funnel, (C) polypropylene Ziploc storage containers, and (D) high density polyethylene 120 mL specimen containers. polyethylene planters. The bottom of each planter was lined with 2.5 L perlite (Goarse, Sunshine, SunGro Horticulture, Bellevue, WA) to support and maintain humidity. A mixture of 2 : 1 potting soil (Moist control, Miracle-Gro Marysville, OH), vermiculite (Goarse, Sunshine, SunGro Horticulture, Bellevue, WA), and 5 g slow release fertilizer (N : P : K = 14:14:14) (vegetable and bedding, Osmocote, Marysville, OH) was mixed and then combined with an equal volume of a mixture of 20 g TeraGel (T-400, The Terawet Gorporation, San Diego, CA), 0.5 g water soluble fertilizer (N:P:K = 24:8: 16) (All-purpose plant food, Miracle-Gro, Marysville, OH), and 2.5 L tap water. The aqueous solution was allowed to equilibrate for 24 h until the water was fully absorbed by the TeraGel crystals and then it was homogeneously incor- porated into the potting soil and vermiculite mixture using a gardening trowel. Seventy seeds were planted and kept for 10 days in each planter for germination. Ten days after ger- mination, planters with young bean plants were transferred to the second half of the divided greenhouse and plants were then massively infested with T. urticae by placing leaves from heavily infested plants on top of them. Spider mite infestation levels were allowed to increase for 5 days after their introduction. Fully infested plants were monitored daily to determine optimal infestation levels. Extreme infestation levels kill the bean plants inducing mas- sive migration of spider mites. Infested bean leaves were col- lected when they were still alive (leaves still green) and sustain a high density of spider mites. Infested leaves were collected daily by cutting them manually using garden scissors and placing them in plastic boxes. Boxes with infested bean leaves were stored at 15° G for 1 to 7 days. 2.2. Predatory Mite Rearing and Cage Design. Although the rearing system presented herein is suitable for any phytoseiid predator of spider mites, P. persimilis was used as the basis to test the system. The rearing system is based on the same principles of Fournier et al.s [16] stacked cage method, but Psyche 3 (a) (b) (c) (d) Figure 2: Basic cage system modular components, (a) Cage bottom, (b) cage cover, (c) connection cup, and (d) cage series stand. we designed a unique modular structure of identical cage units. Stackable modular units were constructed from 473 mL Ziploc storage containers (Ziploc Twist’n Loc, S.C. Johnson & Son, Inc., Racine, WI), 250 mL plastic laboratory fun- nels (Fisherbrand 10-500-2, 10.5cmdia. x 10.3 cm H), plastic 50 mL centrifuge tubes (Corning No. 430897), and 120 mL specimen containers (LSS number 9BC-135972) (Figure 1). Materials for cage construction were chosen based on the quality of a water-tight screwed cover closure. Snap closures tend to fail with continuous use and mites quickly find escape openings. The cage system consisted of 4 basic parts that were modified to fit together: a cage bottom (Figure 2(a)), a cage cover with funnel connection (Figure 2(b)), a con- nection cup (Figure 2(c)), and a multiuse funnel to serve as stand (Figure 2(d)). The covers of the Ziploc containers (Figure 1(c)) were cut to allow the insertion of the lab funnels to the cage covers (Figure 1(b)). The tips (narrow ends) of the funnels were cut to install “male” screw sections of centrifuge tubes to allow closure when required (Figures 2(b) and 2(d)). “Female” screw sections of the covers of specimen containers (Figure 1(d)) were cut and glued to the funnels and bottoms of the cage to allow connection with other cage units (Figures 2(a) and 2(b)). “Male” screw sections of centrifuge plastic tubes (Figure 1(a)) were also glued to the bottom of the connection cups (Figure 2(c)) to allow closure during mite collection and movement when connected to new cage units. Connection cups were also fitted with a second “male” screw section in the bottom allowing connection to both ends (Figure 3(c)). A cage unit consisted of bottom, top, and connection cup (Figure 3(a)). The system stand was used only in the starting cage (Figure 3(b)) and was fitted with a “male” instead of a “female” screw section from specimen containers (Figure 2(d)) to allow connection to the bot- toms (Figure 2(a)) of the cages. Four circular windows (22mmdia.) were cut on the sides of the cages bottom and four more (17 mm dia.) were cut on the sides of the funnels for ventilation. Only one circular window (10 mm dia.) was cut on one side of the connection cups to reduce excessive loss of moisture. Nylon screen 85 fim mesh (Small Parts Inc., U-CMN-85) was used to seal the windows preventing mites 4 Psyche (b) (c) (d) Figure 3: Cage system assembly, (a) Cage unit components, (b) cage unit assembled, (c) connection cup with second cage unit fitted, and (d) cage series assembly of two cage units. from escaping. Cage units were designed to fit together in a modular way by connecting the bottom to the connection cup (Figures 3(c) and 3(d)). Bean leaves heavily infested with T. urticae were placed inside each cage unit stacked vertically to allow mites to move up (Figures 4(a) and 4(c)). A cage series can be started by introducing a few adult predatory mites (10-100) into a cage unit newly filled with infested bean leaves. To start a cage series, a connection cup with mites is fitted to the bottom of a new cage unit (Figure 4(a) arrow and Figure 4(b)). This cup can later be replaced by the stand described in Figure 2(d) to provide better stability. After prey mites had been depleted, a new unit is attached to the top of the old unit by removing the cover of the connection cup (Figure 4(d)) to allow predators to move into the new unit. The cover (full of predators) is placed inside the new unit (Figures 4(e) and 4(f)) and the cage is closed with a new funnel (Figures 4(g) and 4(h)). A new connection cup is attached to the top of the funnel (Figure 4(i)) to complete the system (Figure 4(j)). Nymph and adult predators tend to migrate to the upper end of the cage series and accumulate in the uppermost connection cup feeding on migrating spider mites. 2.3. Evaluation. The cage system was evaluated using P. persimilis as model. Evaluation started on 1 January 2007 by establishing 21 cage series. Each cage series started with approximately 100 adult predators. New cage series were created using predatory mites produced by the initial series. Some cage series had to be terminated and replaced due to contamination by other predatory mite species {Neoseiulus sp.) from the greenhouse spider mite production. Series increased in number to a maximum of 48 at the end of the study on 1 July 2007. The study was conducted in an envi- ronmentally controlled room at 26 ± 1°C, 80 ± 5% RH, 14 h photophase, and 10 h scotophase. Cage units were added to the top of each cage series as described above. Prey mites consisting of T. urticae were reared as described above using P. lunatus Henderson variety. When the connection cup contained a visibly high density of predatory mites, the cup was quickly disconnected from the Psyche 5 Figure 4: Cage system operation, (a) A cage series starts with a connection cup with predators connected to a cage bottom filed with spider mite-infested bean leaves, (b) The starting cage unit is closed and a connection cup is fitted, (c) When spider mites have been depleted by the predators, a new cage unit is prepared, (d) The cover of the connection cup is removed to allow predators to move to the new cage, (e) A new cage bottom is fitted to the connection cup. (f) The connection cup cover is placed inside the new cage, (g) The new cage is covered and (h) sealed, and (i) a new connection cup is fitted to the cover, (j) The process can be repeated by adding a third cage unit when prey has been depleted. At this point, the bottom connection cup can be replaced by the stand piece. series, inverted, taped to make predator fall to the bottom, and filled with 70% ethanol to kill and preserve the predatory mites. Mites were counted and the numbers were recorded. Data consisting of days between harvest, collection date, cage series, and number of P. persimilis collected were recorded. Data were analyzed using single-variable statistics to deter- mine means of mite production per week, mites produced per cage, and mean time from initiation to collection. In this study, predatory mites were collected and killed with 70% ethanol in order to obtain precise numbers. How- ever, live predators can be quantified while alive using less precise methods. One method is based on weight: first, a mean of individual weight is determined by weighing groups of mites in a precision balance; second, an empty collection cup (with cover) is weighed and used to collect predatory mites by attaching it to the top of the cage system. The cup full of mites can be closed (with the previously weighed cover) and weighed a second time. The weight of the live mites can be determined by subtracting the weight of the empty cup from the weight of the full cup. Another method consists of determining the number of mites fitting in a given volume. Mites can be forced by gentle vacuum into receptacles with known volume. When filled, the receptacle can be emptied by reversing the airflow. 3. Results and Discussion During the six-month evaluation period, the mean weekly production was 20, 894.9 ± 10, 482.5 (mean ± standard devi- ation) P. persimilis. Production consisted of 4.1 ± 4.6% deu- tonymphs and 95.9 ± 4.6% adults. Overall production mean was 554.5 ±59.8 mites per harvested cage and a mean of 36.7 ± 17.0 cages were harvested per week. The mean interval of time between harvests was 6.57 ± 6.76 days; however, the length of the harvest intervals did not have a normal dis- tribution and the median was 4 days and the 75% quartile 6 Psyche was 5 days (Figure 6). The top quartile consisted of intervals ranging from 6 to 62 days in length and the 90% percentile was 14 days. Based on this analysis, the time interval between harvests should not exceed 14 days. If by 14 days the pop- ulation of predators is still too low to justify harvest, the cage series should be terminated and a new series should be generated from predator production of a healthy series. The connection cup usually contains large quantities of spider mites, which constantly migrate when they are in high densities. As predatory mites increase in numbers, they consume all the prey in the connection cup. Juvenile preda- tory mites (young adults and deutonymphs) tend to rapidly migrate upwards to the new cage unit. Gravid adult females usually remain in the bottom cage unit until they oviposit. As the last eggs are oviposited and prey is depleted, females move upwards to the new cage unit. As the population of predatory mites increases, it becomes necessary to add new cage units within increasingly shorter periods of time. Empty cage units at the bottom can be removed after all the eggs have hatched and the juveniles have moved to new cage units. The connection cup at the top of the series serves as an indicator of predatory mite population. The predators in the connection cup may be recycled by fitting a new cage unit or harvested by removing the connection cup and closing it with an unmodified cover of a specimen container. Decision to harvest the predatory mites depends on the density of juveniles in the connection cup (Figure 5). Once the predatory mite population is well established in a cage series, it becomes necessary to harvest the predatory mites every 2 to 4 days depending on the quality of prey provided. Harvested mites can be used to start new cage series, for field releases, or for use in experiments. A cage series can be continuously producing predatory mites indefinitely as long as new cage units are added to the top of the series. Peak weekly production occurred during the week between March 11 and 18 with a total of 38,097 adults and nymphs harvested (Figure 7(a)). During this period, 41 cage series were in operation (Figure 7(b)). Mean production per harvested cage was more or less consistent during the exper- imental period (Figure 7(c)). The total weekly production dropped sharply by the end of the experiment even as the number of series increased; however, the number of mites per harvested cage was increasingly consistent evidenced by the decrease of the standard deviation (Figure 7(c)). A study of three-trophic-level impact of secondary chem- icals present in lima beans showed that high levels of linamarin present in Henderson lima beans tend to accu- mulate in the predatory mites after several generations [17]. Accumulation of linamarin may have been the reason the production dropped by the end of the study even as the number of cage series increased. The use of this variety is not recommended for continuous production of phytoseiid mites. Fordhook 242 lima beans provide a better alternative because they contain only trace amounts of linamarin (M. G. Rojas unpublished). Henderson lima beans were selected for this experiment because they are easy to grow and the size of their leaves is optimal to fit inside the cages described in this study. Fordhook 242 lima beans have larger leaves, which must be folded or cut to fit in the cages. Another good choice Figure 5: Connection cup with P. persimilis after prey had been depleted. A piece of paper can be introduced to the connection cup to increase the surface area. Time interval to harvest (days) Figure 6: Frequency distribution of predator harvest intervals. Box plot (top): bars represent 25% and 75% quartiles, line between bars represents the median, dashed line represents the mean, bracket represents the 90% percentile, and dots represent outliers. Bar plot (bottom): bars represent 2-unit classes. of host plant is red beans, which have similar leaf sizes to those in Henderson lima beans. However, a system could be constructed with larger cage units providing more space to accommodate larger leaves. The size limits for the system have not been determined, but 8 liter (2-gallon) size units have been constructed and tested successfully (Figure 8). In theory, the system can be scaled up to accommodate production levels of millions of mites per week. Potential size limits include the structural integrity of currently available materials taking into account the weight of the leaves that must be held by the cage units. The tightness of the closures between cage unit connections can be more difficult as cage size increases. The tolerance of closures between cage units cannot exceed 100 pm to contain Psyche 7 Figure 7: Production of P. persimilis. (a) Total weekly production, (b) Number of cage units in production, (c) Means of predators produced per harvested cage; brackets represent standard deviation. the mites within the cages, and this becomes increasingly difficult as the size of the screw cups increases. 4. Conclusions The modular cage system presented in this study has been shown to be a consistent and robust method to produce phy- toseiid mites. The system is particularly suitable for medium to large scale rearing of P. persimilis. This system provides a good alternative for phytoseiid mites rearing and potentially can be scaled up for mass production. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 8: Larger size cage system design. Acknowledgments The authors thank D. Cahn, Syngenta Bioline, for providing the rearing stocks of P. persimilis and T. urticae and partial funding through CRADA agreement 58-3 K95-0-1428. They acknowledge the peer reviewers for their comments on an earlier version of this paper. The United States Government has the right to retain a nonexclusive, royalty-free license in and to any copyright of this paper. The mention of a com- mercial or proprietary product does not constitute an endorsement of the product by the United States Department of Agriculture (USDA). USDA is an equal opportunity pro- vider and employer. References [1] K. J. F. Bolckmans, “Mass-rearing phytoseiid predatory mites,” in Proceedings of the Working Group AMRQC, C. van Lenteren, P. DeClercq, and M. W. Johnson, Eds., vol. 3, pp. 12-15, Bulletin lOBC Global, 2007. [2] Z. Zhang -Q, Mites of Greenhouses Identification, Biology and Control, CABI, Oxon, UK, 2003. [3] D. A. Chant, “An experiment in biological control of Tetrany- chus telarius (Linneaus) (Acarina: Tetranychidae) in a green- house using the predacious mite Phytoseiulus persimilis Athias- Henriot (Phytoseiidae),” The Canadian Entomologist, vol. 93, no. 6, pp. 437-443, 1961. [4] T. J. Legowski, “Experiments on predator control of the glass- house red spider mite on cucumbers,” Plant Pathology, vol. 15, pp. 34-41, 1966. [5] N. Erench, W. J. Parr, H. J. Gould, J. J. Williams, and S. P. Sim- monds, “Development of biological methods for the control of Tetranychus urticae on tomatoes using Phytoseiulus persimilis” Annals of Applied Biology, vol. 83, no. 2, pp. 177-189, 1976. [6] J. C. van Lenteren, “Commercial availability of biological con- trol agents,” in Quality Control and Production of Biological Con- trol Agents: Theory and Testing Procedures, J. C. van Lenteren, Ed., pp. 167-179, CABI, Oxon, UK, 2003. 8 Psyche [7] J. C. van Lenteren, “The state of commercial augmentative biological control: plenty of natural enemies, but a frustrating lack of uptake,” BioControl, vol. 57, no. 1, pp. 1-20, 2012. [8] L. A. Gilkeson, “Mass rearing of phytoseiid mites for testing and commercial application,” in Advances in Insect Rearing for Research and Pest Management, T. E. Anderson and N. C. Leppla, Eds., pp. 489-506, Westview Press, Boulder, Colo, USA, 1992. [9] S. S. Kamburov, “Methods of rearing and transporting preda- cious mites,” Journal of Economic Entomology, vol. 59, pp. 875- 877, 1966. [10] G. T. Scriven and J. A. McMurtry, “Quantitative production and processing of tetranychid mites for large-scale testing of predator production,” in Journal of Economic Entomology, vol. 64, pp. 1255-1257, 1971. [11] T. Kostiainen and M. A. Hoy, “Egg-harvesting allows large scale rearing of Amblyseiulus finlandicus (Acari; Phytoseiidae) in the laboratory,” Experimental & Applied Acarology , vol. 18, pp. 155- 165, 1994. [12] T. L. Theaker and N. V. Tonks, “A method of rearing the preda- ceous mite Phytoseiulus persimilis (Acarina: Phytoseiidae),” Journal of the Entomological Society of British Columbia, vol. 74, pp. 8-9, 1977. [13] W. P. }. Overmeer, “Rearing and handling,” in Spider Mites: Their Biology, Natural Enemies, and Control, W. Helle and M. W. Sabelis, Eds., pp. 161-170, Elsevier, Amsterdam, The Nether- lands, 1985. [14] J. A. McMurtry, G. T. Scriven, S. N. Newberger, and H. G. Johnson, “Methodologies of rearing, introducing, and estab- lishing phytoseiid mites,” in Proceedings of the ADAP Crop Protection Conference, vol. 134 of Research Extension, pp. 104- 110, HITAHR, Honolulu, Hawaii, USA, 1989. [15] C. I. T. Shih, “Automatic mass-rearing of Amblyseius womersleyi (Acari: Phytoseiidae),” Experimental and Applied Acarology, vol. 25, no. 5, pp. 425-440, 2001. [16] D. Eournier, P. Millot, and M. Pralavorio, “Rearing and mass production of the predatory mite Phytoseiulus persimilisj Ento- mologia Experimentalis et Applicata, vol. 38, no. 1, pp. 97-100, 1985. [17] M. G. Rojas and }. A. Morales-Ramos, “Tri-trophic level impact of host plant linamarin and lotaustralin on Tetranychus urticae and its predator Phytoseiulus persimilisj Journal of Chemical Ecology, vol. 36, no. 12, pp. 1354-1362, 2010. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 783803, 5 pages http://dx.doi.org/10.1155/2014/783803 Research Article Ovicidal Activity of Couroupita guianensis (Aubl.) against Spodoptera litura (Fab.) Kathirvelu Baskar, Chelliah Muthu, and Savarimuthu Ignacimuthu Entomology Research Institute, Loyola College, Chennai 600 034, India Correspondence should be addressed to Kathirvelu Baskar; suribaskar@hotmaiLcom Received 19 August 2013; Accepted 5 November 2013; Published 20 January 2014 Academic Editor: Jacques Hubert Charles Delabie Copyright © 2014 Kathirvelu Baskar 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. Hexane, chloroform, and ethyl acetate extracts of Couroupita guianensis leaves were studied for ovicidal activity against S. litura. All the extracts showed ovicidal activity against S. litura. Maximum activity was noticed in hexane extract and it showed the least LC 50 and LC 90 values; the regression equation was also higher than the other extracts. All the analyzed values showed homogeneity variance. The active hexane extract was fractionated and eight fractions were isolated. The fractions were studied at different concentrations. Among the fractions, fraction 8 showed maximum ovicidal activity with least LCgg and LCgg values. Fraction 8 differed statistically from the other fractions; the regression equation value was higher than the other fractions. All the P values obtained from regression analysis were significant. The results of the present investigation clearly suggest that the active fraction could be purified to isolate active compound(s) and could be used to develop an insecticidal formulation to control economically important agricultural pests. 1. Introduction India is an agricultural country and more than 80% of the population depend on agriculture [1] . Pathogenic organisms and insect pests cause crop loss of 120 billion US dollars worldwide and reduce the yield by 20-40% [2]. In India, approximately 18% of food grains are lost due to pathogens and insect pests. To control the pests and reduce the loss, different chemical pesticides are used. Application of chemi- cal pesticides is polluting the environment, causing ill effects on nontarget organisms, developing resistance, and causing resurgence of pests [3]. These call for an alternative to chemical pesticides through natural means of pest control, including vigorous search for new sources of botanical insecticides [4]. Plant-based pesticides are highly suitable since they have low toxicity, are easily biodegradable, and have multimode of action [5]; they are suitable for organic agriculture [6]. Botanical extracts are used as insecticides for cen- turies and their active compounds reduce the opportunity for the development of insect resistance [7]. Plants have evolved a range of adaptations to increase their survival and reproduction by minimising the impact of phytophagous insects. Plants defend themselves from herbivores with the help of secondary metabolites produced by them and these secondary chemicals can act as repellents or toxins to herbi- vores and affect their behaviour, growth, or survival. Volatile plant signals attract natural enemies of the herbivore insect pests [8]. Presently, botanicals are used as insecticides which constitute only 1% of the world insecticide market [9] . Plant-derived substances have multimode of actions against different agricultural pests and act as antifeedants [10] and larvicidal [1] agents; they reduce adult emergence and increase adult abnormalities [11, 12]; they inhibit larval growth [13] and cause ovicidal and oviposition deterrent activities [14]; and they bring about cytological changes [5]. Couroupita guianensis leaves extracts showed antifeedant, larvicidal, and ovicidal activities against Helicoverpa armigera [15, 16] and antifeedant activity against Spodoptera litura [17]. S. litura is a major polyphagous pest attacking more than 150 host species affecting the yield [18]. It causes serious damage to young plants and the buds of different vegetable crops in Thiruvallur and Kancheepuram districts of Tamil Nadu. The present study was aimed to evaluate the ovicidal activity of 2 Psyche different crude extracts and fractions of C. guianensis against S. litura. 2. Materials and Methods 2.1. Plant Collection. Leaves of C. guianensis were collected from Loyola College Campus, Chennai, Tamil Nadu, India. The plant was identified by Dr. M. Ayyanar, Taxonomist, Entomology Research Institute, Loyola College. The voucher specimen (ERIH: 1310) was deposited at the institute herbar- ium. The plant material was shade- dried at room temperature and powdered coarsely. The plant materials were sequentially extracted using hexane, chloroform, and ethyl acetate. The active hexane extract was fractionated using silica gel column chromatography with increasing polarity of hexane : ethyl acetate combinations. Isolated fractions were concentrated using vacuum rotary evaporator with reduced pressure and the collected fractions were stored at 4°C in the refrigerator [15]. 2.2. Insect Culture. Egg masses of S. litura were collected from groundnut field at Tiruttani in Thiruvallur District of Tamil Nadu. The eggs were surface- sterilized with 0.02% sodium hypochlorite solution, dried, and allowed to hatch. After hatching, the neonate larvae were reared on leaves of castor, Ricinus communis, till prepupal stage. Sterilized soil was provided for pupation at room temperature (27 ± 2°C) with a photoperiod of 14 : 10 (light : dark) and 75 ± 5% relative humidity in insectary. After pupation, the pupae were collected from the soil and placed inside the ovipo- sition chamber. After adult emergence, cotton soaked with 10% (w/v) sugar solution with few drops of multivitamins was provided for adult feeding to increase the fecundity. Potted groundnut plant was kept inside adult emergence cage for egg laying. After hatching, the larvae were fed with tender castor leaves. The eggs laid by the laboratory reared insects were used for the present study [10]. 2.3. Ovicidal Activity. The ovicidal activity of the crude extracts and fractions was studied by spraying them on freshly laid eggs of S. litura. The sprayed concentrations were 5, 10, 25 and 50 mg/mL for crude extracts and 125, 250, 500 and 1000 fig/mL for fractions. Spray solution of 0.5 mL was used per replicate. Azadirachtin was used as positive control [19]. Eive replicates were maintained for each treatment with 20 eggs per replicate (total n - 100). The experiment was conducted at laboratory conditions (room temperature of 27 ± 2°C with 14 : 10 (light : dark) photoperiod and 75 ± 5% relative humidity). The number of eggs hatched in control and treatments was recorded up to 96 hrs. Percent of egg mortality was calculated according to Abbott [20] . 2.4. Statistical Analysis. The ovicidal activity was analysed using one-way ANOVA. Significant differences between treatments were determined using Tukey’s multiple-range HSD tests {P < 0.05). Analyses were performed with the original data after transformation with various approaches (the arcsin, logarithmic, and square root methods). The distribution of the fraction data did not show significant deviations from normality. Shapiro -wilk test for original crude data showed normality. Linear regression analyses were performed for all dose-response experimental data. LC50 and LC90 values were calculated using probit analysis [21]. 3. Result Ovicidal activity of different crude extracts of C. guianensis against S. litura is presented in Table 1. Maximum ovici- dal activity of 67.33% was observed in hexane extract at 50 mg/mL concentration. The chloroform and ethyl acetate extracts showed ovicidal activity of 47 and 42%, respectively. Chloroform and ethyl acetate extracts showed statistically similar activity. Hexane extract was statistically different from chloroform and ethyl acetate extracts. At 25 mg/mL con- centration, hexane extract exhibited 51.17% ovicidal activity against S. litura followed by chloroform and ethyl acetate extracts. All the three extracts statistically differed from each other at 25 and 50 mg/mL concentrations. Hexane extract exhibited 39.52% ovicidal activity at 10 mg/mL concentration against S. litura which was statically similar to chloroform extract that showed 31.20% ovicidal activity (P value 0.63). Lowest concentration of hexane and chloroform extracts showed statistically similar (P value 0.92) ovicidal activity. All the concentrations of ethyl acetate extracts showed minimum ovicidal activity. The homogeneity of variance was significant at all the analyses; also the ANOVA was significant (P value 0). The R indicated that increasing concentration of the extracts increased the activity (Table 1). Regression ANOVA derived from all the three extracts showed significant value (P value 0). The minimum quantity of hexane extract needed to kill 50% eggs of S. litura is shown in Table 1. Ethyl acetate extract required maximum quantity (55.94 mg/mL) for 50% egg mortality of S. litura. The obtained y values were significant for all the tested extracts. The probit analysis clearly indicates that the hexane extract has the potential to kill the eggs of S. litura. Bio assay-guided fractionation of hexane extract was done and finally 8 fractions were obtained; they were screened at different concentrations. Among the fractions tested, fraction 8 showed maximum ovicidal activity of 30.46% at 125 ^wg/mL concentration (Table 2) followed by fractions 3 and 7 which showed ovicidal activity of 28.24 and 23.91%, respectively, fractions 3, 7, and 8 were statistically similar (P value 0.15). Minimum ovicidal activity of 4.32% was noticed in fraction 5. fractions 4 and 2 were statistically similar to fraction 5 (P value 0.15). At 250|Wg/mL concentration, fraction 8 exhibited 51.05% ovicidal activity. Minimum ovicidal activity was noticed in fraction 4. fractions 1, 3, and 7 exhibited more than 30% ovicidal activity. Fraction 8 showed 59.82% ovicidal activity at 500/^g/mL concentration followed by fractions 7, 3, and 1. Maximum ovicidal activity of 71.69% was noticed in fraction 8 at 1000 /^g/mL concentration followed by fraction 7 which exhibited 60.93% ovicidal activity. Minimum ovicidal activity of 19.53% was noticed in fraction 5 which was statistically similar to fraction 4 (P value 1). Fractions Psyche 3 Table 1: Ovicidal activity and effective concentrations (mg/mL) of Couroupita guianensis crude extracts against Spodoptera litura. Solvent extract 5 Concentration (mg/mL) 10 25 50 R R^ Regression equation P value h^50 LC 90 Hexane 21.98 ± 4.03’' 39.52 ± 5.94'’ 51.17 ±5.94" 67.33 ± 4.03’’ 0.92 0.84 26.78 ± 0.899 0.000 28.05 82.50 44.04* Chloroform 23.16 ± 5.27'' 31.20 ± 5.20'’ 41.82 ± 5.52*’ 47.57 ± 4.70^ 0.84 0.70 24.60 ± 0.050 0.000 49.99 145.40 31.02* Ethyl acetate 6.93 ± 4.77^ 16.16 ±4.20^ 24.41 ± 2.12^ 42.85 ± 6.26‘' 0.95 0.89 5.76 ± 0.75 0.000 55.94 108.28 36.31* ANOVA Df2, 12,F.18, Df2, 12, Df2, 12, Df2, 12, 35 PO F.26.15 P 0 F.39.31 P 0 F.32.54 P 0 Homogeneity 0.67 0.52 0.13 0.38 Means followed by the same letter do not differ significantly using Tukey’s test (P < 0.05) and complete regression equations; values are significant. Table 2: Ovicidal activity and effective concentrations (|Mg/mL) of Couroupita g uianensis hexane fractions against Spodoptera litura. Fractions 125 Concentration (ftg/mL) 250 500 1000 R R^ Regression equation P value hCso LC90 a:' 1 17.42 ± 4.6 1"^^ 32.63 ± 4.04"^^ 43.50 ± 4.13‘^" 51.11 ± 3.10" 0.87 0.76 20.11 ±0.034 0.000 871.14 2268.09 43.11* 2 10.81 ±3.62^^" 17.30 ± 4.10^’’ 24.97 ± 4.52’' 32.57 ±5.12’’ 0.87 0.76 10.37 ± 0.024 0.000 1509.42 3137.48 27.91 3 28.24 ± 1.96" 35.84 ± 4.45‘^ 42.39 ± 2.28‘^ 47.83 ± 2.30" 0.88 0.78 28.99 ± 0.020 0.000 1021.37 3426.26 12.92 4 7.60 ± 2.94^'’ 13.04 ± 2.93^ 15.20 ± 2.33^ 19.70 ± 5.79^ 0.81 0.65 7.53 ± 0.015 0.000 2213.23 4273.60 21.80 5 4.32±4.6H 11.98 ±6.14^ 17.42 ± 2 . 7 T 19.53 ± 2.65^ 0.72 0.52 6.21 ±0.015 0.000 2131.16 3997.78 55.51* 6 15.26 ± 4.75'’" 23.85 ± 4.52'"" 32.57 ± 3.29" 42.33 ± 3.66" 0.91 0.83 14.89 ± 0.020 0.000 1167.88 2688.15 25.02 7 23.91 ± 2.94^^" 40.17 ±5.69‘^ 50.00 ± 4.34" 60.93 ± 5.31^^ 0.89 0.79 26.09 ± 0.038 0.000 636.06 1951.39 39.33* 8 30.46 ± 3.24" 51.05 ±4.18" 59.82 ± 4.30^ 71.69 ± 2.90" 0.89 0.79 34.14 ± 0.041 0.000 384.43 1576.55 41.13* Azadirachtin 42.33 ± 3.66^ 54.26 ± 4.01" 65.20 ± 2.98^ 76.02 ± 3.50" 0.92 0.85 42.76 ± 0.036 0.000 206.42 1525.75 18.92 ANOVA Df8,36F 54.93 Df8, 36 F 59.80 Df8, 36 F 126.85 Df8,36F 111.95 Homogeneity 0.74 0.93 0.60 0.001 Means followed by the same letter do not differ significantly using Tukey’s test (P < 0.05) and complete regression equations; values are significant. 1, 3, and 6 showed ovicidal activity between 42 and 51% and were statistically similar (P value 0.065). The data of all the fractions showed homogeneity variance except at 1000 f/g/mL concentration while using one-way ANOVA. The R value exhibited concentration dependent activity. Minimum R value was observed in fraction 5 which showed less than 20% ovicidal activity at maximum concentration. Higher concentration of the fraction increased the ovicidal activity. Maximum regression coefficient was observed in fraction 8 followed by fraction 7. Minimum regression coefficient value was noticed in fraction 5 (Table 2). All these data clearly indicated concentration-dependent activity. All the analysed regression data were significant (P value 0). Minimum LC50 and LC90 values of 384.43 and 1576.55 |Wg/mL, respectively, were obtained in fraction 8 (Table 2). Fraction 4 showed maximum LC50 and LC90 values of 2213.23 and 4273.60 |Wg/mL, respectively. Fraction 5 had lower percent of ovicidal activity than the other fractions; in case of probit analysis, fraction 4 showed lower value than fraction 5. Fractions 1 and 7 showed less than lOOO/rg/mL LC50 values. Fractions 1, 5, 7, and 8 showed significant values. 4. Discussion Hexane, chloroform, and ethyl acetate extracts of C. guia- nensis showed ovicidal activity against S. litura. This finding corroborates with the findings of Deepa and Remadevi [22] who reported that the petroleum ether, chloroform, ethyl acetate, methanol, ethyl alcohol, and acetone extracts of Acacia concinna and Butea monosperma showed ovicidal activity against lepidopteran insect, Hyhlaea puera. Similarly, water extract exhibited ovicidal activity against Sambucus ebulus and Tribolium confusum [23]. Crude extracts with a mixture of compounds showed strong ovicidal activity against S. litura in this study. Similarly, many researchers around the world have reported many plant extracts with ovicidal activity. Myrtus communis, Melaleuca alternifolia, Pimenta dioica, Syzygium aromaticum, Eucalyptus citriodora, and E. globulus exhibited ovicidal activity against Trialeurodes vaporariorum [24]; E. globulus and Syzygium aromaticum showed ovicidal activity against Tribolium castaneum [25, 26]; and E. camaldulensis showed ovicidal activity against T confusum and Ephestia kuehniella [27]. Methanol extract of Celosia argentea, Ricinus communis, Mikania micrantha. 4 Psyche and Catharanthus roseus reduced the egg hatchability in Brontispa longissima and maximum reduction was observed in M. micrantha [28]. Similarly, citronella oil reduced the egg hatchability up to 95% against Helicoverpa armigera [29]. Fractions from hexane extract showed ovicidal activity against S. litura. Fractions exhibited maximum ovicidal activ- ity at lower concentrations than the crude hexane extract. This result corroborates with the findings of Jeyasankar et al. [30] who reported that ethyl acetate extract, its fractions, and isolated compound showed ovicidal activity against S. litura. Maximum activity was noticed at lower dose in the purified compound than the higher dose treated fractions and crudes extracts. In the present study, the presence of alkaloids, coumarin, and quinone in the hexane extract could be responsible for ovicidal activity against S. litura. Similarly, Maciel et al. [31] reported that the presence of different phyto chemicals like tannins, triterpenes, and alkaloids in the ethanol extract of leaves and seeds of M. azedarach is responsible for ovicidal activity. In the present study, partially purified extract (fractions) showed maximum ovicidal activ- ity against S. litura. Similar results were obtained by Alouani et al. [32] against mosquito larvae. Hexane extract and fraction 8 exhibited ovicidal activity against S. litura with least LC50 values than the other extracts and fractions. In this study, hexane extract eluted fractions using hexane : ethyl acetate or ethyl acetate showed ovicidal activity. The present findings coincide with the findings of Baskar and Ignacimuthu [16] who reported that hexane extract fractions eluted with hexane : ethyl acetate from C. guianensis showed maximum ovicidal activity against H. armigera. Hexane extracts derived ethyl acetate fractions from Atalantia monophylla showed maximum ovicidal activ- ity against H. armigera and S. litura [33, 34]. Similarly, fractions eluted using hexane : ethyl acetate from chloroform extract of Clerodendrum phlomidis showed maximum ovici- dal activity against Earias vittella [14] . 5. Conclusion The present study clearly indicates that the hexane extract and its active fraction showed the least LC50 values against the eggs of S. litura. Further study is necessary to identify the active principle(s) responsible for the activity and to develop a new formulation to control the agricultural pests. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgment The authors thank the Department of Science and Technol- ogy (Ref. no. SR/SO/AS-03/2004), New Delhi, for financial support. References [1] A. S. Kandagal and M.C. Khetagoudar, “Study on larvicidal activity of weed extract against Spodoptera litura” Journal of Environmental Biology, vol. 34, pp. 253-257, 2013. [2] C. N. Zhou, “A progress and development foresight of pesticidal microorganisms in China,” Pesticides, vol. 40, pp. 8-10, 2001. [3] L. Jiang and C. S. Ma, “Progress of researches on biopesticides,” Pesticides, vol. 16, pp. 73-77, 2000. [4] C. Regnault-Roger, B. J. R. Philogene, and C. Vincent, Biopesti- cides of Plant Origin, Lavoisier, Paris, France, 2005. [5] S. M. Packiam, K. Baskar, and S. Ignacimuthu, “Insecticidal and histopathological effects of botanical formulations against Helicoverpa armigera (Hub.) (Lepidoptera; Noctuidae),” Journal of Agricultural Technology, vol. 9, pp. 573-583, 2013. [6] M. E A. El-Aziz, “Bioactivities and biochemical effects of marjoram essential oil used against potato tuber moth Phthori- maea operculella Zeller (Lepidoptera: Gelechiidae),” Life Science Journal, vol. 8, pp. 288-296, 2011. [7] R. Feng and M. B. Isman, “Selection for resistance to azadirachtin in the green peach aphid, Myzus persicaej Expe- rientia, vol. 51, no. 8, pp. 831-833, 1995. [8] S. P. Gouinguene and T. C. J. Turlings, “The effects of abiotic factors on induced volatile emissions in corn plants,” Plant Physiology, vol. 129, no. 3, pp. 1296-1307, 2002. [9] V. Rozman, 1. Kalinovic, and Z. Korunic, “Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored-product insects,” Journal of Stored Products Research, vol. 43, no. 4, pp. 349-355, 2007. [10] K. Baskar and S. Ignacimuthu, “Antifeedant, larvicidal and growth inhibitory effects of ononitol monohydrate isolated from Cassia tor a L. against Helicoverpa armigera (Hub.) and Spodoptera litura (Fab.) (Lepidoptera: Noctuidae),” Chemo- sphere, vol. 88, pp. 384-388, 2012. [11] M. C. Khetagoudar and A. S. Kandagal, “Bioefficacy of selected ecofriendly botanicals in management of tobacco cutworm, Spodoptera litura (Fab.) (Lepidoptera: Noctuidae) larvae,” Inter- national Journal of Science Innovations and Discoveries, vol. 2, pp. 12-22, 2012. [12] P. Devanand and P. U. Rani, “Insect growth regulatory activity of the crude and purified fractions from Solanum melongena L., Lycopersicum esculentum Mill, and Capsicum annuum L.,” Journal of Biopesticides, vol. 4, no. 2, pp. 118-130, 2011. [13] D. P. Ray, D. Dutta, S. Srivastava, B. Kumar, and S. Saha, “Insect growth regulatory activity of Thevetia nerifolia Juss. Against Spodoptera litura (Fab.),” Journal of Applied Botany and Pood Quality, vol. 85, pp. 212-215, 2012. [14] C. Muthu, K. Baskar, S. Ignacimuthu, and A. S. Al-Khaliel, “Ovicidal and oviposition deterrent activities of the flavonoid pectolinaringenin from Clerodendrum phlomidis against Earias vittellaj Phytoparasitica, vol. 41, pp. 365-372, 2013. [15] K. Baskar, R. Maheswaran, S. Kingsley, and S. Ignacimuthu, “Bioefficacy of Couroupita guianensis (Aubl) against Helicov- erpa armigera (Hub.) (Lepidoptera: Noctuidae) larvae,” Spanish Journal of Agricultural Research, vol. 8, no. 1, pp. 135-141, 2010. [16] K. Baskar and S. Ignacimuthu, “Ovicidal activity of Couroupita guianensis (Aubl.) against Cotton boUworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae),” Archives of Phytopathology and Plant Protection, vol. 46, pp. 1571-1579, 2013. [17] K. Baskar, S. Kingsley, S. E. Vendan, and S. Ignacimuthu, “Eeeding deterrency of some plant extracts against Asian Psyche 5 army worm Spodoptera litura Fab. (Lepidoptera: Noctuidae),” in Recent Trends in Insect Pest Management, S. Ignacimuthu and S. Jayaraj, Eds., pp. 225-227, Elite Publication, New Delhi, India, 2008. [18] G. V. R. Rao, J. A. Wightman, and D. V. R. Rao, “World review of the natural enemies and diseases of Spodoptera litura (E.) (Lepidoptera; Noctuidae),” Insect Science and Its Application, vol. 14, pp. 273-284, 1993. [19] K. Baskar, S. Kingsley, S. E. Vendan, M. G. Paulraj, V. Duraipandiyan, and S. Ignacimuthu, “Antifeedant, larvicidal and pupicidal activities of Atalantia monophylla (L) Correa against Helicoverpa armigera Hubner (Lepidoptera: Noctu- idae),” Chemosphere, vol. 75, no. 3, pp. 355-359, 2009. [20] W. S. Abbott, “A method of computing the effectiveness of an insecticide,” Journal of Economic Entomology, vol. 18, pp. 65- 266, 1925. [21] D. J. Einney, Probit Analysis, Cambridge University Press, London, UK, 3rd edition, 1971. [22] B. Deepa and O. K. Remadevi, “Insecticidal activity of the phyto-extracts derived from different parts of the trees of Pabaceae family against Hyblaea puera Cramer (Lepidoptera: Hyblaeidae),” Biological Eorum, vol. 3, pp. 1-8, 2011. [23] E. Haghighian and J. Jalali, “Antifeedant, growth inhibitory and ovicidal effect of Sambucus ebulus L. on Tribolium confusum Duv,” Caspian Journal of Environmental Sciences, vol. 3, pp. 159- 162, 2005. [24] W. I. Choi, E. H. Lee, B. R. Choi, H. M. Park, and Y. J. Ahn, “Toxicity of plant essential oils to Trialeurodes vaporariorum (Homoptera; Aleyrodidae),” Journal of Economic Entomology, vol. 96, no. 5, pp. 1479-1484, 2003. [25] M. Mondal and M. Khalequzzaman, “Ovicidal activity of essen- tial oils against red flour beetle, Tribolium castaneum (Herbst) (Coleoptera;Tenebrionidae),” Journal of Bio-Science, vol. 17, no. 1, pp. 57-62, 2009. [26] A. Ebadollahi, M. H. Safaralizadeh, A. A. Pourmirza, and G. Nouri-Ganbalani, “Comparison of fumigant toxicity of Eucalyptus globulus Labill and Lavandula stoechas L. oils against different stages of Tribolium castaneum Herbst,” Indian Journal of Agriculture Research, vol. 44, pp. 26-31, 2010. [27] I. Tunf, B. M. Berger, P. Erler, and E Dagli, “Ovicidal activity of essential oils from five plants against two stored-product insects,” Journal of Stored Products Research, vol. 36, no. 2, pp. 161-168, 2000. [28] C. Lv, B. Zhong, G. Zhong et al, “Pour botanical extracts are toxic to the hispine beetle, Brontis palongissima, in laboratory and semi-field trials,” Journal of Insect Science, vol. 12, article 58, 2012. [29] W. Setiawati, R. Murtiningsih, and A. Hasyim, “Laboratory and field evaluation of essential oils from Cymbopogon nardus as oviposition deterrent and ovicidal activities against Helicoverpa armigera Hubner on chili pepper,” Indonesian Journal of Agri- cultural Science, vol. 12, pp. 9-16, 2011. [30] A. Jeyasankar, K. Elumalai, N. Raja, and S. Ignacimuthu, “Effect of plant chemicals on oviposition deterrent and ovi- cidal activities against female moth, Spodoptera litura (Pab.) (Lepidoptera; Noctuidae),” International Journal of Agricultural Science Research, vol. 2, pp. 206-213, 2013. [31] M. V. Maciel, S. M. Morals, C. M. L. Bevilaqua, A. L. P. Camur^a- Vasconcelos, C. T. C. Costa, and C. M. S. Castro, “Ovicidal and larvicidal activity of Melia azedarach extracts on Haemonchus contortusj Veterinary Parasitology, vol. 140, no. 1-2, pp. 98-104, 2006. [32] A. Alouani, N. Rehimi, and N. Soltani, “Larvicidal activity of a neem tree extract (Azadirachtin) against mosquito larvae in the republic of Algeria,” Jordan Journal of Biological Science, vol. 2, pp. 15-23, 2009. [33] K. Baskar and S. Ignacimuthu, “Ovicidal activity of Atalantia monophylla (L) Correa against Helicoverpa armigera Hubner (Lepidoptera; Noctuidae),” Journal of Agricultural Technology, vol. 8, pp. 861-868, 2012. [34] K. Baskar, C. Muthu, G. A. Raj, S. Kingsley, and S. Ignacimuthu, “Ovicidal activity of Atalantia monophylla (L) Correa against Spodoptera litura Pab. (Lepidoptera: Noctuidae),” Asian Pacific Journal of Tropical Biomedicine, vol. 2, pp. 987-991, 2012. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 253924, 8 pages http://dx.doi.org/ 10 . 1 155/2014/253924 Review Article Sex-Pheromone-Mediated Mating Disruption Technology for the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae): Overview and Prospects Wei N. Kong,‘>^ J. Li,* Ren J. Fan,* Sheng C. Li,‘ and Rui Y. Ma‘ ^ College of Agriculture, Shanxi Agricultural University, Taigu 030801, China ^Institute of Plant Protection, Shanxi Academy of Agricultural Science, Taiyuan 030031, China ^ Pomology Institute, Shanxi Academy of Agricultural Science, Taigu 030815, China Correspondence should be addressed to Rui Y. Ma; maruiyan2004@163.com Received 21 August 2013; Revised 18 October 2013; Accepted 11 November 2013; Published 22 January 2014 Academic Editor: Russell Jurenka Copyright © 2014 Wei N. Kong 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. A great deal of progress has been made over the last three decades in research on pheromone-mediated mating disruption technology for the oriental fruit moth, Grapholita molesta (Busck). Pheromones can interrupt normal orientation, and the most likely mechanism of pheromone disruption, competitive- attraction (false-plume following), invokes competition between point sources of pheromone formulation and females for males. This technology, performed by broadcasting pheromones into orchards to disrupt mate finding, has been successfully implemented in oriental fruit moth control. Reservoir-style dispensers made of polyethylene tubes, which release pheromone throughout the full growing season, are the current industry standard. Although reasonably effective, they require labor-intensive hand application. Recently, a new formulation, paraffin wax, which maximizes competition between point sources of synthetic pheromone and feral females for males, was shown to have high disruption performance. As this formulation is highly effective, inexpensive, and easy to produce, further study and development are advisable. Increased understanding of the principles of mating disruption will aid in the design of more effective dispensers. Continued research is needed to meet grower concerns with regard to risk, efficacy, and cost and to identify other semiochemicals that can be applied to this delivery system. Greater knowledge of the integration of different biological control methods is therefore essential. 1. Introduction The oriental fruit moth (OEM), Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is a key pest of stone and pome fruit in most fruit-growing areas of China, with the exception of Tibet [1-6]. Until recently, this pest has been primarily controlled by use of one or more broad-spectrum insecticides [7]. Issues associated with the widespread use of insecticides, including insecticide resistance, toxicity to natural enemies, worker safety, and food residue, have provided an impetus for research and development regarding alternative control tech- nologies. The application of pheromone-mediated mating disruption technology has resulted in excellent control of this pest and could be an alternative to conventional insecticide use [2, 8]. This review presents the principle of pheromone- mediated mating disruption, summarizes the typical appli- cation of this technology, introduces its pheromone release device (pheromone dispenser), and discusses future direc- tions for research and development. 2. Principles of Pheromone-Mediated Mating Disruption Pheromone- mediated mating disruption controls insect species mainly by using chemicals involved in its own communication system [9]. Although it is not necessary to understand the mechanism underlying mating disruption to verify its efficacy, analysis of probable modes of action is useful to determine the reasons behind success and failure of various formulations [2] . Sex pheromones are specific chemicals released by females into the air to attract conspecific males for mating. Males follow the sex pheromone upwind to locate and mate 2 Psyche with the female [9]. However, if the air in an orchard is filled with synthetic sex pheromone, males would encounter high- dose artificial point sources of pheromone and low-dose call- ing females’ point sources of pheromone during their upwind zigzag flight. As males preferentially orient toward artifi- cial point sources of pheromone than pheromone plumes from calling females, they would be unable to accurately locate calling females and this would greatly modify moth flight tracks. Therefore, synthetic sex pheromone success- fully prevents males from finding and mating with calling females. This sex-pheromone-mediated mating disruption technology can protect orchards from pests, including the OFM, and ultimately achieve long-term reduction of the pest population [2, 9]. Many studies have indicated that competitive attraction; that is, false plume following, may be a leading mechanism underlying disruption of OFM mating by synthetic sex pheromones [10-13] and the most feasi- ble principle of pheromone disruption [2, 14-16] including sensory adaptation or central nervous system habituation on males, while sensory imbalance affects the female plume and the way in which it is perceived by males. When encountering a high concentration of formulated pheromone above that produced by calling females, males may show an increase in response or complete abolition of responsiveness to subsequent pheromone released by females, because of adaptation of peripheral receptors on the antennae or habitu- ation at the central processing level. In lepidopterous insects, pheromones are generally comprised of more than a single component with a very narrow range of ratios. An imbalance in sensory input may be produced and male response to the natural ratio of the components may be decreased by the disproportionality of blends or lack of partial components. In addition, reduction of female copulation propensity may be a secondary mechanism of mating disruption that affects the mating behavior of females in addition to that of males [17] . As an indicator of high competition, the high levels of sex pheromone perceived by females are unsuitable for reproduc- tive success, thus restricting their receptiveness to copulation and causing the loss of female receptivity to mating. Mating of females may be adversely affected following sex pheromone autoexposure due to abnormal behavioral activity, which is thought to be because the preexposed females may be unable to sense the aphrodisiac pheromone of conspecific males and/or antennal sensitivity, which is interpreted as the requirement for male aphrodisiac, which plays a role in courtship, for the occurrence of adaptation of antennal responses, following sex pheromone preexposure. Attention should be paid to the interplay between these principles. With regard to male response threshold raised by encountering a high-dose, artificial source of pheromone, subsequent male responses minimize the odds of detecting females that emit relatively low levels of pheromone, and in turn the enhanced competition effect of the two point sources of pheromone may encourage males to visit a high- dose point-source formulation than to locate and mate with a female. Taken together, these principles are helpful to opti- mize the effect of mating disruption technology by guiding the behavioral peculiarities of the insect to be managed, its spatial distribution, the type of formulation employed, and the rate of formulation application [16, 18]. 3. Application of Sex-Pheromone-Mediated Mating Disruption The sex pheromone of OFM was determined to be a mixture of four components [19] : (Z)-8-dodecenyl acetate (Z8-12:Ac), (T)-8-dodecenyl acetate (T8-12:Ac), (Z)-8-dodecen-l-ol (Z8- 12:OH), and dodecanol (12:OH). As a disruptant, only the (Z)- and (T) -isomers of the acetate were used in early work performed in Australia [20] and USA [21, 22], indicating that OFM was highly susceptible to communication disruption. Later studies [19, 23] indicated that the addition ofZ8-12:OH and 12:OH reduced the amount of pheromone required for disruption and for accurate location of hosts over short distances, respectively. Field results in a number of countries [2, 8, 9, 20, 24-34] indicated that this technology was capable of truly managing pesticide-resistant populations throughout the whole growing season and was therefore fully equiva- lent or superior to conventional pesticides. Therefore, sex- pheromone-mediated mating disruption technology could provide complete crop protection [2, 35]. 4. Pheromone Dispenser Based on the above discussion, use of a good pheromone dispenser plays a key role in achieving high-performance mating disruption. First, an ideal pheromone dispenser should remain effective for a prolonged period, not waste active ingredients, be inexpensive to produce, be easy to use in the field, and be nontoxic [36]. Furthermore, pheromone dispensers should be amenable to use at varying densities and deployment dates according to pest pressure. In addition, it should achieve the availability of a controlled release device to encourage growers to adopt mating disruption technology [37-41]. 4.1. Common Pheromone Dispensers. If competitive attrac- tion is the foremost mechanism underlying mating dis- ruption, as suggested by recent studies [10-12], various pheromone dispensers would be desirable as shown in Table 1. The three most popular types are illustrated here, that is, hollow fiber dispensers, polyethylene tube dispensers, and sprayable formulations of microscopic capsules. In the 1970s, Garde et al. (1977) deployed 1700 hand- applied hollow fiber pheromone dispensers per ha according to this behavioral modification tactic and achieved successful control of OFM, thus indicating that this is a promising alternative to broad-spectrum neurotoxins [21, 42] . In the 1990s, hand-applied polyethylene tube dispensers, such as Isomate-M, M 100, and M Rosso (Pacific Biocontrol Co., Litchfield Park, AZ) became available for commercial use for disruption of OFM [25, 27, 43-47]. These dis- pensers were filled with 75-250 mg of OFM pheromone and applied by hand at 500-1000 units/ha (corresponding to 1-4 dispensers/tree), and pheromone release per dispenser varied between ca. 600-1000-fold as much as that produced by a calling female. 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However, because of the appreciable costs of purchase and labor for hand application, polyethylene tube dispensers have not been widely adopted in some production systems and in many developing countries [53]. Early in the 21st century, sprayable formulations of micro- scopic capsules that release pheromone for prolonged periods were developed and shown to be effective against OEM when properly applied [4, 46, 54]. They were generally applied with standard air-blast sprayers. Not only they were considered a cost-saving alternative to hand-applied dispensers, but also they can be tank-mixed and coapplied with other orchard management chemicals [4, 54]. However, they maintained effectiveness for only 2-4 weeks and required more frequent applications than hand-applied reservoir dispensers [4, 47, 54] . Other drawbacks also included wash-off of microcap- sules by heavy rain and degradation of active ingredients by UV irradiation [55, 56]. In addition, due to their ease of production and constant release rate, rubber septa dispensers are common and effec- tive means of controlling OEM in China. Special lures for mating disruption are directly suspended in the upper third of the tree canopy at 200-400 units/ha without traps. However, the cost associated with this method is ca. US$180/ha per year, so they are unsuitable for common orchards in China. In addition, the rubber septa age rapidly, the duration of pheromone release is short, and hand application is expensive [57, 58] . Although they are mainly used for baiting monitor- ing traps, many research groups spent considerable time and effort to develop more reliable and efficient methods of using rubber septa [32-34, 57, 58]. 4.2. New-Style Pheromone Dispenser: Paraffin Wax. Dis- pensers in which insect sex pheromones are mixed at the required concentrations into paraffin wax emulsions have been used in USA for almost 16 years [59, 60]. Two typical wax-paraffin dispensers, that is, Confuse-OEM and SPLAT- OEM, are described below. Confuse-OEM resembles white, liquid glue and is applied using squirting devices, such as forestry paint marking guns and plastic squirt bottles [53, 61] . Use of this type of dispenser was shown to inhibit capture of male moths in pheromone traps and shoot damage as effective as Hereon (Hereon Environmental, Emigsville, PA) and Consep (Consep, Inc., now Suterra EEC, Bend, OR) hand-applied pheromone dis- pensers [59]. The University of California at Davis patented this emulsion (US. Patent 6,001,346) [62] and it was later commercially developed by Cowan Co. (Yuma, AZ) [53]. SPLAT-OEM consists of microcrystalline wax emulsified in water and so it can be pumped from a storage reservoir and sprayed onto the crop, and it shows long-lasting adhesion of dispensed particles on plant surfaces [63]. Stelinski et al. (2006) reported that mechanical application of SPLAT-OEM could save time and labor for mating disruption of OEM in apple orchards [64]. In 2003, ISCA Technologies, Inc. (Riverside, CA), patented this wax emulsion, and extensive testing of this technology was later performed along with adaptation for a variety of pests and crop systems (ISCA Technologies, Riverside, CA). ISCA Technology’s Specialized Pheromone & Lure Application Technology (SPLAT) has been granted a federal registration for OEM control by the US. Environmental Protection Agency, and it is now commercially available as SPLAT OEM 30 M-1 [53]. 4.2.1. Efficacy. Optimization of mating disruption requires that the density and size of droplets, pheromone release rate, and duration are appropriate for the biology of the targeted pest [13, 14] . Researchers can not only easily manipulate the size, density, and distribution of wax droplets, but also flexibly investigate how moths could be actually disrupted [65]. In 2005, male OEM orientation was shown to be dis- rupted more effectively by deploying ~8,000 0.1-mL drops of SPLAT-OEM per ha (each containing ca. 1% of the total pheromone active ingredient of a standard Isomate dispenser) compared with the label rate of 500 Isomate-M Rosso dispensers per ha, probably because sufficient point sources of pheromone were provided for optimal disruption of OEM with the typical deployment density of pheromone twist ties [51]. In 2006, male OEM orientation was shown to be disrupted by 98% relative to untreated control plots during the whole season using SPLAT-OEM, and either increasing the size of wax drops above the average volume of 0.04 mL achieved by the initial applicator prototype or reformulating the wax to allow for a higher initial pheromone loading concentration for longer release over time, especially in hot temperatures, could maintain efficacy and improve longevity [64]. In 2007, two applications of Confuse-OEM were shown to be as effective against OEM as one application of Isomate- M 100. A new emulsified wax formulation. Wax Dollops, was developed in 2007 with a release rate exceeding a 5 mg/ha/h threshold and duration of action that is twice as long as Confuse-OEM. One application of 3mL dollops (ca. 590 dollops per ha) provided season-long (ca. 15 weeks) control, which was equivalent to the effects of Isomate-M 100 and Confuse-OEM applied as described above [53]. 4.2.2. Advantages. Paraffin wax dispensers are inexpensive and easy to produce. Paraffin wax consists mostly of water and wax, which is a byproduct of petroleum refining, and it is therefore readily available and inexpensive [63, 64]. Wax emulsions can easily be increased proportionally and manu- factured on a large scale with minimal labor [53]. Therefore, commercial production of wax emulsions should be cheaper than other currently available hand- applied formulations. Paraffin wax is a viscous homogenate that hardens on crop foliage or branches once applied and therefore can act as a long-lasting discrete source of pheromone emission. Del- wiche et al. (1998) reported that one of the initial formulations (30% paraffin wax emulsified in water, vitamin E, soy oil, and antioxidant) was as effective as Shin-Etsu, Isomate-M 100 polyethylene-tube dispensers for 75 days in the field [61]. Subsequently, one application of a more viscous version of the above- described paraffin wax dispenser provided the same level of season-long disruption of OEM as Isomate-M 100 dispensers and could be hand- applied once in less time than Isomate-M 100 dispensers [50]. Psyche 5 Paraffin wax dispensers are rapidly applied mechanically, and there is a cost- saving advantage to mechanical appli- cation [51-53, 60, 65]. For example, SPLAT can be easily applied with a machine forming numerous discrete point sources per area of crop [64]. A single operator can treat a hectare of crop with the current mechanized applicator in ca. 20 min, which is approximately 3.4-fold faster than hand-application of Isomate-M Rosso dispensers by three people [50]. Therefore, SPLAT-OFM currently represents an economical alternative to hand-applied reservoir dispensers for high-performance mating disruption of OFM. In addition, the flowable, adhesive, and dispersible emul- sified wax can be applied with a wide range of deposit sizes and spatial distributions [53]. Furthermore, paraffin wax dis- pensers contribute to effective disruption of communication for other moth species [13, 23, 48-52] and are not phytotoxic, so they do not damage foliage and/or mark fruit [66]. In addition, insecticides have been incorporated into emulsified wax to produce effective attracticide formulations (ISCA Technologies). 4.2.3. Drawbacks. Pheromones are costly — for season-long control of OFM, the cost of Confuse- OFM was three times that of Isomate-M 100 (148 g Al/ha vs 57 g Al/ha) [53], and 160 g/ha of pheromone of SPLAT-OFM exceeded the 125 g Al/ha label rate of the Isomate-M Rosso reservoir dispenser [63] . In contrast, hand-application of dispensers is both time- consuming and expensive. Season-long control of OFM requires two applications of Confuse- OFM, and its applica- tion is laborious because this liquid formulation requires care and time for application to the tree bark [53]. In addition, even though SPLAT-OFM is applied mechanically, machine applicators are not affordable for individual growers, so the initial investment in the applicator for application of SPLAT should be provided by the manufacturer and/or distributor [64] . 5. Future Prospects As a major fruit pest [1], OFM is a long-standing target for the development of mating disruption programs [19, 21, 67, 68]. Therefore, accumulating evidence of the reliable and economic applications of pheromone-mediated mating disruption will lead to more widespread adoption of this technology. First, it is necessary to determine the actual costs of OFM pheromone dispensers as well as the relations between the total costs for one or two applications of pheromone dispensers (materials and labor) in comparison with three insecticide and/or miticide applications (materials and labor) [ 2 ]. Second, laboratory assays are required to predict the effects of various types of dispensers on OFM behavior in the field, because the development of mating disruption technology still relies on repeated field trials and therefore remains both costly and slow [69]. Finally, sex-pheromone- mediated mating disruption is currently specific for male OFM, and we should develop other semiochemical-based methods, such as plant volatiles [70, 71], directly targeting females as the most important complement to mating disruption. In addition, more empha- sis should be placed on the integration of different biological control methods, such as use of microbial pesticides [72], to reinforce the effects of behavior-modifying chemicals. All biological methods, such as black light [73], are to some extent species-specific and do not cover all pests associated with a crop, so it will be necessary to develop new mating disruption technology for OFM and other lepidopterous pests in orchards [17, 69, 74]. Conflict of Interests The authors declare that there is no conflict of interests. Acknowledgments The authors thank Dr. Roger Laushman and Dr. Yongliang Fan as well as several anonymous reviewers for improving earlier versions of the original paper. They are grateful to the Public Welfare Project from the Ministry of Agriculture of China (no. 201103024), Shanxi Province Science Foundation for Youths (no. 2013021025-3) and SXAU-BJRC201201 for financial assistance. References [1] G. L. H. Rothschild and R. A. Vickers, “Biology, ecology and control of the oriental fruit moth,” in Tortricid Pests: Their Biology, Natural Enemies and Control, L. P. S. van der Geest and H. H. Evenhuis, Eds., pp. 389-412, Elsevier, New York, NY, USA, 1991. [2] R. T. Garde and A. K. Minks, “Control of moth pests by mating disruption: successes and constraints,” Annual Review of Entomology, vol. 40, pp. 559-585, 1995. [3] A. L. Il’ichev, D. G. Williams, and A. Drago, “Distribution of the oriental fruit moth Grapholita molesta Busck (Lep., Tortricidae) infestation on newly planted peaches before and during 2 years of mating disruption,” Journal of Applied Entomology, vol. 127, no. 6, pp. 348-353, 2003. [4] O. B. Kovanci, C. Schal, f. E Walgenbach, and G. G. Kennedy, “Comparison of mating disruption with pesticides for manage- ment of oriental fruit moth (Lepidoptera: Tortricidae) in North Carolina apple orchards,” Journal of Economic Entomology, vol. 98, no. 4, pp. 1248-1258, 2005. [5] C. T. Myers, L. A. Hull, and G. Krawczyk, “Seasonal and cultivar-associated variation in oviposition preference of ori- ental fruit moth (Lepidoptera: Tortricidae) adults and feeding behavior of neonate larvae in apples,” Journal of Economic Entomology, vol. 99, no. 2, pp. 349-358, 2006. [6] R E. Lu, L. Q. Huang, and C. Z. Wang, “Semiochemicals used in chemical communication in the oriental fruit moth (Grapholitha molesta) (Lepidoptera: Tortricidae),” Acta Ento- mologica Sinica, vol. 53, pp. 1390-1403, 2010. [7] L. H. B. Kanga, D. ]. Free, ]. L. van Lier, and G. M. Walker, “Management of insecticide resistance in oriental fruit moth {Grapholita molesta; Lepidoptera: Tortricidae) popula- tions from Ontario,” Pest Management Science, vol. 59, no. 8, pp. 921-927, 2003. 6 Psyche [8] R. M. Trimble, D. J. Free, and N. J. Carter, “Integrated control of oriental fruit moth (Lepidoptera: Tortricidae) in peach orchards using insecticide and mating disruption,” Journal of Economic Entomology, vol. 94, no. 2, pp. 476-485, 2001. [9] J. W. Du, Insect Pheromones and Its Application, China Forestry Publishing House, Beijing, China, 1988. [10] J. R. Miller, L. J. Gut, F. M. de Lame, and L. L. Stelinski, “Dif- ferentiation of competitive vs. non-competitive mechanisms mediating disruption of moth sexual communication by point sources of sex pheromone — part 2: case studies,” Journal of Chemical Ecology, vol. 32, no. 10, pp. 2115-2143, 2006. [11] L. L. Stelinski, L. J. Gut, K. J. Vogel, and J. R. Miller, “Behaviors of naive vs. pheromone-exposed leafroller moths in plumes from high-dosage pheromone dispensers in a sustained-flight wind tunnel; implications for mating disruption of these species,” Journal of Insect Behavior, vol. 17, no. 4, pp. 533-554, 2004. [12] L. L. Stelinski, L. J. Gut, A. V. Pierzchala, and J. R. Miller, “Field observations quantifying attraction of four tortricid moths to high-dosage pheromone dispensers in untreated and pheromone-treated orchards,” Entomologia Experimentalis et Applicata, vol. 113, no. 3, pp. 187-196, 2004. [13] J. R. Miller, L. J. Gut, F. M. de Lame, and L. L. Stelinski, “Dif- ferentiation of competitive vs. non-competitive mechanisms mediating disruption of moth sexual communication by point sources of sex pheromone — part I; theory,” Journal of Chemical Ecology, vol. 32, no. 10, pp. 2089-2114, 2006. [14] H. M. Flint and J. R. Merkle, “Mating behavior, sex pheromone responses and radiation sterilization of the greater wax moth (Lepidoptera: Pyralidae),” Journal of Economic Entomology, vol. 76, pp. 467-472, 1983. [15] A. K. Minks and R. T. Garde, “Disruption of pheromone communication in moths: is the natural blend really most efficacious?” Entomologia Experimentalis et Applicata, vol. 49, no. 1-2, pp. 25-36, 1988. [16] R. T. Carde, R. T. Staten, and A. Mafra-Neto, “Behaviour of pink bollworm males near high-dose, point sources of pheromone in field wind tunnels; insights into mechanisms of mating disruption,” Entomologia Experimentalis et Applicata, vol. 89, no. 1, pp. 35-46, 1998. [17] E. H. Kuhns, K. Pelz-Stelinski, and L. L. Stelinski, “Reduced mating success of female tortricid moths following intense pheromone auto-exposure varies with sophistication of mating system,” Journal of Chemical Ecology, vol. 38, no. 2, pp. 168-175, 2012. [18] R. T. Carde, “Principles of mating disruption,” in Behaviour Modifying Chemicals for Insects Management, R. L. Ridgway, R. M. Silverstein, andM. N. Inscoe, Eds., pp. 47-71, Marcel Dekker, New York, NY, USA, 1990. [19] A. M. Carde, T. C. Baker, and R. T. Carde, “Identification of a four- component sex pheromone of the female oriental fruit moth, Grapholitha molesta (Lepidoptera: Tortricidae),” Journal of Chemical Ecology, vol. 5, no. 3, pp. 423-427, 1979. [20] O. H. L. Rothschild, “Control of oriental fruit moth (Cydia molesta (Busck) (Lepidoptera, Tortricidae)) with synthetic female pheromone,” Bulletin of Entomological Research, vol. 65, pp. 473-490, 1975. [21] R. T. Carde, T. C. Baker, and P. J. Castrovillo, “Disruption of sexual communication in Laspeyresia pomonella (codling moth), Grapholitha molesta (oriental fruit moth) and G. Pruni- voral (lesser appleworm) with hollow fiber attractant sources,” Entomologia Experimentalis et Applicata, vol. 22, no. 3, pp. 280- 288, 1977. [22] C. R. Gentry, B. A. Bierl-Leonhardt, J. L. Blythe, and J. R. Plimmer, “Air permeation tests with orfralure for reduction in trap catch of oriental fruit moths,” Journal of Chemical Ecology, vol. 6, no. 1, pp. 185-192, 1980. [23] R. E. Charlton and R. T. Carde, “Comparing the effectiveness of sexual communication disruption in the oriental fruit moth (Grapholitha molesta) using different combinations and dosages of its pheromone blend,” Journal of Chemical Ecology, vol. 7, no. 3, pp. 501-508, 1981. [24] H. Audemard, C. Leblon, U. Neumann, and G. Marboutie, “Bilan de sept annees dessais de lutte contre la Tordeuse orientale du pecher Cydia molesta Busck (Lep., Tortricidae) par confusion sexuelle des males,” Journal of Applied Entomology, vol. 108, pp. 191-207, 1989. [25] R. E. Rice and P. Kirsch, “Mating disruption of oriental fruit moth in the United States,” in Behavior-Modifying Chemicals for Insect Management: Applications of Pheromones and Other Attractants, R. L. Ridgway, R. M. Silverstein, and M. N. Inscoe, Eds., Marcel Dekker, New York, NY, USA, 1990. [26] Y. Sakagami, “Remove from marked records recent advances in fruit tree pest managements with pheromones in Japan,” Bulletin of the National Institute of Bruit Tree Science, no. 34, pp. 17-42, 2000. [27] E. Tanaka, “Control of the oriental fruit moth (Grapholita molesta Busk) by communication disruption,” Shokubutsu Boeki, vol. 40, pp. 59-62, 1986. [28] C. Y. Yang, K. S. Han, J. K. Jung, K. S. Boo, and M. S. Yiem, “Control of the oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) by mating disruption with sex pheromone in pear orchards,” Journal of Asia-Pacific Ento- mology, vol. 6, no. 1, pp. 97-104, 2003. [29] R. A. Vickers, G. H. L. Rothschild, and E. L. Jones, “Control of the oriental fruit moth, Cydia molesta (Busck) (Lepidoptera: Tortricidae), at a district level by mating disruption with syn- thetic female pheromone,” Bulletin of Entomological Research, vol. 75, pp. 626-634, 1985. [30] A. L. Il’ichev, D. G. Williams, and A. D. Milner, “Mating disruption barriers in pome fruit for improved control of oriental fruit moth Grapholita molesta Busck (Lep., Tortricidae) in stone fruit under mating disruption,” Journal of Applied Entomology, vol. 128, no. 2, pp. 126-132, 2004. [31] R. A. Vickersa, G. H. L. Rothschilda, and E. L. Jones, “Control of the oriental fruit moth, Cydia molesta (Busek) (Lepidoptera: Tortricidae), at a district level by mating disruption with syn- thetic female pheromone,” Bulletin of Entomological Research, vol. 75, pp. 625-634, 1985. [32] X. Z. Meng, H. Y. Wang, and M. X. Ye, “Controlling oriental fruit moth (Grapholitha molesta) (Lepidoptera: Tortricidae) by mass-trapping of sex pheromone in the field,” Chinese Science Bulletin, pp. 703-704, 1983. [33] L. J. Zhang, X. D. Chen, S. Shuai, B. Yang, J. W. Qiu, and R. Y. Ma, “Experiment in application of sex pheromone to control of oriental fruit moth (Grapholitha molesta) (Lepidoptera: Tortricidae),” Journal of Shanxi Agricultural University, vol. 38, pp. 97-100, 2010. [34] C. He, Y. C. Qin, T. C. Zhou, L. Hua, and R. Zhang, “Experiment of mating disruption control Grapholitha molesta Busck by using sex pheromone,” Acta Agriculturae Boreali-Occidentalis Sinica, vol. 95, pp. 107-109, 2008. [35] P. A. Kirsch, “Concept to commercial reality, confusion among codling moth fellows, with additional notes on oriental fruit moth and grape berry moth,” in Pheromones in Mediterranean Psyche 7 Pest Management, p. 24, International Organization for Biolog- ical Control of Noxious Animals and Plants, Granada, Spain, 1990. [36] J. R. Plimmer, “Formulation and regulation: constraints on the development of semiochemicals for insect pest management,” in Management of Insect Pests with Semiochemicals: Concepts and Practice, E. R. Mitchell, Ed., pp. 403-420, Plenum, New York, NY, USA, 1981. [37] J. R. Plimmer and M. N. Inscoe, “Insect pheromones; some chemical problems involved in their use and development,” in Chemical Ecology: Odour Communication in Animals: Scientific Aspects, Practical Uses, and Economic Prospects, E }. Ritter, Ed., pp. 249-260, Elsevier/North-Holland Biomedical Press, New York, NY, USA, 1978. [38] J. B. Siddall, “Commercial production of insect pheromones- problems and prospects,” in Chemical Ecology: Odour Commu- nication in Animals: Scientific Aspects, Practical Uses, and Eco- nomic Prospects, E J. Ritter, Ed., pp. 389-402, Elsevier/North- Holland Biomedical Press, New York, NY, USA, 1978. [39] G. H. L. Rothschild, “A comparison of methods of dispensing synthetic sex pheromone for the control of oriental fruit moth, Cydia molesta (Busck) (LepidopteraiTortricidae), in Australia,” Bulletin of Entomological Research, vol. 69, pp. 115-127, 1979. [40] G. J. Jackson, “The development and marketing of a pheromone system,” in Insect Pheromones in Plant Protection, A. R. Jutsum and R. E. S. Gordon, Eds., pp. 281-293, Gordon, Wiley, New York, NY, USA, 1989. [41] I. Weatherston, “Principles of design of controlled-release formulations,” in Behavior-Modifying Chemicals for Insect Man- agement: Applications of Pheromones and Other Attractants, R. L. Ridgway, R. M. Silverstein, and M. N. Inscoe, Eds., pp. 93-112, Marcel Dekker, New York, NY, USA, 1990. [42] L. J. Gut, L. L. Stelinski, D. R. Thompson, and J. R. Miller, “Behavior-modifying chemicals: prospects and constraints in IPM,” in Integrated Pest Management: Potential, Constraints and Challenges, O. Koul, G. S. Dhaliwal, and G. Cuperus, Eds., pp. 73-121, CABI Press, Wallingford, UK, 2004. [43] D. G. Pfeiffer and J. C. Killian, “Disruption of olfactory com- munication in oriental fruit moth and lesser appleworm in a Virginia peach orchard,” Journal of Agricultural Entomology, vol. 5, pp. 235-239, 1988. [44] D. J. Pree, R. M. Trimble, K. J. Whitty, and P. M. Vickers, “Gontrol of oriental fruit moth by mating distribution using sex pheromone in the Niagara Peninsula, Ontario,” Canadian Entomologist, vol. 126, no. 6, pp. 1287-1299, 1994. [45] R. M. Trimble, D. J. Pree, and N. J. Carter, “Integrated control of oriental fruit moth (Lepidoptera: Tortricidae) in peach orchards using insecticide and mating disruption,” Journal of Economic Entomology, vol. 94, no. 2, pp. 476-485, 2001. [46] A. Atanasso, P. W. Shearer, G. Hamilton, and D. Polk, “Devel- opment and implementation of a reduced risk peach arthropod management program in New Jersey,” Journal of Economic Entomology, vol. 95, no. 4, pp. 803-812, 2002. [47] R. M. Trimble, D. J. Pree, E. S. Barszcz, and N. J. Carter, “Com- parison of a sprayable pheromone formulation and two hand- applied pheromone dispensers for use in the integrated control of oriental fruit moth (Lepidoptera: Tortricidae),” Journal of Economic Entomology, vol. 97, no. 2, pp. 482-489, 2004. [48] P. Palaniswamy, R. J. Ross, W. D. Seabrook et al, “Mating suppression of caged spruce budworm (Lepidoptera: Tortri- cidae) moths in different pheromone atmospheres and high population densities,” Journal of Economic Entomology, vol. 75, pp. 989-993, 1982. [49] D. M. Suckling, G. Karg, S. J. Bradley, and G. R. Howard, “Eield electroantennogram and behavioral responses of Epiphyas postvittana (Lepidoptera; Tortricidae) under low pheromone and inhibitor concentrations,” Journal of Economic Entomology, vol. 87, no. 6, pp. 1477-1487, 1994. [50] E. M. de Lame, J. R. Miller, C. A. Atterholt, and L. J. Gut, “Development and evaluation of an emulsified paraffin wax dispenser for season-long mating disruption of Grapholita molesta in commercial peach orchards,” Journal of Economic Entomology, vol. 100, no. 4, pp. 1316-1327, 2007. [51] L. L. Stelinski, L. J. Gut, R. E. Mallinger, D. Epstein, T. R Reed, and J. R. Miller, “Small plot trials documenting effective mating disruption of oriental fruit moth by using high densities of wax- drop pheromone dispensers,” Journal of Economic Entomology, vol. 98, no. 4, pp. 1267-1274, 2005. [52] D. L. Epstein, L. L. Stelinski, T. R Reed, J. R. Miller, and L. J. Gut, “Higher densities of distributed pheromone sources provide disruption of codling moth (Lepidoptera: Tortricidae) superior to that of lower densities of clumped sources,” Journal of Economic Entomology, vol. 99, no. 4, pp. 1327-1333, 2006. [53] E. M. de Lame, J. R. Miller, C. A. Atterholt, and L. J. Gut, “Development and evaluation of an emulsified paraffin wax dispenser for season-long mating disruption of Grapholita molesta in commercial peach orchards,” Journal of Economic Entomology, vol. 100, no. 4, pp. 1316-1327, 2007. [54] A. L. Il’ichev, L. L. Stelinski, D. G. Williams, and L. J. Gut, “Sprayable microencapsulated sex pheromone formulation for mating disruption of oriental fruit moth (Lepidoptera: Tort- ricidae) in Australian Reach and Rear Orchards,” Journal of Economic Entomology, vol. 99, no. 6, pp. 2048-2054, 2006. [55] A. L. Knight, T. E. Larsen, and K. C. Ketner, “Rainfastness of a microencapsulated sex pheromone formulation for codling moth (Lepidoptera: Tortricidae)',’ Journal of Economic Entomol- ogy, vol. 97, no. 6, pp. 1987-1992, 2004. [56] D. E. Waldstein and L. J. Gut, “Effects of rain and sunlight on oriental fruit moth (Lepidoptera: Tortricidae) pheromone microcapsules applied to apple foliage,” Journal of Agricultural and Urban Entomology, vol. 21, no. 2, pp. 117-128, 2004. [57] M. Li, J. Liu, J. Li et al., “The method of using high-performance sexual attractant of oriental fruit moth (Grapholitha molesta) (Lepidoptera: Tortricidae),” China Plant Protection, vol. 30, pp. 44-46, 2010. [58] Q. Y. Li, J. L. Liu, L. L. Zhao, and R. Y. Ma, “Applications of slow release technique in the sex pheromone controlling pest,” Chinese Journal of Biological Control, vol. 4, pp. 589-593, 2012. [59] G. A. Atterholt, M. J. Delwiche, R. E. Rice, and J. M. Krochta, “Controlled release of insect sex pheromones from paraffin wax and emulsions,” Journal of Controlled Release, vol. 57, no. 3, pp. 233-247, 1999. [60] C. A. Atterholt, M. J. Delwiche, R. E. Rice, and J. M. Krochta, “Study of biopolymers and paraffin as potential controlled- release carriers for insect pheromones,” Journal of Agricultural and Pood Chemistry, vol. 46, no. 10, pp. 4429-4434, 1998. [61] M. Delwiche, C. Atterholt, and R. Rice, “Spray application of paraffin emulsions containing insect pheromones for mating disruption,” Transactions of the American Society of Agricultural Engineers, vol. 41, no. 2, pp. 475-480, 1998. [62] M. Delwiche, J. M. Krochta, R. E. Rice, and C. Atterholt, “Aqueous emulsion comprising biodegradable carrier for insect 8 Psyche pheromones and methods for controlled release thereof,” U.S. patent no. 6001346, 1999. [63] L. L. Stelinski, J. R. Miller, R. Ledebuhr, P. Siegert, and L. J. Gut, “Season-long mating disruption of Grapholita molesta (Lepi- doptera: Tortricidae) by one machine application of pheromone in wax drops (SPLAT-OFM),” Journal of Pest Science, vol. 80, no. 2, pp. 109-117, 2007. [64] L. L. Stelinski, J. R. Miller, R. Ledebuhr, and L. J. Gut, “Mecha- nized applicator for large-scale field deployment of paraffin-wax dispensers of pheromone for mating disruption in tree fruit,” Journal of Economic Entomology, vol. 99, no. 5, pp. 1705-1710, 2006. [65] P. E. Jenkins and R. Isaacs, “Mating disruption of Paralobesia viteana in vineyards using pheromone deployed in SPLAT- GBM wax droplets,” Journal of Chemical Ecology, vol. 34, no. 8, pp. 1089-1095, 2008. [66] P. Y. Giroux and J. R. Miller, “Phytotoxicity of pheromonal chemicals to fruit tree foliage: chemical and physiological characterization,” Journal of Economic Entomology, vol. 94, no. 5, pp. 1170-1176, 2001. [67] C. R. Gentry, B. Morton, J. L. Blythe, and B. A. Bierl, “Efficacy trials with the pheromone of the oriental fruit moth and data on the lesser appleworm,” Journal of Economic Entomology, vol. 67, pp. 607-609, 1974. [68] C. R. Gentry, B. Morton, J. L. Blythe, and B. A. Bierl, “Captures of the oriental fruit moth, the pecan bud moth, and the lesser appleworm in Georgia field trials with isomeric blends of 8- dodecenyl acetate and air-permeation trials with the oriental fruit moth pheromone,” Environmental Entomology, vol. 4, pp. 822-824, 1975. [69] P. Witzgall, P. Kirsch, and A. Cork, “Sex pheromones and their impact on pest management,” Journal of Chemical Ecology, vol. 36, no. 1, pp. 80-100, 2010. [70] P. E Lu, H. L. Qiao, Z. C. Xu, J. Cheng, S. X. Zong, and Y. Q. Luo, “Comparative analysis of peach and pear fruit volatiles attractive to the oriental fruit moth, Cydia molestaj Journal of Plant Interactions, 2013. [71] P. E. Lu, L. Q. Huang, and C. Z. Wang, “Identification and field evaluation of pear fruit volatiles attractive to the oriental fruit moth, Cydia molesta’,’ Journal of Chemical Ecology, vol. 38, pp. 1003-1016, 2012. [72] E. Riga, L. A. Lacey, N. Guerra, and H. L. Headrick, “Control of the oriental fruit moth, Grapholita molesta, using ento- mopathogenic nematodes in laboratory and fruit bin assays,” Journal of Nematology, vol. 38, no. 1, pp. 168-171, 2006. [73] E. van Langevelde, J. A. Ettema, M. Donners, M. E. WallisDe- Vries, and D. Groenendijk, “Effect of spectral composition of artificial light on the attraction of moths,” Biological Conserva- tion, vol. 144, no. 9, pp. 2274-2281, 2011. [74] K. S. Boo and K. C. Park, “Insect semiochemical research in Korea: overview and prospects,” Applied Entomology and Zoology, vol. 40, no. 1, pp. 13-29, 2005. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 546862, 1 page http://dx.doi.org/ 10 . 1 155/2014/546862 Retraction Retracted: Climatic, Regional Land-Use Intensity, Landscape, and Local Variables Predicting Best the Occurrence and Distribution of Bee Community Diversity in Various Farmland Habitats in Uganda Psyche Received 23 December 2013; Accepted 23 December 2013; Published 28 January 2014 Copyright © 2014 Psyche. 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 article titled “Climatic, Regional Land-Use Intensity, Landscape, and Local Variables Predicting Best the Occur- rence and Distribution of Bee Community Diversity in Var- ious Farmland Habitats in Uganda” [1], published in Psyche, has been retracted as it was found to include erroneous data. Its findings and conclusion cannot be relied on. References [1] T. Munyuli, “Climatic, regional land-use intensity, landscape, and local variables predicting best the occurrence and distri- bution of bee community diversity in various farmland habitats in Uganda,” Psyche, vol. 2013, Article ID 564528, 38 pages, 2013. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 650427, 3 pages http://dx.doi.org/10.1155/2014/650427 Research Article The Effect of Conspecific Density on Emergence of Lestes bipupillatus Calvert, 1909 (Odonata: Lestidae) Ricardo Cardoso-Leite,'’^ Gabriel C. Vilardi,^ Rhainer Guillermo-Ferreira,'’^ and Pitagoras C. Bispo^ ^ Departamento de Ciencias Biologicas, Faculdade de Ciencias e Letras de Assis, Universidade Estadual Paulista, Av. Dom Antdnio, 2100, 19806-900, Assis, SP, Brazil ^ Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirdo Preto, Universidade de Sdo Paulo, Av. dos Bandeirantes, 3900, 14040-901 Ribeirdo Preto, SP, Brazil ^ Departamento de Biologia, Fundagdo Universidade Federal de Ronddnia, 76850-000 Guajard-Mirim, RO, Brazil Correspondence should be addressed to Ricardo Cardoso-Leite; ricardocardosoleite@yahoo.com.br Received 11 October 2013; Accepted 18 November 2013; Published 30 January 2014 Academic Editor: Kleber Del-Claro Copyright © 2014 Ricardo Cardoso-Leite 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. Conspecific density may influence adult recruitment and consequently population dynamics. Several studies have shown the density dependence of larvae growth rates in Odonata. However, few studies studied how conspecific density influence final instar larvae emergence date decisions. Considering that larvae may choose the date of emergence, the present study investigated if density affects larvae choice. Lor this, we reared eight final instar larvae in individual aquaria and other 24 larvae in aquaria with three larvae each. This way, we simulated environments with low and high larval densities. We then noted the days that larvae took to emerge and compared it between low and high density groups. The results showed that larvae seem to emerge earlier when in high densities (Mann-Whitney, U = 10.000, P = 0.03). These results support the hypothesis that damselfly last instar larvae may postpone or hasten emergence in response to the social environment and related constraints. 1. Introduction Natural environments may exhibit large temporal fluctua- tions, which entail a major challenge for animal species. Temporary pools comprise a harsh environment, inhabited by a unique fauna with physiological and behavioral adapta- tions that enable development and survival [1] . Reductions in water levels in temporary pools may affect species population dynamics, since density should increase. Population dynamics are influenced by life history fea- tures such as individual development, survival, fecundity, and dispersal rates amidst environmental fluctuations. Variation in such features may be associated with density- dependent processes [2-6]. In insects, adult population dynamics are usually affected by larval density that may decrease or increase adult emergence rates [7, 8]. In Odonata, increasing density among conspecifics may shorten life cycle [9], influence larval growth rates, and affect species voltinism [1]. The density dependence of larvae growth rates in odonates is well studied [1], but there is no evidence of how conspecific density may determine the emergence rate of final instar larvae. The increased density during the reduction of water level could be an indicator cue of the drying out process. This mechanism could enable some species of Odonata to colonize and complete their life cycles in temporary pools. Since final instar larvae of Odonata may postpone emer- gence, the date of emergence can determine individual body size, fecundity, and reproductive success [1] and may be critical to complete the cycle in temporary ponds. Thus, we tested if conspecific final instar larval density influences the date of emergence in the tropical species Lestes bipupillatus 2 Psyche Calvert, 1909 (Zygoptera: Lestidae). Lestids are good mod- els for this kind of study since they inhabit temporary pools and must carry adaptations to such environment [1, 10-12]. 2. Material and Methods We collected last instar larvae in a temporary pond near the Ecological Reserve Horto Florestal in Assis, SP, Brazil (S 22°37^46.9^Vw 50°24^11.7^^) on 19 May 2007. This reserve is a conservation area with a mixture of native Atlantic Forest vegetation and Neotropical Savanna vegetation. To test whether the density affects the time of emergence, we simulated two situations in laboratory: (i) low density, with one individual per aquarium; and (ii) high density, with three individuals per aquarium. We considered eight replicas for each situation. Each aquarium had 500 mE of capacity and was filled with 300 mL of filtered water collected in the habitat of larvae. The aquaria were wrapped with white paper to prevent visual contact between larvae and were provided with wood sticks for individuals to climb during the rearing process. During the experiment, the aquaria were maintained in a cool room with 12 : 12 photoperiod. The aquaria were placed inside a vial filled with water to guarantee temperature constancy among replicas. The positioning of each aquarium in the vial was randomly sorted. We checked for emergence each 12 hours and we finally compared the number of days that the two groups of larvae took to emerge since the collection date. For the high density group, we sorted eight individuals to represent the group. Differences between the median of emergence time of individuals at high and low densities were assessed using the Mann-Whitney U test. 3. Results and Discussion The results show that high conspecific density decreased the number of days until emergence (Mann-Whitney, U = 10.000, P - 0.03, Figure 1). As the larvae at high densities emerged earlier, we can assume that, when there is low conspecific density, the larvae may delay emergence. These results show how density may influence adult recruitment and the number of flying reproductive individuals in a given time. Based on this information, we can consider the fact that high density may force larvae to hasten emergence and impose a great impact on population dynamics, since larvae that emerge earlier are usually smaller and have a lower reproductive success [1, 12]. We can also consider extrinsic features related to species ecology and the peculiar habitat which they inhabit. Since this study collected L. bipupillatus larvae on a temporary pond, another possible selective force could be pond dryout [12], which may result in larvae aggregation with the decline of water level. In this case, the high density is an indicator that water level is dropping and the early emergence occurs to avoid death due to the low volume of water, high temperatures, and low dissolved oxygen. 18 16 Low density High density Figure 1: Days until emergence of last instar larvae reared with low and high conspecific densities. The earlier emergence when in high densities may also be an evolutionary response to conspecific interactions as can- nibalism, since odonate larvae usually feed on conspecifics [13-16] or competition, since they can be aggressive towards conspecifics and even harm or kill neighboring larvae [1, 16]. 4. Conclusions In conclusion, the experiment allows us to suggest that dam- selfly last instar larvae may postpone or hasten emergence in response to the constraints related to the social environment and water conditions. Although other studies show that many variables may affect development, and consequently emergence [1, 17], here we show that L. bipupillatus last instar larvae make decisions regarding emergence time, indepen- dently of previous development. This can give base for future perspectives, regarding other environmental variables and the intrinsic effects on adult survival and reproduction. We suggest that studies should now focus on the outcomes and handicaps of final instar larvae emergence syndromes in a set of species. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors thank CAPES, FAPESP and CNPq for financial support. They thank two anonymous referees for valuable comments. References [1] P. S. Corbet, Dragonflies Behaviour and Ecology of Odonata, Harley Books, Essex, UK, 1999. [2] T. H. Glutton -Brock, M. Major, S. D. Albon, and F. E. Guinness, “Early development and population dynamics in red deer. Psyche 3 1. Density-dependent effects on juvenile survival,” Journal of Animal Ecology, vol. 56, no. 1, pp. 53-67, 1987. [3] S. D. Albon, T. H. Clutton-Brock, and F. L. Guinness, “Early development and population dynamics in red deer. II. Density- independent effects and cohort variation,” Journal of Animal Ecology, vol. 56, no. 1, pp. 69-81, 1987. [4] R. Lande, S. Engen, and B. Saether, “An evolutionary maximum principle for density-dependent population dynamics in a fluc- tuating environment,” Philosophical Transactions of the Royal Society B, vol. 364, no. 1523, pp. 1511-1518, 2009. [5] A. D. Luis, R. J. Douglass, J. N. Mills, and O. N. Bjornstad, “The effect of seasonality, density and climate on the population dynamics of Montana deer mice, important reservoir hosts for Sin Nombre hantavirus,” Journal of Animal Ecology, vol. 79, no. 2, pp. 462-470, 2010. [6] T. L. Russell, D. W. Lwetoijera, B. G. J. Knols, W. Takken, G. E Killeen, and H. M. Eerguson, “Linking individual phenotype to densitydependent population growth; the influence of body size on the population dynamics of malaria vectors,” Proceedings of the Royal Society B, vol. 278, no. 1721, pp. 3142-3151, 2011. [7] J. E. Gimnig, M. Ombok, S. Otieno, M. G. Kaufman, J. M. Vulule, and E. D. Walker, “Density-dependent development of Anopheles gambiae (Diptera: Gulicidae) larvae in artificial habitats,” Journal of Medical Entomology, vol. 39, no. 1, pp. 162- 172, 2002. [8] K. Hoshino, H. Isawa, Y. Tsuda, and M. Kobayashi, “Laboratory colonization of Aedes japonicus japonicus (Diptera: Gulicidae) collected in Narita, Japan and the biological properties of the established colony,” Japanese Journal of Infectious Diseases, vol. 63, no. 6, pp. 401-404, 2010. [9] D. M. Johnson, T. H. Martin, M. Mahato, L. B. Crowder, and R H. Crowley, “Predation, density dependence, and life histories of dragonflies: a field experiment in a freshwater community,” Journal of the North American Benthological Society, vol. 14, no. 4, pp. 547-562, 1995. [10] C. Utzeri, E. Ealchetti, and G. Carchini, “Alcuni aspetti etologici della ovideposizione di Testes barbarous (Eabricius) presso pozze temporanee (Zygoptera: Lestidae),” Odonatologica, vol. 4, pp. 175-179, 1976. [11] C. Utzeri, G. Carchini, E. Ealchetti, and C. Belfiore, “Philopa- try, homing and dispersal in Lestes barbarous (Eabricius) (Zygoptera: Lestidae),” Odonatologica, vol. 13, pp. 573-584, 1984. [12] M. de Block and R. Stoks, “Pond drying and hatching date shape the tradeoff between age and size at emergence in a damselfly,” Oikos, vol. 108, no. 3, pp. 485-494, 2005. [13] A. Benke, “Interactions among coexisting predators — a field experiment with dragonfly larvae,” Journal of Animal Ecology, vol. 47, pp. 335-350, 1978. [14] D. M. Johnson, P. H. Crowley, R. E. Bohanan, C. N. Watson, and T. FI. Martin, “Competition among larval dragonflies: a field enclosure experiment,” Ecology, vol. 66, no. 1, pp. 119-128, 1985. [15] J. van Buskirk, “Density-dependent cannibalism in larval drag- onflies,” Ecology, vol. 70, no. 5, pp. 1442-1449, 1989. [16] O. M. Fincke, “Population regulation of a tropical damselfly in the larval stage by food limitation, cannibalism, intraguild predation and habitat drying,” Oecologia, vol. 100, no. 1-2, pp. 118-127, 1994. [17] C. Johnson, “Mating and oviposition of damselflies in the laboratory,” The Canadian Entomologist, vol. 97, pp. 321-326, 1965. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 792746, 6 pages http://dx.doi.org/10.1155/2014/792746 Research Article Diversity and Composition of Beetles (Order: Coleoptera) of Durgapur, West Bengal, India Moitreyee Banerjee Department of Zoology, Durgapur Government College, Jawaharlal Nehru Avenue, Durgapur, Burdwan District, West Bengal 713214, India Correspondence should be addressed to Moitreyee Banerjee; miotreyeebanerjee@gmaiLcom Received 31 July 2013; Revised 14 November 2013; Accepted 29 November 2013; Published 30 January 2014 Academic Editor: James C. Nieh Copyright © 2014 Moitreyee Banerjee. 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. A survey of beetle faunal diversity and composition was studied in Durgapur Municipal Corporation, Durgapur, West Bengal, from January to December 2012. Beetles were collected using standard trapping methods from three different sites selected on the basis of their specific habitat differences, identified up to the level of family, and counted monthly. A total of 9 families were reported from the study site. The second site, that is. Site B, showed the highest diversity. It is also noted that the highest diversity was found during monsoon in all the three sites. 1. Introduction Coleoptera is an order of insects commonly called beetles. The word “coleoptera” is from the Greek keleos, meaning “sheath” and pteron, meaning “wing,” thus “sheathed wing.” The reason for the name is that most beetles have two pairs of wings, the front pair, the “elytra,” being hardened and thickened into a sheath-like or shell-like protection for the rear pair and for the rear part of the beetle’s body. The order Coleoptera includes more species than any other order, constituting almost 25% of all known life-forms [1-3]. About 40% of all described insect species are beetles (about 400,000 species) [4] and new species are discovered frequently. Some estimates put the total number of species, described and undescribed, at as high as 100 million, but a figure of 1 million is more widely accepted [5]. The diversity of beetles is very wide. They are found in all major habitats, except marine and the Polar regions. There are particular species that are adapted to practically every kind of diet. The family Scarabaeidae is the largest family of insects which contains more than 30000 species in the world [6]. Coleoptera are found in nearly all natural habitats, that is, vegetative foliage, from trees and their bark to flowers, leaves, and underground near roots, even inside plants like galls, tissue, including dead or decaying ones [7]. About 3/4 of beetle species are phytophagous in both the larval and adult stages, living in or on plants, wood, fungi, and a variety of stored products, including cereals, tobacco, and dried fruits. Because many of these plants are important for agriculture, forestry, and the household, the beetle can be considered a pest [8]. Beetles are not only pests but can also be beneficial, usually by controlling the populations of pests. One of the best, and widely known, examples is the ladybug or ladybird (family Coccinellidae). Both the larvae and adults are found feeding on aphid colonies. Other ladybugs feed on scale insects and mealybugs. If normal food sources are scarce, they may feed on other things, such as small caterpillars, young plant bugs, honeydew, and nectar [9] . Ground beetles (family Carabidae) are common predators of many different insects and other arthropods, including fly eggs, caterpillars, wireworms, and others [10]. Dung beetles (Coleoptera, Scarabaeidae) have been successfully used to reduce the populations of pestilent flies and parasitic worms that breed in cattle dung [9]. Dung beetles are taxonomically as well as functionally very important component of terrestrial ecosystem [11]. This study focuses on the diversity of beetles in Durgapur Municipal Corporation. The area is divided into three study sites in order to get an idea on the variety of beetles found. 2 Psyche The study is restricted to the family level of the order Coleoptera. 9 distinct families of beetles were reported from the three sites over a long one-year survey. Usually diversity studies are conducted in ecologically sound areas with special focus on insects as they are the most diverse group among fauna. Durgapur is an industrial area of major importance in West Bengal. It has large power plants along with cement and iron factories. It has alarming pollution status. Thus it is important to survey the floral and faunal assemblage in this area. A study of the most diverse group of insects, that is, beetles, not only will help to assess the diversity of this area but also will help to carry out further studies to conserve the biodiversity of this industrial belt. 2. Materials and Method 2.1. Study Area. A study was conducted from January to December 2012 at three different sites of Durgapur Munic- ipal Corporation, Durgapur City, West Bengal, India. The geographical location is 23.30 N and 87.20 E with an altitude of 68.9 meters. Durgapur is about 220 Km from Calcutta, capital city of West Bengal. Though Durgapur is an industrial belt, the industrial sector is strictly demarcated from the main city area, which supports good floral assemblage. The metropolitan area is divided into three basic sites for the present study. Site A is the college campus and its surrounding area that is dominated by Shal tree {Shorea robusta). Thus the floral composition is specific and constant. Site B is the township area that mainly has residential complexes with gardens that support diverse floral composition that usually changes with season. Site C is a wetland located near Amravati, surrounded by grasslands supporting different vegetation. 2.2. Field Method. Beetle sampling was done fortnightly from the three sites. For good collection two distinct standard methods were used. Pitfall traps were set up in all the three sites and were monitored every day. Light traps were also used specially in Site B. Apart from these, handpicking is also done. Sometimes shrubs and tree branches were heavily shaken so that beetles may fall on already spread large white sheets. After collection each specimen was preserved in 4% formalin and stored in small vials with proper labeling. Identification up to the family level was done using standard identification manual [12, 13]. 2.3. Data Analysis. As beetles are reported to be the diverse group of insects, main focus of the present study is to estimate the diversity of beetles in this region. Different diversity indices were calculated, of which the widely used Shannon diversity indices were the most important, as it is widely accepted that all species at a site, within and across systematic groups, equally contribute to its biodiversity [14, 15] . A comparison of the diversities in the three different sites was also evaluated. These estimates were calculated using the Table 1: Presence and absence of beetle families in three sites. Site A Site B Site C Scarabaeidae + + - Carabidae + + + Chrysomelidae + + - Coccinellidae + + - Borydae - + - Lycidae - + - Curculionidae - + - Hydrophilidae - - + Derodontidae - - + standard software PAST. Graphical representation of monthly variation of beetle diversity was done using MS Excel. 3. Results and Discussion After a long one-year study, 9 distinct families were identified from the three study sites (Table 1). These are Scarabaeidae, Carabidae, Chrysomelidae, Coccinellidae, Borydae, Lycidae, Curculionidae, Hydrophilidae, and Derodontidae. The last two families were strictly restricted to Site C, that is, the wetland. Families Borydae, Lycidae, and Curculionidae are reported only from Site B. The total numbers of beetle of each family for each site are given in Figures 1, 2, and 3. Diversity analysis study reveals that in Site A the Shannon Diversity indices gradually increase from January (1.18) and reaches the peak by June-July (1.35) and then slowly decrease to the end of the year (Table 2). Lowest value of Shannon Diversity is noted in the month of November (1.10). Similarly the Simpson (D) index is highest in the months of June-July (0.73) and lowest in November (0.59). Evenness values are also in accordance with the other diversity indices. Similar studies in Site B reveal that Shannon Diversity is highest in the month of July (1.85) and lowest in the months of October-November (1.75). The dominance and evenness values also indicate a similar trend, that is, higher values in July and lower values in the months of October-November (Table 3). Diversity analysis of Site C predicts that the Shannon Diversity index is more or less within a range of 1.05-1.10 from January to August but is low in the winter months. The other indices are also similar for this site (Table 4). When the diversity indices were compared for all three sites together (Table 5), it predicted that Site B has the highest diversity, that is. Shannon Diversity (1.81), is and low for site C (0.66); the Dominance_D is low in Site B and high in Site C. The shared species statistics between the three sites is done by Bray- Curtis cluster analysis and the Bray- Curtis similarity index is also calculated (Table 6). The Bray- Curtis similarity index shows 69.3% similarity between Site A and Site B and 12% similarity between Site A and Site C, whereas minimal similarity is seen between Site B and Site C (7%). Psyche 3 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec ■ Scarabaeidae ■ Chrysomelidae ■ Carabidae ■ Coccinellidae Figure 1: Total number of beetles of each family throughout the year at Site A (College Campus and surrounding). ■ Scarabaeidae ■ Borydae ■ Carabidae ■ Lycidae ■ Chrysomelidae ■ Curculionidae ■ Coccinellidae Figure 2: Total number of beetles of each family throughout the year at Site B (residential area). Table 2: Diversity indices of Site A. Site A Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Families 4 4 4 4 4 4 4 4 4 4 4 4 Individuals 37 46 80 90 109 118 128 127 86 79 52 37 Dominance_D 0.37 0.32 0.34 0.31 0.28 0.27 0.27 0.28 0.32 0.34 0.41 0.35 Simpson_l-D 0.63 0.68 0.66 0.69 0.72 0.73 0.73 0.72 0.68 0.66 0.59 0.65 Shannon_H 1.18 1.26 1.23 1.27 1.33 1.35 1.35 1.34 1.27 1.22 1.10 1.21 Evenness_e^H/S 0.81 0.88 0.86 0.89 0.95 0.96 0.96 0.95 0.89 0.84 0.75 0.84 4 Psyche 25 n Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec ■ Hydrophilidae ■ Derodontidae ■ Carabidae Figure 3: Total number of beetle of each family throughout the year at Site C (wetland). 1 . 0 - 0.9- 0 . 8 - 0.7- I 0-5 ■ 0.4- 0.3- 0 . 2 - 0.1 ■ < PQ u ■4— » ■4— » Figure 4: Bray-Curtis cluster analysis (single linkage). The Bray-Curtis cluster analysis data shows that Site A and Site B form a small cluster and Site C is joined to it through a bigger cluster (Figure 4). This also suggests that Site A and Site B are similar in faunal composition rather than Site C. 4. Conclusions It can be concluded that Durgapur though being an industrial city harbors a diverse variety of beetles. The most obvious reason is that being an industrial city, Durgapur has rich floral diversity that can support large growth of fauna. Even the municipality is well aware of the threats of an industry. As a result, the industrial belt is totally cut off from the main city centre. The present study demonstrates that Site A and Site B are much diverse than Site C. Each of the sites shows highest diversity (as obtained from the calculated diversity indices) in June-July (monsoon) compared to Site C. Thus monsoon is the time when maximum beetles are found. It is known that most animals prefer monsoon as their breeding season as it is favorable and resourceful for their proper growth and survival. This study predicts that beetles are no exception to this occurrence. It is also true that insects usually avoid harsh winter through diapause, thus diversity of beetles in all three sites are least in winter months. The floral composition of Site C is distinct from the other two sites. Only three families, namely, Hydrophilidae, Derodontidae, and Carabidae, are reported. The diversity indices also demonstrate a low diversity profile with high Psyche 5 Table 3: Diversity indices of Site B. Site B Jan Eeb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Eamilies 7 7 7 7 7 7 7 7 7 7 7 7 Individuals 77 91 142 160 187 206 225 220 198 172 115 81 Dominance_D 0.22 0.19 0.19 0.19 0.18 0.17 0.17 0.17 0.18 0.20 0.20 0.22 Simpson_l-D 0.78 0.81 0.81 0.81 0.82 0.83 0.83 0.83 0.82 0.80 0.80 0.78 Shannon_H 1.69 1.79 1.78 1.79 1.82 1.84 1.85 1.84 1.82 1.75 1.75 1.66 Evenness_e^H/S 0.78 0.86 0.85 0.85 0.88 0.90 0.91 0.90 0.88 0.83 0.82 0.75 Table 4: Diversity indices of Site C. Site C Jan Eeb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Eamilies 3 3 3 3 3 3 3 3 3 3 3 3 Individuals 7 11 11 18 21 26 34 31 22 35 26 10 Dominance_D 0.35 0.34 0.36 0.36 0.37 0.33 0.36 0.35 0.39 0.43 0.46 0.54 Simpson_l-D 0.65 0.66 0.64 0.64 0.63 0.67 0.64 0.65 0.61 0.57 0.54 0.46 Shannon_H 1.08 1.09 1.07 1.06 1.05 1.10 1.05 1.07 1.00 0.96 0.93 0.80 Evenness_e^H/S 0.98 0.99 0.97 0.96 0.96 1.00 0.96 0.97 0.90 0.87 0.84 0.74 Table 5: Comparison of diversity indices of three sites. Site A Site B Site C Eamilies 4 7 3 Individuals 999 1881 252 Dominance_D 0.30 0.18 0.34 Simpson_l-D 0.70 0.82 0.66 Shannon_H 1.30 1.81 1.09 Evenness_e^H/S 0.92 0.87 1.00 Table 6: Similarity index for three sites. Sample 1 Sample 2 Bray-Curtis index Site A SiteB 0.693 Site A Site C 0.120 Site B Site C 0.070 dominance. Beetles belonging to this site are water beetles and dominate the wetland area thus hindering other beetle families from flourishing. Thus Site C has a different beetle composition from the other two sites. This is also shown in the similarity index and cluster analyses. The only common family of these three sites is Carabidae. The only possible and reasonable conclusion will be that Carabids can explore different habitats. Site B has the highest diversity. It is a residential area and was thought to harbor the least number of beetles, but results obtained were just the opposite. Though being an area of concrete jungle, each and every house has a large expanse of gardens with diverse floral composition, that is, varieties of trees, shrubs, and bushes providing diverse habitat that can support a large variety of beetles. Though the beetle varieties seen in Site A and Site B are more or less similar, diversity indices are high in Site B. This can be explained as Site A has a flxed floral range, mostly Shal trees {Shorea robusta) that can support selective variety of beetles only. Due to expansion of urban areas, more sites that pro- tect biodiversity are required. This study suggests that an industrial town with high pollution threats, Durgapur, can nonetheless harbor a large number of beetles. Keeping this in mind, further studies can be conducted at other industrial sites for different floral and faunal groups. Conflict of Interests The author declares that there is no conflict of interests. References [1] J. A. Powell, “Coleoptera,” in Encyclopedia of Insects, H. Vincent Resh and T. Ring Garde, Eds., p. 199, Academic Press, New York, NY, USA, 2nd edition, 2009. [2] M. L. Rosenzweig, Species Diversity in Space and Time, Cam- bridge University Press, 1995. [3] T. Hunt, J. Bergsten, Z. Levkanicova et al, “A comprehensive phytogeny of beetles reveals the evolutionary origins of a superradiation,” Science, vol. 318, no. 5858, pp. 1913-1916, 2007. [4] P. M. Hammond, “Species inventory,” in Global Biodiversity, Status of the Earths Living Resources, B. Groombridge, Ed., pp. 17-39, Chapman & Hall, London, UK, 1992. [5] A. D. Chapman, Numbers of Living Species in Australia and the World, Department of the Environment, Water, Heritage and the Arts, 2nd edition, 2009. [6] G. T. Eincher, W. G. Monson, and G. W. Burton, “Effect of cattle faeces rapidly buried by dung beetles on yield and quality of Bermudagrass,” Agronomy Journal, vol. 73, pp. 775-779, 1981. [7] P. J. Gullan and P. S. Cranston, The Insects: An Outline of Entomology, John Wiley & Sons, Oxford, UK, 4th edition, 2010. [8] C. Gilliott, Entomology, Springer, New York, NY, USA, 2nd edition, 1995. [9] J. Brown, C. H. Scholtz, J.-L. Janeau, S. Grellier, and P. Podwo- jewski, “Dung beetles (Coleoptera: Scarabaeidae) can improve soil hydrological properties,” Applied Soil Ecology, vol. 46, no. 1, pp. 9-16, 2010. 6 Psyche [10] B. Kromp, “Carabid beetles in sustainable agriculture: a review on pest control efficacy, cultivation impacts and enhancement,” Agriculture, Ecosystems and Environment, vol. 74, no. 1-3, pp. 187-228, 1999. [11] N. Kakkar and S. K. Gupta, “Temporal variations in dung beetle (Coleoptera: Scarabaeidae) assemblages in Kurukshetra, Haryana, India,” Journal of Threatened Taxa, vol. 1, no. 9, pp. 481-483, 2009. [12] P. M. Choate, “Introduction to the Identification of Beetles (Coleoptera). Dichotomous Keys to Some Families of Florida Coleoptera,” pp. 23-33, 1999. [13] Beetles Associated With Stored Products in Canada: An Identi- fication Guide, Yves Bousquet Biosystematics Research Centre Ottawa, Ontario, Canada, 1990. [14] K. N. Ganeshaiah, K. Chandrashekara, and A. R. V. Kumar, “Avalanche index: a new measure of biodiversity based on biological heterogeneity of the communities,” Current Science, vol. 73, no. 2, pp. 128-133, 1997. [15] V. G. Thakare, V. S. Zade, and K. Chandra, “Diversity and abun- dance of scarab beetles (Coleoptera: Scarabaeidae) in kolkas region of Melghat Tiger Reserve (MTR), District Amravati, Maharashtra, India,” World Journal of Zoology, vol. 6, no. 1, pp. 73-79, 2011. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 769021, 4 pages http://dx.doi.org/10.1155/2014/769021 Research Article Notes on the Biology of the Cixiid Planthopper Cixius meridionalis (Hemiptera: Fulgoroidea) M. L. Bowser U.S. Fish & Wildlife Service, Kenai National Wildlife Refuge, P.O. Box 2139, Soldotna, AK 99669, USA Correspondence should be addressed to M. L. Bowser; matt_bowser@fws.gov Received 24 October 2013; Accepted 27 November 2013; Published 3 February 2014 Academic Editor: Martin H. Villet Copyright © 2014 M. L. Bowser. 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. With the exception of a handful of economically important species, the biology of cixiid planthoppers (Hemiptera: Fulgoroidea: Cixiidae) is poorly known. The host plants and life history of Cixius meridionalis Beirne were investigated in a wetland in Soldotna, Alaska. Specimens were collected over the course of the growing season by hand, aspirator, Berlese funnel, and sweep net. A handful of live nymphs were placed in a terrarium containing potential host plants for direct observation of feeding. C. meridionalis was found to feed on roots of Picea mariana (Mill.) Britton, Sterns & Poggenb, Empetrum nigrum L., Chamaedaphne calyculata (L.) Moench, and Vaccinium vitis-idaea L. At least within the study area, C. meridionalis appears to require multiple years to reach adulthood, with overwintering in nymphal instars. C. meridionalis was occasionally tended by Myrmica alaskensis Wheeler. 1. Introduction With the exception of a handful of economically important species that transmit phytoplasmas [1-4] , the biology of cixiid planthoppers (Hemiptera: Fulgoroidea: Cixiidae) is largely unknown. Nymphs are subterranean, feeding on plant roots or fungi [3] . Adults generally live aboveground but have been collected underground [2, 3]. Some species have a completely cavernicolous life cycle, with adults feeding on roots along with nymphs [5, 6]. Cixiids are usually univoltine, with diapause in nymphal instars [3], although at least one species requires two years to complete its life cycle [7]. Cixiids are sometimes associated with ants [3, 8, 9], and at least one species appears to be an obligate guest of ants [10] . Cixius meridionalis Beirne is the most widespread and frequently collected cixiid in Alaska based on material in the collection of the University of Alaska Museum (Fairbanks, Alaska) and is broadly distributed in northern North Amer- ica [11]. No biological data are available for this species other than localities and collection dates of adults [11] . Other Cixius species feed on roots of vascular plants (Table 1). I first observed C. meridionalis as adults swept from low vegetation at Headquarters Lake, Soldotna, Alaska, on October 20, 2006. In 2008, I observed cixiid nymphs in a Berlese sample from a wetland near Headquarters Lake (KNWR:Ento:8917, http://dx.doi.org/10.7299/X72807QZ) and in a sample of Hylocomium splendens (Hedw.) Schimp. moss from a black spruce forest in the vicinity of the Chickaloon River near Chickaloon Flats. Over the summer of 2013, I sought to determine the host plants and life history of C. meridionalis. 2. Materials and Methods 2.1. Field Sampling. Adult and nymphal cixiids were sam- pled by hand, aspirator, and sweep net in the wetland around Headquarters Lake, Soldotna, Alaska (N 60°27^35^^ W 15r03^58^^), at least weekly from May 28, when soils in the muskeg were still partly frozen, until the first fall frost on September 20, 2013. Moss samples were collected at least weekly from July 31, 2013, to September 20, 2013, and cixiids were extracted with a BioQuip model 2831 Berlese funnel. Nymphs of C. meridionalis were mainly collected by carefully pulling apart moss and duff by hand and extracting nymphs with a BioQuip model 1135A aspirator. Feeding behavior was difficult to observe in the field because nymphs quickly hopped away or crept down into the substrate to avoid capture, but nymphs were occasionally found in dense moss or duff where they could not easily move away from their 2 Psyche Table 1: Known nymphal hosts of Cixius species. Cixius species Nymphal host Reference C. pallipes Fieber C. pilosus (Olivier) C. wagneri China Roots (species unknown) Grasses Fragaria x ananassa (Weston) Duchesne ex Rozier (pro sp.) (chiloensis x virginiana) Vadell and Hoch [6] China [12] Salar et al. [4] feeding sites. Roots were identified to species by excavating them back to the aboveground parts of the plants. Infested roots were brought to the laboratory and feeding sites were examined for evidence of feeding. 2.2. Rearing and Identification. Adults were reared from fifth- instar nymphs by placing nymphs in vials or plastic bags with damp Sphagnum moss, and then adults were identified using the key of Kramer [11] . To identify the nymphal instars of C. meridionalis, I devel- oped a key through examination of the nymphs collected and by comparison with existing descriptions and keys [2, 13, 14]. 2.3. Direct Observation of Feeding. In an attempt to observe feeding, several C. meridionalis nymphs were placed in a clear plastic terrarium (27 cm length x 17 cm width x 17 cm depth) filled with a correspondingly sized divot of moss and plants from the Headquarters Lake wetland including Sphagnum moss, a Picea mariana (Mill.) Britton, Sterns & Poggenb (Pinaceae) seedling, Empetrum nigrum L. (Ericaceae), Ledum palustre L. (Ericaceae), Vaccinium vitis-idaea L. (Ericaceae), Andromeda polifolia L. (Ericaceae), Vaccinium oxycoccos L., Rubus chamaemorus L. (Rosaceae), and Drosera rotundifolia L. (Droseraceae). The moss was kept moist. Roots visible on the sides and bottom of the terrarium were checked often for feeding activity. 2.4. Specimen Records and Specimen Deposition. Specimens were deposited in the entomology collection of the Kenai National Wildlife Refuge, Soldotna, Alaska (KNWR). Most specimens will be transferred to the entomology collection of the University of Alaska Museum (UAM), Fairbanks, Alaska. Specimen records for both collections are avail- able via Arctos (http://arctos.database.museum/). Complete specimen records for specimens used in this study can be found in Supplementary Table 1 available online at http://dx.doi.org/10.1155/2014/769021. 3. Results 3.1. Habitat and Feeding Behavior. Nymphs were often closely associated with roots of P. mariana, but they also appeared to be on roots of E. nigrum, V. vitis-idaea, and Chamaedaphne calyculata (L.) Moench (Ericaceae) in black spruce muskeg. No nymphs were found farther than about 2 m from boles of black spruce trees, even where the microhabitat contained similar moss communities and ericaceous shrubs. Figure 1: Fourth instar C. meridionalis nymph in situ, luly 12, 2013. The disturbed nymph had moved from its feeding site on a root of Chamaedaphne calyculata. Nymphs were found exclusively in loose, moist moss and duff, where they could move relatively freely through voids (Figure 1). In some places, there was evidence of abundant activity of C. meridionalis nymphs judging from the quanti- ties of nymphs collected, copious amounts of waxy secretions, and nymphal exuviae, but it was often difficult to distinguish between feeding sites and waxy secretions of cixiids and other Hemiptera including eriosomatines (Aphididae) and orthezi- ids (Coccoidea). Feedings sites of cixiids were generally less conspicuous than those of eriosomatines and ortheziids, with sparser waxy secretions and imperceptible damage to roots. One nymph kept in the terrarium fed on a root of E. nigrum (Figure 2). No other feeding was observed in the terrarium. 3.2. Life History. Sampling by all methods yielded 288 speci- mens of C. meridionalis (Figure 3). Despite targeted searching, only one egg was found that was thought to belong to C. meridionalis. This had been in moss inadvertently taken with nymphs on September 4. Nymphs were collected at Headquarters Lake over the entire sampling season. Only three first-instar nymphs were found between June 7 and August 7. Second-instar nymphs were found from June 7 to September 12 and third-instar nymphs were found from June 5 to September 20. Fourth-instar and fifth-instar nymphs were found for the entire portion of the season sampled (May 28 to September 20). My sampling methods appeared to be biased, yielding more large, late- instar nymphs than early instars. Adult males appeared by July 8 and were observed until September 4. Adult females were found from July 12 to August 27. Psyche 3 Figure 2: Fifth instar C. meridionalis nymph feeding on a root of Empetrum nigrum in a terrarium. Adult female (n = 30) Adult male (n = 10) • • •• • •• 0 ■ • • •• • • 5th instar (n = 88) 4th instar (n = 92) • •• • 0 m 0 00 0 3rd instar (n = 53) m m 0 0 •i •• • • • • • 2nd instar (n = 11) 0 0 0 • • • • 1st instar (n = 3) Egg(n = 1) 0 0 - • • • June July August September Figure 3: Distribution of life history stages of 288 specimens of C. meridionalis collected at Headquarters Lake from May 28 through September 20, 2013. 3.3. Key to the Nymphal Instars ofCixius meridionalis (1) Metatarsi 3-segmented: mesonotum with a longitudi- nal row of 2-4 pits near anteromedial corner (2); Metatarsi 2-segmented: mesonotum with 0-1 pits in anteromedial corner (3); (2) Mesonotal wingpads extending nearly to apex of metanotal wingpads; mesonotum with a longitudinal row of 2-4 (usually 3-4) pits near anteromedial corner (fifth instar); Mesonotal wingpads not approaching apex of metan- otal wingpads; mesonotum with a longitudinal row of 1-2 (usually 2) pits near anteromedial corner (fourth instar); (3) Mesonotum with 0-1 (usually 1) pits in anteromedial corner, length 2. 1-2.2 mm (third instar); Mesonotum with no pits in anteromedial corner, length 1.7 mm or less (4); (4) Abdominal wax plates clearly evident, length 1.6- 1.7 mm (second instar); Abdominal wax plates not apparent, length about 1.2 mm (first instar). 3.4. Additional Observations. None of the nymphs collected were obviously parasitized. C. meridionalis nymphs, although seldom associated with ants, were not averse to close proximity. When a large colony of Formica aserva Forel (including slaves of Formica neorufibarbis Emery) in a peaty hummock was excavated, C. meridionalis nymphs were found within 2-3 cm of ant tunnels. There was no obvious communication between the ants’ tunnels and the feeding places of the nymphs. In one case, a Myrmica alaskensis Wheeler worker appeared to be attending a C. meridionalis nymph. In another case, M. alaskensis workers were found in the same wax-lined cavity with C. meridionalis nymphs. When disturbed, the nymphs would forcibly squirt hon- eydew from their posterior ends, sending a narrow stream up to about 1 cm away. This was not obvious in the field but was observed when handling live nymphs under magnification. 4. Discussion Within the area sampled, C. meridionalis nymphs appear to be polyphagous on roots of P. mariana and ericaceous dwarf shrubs in moist moss and duff. However, the species’ consistent proximity to P. mariana suggests that it is either dependent on P. mariana at some stage in its life history or the two species share similar microhabitat requirements. In addition to the wetland habitat at Headquarters Lake, C. meridionalis has been collected from moist Sphagnum moss in well-drained black spruce forest in Kasilof, Alaska (KNWR:Ento:8918, http://dx.doi.org/10.7299/X7XG9R8K). Phytoplasmas were recently reported from Poland in Picea abies (L.) Karst, and the two imported Nearctic species Piceaglauca (Moench) Voss and Picea pungens Engelm. [15]; none are currently known from P. mariana. As potential vectors, it would be interesting to check for the presence of phytoplasmas in cixiids associated with Picea spp., especially Cixius beieri Wagner, whose adult hosts include Picea in central Europe [16]. Based on the observed phenology at Headquarters Lake, the life history of C. meridionalis is suggested to be as follows. Eggs are deposited in the late summer, hatching in the fall or spring. The nymphs appear to take multiple years to reach adulthood, overwintering in place as nymphs. Adults emerge in early July and are active until late September. However, adults can be found over a longer season. The University of Alaska Museum has adults collected as early as June 23 from interior Alaska (UAM:Ento:176723) and I have observed adults at Headquarters Lake as late as October 20. The unusually long nymphal stage of C. meridionalis at Headquarters Lake as compared to most other cixiids maybe due to the cold soil temperature of this wetland, a protracted 4 Psyche life cycle being a common adaptation of insects to cold climates [17]. As with some other cixiids, C. meridionalis is at least occasionally tended by ants, but the association is facultative and infrequent, fitting best into the “opportunistic and occasional” category of ant attendance defined by Bourgoin [18]. This species’ lack of spines on the fore tibiae, common on many cixiid nymphs, may be an adaptation to the loose, mossy microhabitat in which it lives, where such spines would not be necessary for digging. Conflict of Interests The author declares that there is no conflict of interests regarding publication of this paper. Acknowledgments Shanice Bailey assisted with field work. Dr. Andre Francoeur provided identifications for ant specimens. Charles Bartlett, Lois O’Brien, Stephen Wilson, Andy Hamilton, Elizabeth Bella, and two anonymous reviewers provided helpful com- ments on this paper. References [f] S. W. Wilson and L. B. O’Brien, “A survey of planthopper pests of economically important plants (Homoptera: Fulgoroidea),” in Proceedings of the 2nd International Workshop on Leafhoppers and Planthoppers of Economic Importance, M. R. Wilson and L. R. Nault, Eds., pp. 343-360, Brigham Young University, CAB International Institute of Entomology, Provo, Utah, USA, July- August 1986. [2] R. Sforza, T. Bourgoin, S. W Wilson, and E. Boudon-Padieu, “Eield observations, laboratory rearing and descriptions of immatures of the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae),” European Journal of Entomology, vol. 96, no. 4, pp. 409-418, 1999. [3] W. E. Holzinger, A. E. Emeljanov, and I. Kammerlander, “The family Cixiidae Spinola 1839 (Hemiptera; Eulgoromorpha) — a review,” in Zikaden: Leafhoppers, Planthoppers, and Cicadas (Insecta: Hemiptera: Auchenorrhyncha), W. Holzinger, Ed., vol. 4 of Denisia 4 (Zugleich Kataloge des Oberosterreichischen Landesmuseums), pp. 113-118, 2002. [4] P. Salar, J. L. Danet, J. J. Pommier, and X. Poissac, “The biology of Cixius wagneri, the planthopper vector of ‘Candidatus Phlomobacter fragariae in strawberry production tunnels and its consequence for the epidemiology of strawberry marginal chlorosis,” Julius-Kuhn-Archiv, vol. 427, pp. 24-26, 2010. [5] G. S. Taylor and P. Weinstein, “Confirmation of host plant of cave-dwelling Cixiid Planthoppers (Hemiptera: Cixiidae) by histological sectioning of fig roots,” Australian Journal of Entomology, vol. 35, no. 2, pp. 115-118, 1996. [6] M. Vadell and H. Hoch, “Cixius (Ceratoxicius) pallipes Eieber, 1876 (Hemiptera: Auchenorrhyncha; Eulgoromorpha: Cixi- idae): first record for Spain,” Bolleti de la Societat d’Historia Natural de les Balears, vol. 52, pp. 123-128. [7] R. A. Cumber, “Studies on Oliarus atkinsoni Myers (Hem.: Cixiidae), vector of the ‘Yellow-leaf’ disease of Phormium tenax Eorst. II. — the nymphal instars and seasonal changes in the composition of nymphal populations,” New Zealand Journal of Science and Technology B, vol. 34, pp. 160-165, 1952. [8] J. G. Myers, “Observations on the biology of two remarkable cixiid plant-hoppers (Homoptera) from Cuba,” Psyche, vol. 36, pp. 283-292, 1929. [9] C. R. Thompson, J. C. Nickerson, and E. W. Mead, “Nymphal Habitat of Oliarus vicarius (Homoptera: Cixiidae), and Possible Association with Aphaenogaster and Paratrechina (Hymenoptera; Eormicidae),” Psyche, vol. 86, pp. 321-326, 1979. [10] C. R. Thompson, “Association of Paratrechina arenivaga (Hymenoptera: Eormicidae), with Nymphs of Oecleus borealis (Homoptera: Cixiidae),” Journal of the New York Entomological Society, vol. 92, no. 1, pp. 35-41, 1984. [11] J. P. Kramer, “Taxonomic study of the planthopper genus Cixius in the United States and Mexico (Homoptera: Pulgoroidea: Cixiidae),” Transactions of the American Entomological Society, vol. 107, pp. 1-68, 1981. [12] W. E. China, “A revision of the British species of Cixius Latr. (Homoptera), including the description of a new species from Scotland,” Transactions of the Society Eor British Entomology, vol. 8, pp. 79-110, 1942. [13] S. W. Wilson and J. H. Tsai, “Descriptions of the immature stages oIMyndus crudus (Homoptera; fulgoroidea: Cixiidae),” Journal of the New York Entomological Society, vol. 90, pp. 166-175, 1982. [14] S. W. Wilson, J. H. Tsai, and C. R. Thompson, “Descriptions of the nymphal instars of Oecleus borealis (Homoptera: Eulgo- roidea; Cixiidae),” Journal of the New York Entomolgical Society, vol. 91, no. 4, pp. 418-423, 1983. [15] M. Kaminska and H. Berniak, “Detection and identification of three ‘Candidatus phytoplasma species in Picea spp. trees in Poland,” Journal of Phytopathology, vol. 159, no. 11-12, pp. 796- 798, 2011. [16] H. Nickel, The Leafhoppers and Planthoppers of Germany: Pat- terns and Strategies in a Highly Diverse Group of Phytophagous Insects, Pensoft Publishers, Sofia, Bulgaria, 2003. [17] S. E MacLean Jr., “Ecological adaptations of tundra inver- tebrates,” in Physiological Adaptation to the Environment, J. Vernberg, Ed., pp. 269-300, Intext Educational Publishers, New York, NY, USA, 1975. [18] T. Bourgoin, “Habitat and ant -attendance in Hemiptera: a phylogenetic test with emphasis on trophobiosis in Eulgoro- morpha,” in The Origin of Biodiversity in Insects: Phylogenetics Tests of Evolutionary Scenarios, P. Grandcolas, Ed., vol. 173, pp. 109-124, Memoires du Museum National d’Histoire Naturelle, 1997. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 652518, 9 pages http://dx.doi.org/ 10 . 1 155/2014/6525 1 8 Research Article An Ultrastnictural and Fluorescent Study of the Teratocytes of Microctonus aethiopoides Loan (Hymenoptera: Braconidae) from the Hemocoel of Host Alfalfa Weevil, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) Kent S. Shelby,' Javad Habibi,^ and Benjamin Puttier^ ^ USDA Agricultural Research Service, Biological Control of Insects Research Laboratory, 1503 S. Providence Road, Columbia, MO 65203, USA ^ Division of Endocrinology, Department of Internal Medicine, Health Sciences Center, University of Missouri, Columbia, MO 65203, USA ^ Division of Plant Sciences (Entomology), University of Missouri, Columbia, MO 65203, USA Correspondence should be addressed to Kent S. Shelby; shelbyk@missouri.edu Received 27 September 2013; Revised 6 November 2013; Accepted 15 November 2013; Published 4 February 2014 Academic Editor: Russell Jurenka Copyright © 2014 Kent S. Shelby 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 braconid wasp Microctonus aethiopoides Loan is an idiobiont endoparasitoid of alfalfa weevil adults Hypera postica (Gyllenhal). After oviposition and subsequent egg maturation, large trophic cells called teratocytes dissociate from the serosa and are released into the host hemocoel. These teratocytes are present in large numbers and are visible to the naked eye. It is thought that they accumulate host hemocoelic metabolites for later consumption by the parasitoid larvae. We have undertaken a microscopic study of these gargantuan and complex cells at approximately seven months after parasitization. Parasitized adult weevils were dissected into medium and teratocytes were fixed, embedded, and sectioned at 1 pm. Teratocytes were stained with various specific fluorescent dyes for plasma membrane, Golgi, nuclei, lysosomes, mitochondria, and endoplasmic reticulum (ER). The surface of each cell is covered with a dense microvillar layer. Analysis of fluorescent images showed that these cells do not have condensed nuclei. ER was abundant around the nuclear envelope. Lysosomes were positioned around the periphery of the nucleus and the Golgi apparatus was significantly enlarged, being located around the nuclear envelope. 1. Introduction Microctonus aethiopoides Loan (Hymenoptera: Braconidae) was found to be an effective biological control agent for the adult alfalfa weevil Hypera postica (Gyllenhal) [1, 2]. M. aethiopoides is an idiobiont parasitoid which prevents further host development after parasitization. Following oviposition into the hemocoel of the adult alfalfa weevil host the M. aethiopoides egg hatches, releasing both a first instar parasitoid larva and numerous free floating extraembryonic serosal cells which subsequently develop and differentiate into teratocytes [3]. Teratocytes undergo significant hyper- trophic growth within the hemocoel of the host. The presence of these large, white opaque cells has been implicated in the determination of host range ofM. aethiopoides [4-6]. Teratocytes or teratocyte-like cells have been documented in developmental studies of four hymenopteran families: Bra- conidae, Ichneumonidae, and Platygastridae [7, 8] and have been reported in a single species of chalcids [9]. Teratocytes are thought to function primarily as trophic cells which assimilate host metabolites for later ingestion by the develop- ing parasitoid larvae [7] . However, teratocytes secrete a num- ber of proteins in vivo and in vitro which may be responsible for the observed alterations of the host endocrine system [10- 15] and suppression of the host’s immune response against the parasitoid egg and larvae [16-18]. Despite their importance to successful parasitization by braconid parasitoids and their unique functions in suppression of the host immune system and alterations of host endocrinology, the ultrastructure of 2 Psyche teratocytes has been rarely studied [9, 19-21]. The present study was undertaken to elucidate and to document the ultrastructure of these extremely large and important cells. 2. Materials and Methods 2.1. Insects and Teratocyte Collection. Nondiapausing alfalfa weevils, Hypera postica parasitized by Microctonus aethiopoides, used in this study were obtained by sweeping adults from an alfalfa field near Boonville, Missouri (Cooper Co.) on April 29, 2002. These weevils were collected in late spring and early summer. The weevils were returned to the laboratory and maintained at 5°C on a bouquet of alfalfa in a 0.275 L ice cream carton with a Petri dish as a lid until May 1, at which time some were initially dissected under a dissecting microscope in a Stender dish cover in distilled water to assess the extent of parasitization. Adult weevils were grasped with forceps and a needle inserted in anus and gently teased to expose the contents of the abdomen. A careful search was made to locate the parasite larvae to confirm parasitization. If the host was parasitized teratocytes floated into the dissection medium. The identity of the parasite larvae as M. aethiopoides was confirmed by emergence of adult parasites reared from adult weevils collected from the same site. Dissections from weevils maintained in the same manner but for fixing, staining and sectioning of teratocytes Excel 401 tissue culture medium (Gibco Invitrogen; Carlsbad, CA) was used as dissection medium. Dissections were completed 2 days later (May 3), and again on May 6, May 8, and May 10. Teratocytes were collected from the medium for processing as detailed below. 2.2. Light Microscopy. Teratocytes were immediately fixed in 3% paraformaldehyde in HEPES wash buffer for 10 min in a gentle vacuum and 2hrs at room temperature for fluorescent studies. For light microscopy and TEM studies, the teratocytes were placed in fixative solution (2.5% glu- taraldehyde/2% paraformaldehyde, 70 mM HEPES, and pH 7.4) for 3hrs. Then, the teratocytes were dehydrated in an ethanol series, infiltrated, and embedded in methacrylate for fluorescent studies and in Epon/Spurr’s resin for light microscopy. Semithin sections of the teratocytes were stained with 0.5% acid fuchsin/0.5% toluidine blue in ultrapure water. The slides were air-dried at room temperature, mounted with Permount, and viewed with an Olympus (Melville, NY) or Nikon (Melville, NY) microscope. Images were captured using ImagePro (Media Cybernetic, Silver Spring, MD) and Spot (Diagnostic Instruments, Inc.) software and further edited using Adobe Photoshop 6.0 software. 2.3. Fluorescence Microscopy. Using a modification of the technique of Baskin et al. [22-24] teratocytes were encased in a sandwich of Formvar supported on a copper wire loop. A copper wire loop (36 ga) was made and flattened between two flat pieces of steel. Small rectangular films of 0.25% of Formvar in ethylene chloride were floated on water and the loop plunged into the middle of rectangle so that a film of Formvar surrounded the wire. A number of loops were made in advance. The loops covered with a film of Formvar were placed on a drop of water on a piece of clean glass separately. The fixed teratocytes were gently placed on the center of the Formvar surface. This assembly was coated with another layer of Formvar, sandwiching teratocytes between two layers of Formvar. Teratocytes sandwiched between layers of Formvar were dehydrated in an ethanol series at -20° C for 30 minutes for each step and then infiltrated with methacrylate (80% butyl methacrylate, 20% methyl methacrylate, and 0.5% benzoin ethyl ether; Aldrich) and 10 mM DTT. Teratocytes were embedded in fresh methacrylate mix in BEEM capsules and the plastic was polymerized under UV light in a cold room (4°C) overnight. Blocks were sectioned at 1 pm using a Reichert Cut S ultramicrotome (Eeica, Wien, Austria). Serial sections of teratocytes were placed on drops of 5% ammonium hydroxide on silane-coated slides. The sections were annealed onto the slide by gentle heating on a slide warmer and the slides were stored at 4°C for further staining. Entire 1 pm sections of eight teratocytes were deplasticized with acetone for 10 minutes, washed with HEPES buffer for 30 minutes, and cleared with 0.1% Tween 20 in PBS for 15 minutes. Nonspecific binding was minimized by incubation in blocking buffer (5% BSA, 1% nonfat dry milk, 1% gelatin, and 0.01% sodium azide). Organelle specific stains purchased from Molecular Probes (Eugene, OR) were incubated with semithin sec- tions according to manufacturer’s recommendations (http:// www.probes.com). Initial range finding experiments with fluorescent dyes, over several orders of magnitude, allowed us to derive an optimal concentration for each fluorescent dye. The sections of two cells were stained with 500 nM of Dil (a plasma membrane specific stain) in HEPES wash buffer for 90 min. Then the sections were washed with the same buffer for 30 min and were counterstained with 500 nM TO-Pro3 (a DNA specific stain) for 90 min. Sections of two other teratocytes were stained with 2 pM of NBDCg (a Colgi apparatus specific stain) for 90 min and counter- stained with 100 nM of Lysotracker (a lysozyme specific stain) for 90 min. The sections of two more teratocytes were stained with 250 nM of Mitotracker (a mitochondrial specific stain) for 90 min and counterstained with 500 nM of DioCg (an endoplasmic reticulum specific stain) for 90 min. Slides were mounted with Mo viol and were imaged by confocal microscopy. 2.4. Transmission Electron Microscopy (TEM). Teratocytes encased between two Formvar layers were fixed in 2% glu- taraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer. After fixation, the samples were rinsed three times in buffer and then postfixed in 1% OSO4 in the same buffer. Samples were then rinsed three times for 20 min each with ultrapure water. Tertiary fixation was done in 1% aqueous uranyl acetate, followed by three rinses of 20 min each in ultrapure water. The samples were dehydrated in an ethanol series and infiltrated with Epon/Spurr’s resin, and the resin was then polymerized at 55° C for two days after which the blocks were stored in a desiccator until sectioning. Ultrathin sections were Psyche 3 Figure 1: Bright-field image of M aethiopoides larva and teratocytes collected from a single host adult H. postica hemocoel at approximately seven months after parasitization showing hypertrophied M. aethiopoides teratocytes. PL: parasitoid larvae, T; teratocytes, scale bar = 1mm. (c) (d) Figure 2: Bright-field images of different 1 pm (semithin) sections from different areas of teratocytes of M. aethiopoides were stained with 0.5% toluidine blue and counterstained with 0.5% acid fuchsin showing amorphous nuclei. N: nuclei. Large unstained areas (arrow). Scale bar = 50 pm. cut with a Reichert Ultracut S ultramicrotome (Leica, Wien, Austria). Standard TEM procedures were applied to ultra- thin sections of the teratocytes on copper grids. 3. Results Teratocytes collected from the hemolymph of field-collected adult alfalfa weevils at approximately seven months after par- asitization were examined by bright-field microscopy. When parasitized weevils were dissected under saline or tissue culture medium numerous teratocytes were easily visualized. No attempt was made to definitively determine the mean number of teratocytes per host. Teratocytes were extremely large hypertrophied, opaque buoyant spherical cells, visible to the naked eye (Figure 1). Since only a single developmental stage was available for study, earlier stages of teratocyte development, release, and growth were not observed and are not presented in this study. At the time of collection, all teratocytes appeared to have attained maximal diameter. A cytological investigation of teratocyte internal structures 4 Psyche (c) (d) Figure 3; Semithin sections of teratocytes were dual-stained with Dil for plasma membrane and with TO-Pro3 for nucleus, (a) Transmitted light micrograph of teratocyte showing internal structure of the cell, (b) Same cell section stained with Dil showing strong staining of plasma membrane and an unidentified internal membranous network, (c) Same section stained with TO-Pro3 showing intense staining of chromosomal DNA within the large amorphous nucleus, (d) Overlaid images from (a) to (c) showing red plasma membrane and blue amorphous nucleus. N: nucleus, PM: plasma membrane, UIM: unidentified internal plasma membrane network. Scale bars = 20 ^m. was undertaken based on light and fluorescent microscopic examination of a large number of semithin sections. Semithin sections were stained with toluidine blue/acid fuchsin to obtain resolution of intracellular structures (Fig- ures 2(a)-2(d)). A large stellate nucleus occupied the center of each teratocyte, with ramifications extending throughout the cytoplasm to the plasma membrane. The remainder of the interior was occupied by densely staining granules. In all teratocytes examined, a curious unstained internal membra- nous network at one end of each teratocyte and occupying a substantial volume was present in close proximity to the plasma membrane (Figures 2(b)-2(d)). Further examination of teratocyte intracellular structure was undertaken by fluorescence microscopy of semithin sections using dyes chosen for their ability to stain specific organelles (Figures 3(a)-3(d)). Sections were dual-stained with the plasma membrane specific stain, Dil, and with the nuclear stain TO-Pro3. A wide plasma membrane surround- ing each cell was observed, and a polar structure comprised of an internal membranous network heavily stained by Dil was present at the periphery of each teratocyte examined (Figure 3(b)). The same sections stained with TO-Pro3 exhib- ited intense staining of chromosomal DNA within the large stellate nucleus (Figure 3(c)). The nucleus did not appear to overlap with the unidentified internal membranous network (Figure 3(d)). Semithin sections were dual-stained with the Golgi spe- cific stain NBDCg and with the lysosomal specific stain Lysotracker (Figures 4(a)-4(d)). Within each teratocyte multiple strongly staining Golgi structures were observed distributed along the periphery of the nucleus (Figure 4(b)). Numerous lysosomal-staining structures were distributed throughout the volume of the teratocyte (Figure 4(c)). When the images were digitally overlaid, the perinuclear position of Psyche 5 Figure 4; Semithin sections of teratocytes were dual-stained with NBDCg for Golgi apparatus and Lysotracker for lysosomes. (a) Transmitted light micrograph of teratocyte showing internal structure of the cell, (b) Same cell section stained with NBDCg showing strong staining of the Golgi distributed mostly around the periphery of the nucleus, (c) Same section stained with Lysotracker showing distribution of lysosomes (red) within the teratocyte. (d) Overlaid images of (a)-(c) showing Golgi around nucleus. G: Golgi apparatus, L: lysosomes. Scale bars = 20 (A.m. the multiple Golgi was observed (Figure 4(d)). Sections dual- stained with the mitochondrial specific dye Mitotracker and the ER specific dye DiOCg revealed the close proximity of protein synthesis with energy production (Figure 5(a)). Dark unstained regions were seen within lobes of the teratocyte bordered by intense ER staining (Figure 5(b)). Mitochondria are present throughout the same volume of teratocyte occu- pied by intensely staining ER (Figure 5(c)). Ultrastructural studies were undertaken to obtain a more detailed view of the highly complex teratocyte cytoplasm. The plasma membrane appeared to be composed of a dense lawn of microvilli. Substantial amounts of rough ER cister- nae and mitochondria were closely associated with plasma membrane in close proximity to the microvilli (Figures 6(a) and 6(d)). The membranous network located at one end of the teratocytes was composed of highly complex and folded membranes with a tubular appearance that could perhaps be invaginations of the plasma membrane involved in uptake of materials from the host hemocoel (Figure 6(b)). The function of this structure has yet to be determined. The stellate nucleus was surrounded by rough ER, Golgi (Eigure 6(c)), and lipid droplets (Figure 6(d)). Higher magnification revealed mitochondria, lipid droplets, and darkly staining granules throughout the cytoplasm between the nucleus and the plasma membrane (Figure 6(e)). The internal structure of the same area of the teratocyte showed large numbers of starch and pigment granules and of lipid droplets (Figure 6(f)). 4. Discussion Teratocytes collected from the hemolymph of field-collected adult alfalfa weevils at approximately seven months after par- asitization were examined by bright-field microscopy. When parasitized weevils were dissected under saline or tissue culture medium numerous teratocytes were easily visualized. No attempt was made to determine the mean number of 6 Psyche (c) Figure 5: Semithin sections of teratocytes were dual-stained with Mitotracker for mitochondria and with DiOCg for endoplasmic reticulum, (a) Transmitted light micrograph of teratocyte showing internal structure of the cell, (b) Same cell section stained with DiOCg showing green staining of ER surrounding the dark unstained bodies, (c) Same section stained with Mitotracker (red) showing distribution of mitochondria within the teratocyte. ER: endoplasmic reticulum, M: mitochondria, N: nucleus. Scale bars = 20 ^m. teratocytes per host. In comparison to the developing M. aethiopoides larva from the same host, the teratocytes were extremely large hypertrophied, opaque buoyant spherical cells, visible to the naked eye. Since only a single age was available for study, the initial stages of teratocyte release and growth were not observed. At the time of collection, all teratocytes appeared to have attained maximal diameter. No earlier teratocyte developmental stages were examined. An initial cytological investigation of the internal structures was undertaken. Semithin sections of the same material were stained with toluidine blue/ acid fuchsin to obtain resolution of intracellular structures. In a manner similar to the terato- cytes of Toxoneuron {-Cardiochiles) nigriceps (Viereck) [20], a large stellate nucleus occupied the center of each teratocyte, with ramifications extending throughout the cytoplasm to the plasma membrane. The remainder of the interior was occupied by densely staining granules. An unstained internal membranous network occupying a substantial volume was present in close proximity to the plasma membrane. Ultrastructural studies were undertaken to obtain a more detailed view of the highly complex teratocyte cytoplasm. The plasma membrane appeared to be composed of a dense lawn of microvilli, similar in appearance to teratocytes of another braconid wasp, Microplitis croceipes (Cresson) [19]. Substantial amounts of rough ER cisternae and mitochon- dria were closely associated with plasma membrane. Close association of the microvilli, rough ER, and mitochondria at the plasma membrane indicates that both uptake of nutrients from and the synthesis/secretion of proteins into the host hemocoel are the primary activities occurring at this stage of teratocyte development [20, 25]. The membranous network located at one end of the teratocytes was composed of highly complex and folded membranes with a tubular appearance that could perhaps be invaginations of the plasma membrane involved in uptake of materials from the host hemocoel. The function of this structure has yet to be determined. The stellate nucleus was surrounded by rough ER, Golgi, and lipid droplets. Higher magnification revealed mitochondria. Psyche 7 (e) (f) Figure 6: Ultrathin sections of teratocytes were stained with uranyl acetate and Lead citrate, (a) Dense microvilli, rough endoplasmic reticulum, and mitochondria associated with plasma membrane. Scale bar = 500 nm. (b) TEM image of unidentified membranous network located at opposite sides of the teratocyte. Scale bar = 1 ^m. (c) TEM image of one arm of amorphous nucleus showing intense staining of heterochromatin, numerous lipid droplets, and pigment. Scale bar = 1 jw. (d) Same cell showing microvilli, rough ER, and associated mitochondria. Scale bar = 500 nm. (e) Same cell with higher magnification showing mitochondria and lipid droplets. Scale bar = 100 nm. (f) Electron micrograph of the internal structure of the same area of the teratocyte showing starch and pigment granules and lipid droplets. Scale bar = 500 nm. PM: plasma membrane; MV: microvilli; UIM: unidentified membranous network; N: nucleus; L: lipid droplets; RER: rough endoplasmic reticulum; M: mitochondria; S: starch; P: pigment granules. Scale bar = 20 (dm. lipid droplets, and darkly staining granules throughout the cytoplasm between the nucleus and the plasma membrane. Within this area of the teratocyte adjacent to mitochondria were putative energy storage depots such as lipid droplets and starch granules. In conclusion, the extremely large M. aethiopoides ter- atocytes possess an internal structure well suited to their putative trophic and secretory function. The plasma mem- brane is studded with a dense array of microvilli which would facilitate efficient uptake of nutrients from the host hemocoel. Lysosomes, numerous lipid droplets, and granules of protein and starch crowd the cytoplasm. An amorphous membranous network staining heavily with an ER specific dye is present at one end of the cell. Arms of the large stellate 8 Psyche nucleus ramify throughout the volume of the cell abutting against rough ER and mitochondria. Finally, these teratocytes contain large amounts of Golgi, rough ER, and mitochondria that would be required for the synthesis and export of proteins into the host hemocoel that may be responsible for suppression of the host immune system. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors wish to thank Tom Phillips of the University of Missouri, Department of Biological Sciences, for consultation and technical assistance. They also wish to thank Tobias Baskin of the Department of Biological Sciences of the University of Massachusetts, Amherst (formerly of the Uni- versity of Missouri), for consultation and technical assistance. Names are necessary to report factually on available data; however, the USD A neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. All programs and services of the US. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. References [1] L. W. Coles and B. Puttier, “Status of the alfalfa weevil biological control program in the Eastern United States,” Journal of Economic Entomology, vol. 56, pp. 609-611, 1963. [2] W. H. Day, L. W. Coles, J. A. Stewart, and R. W. Fuester, “Distribution of Microctonus aethiops and M. colesi, parasites of the alfalfa weevil, in the Eastern United States,” Journal of Economic Entomology, vol. 64, pp. 190-193, 1971. [3] O. J. Smith, “Biology and behavior of Microctonus vittatae Mue- sebeck (Braconidae),” in University of California Publications in Entomology, E. G. Linsley, S. B. Freeborn, E. A. Steinhaus, and R. L. Usinger, Eds., pp. 315-343, University of California Press, Los Angeles, Calif, USA, 1952. [4] B. I. P. Barratt and P. D. Johnstone, “Factors affecting parasitism by Microctonus aethiopoides (Hymenoptera; Braconidae) and parasitoid development in natural and novel host species,” Bulletin of Entomological Research, vol. 91, no. 4, pp. 245-253, 2001. [5] B. I. P. Barratt and M. Sutherland, “Development of teratocytes associated with Microctonus aethiopoides Loan (Hymenoptera: Braconidae) in natural and novel host species,” Journal of Insect Physiology, vol. 47, no. 3, pp. 251-262, 2001. [6] B. I. P. Barratt, A. A. Evans, D. B. Stoltz, S. B. Vinson, and R. Easingwood, “Virus-like particles in the ovaries of Microctonus aethiopoides Loan (Hymenoptera: Braconidae), a parasitoid of adult weevils (Coleoptera: Curculionidae),” Journal of Inverte- brate Pathology, vol. 73, no. 2, pp. 182-188, 1999. [7] N. E. Beckage and D. B. Gelman, “Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control,” Annual Review of Entomology, vol. 49, pp. 299-330, 2004. [8] F. Rouleux-Bonnin, S. Renault, A. Rabouille, G. Periquet, and Y. Bigot, “Free serosal cells originating from the embryo of the wasp Diadromus pulchellus in the pupal body of parasitized leek-moth, Acrolepiosis assectella. Are these cells teratocyte- like?” Journal of Insect Physiology, vol. 45, no. 5, pp. 479-484, 1999. [9] P. A. Pedata, A. P. Garonna, A. Zabatta, P. Zeppa, R. Romani, and N. Isidore, “Development and morphology of teratocytes in Encarsia berlesei and Encarsia citrina: first record for Ghal- cidoidea,” Journal of Insect Physiology, vol. 49, no. 11, pp. 1063- 1071, 2003. [10] R. L. Rana, D. L. Dahlman, and B. A. Webb, “Expression and characterization of a novel teratocyte protein of the braconid, Microplitis croceipes (cresson),” Insect Biochemistry and Molecu- lar Biology, vol. 32, no. 11, pp. 1507-1516, 2002. [11] K. Kadono-Okuda, F. Weyda, and T. Okuda, “Dinocampus (= Perilitus) coccinellae teratocyte-specific polypeptide: its accu- mulative property, localization and characterization,” Journal of Insect Physiology, vol. 44, no. 11, pp. 1073-1080, 1998. [12] F. Pennacchio, S. B. Vinson, E. Tremblay, and A. Ostuni, “Alteration of ecdysone metabolism in Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae induced by Cardiochiles nigri- ceps Viereck (Hymenoptera: Braconidae) teratocytes,” Insect Biochemistry and Molecular Biology, vol. 24, no. 4, pp. 383-394, 1994. [13] M. Hotta, T. Okuda, and T. Tanaka, “Cotesia kariyai teratocytes: growth and development,” Journal of Insect Physiology, vol. 47, no. 1, pp. 31-41, 2001. [14] Q. Qin, H. Gong, and T. Ding, “Two coUagenases are secreted by teratocytes from Microplitis mediator (Hymenoptera: Bra- conidae) cultured in vitroj Journal of Invertebrate Pathology, vol. 76, no. 1, pp. 79-80, 2000. [15] Y. Nakamatsu, S. Fujii, and T. Tanaka, “Larvae of an endopara- sitoid, Cotesia kariyai (Hymenoptera: Braconidae), feed on the host fat body directly in the second stadium with the help of teratocytes,” Journal of Insect Physiology, vol. 48, no. 11, pp. 1041- 1052, 2002. [16] D. L. Dahlman, “Teratocytes and host/parasitoid interactions,” Biological Control, vol. 1, no. 2, pp. 118-126, 1991. [17] D. L. Dahlman and S. B. Vinson, “Teratocytes. Developmental and biochemical characteristics,” in Parasites and Pathogens of Insects, vol. I, pp. 145-165, Academic Press, New York, NY, USA, 1993. [18] T. Tanaka, “Effect of the venom of the endoparasitoid, Apanteles kariyai Watanabe, on the cellular defence reaction of the host, Pseudaletia separata Walker,” Journal of Insect Physiology, vol. 33, no. 6, pp. 413-420, 1987. [19] D. Zhang, D. L. Dahlman, U. E. Jarlfors, H. H. Southgate, and S. P. Wiley, “Ultrastructure of Microplitis croceipes (Gresson) (Braconidae: Hymenoptera) teratocytes,” International Journal of Insect Morphology and Embryology, vol. 23, no. 3, pp. 173-187, 1994. [20] S. B. Vinson and J. R. Scott, “Ultrastructure of teratocytes of Gardiochiles Nigriceps Viereck (Hymenoptera: Braconidae),” International Journal of Insect Morphology and Embryology, vol. 3, no. 2, pp. 293-304, 1974. [21] F. Pennacchio, S. B. Vinson, and E. Tremblay, “Morphology and ultrastructure of the serosal cells (teratocytes) in Cardiochiles Psyche 9 nigriceps Viereck (Hymenoptera: Braconidae) embryos,” Inter- national Journal of Insect Morphology and Embryology, vol. 23, no. 2, pp. 93-104, 1994. [22] T. I. Baskin and }. E. Wilson, “Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules,” Plant Physiology, vol. 113, no. 2, pp. 493-502, 1997. [23] T. I. Baskin, J. E. Wilson, A. Cork, and R. E. Williamson, “Morphology and microtubule organization in arabidopsis roots exposed to oryzalin or taxol,” Plant and Cell Physiology, vol. 35, no. 6, pp. 935-942, 1994. [24] T. I. Baskin, C. H. Busby, L. C. Eowke, M. Sammut, and E. Gubler, “Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa,” Planta, vol. 187, no. 3, pp. 405-413, 1992. [25] S. B. Vinson, “Development and possible functions of terato- cytes in the host-parasite association,” Journal of Invertebrate Pathology, vol. 16, no. 1, pp. 93-101, 1970. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 396095, 9 pages http://dx.doi.org/10.1155/2014/396095 Research Article Inflorescences of the Bromeliad Vriesea friburgensis as Nest Sites and Food Resources for Ants and Other Arthropods in Brazil Volker S. Schmid,^ Simone Langner,^ Josefina Steiner,^ and Anne Zillikens^’^ ^ Department of Evolution, Behavior and Genetics, University of Regensburg, 93040 Regensburg, Germany ^ Justus-Liebig-Universitdt Giefien, Karl-Glockner-Strafie 21 G, 35394 Giefien, Germany ^ Departamento de Biologia Celular, Embriologia e Genetica, CCB, Campus Universitdrio Trindade, Universidade Eederal de Santa Catarina, 88040-900 Elorianopolis, SC, Brazil Med.-Naturwissenschaftliches Eorschungszentrum, Universitdt Tubingen, Ob dem Himmelreich 7, 72074 Tubingen, Germany Correspondence should be addressed to Volker S. Schmid; volker.schmid@biologie.uni-regensburg.de Received 17 September 2013; Accepted 1 December 2013; Published 2 March 2014 Academic Editor: Jacques Hubert Charles Delabie Copyright © 2014 Volker S. Schmid 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. For the first time, the usage of bromeliad inflorescences as nesting sites for ants and other arthropods was studied. Frequencies of occurrence of nests were recorded from hollow stems of dried infructescences of the bromeliad Vriesea friburgensis on Santa Catarina Island, southern Brazil. Three habitat types were studied; miconietum and two types of restinga, one with low (restinga- low) and one with high vegetation cover (restinga-high). Additionally, flower visitation by ants was examined in restinga-low. Out of 619 infructescences, 33% contained nests. Ants were the most frequent occupants (82-96% of nests), followed by termites (3-18%) and bees (0-0.6%). Species accumulation curves and diversity indices indicate that the diversity of stem-occupying ant species is highest in restinga-low (eight species observed, 18 predicted) and lowest in restinga-high (four observed and predicted). Highest similarity of compositions of infructescence-inhabiting ant species was recorded between miconietum and restinga-high, lowest between restinga-low and restinga-high. Similarity between compositions of inflorescence-visiting and infructescence-inhabiting species in restinga-low was even higher (compared with the cases described in the previous sentence) although 50% of the involved species were present in only one of the samples. Altogether, our results indicate that inflorescences are important resources for ants and other nest-building insects from flowering season to past-fruiting season. 1. Introduction Bromeliads (Bromeliaceae) are monocot plants occurring almost exclusively in the neotropics [1]. Animal-bromeliad interactions are highly diverse and have been in the focus of intensive research during the last decades [2]. Consequently, several aspects of these associations are well studied, for example, pollinator systems [3-5] or usage of bromeliad rosettes as nest sites by ants [6, 7] and bees [8]. Additionally, bud and fruit capsules are known to nourish and shelter developmental stages of wasps [9] and butterflies [10] . The stalks of bromeliad inflorescences or infructescences have mainly been reported as subjects of insect larval her- bivory. For example, inflorescence stems may be infested by several species of Curculionidae (Coleoptera) [2]. In the bromeliad species Vriesea friburgensis Mez van paludosa (L. B. Smith) L. B. Smith 1952, flower buds are sterilized by the feeding behavior of a eurytomid wasp larva. Affected flowers do not open and eventually dry up forming a resistant pupal chamber for the developing larva [9] . A similar case is that of Strymon serapio (Godman and Salvin 1887) [10] whose larvae attack the ripening fruit capsule of V. friburgensis. During feeding, the larva enters the capsule and finally pupates within. In both cases, the imagines emerge from their pupal chambers after weeks or even months. This is enabled by the fact that the stalk of the drying infructescence usually remains standing erect in the rosettes for one year or longer. 2 Psyche instead of wilting and decomposing rapidly (pers. obs.). This feature is typical for many species in the genus Vriesea which has an anemogamous seed dispersal syndrome, and allows the small seeds provided with pappi to take flight with the wind. This is in sharp contrast to other bromeliads in the subfamily Bromelioideae whose seeds are embedded into a fleshy pulp and whose infructescence stalks wilt and collapse soon after the colorful berries have been eaten by birds and small mammals. In the course of a study on the diversity and interactions of flower visitors of bromeliads in the Atlantic rainforest of southern Brazil, we discovered stems of old infructescences of V. friburgensis to be inhabited by ant species. This, together with the other mentioned characteristics of the infructes- cence stalk, suggests that it might play another important role in the ecosystem by providing shelter or nest sites with a beneficial environment for perennial arthropods and social insects. Among the latter, ants constitute the dominant ani- mal group in most terrestrial ecosystems [11], and numerous species that are unspecialized nesters could benefit from the properties of infructescence stems. Therefore, assessing the use of stalks as nesting sites was the main purpose of our study. Not only infructescences but also inflorescences are, at least during flowering, attractive to ants: in a preliminary census, about 50% of 159 open flowers (distributed over 68 inflorescences) were visited by at least one ant (S. Langner, unpubl. data). The inflorescences are visited by a high diversity of animals, mainly bees [12] . Yet, ants have not been systematically registered so far; hence, an additional goal of our study was to survey the spectrum of ant species associated with inflorescences and infructescences. In particular, we determined frequencies of occurrence as well as alpha and beta diversity (i.e., diversity within and among habitats) of ants and other arthropods in old infructescence stems of 14 friburgensis. We expected differ- ences in ant species richness (which is one component of diversity) and composition among habitat types due to dif- ferent species communities as was reported for inflorescence - visiting ants of other bromeliad species [13] . At one site, we also recorded ants visiting inflorescences for comparison with the stem- inhabiting ants, testing the hypothesis that species richness and composition should be similar because ants living within the bromeliads can be expected to visit nearby flowers. 2. Material and Methods 2.1. Study Plant. Vriesea friburgensis is a common, mostly soil-growing but facultatively epiphytic tank bromeliad occurring in forest and restinga habitats in southern Brazil [12, 14, 15]. Its inflorescences, flowering from November to March [9, 12], reach a height of about 0.5-1.5 m (for habitus see Supplementary Figures SI and S2 in Supplementary Mate- rial available online at http://dx.doi.org/10.1155/2014/396095). The dry infructescences frequently remain standing erect in the rosettes for one year or longer, and even when bent or broken, they may stay more or less intact for a long period of time (pers. obs.). Only terrestrially growing plants were examined. 2.2. Study Sites and Period. The study was conducted from January 2006 to January 2009 in the municipality of Florianopolis on Santa Catarina Island, southern Brazil. Infructescence occupation was examined between August and February, inflorescence visitation in December and January. Observations and sampling were performed in the two habitat types “miconietum” (a pre-forest succession stage, [8]) and “restinga” (a xerophilous vegetation forma- tion on sand dunes, [16]). Four sites were studied: (i) a mountainside in Santo Antonio de Lisboa (miconietum; 27°30'26"S, 48“30'28"W); (ii) Joaquina beach (27°37'37"S, 48°26^59^^W) (see Supplementary Figure S2) and (iii) Campeche beach (27°40^38^^S, 48°28^48^^W), both similar low-vegetation restingas and pooled as “restinga-low”; (iv) Reserva Ecologica do Morro das Aranhas (high-vegetation restinga, termed “restinga-high”; 27°28^11^^S, 48°23^06^^W). An overview of most samples described in the following subsections is provided in Supplementary Figure S3. 2.3. Assessing Infructescence Occupation. We examined a total of 619 infructescences (defined as the stem and remains of buds and fruits above the level of the water reservoir in the rosette) for nests of ants and other social insects by breaking them apart. Criterion for the record of a nest was the presence of brood. Other arthropods were occasionally registered too. From a subset of 131 infructescences (restinga-low: 54; restinga-high: 28; miconietum: 49), inhabitants of the interior of the stem as well as of cavities under bracts were hand- collected and identified in the laboratory. For the other frac- tion (488 infructescences, only restinga-low), we identified the inhabitants directly in the field and additionally noted whether the stems were solid or hollow. Cavities under bracts were not examined in this case. To estimate cavity volume, five hollow infructescences were randomly chosen and the lengths and inner diameters (at base and apex) of their cavities were measured. In addition to the samples described above, we recorded arthropods in infructescences during occasional field trips that were not specifically associated with this study. Some- times, we also examined the most basal, humid part of the stems. Those findings are separately presented in the results section and Supplementary Table S9 but were not taken into account for the remaining data presentation and analysis. Generally, sets of infructescence stems that served as references for calculation of percentages included those that were not hollow. This is because we regard massive stems to be a resource for ants too, at least for those that are capable of gnawing holes into the plant material themselves. Three samples of non-social insect brood were taken to the laboratory and reared to adult stages for identification. Voucher specimens of all collected species were deposited in the entomological collection of the Native Bee Laboratory, BEG, Eederal University of Santa Gatarina, Elorianopolis, Santa Gatarina, Brazil. Psyche 3 2.4. Inflorescence Visitation by Ants. For identification of ants foraging on inflorescences, specimens were hand-collected from a set of 33 flowering inflorescences (from flower-bearing branches or the nearby stalk; see Supplementary Figure SI) at Joaquina. In two cases, ants were present but could not be identified because they escaped collection. Moreover, the presence of ants within flowers was recorded at the same site, where a further sample of 101 randomly chosen inflorescences was defined. On five days in weekly intervals, all available open flowers on these inflorescences were examined for ants. Because inflorescence lifetime of 14 friburgensis usually includes days without open flowers [12], the number of inflorescences with open flowers varied between 40 and 79 (out of 101). For investigating whether the attractiveness of inflores- cences begins with the emergence of open flowers or earlier, we assessed ant presence for a separate set of 26 inflorescences with buds only. 2.5. Species Richness and Diversity. We compared the diver- sity of ants occupying infructescences among habitats (three comparisons) as well as infructescence occupation with inflo- rescence visitation in restinga-low. Alpha diversity (diversity within each locality) was assessed by computing species accumulation curves (generated with the “Mao Tau” binomial mixture model by Colwell et al. [17]), the Chao2 species richness estimator, and the reciprocal Simpson index. Since Simpson diversity is a measure that combines species richness with the evenness of the species abundance distribution, we also calculated Simpson evenness by dividing the Simpson index by the number of observed species (as given by the species accumulation curves) [18]. Accumulation curves and means (obtained by 1000-fold resampling) of estimators and indices were plotted against the cumulative number of species occurrences as a measure of sampling effort [19]. If such a curve reached a plateau over a logarithmically scaled x- axis, we regarded the corresponding index or species richness as stable [19]; if not, then it was expected to change with increased sampling. For evaluating beta diversity (i.e., complementarity between sites, [18]) of ant species inhabiting infructescences and visiting inflorescences among the three habitats we calculated Chaos estimator for the Jaccard similarity index (“Chao-Jaccard”) with 95% confidence intervals for statistical comparisons. Since similarity is reciprocally related to beta diversity [18], a low similarity index indicates high beta diversity among the sites compared. All species richness and diversity computations were performed with the software package Estimates 8.2 [20]. 3. Results 3.1. Infructescence Occupation. Overall, 205 of all 619 exam- ined infructescences (33%) were occupied by nests of ants (Supplementary Figure S4), termites, or bees. Depending on habitat, ant nests were found in at least 18% of the stems (Figure 1) and made up the majority of nesting occupants (miconietum: 82%; restinga-low: 97%; restinga-high: 94%). □ Restinga-low (n = 542) □ Restinga-high (n = 28) □ Miconietum (n = 49) Figure 1: Percentages of infructescences of Vriesea friburgensis occupied by ant, termite, or bee nests in different habitat types (whole dataset, n = 619) on Santa Catarina Island, southern Brazil. Under bracts, ant nests were present on 4-11% of infructes- cences (Supplementary Table S7). The most frequent stem inhabitants were ants of the genera Camponotus, Pseu- domyrmex, and Solenopsis (Tables 1 and 2). During regular collections, we found nests of 14 ant species, at least two termite species (Termitidae: Nasutitermitinae: Cortaritermes sp. and Velocitermes sp.; habitat types miconietum and restinga-low; all nests occupied stem and rosette), and one bee species (Tables 1 and 2). Ant species composition differed among the habitat types (Table 1). We encountered eight cases of two social insect species occupying different sections of the same infructescence stem (Supplementary Table S8). Occasional findings outside of the regular dataset comprised nests of further ant species; brood of a megachilid bee, a castniid moth, and syrphid flies; several coleopterans, pseudoscorpions, collembolans, and spiders (Supplementary Table S9). Out of 488 infructescence stems collected at restinga- low sites, 402 (82%) were hollow, and of these, 161 (40%) were occupied by ants, termites, or bees (Table 2). Length of cavities in infructescences was 92 ± 22 cm (mean ± SD, n - 5), and inner diameter was 1.9 ± 1.3 mm at apex and 4.6 ± 0.9 mm at base, yielding an estimated volume of 37 ± 30 cm (assuming a truncated-cone shape). 3.2. Inflorescence Visitation by Ants. We found ants foraging on 22 of 33 (67%) flowering inflorescences. Furthermore, at 36-70% (median 55%, n = 5) of the weekly examined flower- ing inflorescences (40 < n < 79), ants were observed inside flowers. In contrast, ants were present at one of 26 (4%) inflo- rescences with buds only. Nine ant species/morphospecies were identified; most species and visitor records belonged to the genera Camponotus and Pseudomyrmex (Table 1). 4 Psyche Table 1: Absolute frequencies of occurrence of ant species recorded at 33 flowering inflorescences as well as of ant nests in infructescence stems (subset of 131 infructescences) of Vriesea friburgensis in three habitat types on Santa Catarina Island, southern Brazil. Differences between sum of ant records and number of occupied infructescences are due to occupations of stems with two ant species. Ant species At inflorescences Nests in infructescences (restinga-low) Restinga-low Restinga-high Miconietum Acromyrmex rugosus (Smith 1858) 1 Camponotus arboreus (F. Smith 1858) 1 Camponotus bonariensis Mayr 1868 4 Camponotus novogranadensis Mayr 1870 11 1 Camponotus rufipes (Fabricius 1775) 2 Camponotus sexguttatus (Fabricius 1793) 4 1 Camponotus trapezoideus Mayr 1870 1 2 Camponotus sp. 13 1 Camponotus sp. 14 1 Cephalotes minutus (Fabricius 1804) 1 Crematogaster curvispinosa Mayr 1862 3 Crematogaster limata F. Smith 1858 4 3 Nesomyrmex spininodis (Mayr 1887) 2 Procryptocerus convergens (Mayr 1887) 2 Pseudomyrmex gracilis (Fabricius 1804) 6 6 1 Pseudomyrmex phyllophilus (F. Smith 1858) 6 4 Pseudomyrmex sp. PSW05^ 1 Solenopsis sp. 2 2 1 lO’' 1 Sum of ant records 34 19 18 11 Number of occupied stems — 18 17 9 Number of examined stems 33 54 28 49 species of the P. flavidulus species complex which “might actually correspond to P. flavidulus itself” (Philip Ward, pers. comm.). '^Once three stems very close to each other were occupied by this species, so it was presumably the same colony, resulting in eight independent findings in restinga-high. Therefore, the value “8” was used for computation of similarity and diversity indices. 3.3. Species Richness and Diversity. The species accumula- tion curve of the restinga-high habitat reached a plateau (Supplementary Figure S5) and was significantly lower than the curve of miconietum, indicated by non-overlapping 95% confidence limits at the end of the shorter curve (Figure 2(a)). Moreover, the confidence limits of the restinga-high curve almost fell below those of the restinga-low curve. The other three curves lay near to each other without significant differences and without stabilizing. The Chao2 species rich- ness estimator predicted highest (and even rising) richness for restinga-low and (according to the 95% confidence limits) significantly lowest for restinga-high (Figure 2(b)). The Simpson diversity index showed the same trend as the species accumulation curves (Figure 2(c)): highest diversity in miconietum and lowest in restinga-high. Finally, evenness was highest for miconietum (Figure 2(d)). Comparing the ant communities that nested in infructes- cences, the Chao-Jaccard similarity index was highest for the habitat type pair restinga-high/miconietum (0.58), fol- lowed by restinga-low/miconietum (0.19) and restinga- low/ restinga-high (0.05) (the latter significantly different from the first, according to 95% CIs), that is, beta diversity ascended in that order (Figure 3). Similarity between the inflorescence and infructescence samples in restinga-low was highest overall and significantly higher than between infructescence occupation in restinga-low and the other two habitats (Figure 3). 4. Discussion 4.1. Species Accounts. Altogether, we recorded 22 ant species (three subfamilies, nine genera) and at least 12 other arthro- pod species associated with inflorescences and infructes- cences of 3/ friburgensis. Even these high numbers must still be regarded as underestimations because species richness did not reach saturation in any habitat. Considering this inventory incompleteness and our sampling bias (focusing the search on social insects), there must be a lot more to discover in terms of animal associations with V. friburgensis. This is especially true if the view is extended from the inflorescence to the whole plant. The rosettes, which were not systematically examined in this study, might harbor a high diversity of macroinvertebrates as indicated by Zanin and Psyche 5 3 n C C aj > aj C o I/) Dh ao 2 - 1 - ♦♦♦♦♦♦♦♦ ~i — 10 —\ — 20 “I — 30 40 Number of species occurrences (d) Main curves (symbols): ♦ Inflorescences (restinga-low) a Infructescences (restinga-high) ■ Infructescences (restinga-low) • Infructescences (miconietum) 95% confidence limits: Inflorescences (restinga-low) Infructescences (restinga-high) Infructescences (restinga-low) Infructescences (miconietum) Figure 2; Diversity of ants inhabiting infructescences or visiting inflorescences of Vriesea friburgensis, according to habitat type on Santa Catarina Island, southern Brazil. Plotted against number of species occurrences as measure of sampling effort are (a) species accumulation curves and their 95% confidence limits; (b) Chao2 species richness estimator including 95% confidence limits; (c) rarefaction curves of Simpson diversity index; and (d) Simpson evenness (Simpson diversity divided by the number of species observed). Diagrams with the same data but with logarithmically scaled x-axes are included in the Supplementary Material (Supplementary Figure S5). Tusset [15] for 14 friburgensis and as reported for the related bromeliad species 14 inflata (Wawra 1883) [21]. At least two ant species were for the first time reported for Santa Catarina Island {Procryptocerus convergens) or even Santa Catarina state {Cephalotes minutus) since they do not appear in previous inventories [7, 13, 22-30]. The termites we found living in the bromeliads probably belong to two undescribed species (E. Marques Cancello, pers. comm.). Since their nests were mostly located both in the rosettes and infructescence stems, their relation to the bromeliads might be similar to the association between the termite Cortaritermes silvestrii (Holmgren 1910) and the bromeliad Dyckia maritima Baker where the plants appear to grow on termite nests because of beneficial nutrition [31]. But this assumption needs further investigation to be confirmed. 4.2. Arthropods Living in Infructescences. We consider infruc- tescences of 14 friburgensis to be attractive nest sites for certain groups of arthropods (e.g., small ant colonies and small, serially nesting bees) because they 6 Psyche Table 2: Nests of ants and other insects in 488 infructescences (161 occupied) of Vriesea friburgensis in Joaquina (habitat type restinga- low) on Santa Catarina Island, southern Brazil, n: number of findings (total of 164 due to three cases with two nests in the same stem); % (occ.): percent fraction of the number of occupied stems; % (total): percent fraction of the number of examined infructescences. Taxon n % (occ.) % (total) Apidae Ceratina (Rhyssoceratina) sp. (Xylocopinae)^ 1 0.6 0.2 Formicidae Brachymyrmex 3 1.9 0.6 Pseudomyrmex 63 39.1 12.9 Pseudomyrmex gracilis 57 35.4 11.7 Pseudomyrmex sp. PSW05 6 3.7 1.2 Camponotus 68 42.2 13.9 Myrmelachista 1 0.6 0.2 Solenopsis 24'’ 14.9 4.1 Small yellow formicine or dolichoderine ants 1 0.6 0.2 Isoptera^^ 3 1.9 0.6 ‘^Three females reared from brood cells. '’Once, five stems very close to each other were occupied by this species, so it was presumably the same colony, resulting in 20 independent findings. ‘^Probably Cortaritermes sp. according to another termite sample from the same location. Increasing beta diversity Increasing similarity 1 M 0.5 a X 1 Rest. -low/ Rest.-low/ Rest. -high/ Infl. (rest.-low)/ rest. -high micon. micon. rest.-low Figure 3: Chao-Jaccard similarity index for comparisons of infructescence occupation of Vriesea friburgensis by ants among habitats and of infructescence- with inflorescence-associated ants in restinga-low. Whiskers represent 95% confidence intervals; for convenience their lower bounds were cut when crossing the x-axis. Calculations were based on the data given in Table 1. (1) provide a long, narrow space which can be used and defended more efficiently than a compact space of the same volume; (2) dry up and frequently remain stable for more than a year, a feature which is important for bees and wasps with annual or bivoltine life cycles [9, 32]; (3) are mostly connected to a water reservoir in the leaf rosette, building kind of an oasis especially in well drained sandy habitats such as restingas; (4) grow near to food sources, such as future inflores- cences emerging from adjacent rosettes of the same bromeliad clone, providing floral nectar and flower visitors. Not only can arthropods profit from nest space provided by VI friburgensis but also the plant may gain benefits. As known from former studies, ants frequently protect the plants they live in from herbivores (e.g. [33-36]). Whether VI friburgensis actually is protected from its inflorescence herbivores (e.g. Eurytoma sp. [9], Strymon serapio [10]) is unclear since many of them might already be active before the first flowers open; that is, when ants are not present yet. This might be assessed with exclusion experiments (e.g., [37]). In most of the habitats studied, Vriesea infructescences appear to support a similar alpha diversity of ants. Whereas all diversity measures employed accord that restinga-high had lowest alpha diversity, with no more than four species predicted, species accumulation curves do not allow distin- guishing among the other habitat types because they were too close to each other and did not stabilize. Moreover, the habitat ranking derived from the species richness estimator contra- dicts that indicated by the Simpson diversity index. Since the latter is influenced by species abundance distributions [18] we assume that restinga-low contains more species but with a rather uneven distribution in contrast to miconietum. This is indeed confirmed by estimated evenness. The restinga-high habitat, in spite of its relatively low species richness, complements the ant species composition of the other habitats, especially low- vegetation restinga, demon- strated by the low similarity index value. Hence, occurring in such different habitats, V. friburgensis also supports a high beta diversity of ants. We do not expect ants to be exclusively dependent on the infructescence stems as nest sites because similar cavities can also be found in other plants. For example, we discovered nests of Pseudomyrmex gracilis and Ps. sp. PSW05 in twigs of Epidendrum fulgens Brongn. 1834 (Orchidaceae) as well as Nesomyrmex spininodis in twigs of shrubs (V. S. Schmid, unpubl. data), and Cereto et al. [38] collected, at another restinga-low site on Santa Catarina Island, nests of eight ant species from postreproductive plants of Actinocephalus polyanthus (Bong.) Sano (Eriocaulaceae), a plant with a habitus similar to bromeliads and occurring sympatrically with Vriesea at our restinga-low study sites. Cereto et al. [38] reported that 79.1% of A. polyanthus plants contained ant nests with up to four species living in the same plant. On the one hand, comparison with our study is difficult because sample sizes greatly differ, no accu- mulation curves were provided by Cereto et al. [38], and they examined whole plants while we only systematically examined the infructescence stems. It is well known that bromeliad rosettes are frequently used by ants as nest sites [6, 7, 13] , so the percentage of V. friburgensis plants containing ant nests will most likely be higher than the occupation ratio of infructescence stems. On the other hand, both Psyche 7 Cereto et al. [38] and we report the occurrence of several ant species sharing the same plant, indicating that there might be high competition for nest sites in the restingas (see also Livingston and Philpott [39] arguing for generally high competition among ants). If this is true, it seems strange that a high proportion (61%) of hollow V. friburgensis infructescence stems was found unoccupied. The causes of this phenomenon remain to be studied in more detail, taking into account aspects such as dynamics of cavity development in plants and of ant colony movements, that is, turnover of site occupation. 4.3. Ants Visiting Inflorescences. Simpson diversity of ants inhabiting infructescence stems in restinga-low was similar to that of those visiting inflorescences in the same habitat type, although the estimated species richness differed sig- nificantly. Similarity in alpha diversity goes in line with the compositional similarity between these samples being higher than among the habitats, thus supporting our hypothesis that there is a great overlap between ant species that live in the bromeliads and those that visit their flowers. However, individuals of some ant species visited the inflorescences, whereas the same species were not found living in infructescences. This may be due to nesting pref- erences; for example, Camponotus rufipes has large workers and colonies that construct nest mounds using plant material, sometimes within groups of Vriesea rosettes but apparently not extending into infructescence stems emerging from those rosettes; and Acromyrmex species generally nest in the soil [40]. Three Camponotus species nested in infructescence stems but were not observed on inflorescences. It might turn out interesting to find out the causes for this pattern, that is, whether it was mere chance owing to low sample size or whether these ants systematically avoid inflorescences, be it due to interspecific competition or because of their foraging habits. We found five ant genera at inflorescences of V. friburgen- sis. However, within flowers, almost exclusively Camponotus ants were present (mainly Ca. novogranadensis and Ca. rufipes, probably also Ca. sexguttatus; V. S. Schmid, pers. obs.). They do not entirely monopolize the inflorescences since we mostly found unoccupied flowers near the occupied ones on the same inflorescences. Occasional behavioral observations indicate that Camponotus workers visit flowers to take up floral nectar, sometimes apparently guarded by a conspecific worker (Supplementary Figure S6). They might additionally hunt flower mites which we found along our examinations within flowers of 29 out of 32 (91%) inflores- cences in Joaquina and also recorded them in miconietum (V. S. Schmid, unpubl. data), as similarly reported by Schmid et al. [13] for the bromeliad species Aechmea lindenii (E. Morren) Baker and Ae. nudicaulis (L.) Grisebach. We expect the mites in 14 friburgensis to belong to the same species {Proctolaelaps sp. and Tropicoseius sp.) as in Ae. lindenii because they are phoretically transported by hummingbirds (see Video S6 in [13]) which occur on the whole island and visit flowers of species of Aechmea [3-5, 41] and Vriesea [4, 12]. As for infructescence occupation, we regard it as unlikely that any of the ant species reported here is specifically associated with inflorescences of V. friburgensis. Its flowers are accessible only during a limited period throughout the year and are not completely monopolized by one or a few ant species; thus ants do not completely depend on the floral resources. The presence of ants on plants is frequently accompa- nied by a mutually beneficial association where the ants are attracted by food and/or shelter and in turn provide protection to the plant by deterring herbivores and/or cutting other vegetation that competes with the host plant for resources (e.g., [33-36]). In bromeliads, such a mutualism was reported for Dyckia floribunda where exclusion of ants yielded a significant decrease in total seed production per plant [37]. Unlike D. floribunda, V friburgensis does not produce extrafloral nectar on its inflorescences (V. S. Schmid, unpubl. data: six plastic-bagged inflorescences inaccessible to animals did not show signs of secreted fluids). The ants are apparently attracted mainly by the nectar contained inside the flowers. They might have both positive and negative effects on the plants’ reproductive success by interfering with herbivores and/or pollinators, respectively. Hence, whether V friburgensis benefits from the presence of the ants cannot be judged without appropriate manipulative experiments. 5. Conclusion Even though there are probably no specific associations with V friburgensis, this bromeliad species does support a high level of alpha and beta diversity of arthropods, mainly ants. Regarding the high potential for competition for nest sites among ant species [39], V. friburgensis likely plays an important role for the species communities of the Atlantic Eorest region, confirming former studies which stressed the ecological significance of bromeliads (e.g., [10, 13, 42, 43]; see also [2] and references therein). Beyond the scope of our study, there are certainly other bromeliad species (e.g., Dyckia spp.; Hohenbergia spp.; other Vriesea spp.) whose infructescences are worth a closer examination with respect to inhabiting arthropods. Concluding, we recommend that bromeliads should be taken into special consideration for biodiversity conservation efforts. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments We thank late M. Hering de Queiroz for access to the study site at Santo Antonio; Eliana Marques Cancello and Carolina Cuezzo for termite identification; Jacques Delabie, Manfred Verhaagh, and Philip Ward for ant identification; Daniel Bartsch and Christoph Hauser for castniid identification; Robert Colwell for multiple advices in diversity analysis; and two anonymous referees for helpful comments on the paper. Research was authorized by IBAMA, permits 090/2005 8 Psyche and 260/2006, and by SISBIO, permits 12486-1, 12826-1, and 12826-2. This study was part of the projects “Internal dynamics of rain forests: specificity of animal-plant inter- actions” (BMBF, Germany, 01LB0205A1) and “Importancia das bromelias para a manuten^ao da diversidade da fauna associada na Mata Atlantica” (CNPq, Brazil, 590040/2006-5) within the Brazilian -German program “Mata Atlantica.” All authors acknowledge financial support by BMBF and GNPq within this program. References [1] D. H. Benzing, Bromeliaceae — Profile of an Adaptive Radiation, Cambridge University Press, Cambridge, UK. [2] J. H. Frank and L. P. Lounibos, “Insects and allies associated with bromeliads: a review,” Terrestrial Arthropod Reviews, vol. 1, pp. 125-153, 2008. [3] L. L. Dorneles, A. Zillikens, B. Flarter-Marques, and J. Steiner, “Effective pollinators among the diverse flower visitors of the bromeliad Aechmea lindenii in south Brazilian Atlantic rain forests,” Entomologia Generalis, vol. 33, no. 3, pp. 149-164, 2011. [4] R. Kamke, S. Schmid, A. Zillikens, B. C. Lopes, and J. Steiner, “The importance of bees as pollinators in the short corolla hromelmd Aechmea caudata in southern Brazil,” Flora, vol. 206, no. 8, pp. 749-756, 2011. [5] S. Schmid, V. S. Schmid, A. Zillikens, B. Harter- Marques, and J. Steiner, “Bimodal pollination system of the bromeliad Aechmea nudicaulis involving hummingbirds and bees,” Plant Biology, vol. 13, no. si, pp. 41-50, 2011. [6] N. Bliithgen, M. Verhaagh, W. Goitia, and N. Bliithgen, “Ant nests in tank bromeliads — an example of non-speciflc interac- tion,” Insectes Sociaux, vol. 47, no. 4, pp. 313-316, 2000. [7] E B. Rosumek, M. A. Ulyssea, B. C. Lopes, J. Steiner, and A. Zil- likens, “Eormigas de solo e de bromelias em uma area de Mata Atlantica, Ilha de Santa Catarina, sul do Brasil: Levantamento de especies e novos registros,” Biotemas, vol. 21, no. 4, pp. 81-89, 2008. [8] A. Zillikens, J. Steiner, and Z. Mihalko, “Nests ofAugochlora (A.) esox in bromeliads, a previously unknown site for sweat bees (Hymenoptera: Halictidae),” Studies on Neotropical Fauna and Environment, vol. 36, pp. 137-142, 2001. [9] S. Grohme, J. Steiner, and A. Zillikens, “Destruction of flo- ral buds in the bromeliad Vriesea friburgensis by the phy- tophagous larvae of the wasp Eurytoma sp in southern Brazil (Hymenoptera: Eurytomidae),” Entomologia Generalis, vol. 30, no. 2, pp. 167-172, 2007. [10] S. Schmid, V. S. Schmid, R. Kamke, J. Steiner, and A. Zillikens, “Association of three species of Strymon Hiibner (Lycaenidae: Theclinae: Eumaeini) with bromeliads in southern Brazil,” Journal of Research on the Lepidoptera, vol. 42, pp. 50-55, 2010. [11] B. Holldobler and E. O. Wilson, The Ants, Belknap Press of Harvard University Press, Cambridge, Mass, USA; Springer, Berlin, Germany, 1990. [12] S. Schmid, V. S. Schmid, A. Zillikens, and J. Steiner, “Diver- sity of flower visitors and their role for pollination in the ornithophilous bromeliad Vriesea friburgensis in two different habitats in Southern Brazil,” Ecotropica, vol. 17, no. 1, pp. 91-102, 2011. [13] V. S. Schmid, S. Schmid, J. Steiner, and A. Zillikens, “High diver- sity of ants foraging on extrafloral nectar of bromeliads in the Atlantic rainforest of southern Brazil,” Studies on Neotropical Fauna and Environment, vol. 45, no. 1, pp. 39-53, 2010. [14] R. Reitz, Bromelidceas e a Maldria-Bromelia Endemica, flora Ilustrada Catarinense, Itajai, Brazil, 1983. [15] E. M. Zanin and C. Tusset, “Vriesia [sic!] friburgensis Mez.: distribui^ao vertical da especie e fauna associada,” Revista Brasileira de Biociencias, vol. 5, supplement 1, pp. 138-140, 2007. [16] M. C. Sampaio, L. E. Perisse, G. A. De Oliveira, and R. I. Rios, “The contrasting clonal architecture of two bromeliads from sandy coastal plains in Brazil,” Flora, vol. 197, no. 6, pp. 443-451, 2002. [17] R. K. Colwell, C. X. Mao, and J. Chang, “Interpolating, extrap- olating, and comparing incidence-based species accumulation curves,” Ecology, vol. 85, pp. 2717-2727, 2004. [18] A. E. Magurran, Measuring Biological Diversity, Blackwell Sci- ence, Oxford, UK, 2004. [19] I. T. Longino, J. Coddington, and R. K. Colwell, “The ant fauna of a tropical rain forest; estimating species richness three different ways,” Ecology, vol. 83, no. 3, pp. 689-702, 2002. [20] R. K. Colwell, “EstimateS: statistical estimation of species richness and shared species from samples, version 8.2,” Users Guide and application, 2009, http://purl.oclc.org/estimates/. [21] L. A. M. Mestre, ]. M. R. Aranha, and M. L. P. Esper, “Macroin- vertebrate fauna Associated to the Bromeliad Vriesea infiata of the Atlantic forest (Parana State, Southern Brazil),” Brazilian Archives of Biology and Technology, vol. 44, no. 1, pp. 89-94, 2001. [22] A. Bonnet and B. C. Lopes, “Eormigas de dunas e restingas da Praia da loaquina, Ilha de Santa Catarina, SC, (Insecta: Hymenoptera),” Biotemas, vol. 6, pp. 107-114, 1993. [23] D. C. Cardoso and M. P. Cristiano, “Myrmecofauna of the southern Catarinense Restinga Sandy Coastal Plain: new records of species occurrence for the State of Santa Catarina and Brazil,” Sociobiology, vol. 55, no. IB, pp. 229-239, 2010. [24] D. C. Cardoso, T. G. Sobrinho, and ]. H. Schoereder, “Ant community composition and its relationship with phytophys- iognomies in a Brazilian Restinga,” Insectes Sociaux, vol. 57, no. 3, pp. 293-301, 2010. [25] B. C. Lopes and H. G. fowler, “fungus-growing ants (Hymenoptera; formicidae) on Santa Catarina Island, Brazil: patterns of occurrence,” Revista de Biologia Tropical, vol. 48, pp. 643-646, 2000. [26] I. A. Lutinski and E. R. M. Garcia, “Analise faunistica de Eormi- cidae (Hymenoptera: Apocrita) em ecossistema degradado no munidpio de Chapeco, Santa Catarina,” Biotemas, vol. 18, pp. 73-86, 2005. [27] I. A. Lutinski, E. R. M. Garcia, C. ]. Lutinski, and S. lop, “Diver- sidade de formigas na floresta Nacional de Chapeco, Santa Catarina, Brasil,” Ciencia Rural, vol. 38, pp. 1810-1816, 2008. [28] R. R. d. Silva, “formigas (Hymenoptera: formicidae) do oeste de Santa Catarina: historico das coletas e lista atualizada das especies do Estado de Santa Catarina,” Biotemas, vol. 12, pp. 75- 100, 1999. [29] R. R. d. Silva and R. Silvestre, “Diversidade de formigas (Hymenoptera: formicidae) em Seara, oeste de Santa Catarina,” Biotemas, vol. 13, pp. 85-105, 2000. [30] R. R. d. Silva and R. Silvestre, “Riqueza da fauna de formigas (Hymenoptera: formicidae) que habita as camadas superflciais do solo em Seara, Santa Catarina,” Papeis Avulsos de Zoologia, vol. 44, pp. 1-11, 2004. Psyche 9 [31] C. C. Waldemar and B. E. Irgang, “A ocorrencia do mutualismo facultative entre Dyckia maritima Backer [sic!] (Bromeliaceae) e o cupim Cortaritermes silvestrii (Holmgren), Nasutitermiti- nae, em afloramentos rochosos no Parque Estadual de Itapua, Viamao, RS,” Acta Botanica Brasilica, vol. 17, no. 1, pp. 37-48, 2003. [32] A. Zillikens and J. Steiner, “Nest architecture, life cycle and cleptoparasite of the neotropical leaf-cutting bee Megachile (Chrysosarus) pseudanthidioides Moure (Hymenoptera: Mega- chilidae),” Journal of the Kansas Entomological Society, vol. 77, no. 3, pp. 193-202, 2004. [33] B. L. Bentley, “The protective function of ants visiting the extrafloral nectaries of Bixa orellanaj The Journal of Ecology, vol. 65, no. 1, pp. 27-38, 1977. [34] B. Eiala and U. Maschwitz, “Studies on the south east Asian ant- plant association Crematogaster horneensis! Macaranga: adapta- tions of the ant partner,” Insectes Sociaux, vol. 37, no. 3, pp. 212- 231, 1990. [35] D. H. Janzen, “Coevolution of mutualism between ants and acacias in Central America,” Evolution, vol. 20, pp. 249-275, 1966. [36] D. H. Janzen, “Allelopathy by myrmecophytes: the ant Azteca as an allelopathic agent of CecropiaJ Ecology, vol. 50, pp. 147-153, 1969. [37] J. L. Vesprini, L. Galetto, and G. Bernardello, “The beneficial effect of ants on the reproductive success of Dyckia floribunda (Bromeliaceae), an extrafloral nectary plant,” Canadian Journal of Botany, vol. 81, no. 1, pp. 24-27, 2003. [38] C. E. Cereto, G. O. Schmidt, A. G. Martins, T. T. Castellani, and B. C. Lopes, “Nesting of ants (Hymenoptera, Eormicidae) in dead post-reproductive plants of Actinocephalus polyanthus (Eriocaulaceae), a herb of coastal dunes in southern Brazil,” Insectes Sociaux, vol. 58, no. 4, pp. 469-471, 2011. [39] G. E. Livingston and S. M. Philpott, “A metacommmunity approach to co-occurrence patterns and the core-satellite hypothesis in a community of tropical arboreal ants,” Ecological Research, vol. 25, no. 6, pp. 1129-1140, 2010. [40] M. Bollazzi, J. Kronenbitter, and E. Roces, “Soil temperature, digging behaviour, and the adaptive value of nest depth in South American species of Acromyrmex leaf-cutting ants,” Oecologia, vol. 158, no. 1, pp. 165-175, 2008. [41] M. Lenzi, J. Z. De Matos, and A. I. Orth, “Varia^ao morfologica e reprodutiva deAechmea lindenii (E. Morren) Baker var. lindenii (Bromeliaceae),” Acta Botanica Brasilica, vol. 20, no. 2, pp. 487- 500, 2006. [42] A. C. Araujo, E. Eischer, and M. Sazima, “As bromelias da regiao do Rio Verde,” in Estagdo Ecologica Jureia-Itatins: ambiente fisico, flora e fauna, A. V. Marques and W. Duleba, Eds., pp. 162- 171, Holos Editora, Ribeirao Preto, Brazil, 2004. [43] S. Buzato, M. Sazima, and I. Sazima, “Hummingbird-pollinated floras at three Atlantic forest sites,” Biotropica, vol. 32, no. 4B, pp. 824-841, 2000. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 793298, 6 pages http://dx.doi.org/10.1155/2014/793298 Research Article Volatile Organic Compounds from the Clone Populus x canadensis “Conti” Associated with Megaplatypus mutatus Attack Alejandro Lucia, Paola Gonzalez-Audino, and Hector Masuh Centro de Investigaciones de Plagas e Insecticidas (UNIDEF (MINDEF-CONICET), J. B. de La Salle 4397, Villa Martelli B1603ALO, Provincia de Buenos Aires, Argentina Correspondence should be addressed to Paola Gonzalez-Audino; pgonzalezaudino@citedef.gob.ar Received 5 December 2013; Accepted 27 January 2014; Published 6 March 2014 Academic Editor: Taya Chermenskaya Copyright © 2014 Alejandro Lucia 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. Megaplatypus mutatus (Chapuis) (Coleoptera, Platypodidae) is an ambrosia beetle native to South America. It builds internal galleries that weaken the tree trunks, causing them severe stem breakage and mortality in commercial poplar plantations. The host selection by male M. mutatus has previously been correlated with the increasing diameter. This work explores the possibility that differential susceptibility of individual plants to M. mutatus could be associated with volatiles emitted. The comparison of the VOCs profiles of attacked and nonattacked P. x canadensis “Conti” 12 during M. mutatus flying season showed both qualitative and quantitative differences. The attacked plants, but not the nonattacked ones, showed the following compounds: a long chain aldehyde, a-ylangene, d-cadinene, a-gurjunene, and ^-cubebene; on the other side, ^-sesquiphellandrene and j3-chamigrene were detected only in nonattacked plants. a-Copaene is a common component of all the samples analyzed, but its proportion is increased in attacked individuals. Behavioral bioassays showed that males but not females M. mutatus are attracted to a-copaene. The relative increase of a-copaene in attacked individuals and the positive behavioral answer of males to it suggest that this compound could play a role in the orientation of the pioneer male towards the most suitable host. 1. Introduction Ambrosia beetles are an important insect group in forest ecosystems affecting weakened or felled trees. Megaplatypus mutatus (Syn. Platypus mutatus) (Chapuis) (Coleoptera, Platypodidae) is an ambrosia beetle native to South America. Unlike most ambrosia beetles, it attacks only living trees, penetrating the xylem of its host by boring long tunnels. The attack is initiated by pioneer males selecting a host tree to build a short nuptial gallery, from which they attract females using a sexual pheromone [1] . Following copulation, they extend their gallery in order to lodge their new brood. These galleries weaken the tree trunks, causing severe stem- breakage and mortality in commercial poplar plantations of Populus deltoides [2-4]. Additionally, the dark tunnels caused by the Ambrosia mycelium of the associated symbiotic fungi seriously affect the quality of the wood. The prevalence of attacks by M. mutatus has been cor- related with the tree diameter. Etiennot et al. [5] found that 86% of the attacked trees in a plantation had a diameter breast height (DBH) >20 cm. Also, other authors found a preference of M. mutatus for bigger diameters [6-9], probably because there is more room available to develop their brood [1] . Concerning the susceptibility associated with the clone, although it is well known that some clones are less susceptible than others, this differential susceptibility is more likely to be associated with the average DBH of the particular clone characteristic for its growing rate than to the clone itself [6- 9] . Also, there is a strong association between the site quality and the prevalence of attacks [10]. Again, this phenomenon can be correlated with the productivity of the plantation. With the aim of implementing an environmentally friendly management programme, a large amount of work has been done with traps baited with sexual pheromone that 2 Psyche attract females [11-13]. However, the existence of chemical cues involved in the host selection by the male has not been explored and it gains interest in the search of synthetic attractants to be incorporated in baited traps. In this work we explore the possibility that differential susceptibility of individual plants at M. mutatus could be associated with VOC emitted, so we collected and analyzed VOC emitted by wood bark of the clone P. x canadensis “Conti” 12 attacked and nonattacked during M. mutatus flying season. 2. Materials and Methods 2.1. Plant Material. Populus x canadensis Monch (Syn. P. x euramericana (Dode) Guiner) plants were selected from 10- year-old commercial poplar plantations {Populus x euramer- icana cv. “Conti 12”). The plantation had a density of 1,111 trees/ha (square of plantation 3 m x 3 m), average DBH 23,2 cm and is located at Alberti, Buenos Aires Province, Argentina (35°10^S, 60°17^W, 68 m.a.s.L). All the selected individuals had the same age, site, clone, and history of plantation. The selection methodology was the following: we ran- domly selected an attacked plant and a nearby nonattacked one with a similar diameter. We also tried to select attacked plants close to nonattacked one and vice versa. Trees were considered attacked if they had a visible pioneer calling male, characterized by the presence of a crown-like arrangement surrounding the entrance to the gallery (Figure 1). Four replicates of attacked and nonattacked trees were analyzed. Samples were collected during the flying season of M. mutatus (November). Figure 1: Crown-like arrangement in P. canadensis surrounding the entrance to the nuptial gallery built with the particles of boring dust (frass) produced by the male M. mutatus from where volatiles are emitted to attract individuals of the opposite sex. The identities of compounds observed were assigned by comparison with spectral data of commercial libraries NIST and Wiley (tentative identifications) or with the authentic compound in the case of a-copaene. 2.3. Insects. The insects were collected shortly after their emergence (maximum 3 hours) from infested Populus sp. and Quercus palustris (Miinchh) located at our institute planta- tion (34°33^ south, 58°30^ west). Emergence traps specifically designed for this beetle were used to avoid antagonistic interactions between emerged insects [14]. 2.2. Volatile Organic Compounds Emitted by Populus x eura- mericana cv. “Conti 12”. Using a cork borer we extracted a wood bark cylinder vicinal (1.5 cm x 1 cm) to the M. mutatus entrance hole and placed it in a 20 mL vial standard clear glass (Scientific Specialties Service, Inc., Baltimore, MD, USA and Reno, NV, USA) with a teflon-coated cap (teflon septum with glass reinforced polypropylene resin open cap) adequately refrigerated. All the samples were collected between 10 and 12 a.m. Once in the lab, the volatiles from the vial headspace were collected at 29 ± 2°C for 30 minutes using a solid phase microextraction fiber (SPME) covered with a 100 pm PDMS (Supelco Bellefonte, PA, USA) nonpolar phase. This coating is of general use to adsorb low molecular weight compounds. Samples were immediately analysed by GC-MS. GC-MS analyses were performed with a Shimadzu QP-5050A spectrometer in the electron impact mode, equipped with a polar fused CP wax 52CB column (30 m x 0.32 mm ID x 0.25 pm film thickness). Samples were injected in the splitless mode. Volatiles from the SPME fibres were desorbed in the injector port at 250°C during 1.5 min. The GC column was kept at 50° C for 5 min after which the temperature was programmed to increase 10°C/min up to 220° C, where it was maintained for 5 min. The carrier gas was helium with a head pressure of 30 kPa. The MS detector was set on at 70 eV. 2.4. Behavioral Bioassays. Walking behavior of female M. mutatus was evaluated in an experimental arena with a video tracking technique [15] adapted for M. mutatus [16]. The floor of the test arena was covered with a round piece of Whatman No. 1 filter paper (125 mm diameter, Whatman Ltd., Maidstone, UK), and a glass cover (20 x 20 mm) was placed in the center of the paper. Next, the filter paper and glass cover were both covered with a rectangular piece of wire mesh (100 x 100 mm, 1 mm mesh size). A colorless glass ring (100 mm diameter, 50 mm high) was used to confine the insects. A new glass cover and filter paper were used in each replicate. A closed circuit video camera providing black and white images (VC 1910, Sanyo Electrical Co., Tokyo, Japan) was suspended 22 cm over the center of the test arena. A circular fluorescent tube (22 W, OSRAM, Buenos Aires, Argentina) was placed 64 cm above the video camera. An image analyzer (Videomex V, Columbus, OH, USA) received input from the video camera, converting the analog signal into digital data. The resolution was 256 x 192 pixels and the acquisition and processing speed was 30 frames/sec. The presence of insects in the arena was determined by visual contrast between the individuals (white) and the arena background (dark) and scored as the number of “ON” pixels. The area occupied by the insects was recorded by using the Multiple Zone Motion Monitor for Videomex software. Psyche 3 The arena image was divided into a central square (4 cm , 5% of the total area) and a circular outer area. The cen- ter of the glass cover was located in the center of the virtual central square. A male M. mutatus was placed on the wire mesh and allowed to acclimatize for 5 min before starting the bioassay. During this time, the insect moved all around the arena. Insect movement was recorded for 60 min. During the first 30 min, the glass cover was clean. Then, 1.5 ffL of a-copaene was placed on the cover. Tem- perature varied between 25 and 30°C. The first 30 min of each test was the control, and the remaining 30 min was the experimental treatment. Thus, the occupation level of the central circle during the first 30 min (control) was compared to the occupation level during second 30 min (following the introduction of the test substance). The exper- iment was replicated 10 times with independent males and females. We used the central area of occupation (CAO) parameter, previously defined as the total number of “OiV” pixels in the central circle (where the test compound is placed) during a replicate [15, 16], to quantify insect behavior. A mean CAO value was obtained for each treatment and compared to its respective control. 2.5. Chemical. (-)-a-Copaene (Technical grade > 90%, GC sum of enantiomers) was purchased from Fluka (Milwaukee, USA). 2. 6 . Statistical Analysis . Data from the behavioral assay were analyzed by Kruskal-Wallis Test (nonparametric ANOVA) using STATISTICA software. A mean CAO values were obtained for a-copaene and compared to its respective control. The accepted level of significance was P value < 0.01, meaning highly different from control group (Kruskal-Wallis Test). The values of relative concentration of the compounds for each sample were transformed (log) and analyzed using one- way analysis of variance (ANOVA), and means were com- pared a posteriori by Tukey HSD mean multiple comparison test using STATGRAPHICS Plus Software. A value of P < 0.01 was considered for a significant highly difference and P < 0.05 for a significant difference. 3. Results All the specimens of Populus x canadensis clone “Conti 12” whose volatiles where analyzed have the same age, site, clone, diameter, and history of plantation. The attacked ones had a DBH 25.5 ± 1.63 cm and the nonattacked ones 20.7 ± 1.96 cm. This means that among a similar diametrical class, the insect prefers the larger diameters (P value < 0.05). 3.1. Volatile Organic Compounds Emitted by Nonattacked Populus X canadensis Clone “Conti 12”. The volatile blend emitted by the wood and bark sample of the P x canadensis “Conti 12” nonattacked by M. mutatus was dominated by f3- selinene (36.9 ± 2.6%), followed by a-selinene (27 ± 3.0%), jS-chamigrene (7.1 ± 2.6%), a long chain aldehyde with Rt2i.82 (6.3 ± 1.8%), jS-elemene (5.0 ± 2.6%), salicylic aldehyde (3.5 ± 2.0%), and a-copaene (1.8 ± 0.2%) (Figure 2(a)). 3.2. Volatile Organic Compounds Emitted by Attacked Populus X canadensis Clone “Conti 12”. Figure 2(b) shows the typical GC trace of the volatiles emitted by the wood and bark sample of the P X canadensis “Conti” 12 attacked by M. mutatus. In this case a-copaene was the major component (34.4 ± 23.9%), followed by a long chain aldehyde of Rt 20.57 (30.7 ± 15.8%), /3-selinene (9.1 ± 3.8%), a long chain aldehyde of Rt 21.82 (8.6 ±4.7%), a-selinene (4.4 ±3.0%), j5-cubenene (2.2 ±0.6%), salicylic aldehyde (2.1 ± 1.8%), a-gurjunene (1.8 ± 0.5%), [3- elemene ( 1 .7 ± 0.9%) , a-ylangene ( 1 .0 ±0.6%), and d-cadinene (0.9 ± 0.4%). 3.3. Behavioral Response to a-Copaene. The occupation level of the central circle during the first 30 min (control) did not reveal a significant behavioral response when compared with their second 30 min (following the introduction of the test substance) (P value: 0.001). Results were analyzed based on the central area of occupation (CAO) parameter. Significant occupation of the central area can be interpreted as an effective attraction to the source followed by an arrestment in the area [17]. CAO values of female M. mutatus exposed to a- copaene did not reveal a significant behavioral response (P value: 0.62) when compared with their respective controls (Figure 3). Thus, females were not attracted to the stimulus source. CAO values of male M. mutatus exposed to a-copaene revealed a significant behavioral response (P value: 0.0042) (Figure 4) when compared with their respective controls. Thus, males were attracted to the stimulus source. 4. Discussion The comparison of the volatile profiles of attacked and nonattacked trees showed both qualitative and quantitative differences(Figure 5). The attacked plants, but not the nonat- tacked ones, showed the following compounds: a long chain aldehyde of Rt2o.57, tx-ylangene, d-cadinene, a-gurjunene, and jS-cubebene; on the other side, j5-sesquiphellandrene and (3- chamigrene were detected in nonattacked plants but not in attacked ones. A quantitative analyses showed that a-copaene is present in 1-2% in nonattacked plants but in 34, 4% in attacked ones (P value < 0.05). Also, the long chain aldehyde of Rt2i.8o shows the same pattern: it varies from 6.3% in nonattacked plants to 30.7% in the attacked ones (significant difference, P value < 0.05). Instead, a-selinene, /5-selinene, and j5-elemene decrease their relative concentrations in attacked trees with respect to nonattacked ones (P value < 0.01, P value < 0.01, and P value > 0.05, resp.). Overall, we can conclude that although a-copaene is a common confirmed component of all the samples analyzed, its proportion is increased in attacked individuals and males 4 Psyche (a) (b) Figure 2: (a) Typical GC trace of volatile organic compounds emitted by nonattacked Populus x canadensis clone “Conti 12” (1: Salicylic aldehyde**, 2; a-copaene***, 3: jS-elemene**, 4: N.I., 5: jS-chamigrene**, 6: jS-selinene* * , 7: a-selinene**, 8: N.L, 9: aldehyde of Rt 2 ig 2 **, 10: N.L, and 11: N.L). **: Tentatively identified against GC-MS library, ***: identified against authentic standard, and N.L: nonidentified. (b) Typical GC trace of volatile organic compounds emitted by attacked Populus x canadensis clone “Conti 12” (1: salicylic aldehyde**, 2: a-ylangene , 3: a-copaene , 4: p-elemene , 5: N.L, 6: p-cubebene , 7: a-gurjunene , 8: p-selmene , 9: a-selmene , 10: 0-cadmene , 11: N.L, 12: aldehyde ofRt 20 57 **, 13: aldehyde of Rt 2 i § 2 **, 14: N.L, and 15: N.L). **: Tentatively identified against library, ***: identified against authentic standard, and N.L: nonidentified. Figure 3: Response of female Megaplatypus mutatus measured as the central area occupation (=on pixels) for a-copaene compared to its respective control. Each bar represents the mean of 10 independent replicates ± SE. NS: not significant differences between treatment and control group (Kruskal-Wallis Test, P > 0.01). M. mutatus are attracted to it at short range but females are not. The relative increase of a-copaene in attacked individuals and the positive behavioral answer of males to it suggest that this compound could play a role in the orientation of the pioneer male towards the most suitable host. a-Copaene and its stereoisomer a-ylangene are active kairomones of Archangelica officinalis essential oil; however, their proportion goes from 0.5 to 1% and pure a-copaene is quite more active. The Angelica essential oil has been used in baited traps to catch fruit flies in Florida [18] . Also, extracts of Litchi chinensis, Ficus retusa, and Ficus benjamina were active for males of the same species being this response attributed to the presence of a-copaene [19]. Our result is interesting for our goal of finding nat- ural attractants to be set up in baited traps in the field. 2 " 120 Mov. first 1/2 hour (control) Mov. second 1/2 hour (treatment) Eigure 4: Response of male Megaplatypus mutatus measured as the central area occupation (=on pixels) for a-copaene compared to its respective control. Each bar represents the mean of 10 independent replicates ± SE. *: significant differences between treatment and control group (Kruskal-Wallis Test, P < 0.01). Attraction of bark beetles to pheromone baited traps is increased by the addition of host volatiles as monoterpenes to pheromone baits [20, 21] and commercial lures based on the combination of synthetic attractants are available. In this sense, the introduction of a-copaene to pheromone baited traps could be a promising tool that optimizes adult trapping, leading to improve monitoring and control systems in infested plantations. 5. Conclusions The volatile profiles of attacked and nonattacked trees showed both qualitative and quantitative differences. a-Copaene is a common confirmed component of all the samples analyzed, but its proportion is increased in attacked individuals. Psyche 5 Compounds □ Nonattacked Populus x Canadensis clone “Conti 12” ■ Attacked Populus x Canadensis clone “Conti 12” Figure 5; Volatile organic compounds emitted by attacked and nonattacked Populus x canadensis clone “Conti 12.” The numbers represent the major compounds, 1; ^-selinene, 2: a-selinene, 3: chamigrene, 4; j3-elemene, 5: salicylic aldehyde, 6: ^-cubebene, 7; j3- sesquiphellandrene, 8; a-gurjunene, 9: d-cadinene, 10: a-ylangene, 11: a-copaene, 12: aldehyde of Rt 2 o. 57 , and 13: aldehyde of Rt 2 i g 2 . The area normalization performed only on identified compounds and the values are the mean of four replicates ± SD. NS: not significant differences between relative concentration in attacked and non- attacked plants (P > 0.05). * and **: relative concentration in the attacked plants is significantly different (P < 0.05) or highly different (P < 0.01) respectively, from non-attacked plants (ANOVA-Tukey HSD mean multiple comparison test). AT: compounds only present in attacked plants and NA: only present in nonattacked plants. In behavioral bioassays, males M. mutatus are attracted at short range to a-copaene, while females are not. Introduction of a-copaene to pheromone baited traps could optimize adult trapping. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work was supported by the Agencia Nacional de Pro- mocion Cientifica y Tecnica (PICT 2005). Alejandro Lucia, Paola Gonzalez -Audino, and Hector Masuh are members of CONICET. References [1] P. G. Audino, R. Villaverde, R. Alfaro, and E. Zerba, “Identifi- cation of volatile emissions from Platypus mutatus (= sulcatus) (Coleoptera: Platypodidae) and their behavioral activity,” Jour- nal of Economic Entomology, vol. 98, no. 5, pp. 1506-1509, 2005. [2] F. H. Santoro, “Bioecologia de Platypus sulcatus Chapuis (Coleoptera, Platypodidae),” Revista de Investigaciones Eore- stales IV, vol. 1, pp. 47-79, 1963. [3] F. G. Achinelli, G. Liljersthrom, A. Aparicio, M. Delgado, M. louanny, and C. Mastandrea, “Danos por taladriUo (Megaplaty- pus mutatus (= Platypus sulcatus)) en plantaciones de alamo (Populus spp.) de Alberti, Buenos Aires: analisis preliminar de la magnitud y disrtibucion de fustes quebrados,” Revista de la Asociacion Porestal Argentina, vol. 59, pp. 8-11, 2005 (Spanish). [4] R. 1. Alfaro, L. M. Humble, P. Gonzalez, R. Villaverde, and G. Allegro, “The threat of the ambrosia beetle Megaplatypus mutatus (Chapuis) (= Platypus mutatus Chapuis) to world poplar resources,” Eorestry, vol. 80, no. 4, pp. 471-479, 2007. [5] A. E. Etiennot, R. A. Gimenez, and M. E. Bascialli, “Platypus sulcatus Chapuis (Col. Platypodidae): distribucion del ataque segun el DAP de Populus deltoides y evaluacion de insecticidas,” in I Simposio Argentino-Canadiense de Proteccion Porestal, Buenos Aires, Argentina, 1998. [6] T. Cerrillo, “Revision bibliografica sobre Platypus sulcatus Chapuis y ortos coleopteros del genero,” Revista de la Asociacion Porestal Argentina, vol. 50, pp. 59-70, 1996. [7] E. Casaubon, G. Cueto, K. Hodara, and A. Gonzalez, “Inter- acciones entre sitio, plaga y una enfermedad del fuste en una plantacion de Populus deltoides cv. Catfish-2 en el bajo delta del Rio Parana (Argentina),” Investigacion Agraria. Sistemas y Recursos Porestales, vol. 2, no. 1, pp. 29-38, 2002. [8] E. A. Casaubon, G. R. Cueto, K. Hodara, and A. C. Gonzalez, “Influence of site quality on the attack of Platypus mutatus Chapuis (Coleoptera, Platypodidae) to a willow plantation (Salix babylonica x Salix alba cv 131/27),” Ecologia Austral, vol. 14, no. 1, pp. 113-120, 2004. [9] f L. Marquina, R. Marlats, and M. N. Cresto, “Cloning suscepti- bility of Poplar (Populus sp.) when attacked by Platypus mutatus in Buenos Aires, Argentina,” Bosque, vol. 27, no. 2, pp. 92-97, 2006. [10] E. Casaubon, G. Cueto, and C. Spagarino, “Diferente com- portamiento de Megaplatypus mutatus (= Platypus sulcatus) (Chapuis, 1865) en un ensayo comparativo de rendimiento de 30 clones de Populus deltoides Bart. En el bajo delta bonaerense del Rfo Parana,” RIA INTA Argentina, vol. 35, no. 2, pp. 103-115, 2006. [11] H. Funes, E. Zerba, and P. G. Audino, “Comparison of three types of traps baited with sexual pheromones for ambrosia beetle Megaplatypus mutatus (coleoptera: Platypodinae) in poplar plantations,” Journal of Economic Entomology, vol. 102, no. 4, pp. 1546-1550, 2009. [12] H. Funes, E. Zerba, and P. Gonzalez-Audino, “Effect of release rate and enantiomeric composition on response to pheromones of Megaplatypus mutatus (Chapuis) in poplar plantations of Argentina and Italy,” Bulletin of Entomological Research, vol. 103, pp. 564-569, 2013. [13] P. Gonzalez-Audino, P. Gatti, and E. Zerba, “Traslucent phe- romone traps increase trapping efficiency of ambrosia beetle Megaplatypus mutatus,” Crop Protection, vol. 30, no. 6, pp. 745- 747, 2011. [14] P. G. Liguori, E. Zerba, and P. G. Audino, “New trap for emergent Megaplatypus mutatusj Canadian Entomologist, vol. 139, no. 6, pp. 894-896, 2007. [15] R. A. Alzogaray, A. Fontan, and E. N. Zerba, “Repellency of deet to nymphs of Triatoma infestansj Medical and Veterinary Entomology, vol. 14, no. 1, pp. 6-10, 2000. [16] P. G. Liguori, E. Zerba, R. A. Alzogaray, and P. G. Audino, “3- Pentanol: a new attractant present in volatile emissions from the ambrosia beetle. Megaplatypus mutatusj Journal of Chemical Ecology, vol. 34, no. 11, pp. 1446-1451, 2008. 6 Psyche [17] A. Fontan, P. G. Audino, A. Martinez et al, “Attractant volatiles released by female and male Triatoma infestans (Hemiptera: Reduviidae), a vector of chagas disease: chemical analysis and behavioral bioassay,” Journal of Medical Entomology, vol. 39, no. 1, pp. 191-197, 2002. [18] L. R. Metcalf and E. R. Metcalf, “Plant kairomones in insect ecology and control,” in Contemporany Topics in Entomology 1, pp. 117-118, Chapman & Hall, New York, NY, USA, 1992. [19] J. D. Warthen Jr. and D. O. Mclnnis, “Isolation and identification of male medfly attractive components in Litchi chinensis stems and Eicus spp. stem exudates,” Journal of Chemical Ecology, vol. 15, no. 6, pp. 1931-1946, 1989. [20] G. B. Pitman, “Trans-verbenol and alpha-pinene: their utility in manipulation of the mountain pine beetle,” Journal of Economic Entomology, vol. 64, no. 2, pp. 426-430, 1971. [21] D. R. Miller and J. H. Borden, “Dose-dependent and species- specific responses of pine bark beetles (Coleoptera: Scolytidae) to monoterpenes in association with pheromones,” Canadian Entomologist, vol. 132, no. 2, pp. 183-195, 2000. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 930584, 7 pages http://dx.doi.org/10.1155/2014/930584 Research Article Efficacy of Neem Oil on Cardamom Thrips, Sciothrips cardamomi Ramk., and Organoleptic Studies Johnson Stanley,'’^ G. Preetha,^ S. Chandrasekaran,^ K. Gunasekaran,^ and S. Kuttalam^ ^ Tamil Nadu Agricultural University, Coimbatore 641 003, India ^ Vivekananda Institute of Hill Agriculture, Almora 263 601, India Correspondence should be addressed to Johnson Stanley; stanley_icar@redifFmail.com Received 23 November 2013; Revised 23 January 2014; Accepted 30 January 2014; Published 13 March 2014 Academic Editor: Roman Pavela Copyright © 2014 Johnson Stanley 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 neem tree contains promising pest control substances which are effective against many pests. Oil extracted from neem seeds was used against cardamom thrips, Sciothrips cardamomi, a severe and economic pest of cardamom. Neem oil formulations, namely, Tamil Nadu Agricultural univeristy neem oil (TNAU NO) (acetic acid & citric acid), were found effective against the pest with a overall damage reduction of 30% after 14 days of treatment. The percent damage reduction in capsules over control after three consecutive sprays of TNAU NO(C) 2% and TNAU NO(A) 2% was 78.3 and 75.2 percent, respectively. The newly extracted and unformulated neem oil, though found inferior to the formulated one, still found to cause 50% and 70% reduction in damage caused by thrips at two and three rounds of sprays, making it useful in pest management. Organoleptic tests conducted on cardamom capsules sprayed with neem oil revealed no significant difference in taste, aroma, and overall acceptability of cow milk boiled with cardamom. Thus, TNAU NO (A and C) 2% was found effective against cardamom thrips with no adverse organoleptic properties and can be recommended. 1. Introduction The backlash of synthetic pesticides because of the residual, resistance, and nontarget effects has led to the exploration of ecologically safe pest control alternatives in crop production. Among the different plant species with insecticidal proper- ties, neem {Azadirachta indica A. Juss) is the well- studied and most commercially exploited one for pest management. Azadirachtin, a tetranortriterpenoid, was reported active over nearly 550 insect species [1]. Neem based insecticides especially those having azadirachtin are very much required for 1PM programmes because they are selectively toxic, nonbioaccumulating, less persistent, and a natural source of insecticides [2] . Mode of action of neem on insect pests include direct effects on insect reproduction and secondary antifeedancy, and the physiological effects, measured as growth reduction, increased mortality and abnormal and delayed moults [3]. Neem seed kernel extract (NSKE), neem oil (NO) and neem cake (NC) are used in various field and horticultural crop pest managements. Neem oil cannot be used as such and has to be formulated to increase its efficacy and to decrease the potential phytotoxicity and to increase the storability. Neem oil per se is less systemic because it is insol- uble in water. It should be formulated to make it systemic, to enhance its efficacy on sucking pests. To overcome these hurdles, better formulations are being developed [2]. Two neem oil formulations were made available by Tamil Nadu Agricultural University namely, TNAU NO(C) and TNAU NO(A) which are being tested for the efficacy on different insect pests. The neem product, TNAU NO(C) 30 mL/L, was reported effective against okra leaf hopper, Amrasca devas- tans, and reduced the population by 90% in one week period [4]. Both the formulations A and C at 3% were reported effective against sesame shoot webber and capsule borer, Antigastra catalaunalis, also [5]. TNAU NO is found effective against many other pests like Liriomyza trifolii on cotton [6], Amrasca higuttula and Aphis gossypii in okra [7], Hypothen- emus hampei in coffee [8], Pseudodendrothrips mori in mul- berry [9], and Tetranychus urticae in bhendi and brinjal [10]. Application of neem based formulations effectively checks insect pests of cardamom [11] and neem based 1PM was also developed for cardamom borer and thrips [12]. 2 Psyche Spraying neem oil 0.03% was found effective and caused 47% reduction in cardamom borer infestation [13]. Neem oil suspension at 0.5% sprayed on the lower surface of the leaf is very effective for the control of whitefly nymphs [11] . Margocide CK 0.1% effectively reduced root grubs in the field [14]. Neem cake 600Kgacre~^ is also effective in control- ling the grubs [15]. Moreover, neem cake was reported to significantly reduce the incidence of shoot fly of cardamom and also to enhance the production of side suckers [16]. The neem formulation under study is new, easy to make, cheap, and reported effective against many sucking pests of crops and thus needs to be evaluated against important pests of cardamom. The reports of Jood et al. [17] stated that maize treated with neem oil, neem leaf, and kernel powder adversely affected the taste, aroma, and overall acceptability of Chapati rendering it unsuitable for consumption makes the neces- sity of organoleptic test especially for botanical pesticides. However, organoleptic tests conducted with broiler chicken fed with diets containing urea ammoniated neem seed kernel cake revealed no bitter taste in the cooked meat [18]. With these views, a study was carried out to find the efflcacy of TNAU NO (A and C) along with unformulated neem oil and a commercial neem product (Vijay Neem) on cardamom thrips and organoleptic test on capsules collected from neem sprayed cardamom plants. 2. Materials and Methods 2.1. Efficacy Studies. Two field trials were laid out in car- damom plantations in Bodimettu, Bodi, during March to May, 2006, and Devarshola, Gudalur, Tamil Nadu, during September to November, 2006, to find out the efficacy of neem formulations on cardamom thrips. The trials were laid in randomized block design as per the treatments given in Tables 1 and 2 with three replications. The new neem formu- lations, TNAU NO (A and C), were made and standardized by Tamil Nadu Agricultural University, Coimbatore. The formulations are of 60% a.i and the first formulation contains acetic acid and thus is denoted as A and the other has citric acid and is denoted as C. The TNAU neem oil formulations A and C were tested at the rate of 2 and 3% each and compared with unformulated neem oil and a commercially available neem formulation (Vijay Neem). Field trials were laid out in randomized block design (RBD) in the farmers’ holdings in Bodimettu, Bodi and Devarshola, Gudalur, to test the efficacy of neem oil against thrips. Both the trials were conducted in a ruling variety of cardamom, namely, Njellani Green Gold, as per the treat- ments given in Tables 1 and 2 and replicated thrice. Spray treatment was given using backpacked knapsack sprayer with hollow cone nozzle at a rate of 750Lha~^ (1500 cardamom clumps). Three sprays were given at 15 days interval and observations were made on the capsule damage. A control treatment was made by spraying only water. The thrips incidence in cardamom was assessed on capsule basis and expressed as percent damage. Percent damage was assessed by counting total number of capsules per ten panicles in four clumps in a treatment and capsules showing scabs 3, 7 and 14 days after each application and also prior to the treatment. A clump consists of 5-6 cardamom plants planted/grown together, which covers an area of 0.8 to 1 m , demanding 0.5 L of spray fluid per clump. The percent damage thus recorded was subjected to statistical analysis adopting randomized block design using IRRISTAT version 3/93 after converting it to arcsine values. The mean values of treatments were then separated by Duncan’s multiple range test (DMRT) after being transformed into arcsin values [19]. 2.2. Organoleptic Test for Neem Sprayed Cardamom. Samples were collected 10 days after treatment from different treat- ments as given in Table 3 for TNAU neem oil sprayed plants from the field. Milk was boiled after putting these cardamom capsules separately for each treatment at 20 capsules per L of milk. To obtain unbiased scores each sample was coded. Organoleptic properties of milk for colour, aroma, taste, and overall acceptability were done by a panel of 10 judges. All are untrained panelists but well educated and most of them are agricultural professionals aged between 24 and 55 years. Using a well- structured questionnaire, the panelists independently assessed the samples for appearance (colour), taste, aroma/flavor, and overall acceptability employing 9.0 point hedonic scale [20] as given in Table 4. 3. Results 3.1. Field Trial I-Bodimettu. The mean damage by thrips prior to neem application ranged from 12.0 to 14.6 percent (Table 1). Three days after spraying, the capsule damage ranged between 11.5 and 13.3 percent in different treatments, while in the control it was 15.0 percent. The maximum mean reduc- tion in capsule damage over check being 32.9 percent was recorded in TNAU NO(G) 3% followed by TNAU NO(A) 3% (31.5%) at the end of first spray. Ordinary neem oil 0.2% recorded the least reduction of damage over check (21.5%). Plots treated with TNAU NO(G) 3% and 2% registered a damage score of 10.8 and 9.6 percent and 10.7 and 9.6 percent 7 and 14 days after treatment, respectively, which were not significantly different from each other. The check, Vijay Neem at 2 mL recorded 11.8 7 days after treatment and 10.6 per- cent damage 14 days after treatment. Second spray was given fifteen days after the first spray when the damage ranged from 9.4 to 16.8 percent. At 7 days after treatment, TNAU NO(G) 3% and 2% recorded a damage of 7.8 to 8.1 percent which were on par with each other (Table 1). Though the reductions in capsule damage were low, the thrips population was reduced significantly in all the treatments after the sprays except untreated check. The same trend of efficacy was observed in the third spray also. TNAU NO(G) 3% recorded thrips damage to a level of 6.0, 4.9, and 3.8 percent 3, 7, and 14 days after treatment, respectively, and was found to be sta- tistically superior to other treatments. TNAU NO(G) 3% was found superior in reducing the damage to a level of 80.1 percent at the end of three applications. The percent reduc- tion over control after three sprays of TNAU NO(G) 2% and TNAU NO(A) 2% was 78.3 and 75.2 percent, respectively (Table 1). 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SED: standard error of a difference between 2 means; CD: critical difference. Table 4 Category Scale Like extremely 9 Like very much 8 Like slightly 7 Neither like nor dislike 6 Dislike slightly 5 Dislike moderately 4 Dislike very much 2 Dislike extremely 1 The data collected were subjected to analysis of variance (ANOVA) using completely randomized block design (CRD) using ACRES Version 7.01. 3.2. Field Trial II-Devarshola. The mean damage of thrips to cardamom capsules was high prior to spraying which ranged from 29.1 to 31.0 percent (Table 2). Three days after spraying, the capsule damage ranged between 25.6 and 28.9 percent in different treatments. Plots treated with TNAU NO(C) 3% registered a damage of 23.8 and 21.0 percent 7 and 14 days after treatment against 34.7 and 36.7 percent in control, respectively, and were found to be the best but not statistically superior to TNAU NO(C) 2% and TNAU NO(A) 3%. The thrips damage in TNAU NO(A) 3% treatment was on par with TNAU NO(C) 2% in all the days of observations. The maximum mean reduction in capsule damage over check of 32.3 percent was recorded in TNAU neem oil (C) at 3% followed by Vijay Neem at 2 mL (27.3%) at the end of first spray. Second spray was given 15 days after the first spray. Seven and 14 days after treatment, the thrips damage was found to be 15.8 and 14.0 percent in TNAU NO(C) 3% treat- ment, respectively, while the standard check Vijay Neem reg- istered 17.7 and 15.3 percent, respectively. Ordinary neem oil was also somewhat effective by reducing the thrips damage, namely, from 24.7 percent before spray down to 18.9 percent, 14 days after treatment. After the third application, the thrips damage was 7.7 and 10.2 in TNAU NO(C) 3 and 2% treatments 7 days after treatment, respectively. TNAU NO(A) 3% regis- tered 6.5 percent thrip damage 14 days after treatment while that in the standard check, Vijay Neem, was 7.3 percent. The mean reduction of thrips damage was 81.5 and 75.9 percent in TNAU NO(C) 3 and 2% sprays, respectively (Table 2). Vijay Neem registered 75.8 percent mean reduction in thrips damage when compared to control at the end of three appli- cations. 3.3. Phytotoxicity. The treatments irrespective of the doses given did not inflict any phytotoxicity symptoms like epinasty, hyponasty, leaf injury, wilting, vein clearing, and necrosis on cardamom. 3.4. Organoleptic Tests. The mean scores graded based on the sensory perception are furnished in Table 3. There was no signiflcant variation in the quality parameters assessed, namely, colour, aroma, taste, and overall acceptability. The standard error differences between two means of all the parameters assessed are approximately 0.2 and none of the treatments in any of the parameters evaluated are found to be statistically significant from each other. 4. Discussion Though many chemical insecticides were reported to be effec- tive for the management of cardamom pests [21, 22] , it cannot be recommended for spraying continuously all the year. At the same time, control measures cannot be stopped because thrips will begin to infest the crop as soon as the treatment is stopped. So an effective botanical pesticide for thrips to be sprayed in between the chemical sprays can minimize the pesticide load. Particularly in the mountain ecosystem where cardamom is grown, the dislodgeable pesticides will be washed off from the plants, soil, and so forth and collected in the ponds and rivers contaminating the elixir of life — the “water.” Moreover, cardamom is an export oriented crop and needs to be free of pesticide residues, and if a botanical pesticide is found to be effective, it will be an added advantage to the cardamom producers and exporters. The extent of reduction in the thrips damage in TNAU NO(A) 3% was 31.5-76.0 percent and that of TNAU NO(C) 3% was 32.9-80.1 percent. TNAU NO(C) 2% was on par with its higher dose 3% in all the days of observations. So the two formulations were found to have no significant difference in reducing the thrips damage in cardamom. Generally, the per- cent reduction was low initially since the reduction of scabs in the capsules cannot be realized at once but in due course. This is evident from the continuous reduction in percent damage counts. TNAU NO(C) 3% is the best of the treatments imposed in terms of reduction in damage. The unformulated neem oil was also found effective against the thrips since it was used immediately after extraction. An overall reduction of 21% in cardamom thrips damage was reported by spraying neem oil 0.03% [16]. 6 Psyche The reduced infestation of the cardamom pest in neem formulations sprayed field might be due to antifeedant, ovi- positional deterrent or growth disturbing actions and also repellency effect. It is evident from the results that TNAU NO(C) when evaluated against different insect pests like Cnaphalocrocis medinalis is found to reduce the food con- sumption, pupal weight, adult emergence, pupation rate, and egg hatchability and to increase larval mortality [23]. The diverse biological effects of neem are also reported as it poses repellency, phagodeterrence, growth inhibition, abnormal development [24], and ovipositional suppression [25]. TNAU NO is reported as a potent ovipositional deterrent and it was found up to 90.81 percent in the laboratory. TNAU NO 0.3% causes a reduction up to 60.38 percent of thrips damage in cardamom capsules [26]. The neem sprays which were given to reduce the thrips damage and thereby to reduce the quality deterioration by the pest should not deteriorate the quality of the capsules by itself through its characteristic bitter taste or smell. Thus organoleptic test was carried out to know if there is any unac- ceptability for the cardamom harvested from neem sprayed field and blended with milk. Milk was taken as the medium so that any slight change in taste, aroma, or colour can be easily detected. Table 3 depicts that the scores given by the judges were between 8 and 9 which implies that the product is accepted by the consumers. This finding is in accordance with the reports of Shivashankar et al. [27], who reported no change in taste in tender coconuts harvested from soluneem (water soluble neem formulation) treated palms for the control of coconut black headed caterpillar. The present investigation clearly indicated that there are no disagreeable attributes in the harvested product of cardamom due to the application of neem which is effective in reducing the thrips and thus can be recommended for spray since it will not hamper the export also. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. References [1] A. Anuradha and R. S. Annadurai, “Biochemical and molecular evidence of azadirachtin binding to insect actins,” Current Science, vol. 95, no. 11, pp. 1588-1593, 2008. [2] R. R Soundararajan and V. Lakshmanan, “Neem in pest man- agement of horticultural crops,” in Neem, A Treatise, K. K. Singh, Ed., p. 546, 1. K. International Pvt Ltd, New Delhi, India, 2009. [3] A. J. Mordue and A. J. Nisbet, “Azadirachtin from the neem tree Azadirachta indica: its action against insects,” Anais da Sociedade Entomologica do Brasil, vol. 4, pp. 615-632, 2000. [4] T. Senguttuvan and R. Rajendran, “Plant products for the control of bhendi fruit borers,” in Proceedings of the 2nd National Symposium on Integrated Pest Management (IPM) in Horticultural Crops: New Molecules, Biopesticides and Environ- ment, pp. 17-18, Bangalore, India, October 2001. [5] R. Vishnupriya, A. Annie Bright, V. Paramasivam, and V. Manoharan, “Field Evaluation of some Plant Products against the Sesame Shoot Webber and Capsule Borer, Antigastra catalaunalis Dup,” Sesamum and Safflower Newsletter, 2004, http://ecoport.org/ep?SearchType=earticleView&earticleId=209 &page=-2#section3158. [6] P. Jeyakumar and S. Uthamasamy, “Bio-efficacy of some syn- thetic insecticides and botanicals against Liriomyza trifolii” Indian Journal of Entomology, vol. 59, no. 4, pp. 347-350, 1997. [7] G. P. Indira, K. Gunasekaran, and T. Sa, “Neem oil as a potential seed dresser for managing Homopterous sucking pests of Okra (Abelmoschus esculentus (L.) Moench),” Journal of Pest Science, vol. 79, no. 2, pp. 103-111, 2006. [8] S. Irulandi, R. Rajendran, S. D. Samuel, A. Ravikumar, P. K. Vinodkumar, and K. Sreedharan, “Effect of botanical insecti- cides on coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae),” Journal of Biopesticides, vol. 1, no. 1, pp. 70-73, 2008. [9] A. Subramanian, “Management of mulberry thrips, Pseudoden- drothrips mori (Nawa) by chemical method,” Madras Agricul- tural Journal, vol. 90, no. 10-12, pp. 746-748, 2003. [10] K. Ramaraju, “Evaluation of acaricides and TNAU neem oils against spider mite, Tetranychus urticae (Koch) on bhendi and brinjal,” Madras Agricultural Journal, vol. 91, no. 7-12, pp. 425- 429, 2004. [11] http://www.indianspices.com/. [12] D. Rajabaskar and A. Regupathy, “Neem based IPM modules for control of Sciothrips cardamomi Ramk and Conogethes punctiferalis Gunee in small cardamom,” Asian Journal of Biological Sciences, vol. 6, no. 3, pp. 142-152, 2013. [13] D. J. Naik, V. V. Belavadi, D. Thippesha, M. D. Kumar, and D. Madaiah, “Field efficacy of neem products against thrips and capsule borer of small cardamom,” Karnataka Journal of Agricultural Sciences, vol. 19, no. 1, pp. 144-145, 2006. [14] S. Varadarasan, T. Sivasubramanian, and R. Manimegalai, “Evaluation of some insecticides on cardamom root grub, Basileptafulvicornis Jacoby in field condition,” Pestology, vol. 16, pp. 5-7, 1992. [15] N. E. Thyagaraj, A. K. Chakravarthy and D. Rajagopal, “Evalu- ation of insecticides for suppression of the root grubs, Basilepta fulvicornis Jacoby and Mimela xanthorrhina Hope on small car- damom, Elettaria cardamomum Manton in Karnataka,” Journal of Plantation Crops, vol. 21, pp. 118-120, 1993. [16] D. J. Naik, V. V. Belavadi, and D. Thippesha, “Efficacy of neem products and insecticides for the control of shoot fly Pormosina flavipes Mall, of Cardamom (Elettaria cardamomum Maton.),” Communications in agricultural and applied biological sciences, vol. 71, no. 2, pp. 483-487, 2006. [17] S. food, A. C. Kapoor, and R. Singh, “Evaluation of some plant products against Trogoderma granarium everts in stored maize and their effects on nutritional composition and organoleptic characteristics of kernels,” Journal of Agricultural and Eood Chemistry, vol. 41, no. 10, pp. 1644-1648, 1993. [18] D. Nagalakshmi, V. R. B. Sastry, R. C. Katiyar, D. K. Agrawal, and S. V. S. Verma, “Performance of broiler chicks fed on diets containing urea ammoniated neem (Azadirachta indica) kernel cake,” British Poultry Science, vol. 40, no. 1, pp. 77-83, 1999. [19] K. A. Gomez and A. A. Gomez, Statistical Procedures for Agricultural Research, Wiley International Science Publication, John Wiley and Sons, New York, NY, USA, 1984. Psyche 7 [20] B. M. Watts, G. L. Jlimake, L. E. Jeffery, and L. G. Elias, Basic Sen- sory Methods for Food Evaluation, International Development Research Centre (IDRC), Ottawa, Canada, 1989. [21] S. Varadarasan, D. Kuamresan, and B. Gopakumar, Bi- Annual Report 1987-1988 and 1988-1989, Indian Cardamom Research Institute, Spices Board, Myladumpara, India, 1990. [22] J. Stanley, S. Chandrasekaran, G. Preetha, and S. Kuttalam, “Physical and biological compatibility of diafenthiuron with micro/macro nutrients fungicides and biocontrol agents used in cardamom,” Archives of Phytopathology and Plant Protection, vol. 43, no. 14, pp. 1396-1406, 2010. [23] P. Saikia and S. Parameswaran, “Evaluation of EC formulations of plant derivatives against rice leaf folder, Cnaphalocrocis medinalis Guenee,” Annals of Plant Protection Sciences, vol. 11, pp. 204-206, 2003. [24] H. Rembold, G. K. Sharma, C. H. Czoppelt, and H. Schmutterer, “Evidence of growth disruption in insects without feeding inhibition by neem seed extractions,” Z. Pflkrankh. Pflschutz, vol. 87, pp. 287-297, 1980. [25] B. G. Joshi and S. Sitaramaiah, “Neem kernel as an ovipositional repellent for Spodoptera litura E. moths,” Phytoparasitica, vol. 7, no. 3, pp. 199-202, 1979. [26] D. Rajabaskar, Studies on the evaluation ofIPM modules against Conogethes punctiferalis Guenee and Sciothrips cardamom! Ramk. on cardamom [Ph.D. thesis], Tamil Nadu Agricultural University, Coimbatore, India, 2003. [27] T. Shivashankar, R. S. Annadurai, M. Srinivas et al, “Control of coconut black-headed caterpillar (Opisina arenosella Walker) by systemic application of “Soluneem”-a new water-soluble neem insecticide formulation,” Current Science, vol. 78, no. 2, pp. 176-179, 2000. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 642908, 13 pages http://dx.doi.org/ 10 . 1 155/2014/642908 Review Article Preemptive Circular Defence of Immature Insects: Definition and Occurrences of Cycloalexy Revisited Guillaume J. Dury,^’^ Jacqueline C. Bede,^ and Donald M. Windsor^ ^ Department of Plant Science, McGill University, 21 111 Lakeshore Road, Sainte- Anne- de-Bellevue, QC, Canada H9X 3V9 ^Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancon, Panama City, Panama Correspondence should be addressed to Guillaume J. Dury; guillaume.dury@maiLmcgilLca Received 6 December 2013; Accepted 13 February 2014; Published 24 March 2014 Academic Editor: Jacques Hubert Charles Delabie Copyright © 2014 Guillaume J. Dury 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. Cycloalexy was coined by Vasconcellos-Neto and Jolivet in 1988 and further defined by Jolivet and collaborators in 1990 in reference to a specific type of circular defence. The term has been applied to numerous organisms, including adult insects, nymphs, and even vertebrates, but has lost precision with the accumulation of anecdotal reports not addressing key elements of the behaviour as first defined. We review the literature and propose three criteria that are sufficient and necessary to define the behaviour: (1) individuals form a circle; (2) defensive attributes of the individuals are positioned on the periphery of the circle, and as a result, the periphery of the circle uniformly contains either heads or abdomens; (3) animals preemptively adopt the circle as a resting formation, meaning it is not necessary to observe predation. When these considerations are taken into account, cycloalexy appears less common in nature than the literature suggests. We argue that unequivocal cases of cycloalexy have been found only in sawflies (Tenthredinoidea: Pergidae, Argidae), leaf beetles (Chrysolemidae: Galerucinae, Cassidinae, Chrysomelinae, Criocerinae), weevils (Curculionidae: Phelypera distigma), and midges (Diptera; Ceratopogonidae, Forcipomyia). Reports of cycloalexy in caterpillars (Saturniidae: Hemileucinae: Lonomia, Papilionidae) require further documentation. We report one new case of cycloalexy in thrips (Thysanoptera) and question reports of cycloalexic behaviour in other taxa. 1. Introduction Some animals show a remarkable behaviour: they group in a tight circle for defence [1]. This behaviour is reminiscent of Carl von Clausewitz’s 1812 Principles of War: “In strategy (. . .) the side that is surrounded by the enemy is better off than the side which surrounds its opponent, especially with equal or even weaker forces” [2] . Many animal species employ this strategy. For example, among vertebrates, muskoxen {Ovibos moschatus, Blainville, 1816) form a circle enclosing the young calves when attacked by wolves, their principal natural predators [3, 4]. Their circular formation protects the most vulnerable body parts while the extremity that is best defended or involved in attack is at the periphery. Vasconcellos-Neto and Jolivet [5] coined the term “cycloalexy (kuklos = circle, alexo = defend)” to describe a particular behaviour of gregarious insect larvae. They defined their new term as “the attitude adopted at rest by some insect larvae, both diurnal and nocturnal, in a tight circle where either the heads or ends of the abdomen are juxtaposed at the periphery, with the remaining larvae at the center of the circle. Coordinated movements such as the adoption of threatening attitudes, regurgitation, and biting, are used to repel predators or parasitoids.” [1]. Several elements of the original definition distinguish cycloalexy from other circular formations occurring in nature. However, as new examples of the behaviour have been proposed in the literature without addressing key aspects of the original definition, the distinctions between cycloalexic behaviour and other circular formations have become impre- cise and weakened the concept of cycloalexy. Here, we will review reported examples of cycloalexy and question whether they meet the criteria of a revised definition or are alternative forms of aggregation. Our revised definition 2 Psyche strives to adhere to the key aspects of cycloalexy as originally defined, while removing arbitrary and unnecessary criteria. This way, cycloalexy can be recognized as an evolutionarily convergent behaviour rather than several superficially similar behaviours. 1.1. Redefining Cycloalexy. The defensive nature of cycloalexy is paramount as it is found in the etymology of the term: “to defend” [11] . However, this key aspect of the behaviour can be problematic since cycloalexy has often been invoked upon fragmentary observations of groups rather than con- trolled ecological studies. Nevertheless, until species can be more thoroughly studied, we suggest that three criteria are sufficient to distinguish cycloalexy from other behaviours. Criterion 1: Individuals Are Arranged in a Circle. The original definition specified “in a tight circle” [1], but we argue that tightness is subjective and should be removed from the definition. Criterion 2: The Extremity Bearing Defensive Attribute Is Positioned Outwards. In the original definition, the periphery of the circle is uniform: “either the heads or ends of the abdomen are juxtaposed at the periphery” [1], sometimes with individuals at the centre with neither head nor abdomen reaching the periphery. This means that peripheral individu- als in a given group face outwards or inwards, but not both. We argue that, in this statement, it was implicit that the best defended extremity is at the periphery since it is unlikely that individuals with their vulnerable side out could ever be at an advantage. Uniformity of the circle in this respect, then, becomes a corollary of our second criterion. Criterion 3: The Circle Is Adopted as a Resting Formation. Following the original definition, we limit cycloalexy to cases when individuals are in resting or quiescent, nonfeeding periods. This makes cycloalexy a preemptive behaviour. These criteria allow for the initial identification of cycloalexy by rapid, visual assessment. If later studies dis- prove defensiveness, then the behaviour studied is not cycloalexy. Additionally, although not specified in the orig- inal definition, we suggest adding the distinction that the behaviour is for the defence of the individuals themselves and others within the group, as opposed to the defence of a resource or nest. We also suggest removing the criterion that “Coordinated movements such as the adoption of threatening attitudes, regurgitation, and biting, are used to repel predators or parasitoids” [1], for several reasons. Cycloalexy is notably preemptive, taken regardless of the immediate presence of a threat; in some species, immature animals have passive defences made more efficient by a circular formation; and the second criterion of our amended definition already includes defence, either passive or active. Finally, although the original definition was limited to insect larvae, in this paper, we review all reports of cycloalexy and suggest removing the limitation altogether. 2. Results See Table 1. 3. Discussion 3.1. Strict Cycloalexy. Among the records in the literature, some species demonstrate behaviour that precisely fit the revised definition and our three essential criteria of cycloal- exy (Table 1). This is the case of Coelomera spp. (Coleoptera: Chrysomelidae: Galerucinae) and spitfire grubs Perga dor- salis Leach, 1817 (Hymenoptera: Tenthredinoidea: Pergidae) [ 1 ]. Approximately 35 species of genus Coelomera are cycloalexic and feed on Cecropia (Urticaceae). Most Cecropia plants are myrmecophytes protected by mutualistic Azteca ants (Formicidae: Dolichoderinae). The gregarious leaf beetle larvae feed during the day and rest at night, in a tight circular cluster with heads inside and abdomens at the periphery [7, 8]. Their rear end is protected by a supra-anal shield and, when threatened, these larvae excrete a nauseating fluid from the anus. Therefore, the better protected part of the insect, the posterior, is facing outwards in the circle, with the more vulnerable head inside [7, 8]. Thus, by orienting the same way, individual larvae protect themselves and other members of the group (Figures 1 and 2). Spitfire grubs Perga dorsalis feed on Eucalyptus during the night and rest during the day in a circular formation [47]. The larvae rest with their heads at the periphery of the circle, with some larvae in the middle of the aggregation. When threatened, the larvae rear their heads and abdomens and regurgitate oils sequestered from their host Eucalyptus [47]. The oils are an effective deterrent of potential predators, including ants, birds, and mice [49]. The heads are the better protected part of the insects and, again, form the periphery of the circle. 3.2. Examples of Cycloalexy That Do Not Agree with the Revised Definition 3.2.1. The Oxymoron of Noncircular Cycloalexy. Gregarious caterpillars of genus Arsenura (Saturniidae: Arsenurinae) are reported to “show a kind of cycloalexy when resting on tree trunks during the day” [1]. The caterpillars align side- by-side or head-to-abdomen or both, in an elongated oval cluster [11]. The posture of these caterpillars with their heads, sides, and abdomens at the periphery in a linear mass rather than a circle does not meet the first criterion of the revised definition of cycloalexy. The circle formation with the best defended extremity outwards is an important characteristic of cycloalexic behaviour. Arsenura are gregarious and rest in a tightly aggregated mass, but they are not cycloalexic. Santiago -Blay et al. [11] suggest that, on a tree trunk, “the available background surface makes the shape of the larval aggregation distorted.” However, on the scale of a caterpillar, and depending on the diameter of the tree, a tree trunk can be quite large and nearly flat. In addition, probable cycloalexy on tree trunks has been observed in Lonomia sp. (Figure 3(b)). We suggest the caterpillar aggregations described by Santiago-Blay et al. [11] are less circular and compact not because of the shape of tree trunks but because Arsenura caterpillars’ resting positions are not cycloalexic. Psyche 3 Table 1: Reported cases of cycloalexy and how they fit within the revised definition. Criteria 1 2 3 Aggregation is Is the Organism Best defended Default rest position defensive (not behaviour Reference Circular formation extremity at the periphery for nest protection) cycloalexy? Coleoptera: Chrysomelidae: Galerucinae Coelomera spp.; for example, C. ruficornis Baly, 1865; C. helenae Jolivet, 1987; Yes Yes, abdomens Yes Yes Yes [1, 5-8] C. raquia Bechyne, 1956; and so forth Not Not observed Not Not observed Nnnp [6.7] Dircema spp. observed observed observed Coleoptera: Chrysomelidae: Criocerinae Lema sp.; Lema apicalis Lacordaire, 1845 and L. reticulosa Clark, 1866 Yes Yes, heads Yes Yes Yes (Figure 1(a)) [9, 10] Lilioceris nigropectoralis (Pic, 1928), Yes Yes, heads Yes Yes Yes (Figure 1(b)) L. formosana Heinze, 1943 [11] Coleoptera: Chrysomelidae: Chrysomelinae Not observed Not observed None observed Agrosteomela chinensis (Weise, 1922) Not observed Not observed [11-13] Chrysophtharta obovata (Chapuis, 1877) Yes Yes, abdomens Yes Probably Yes [11, 14] Doryphora paykulli (Stal, 1859), D. Yes Unclear, Yes Yes No* [15] reticulata Fabricius, 1787 abdomens Eugonycha melanostoma (Stal, 1859) Yes Unclear, abdomens Not reported Not reported Tentatively [7, 11] Gonioctena sibirica Kimoto, 1994 Roughly Unclear, mostly abdomens Mostly Unclear Unclear [11, 12, 16] Labidomera suturella Cuerin-Meneville, Not Not observed Not Not observed None [11, 17-19] 1838 observed observed observed Paropsis spp.; for example, P. aegrota Boisduval, 1835, No mi vpH [7, 11, 14, 20, 21] P maculata (Marsham, 1908), P atomaria Olivier, 1807 and P. tasmanica Baly, 1864 Not circular extremities No Perhaps, unclear No Paropsisterna spp. Not reported Not reported Not reported Not reported Not enough information [11, 12, 14, 20] Plagiodera spp. for example. Not circular No, mixed extremities No No No [7, 22-25] P versicolora (Laicharting, 1781) Phratora spp. Not Not observed Not Not observed Nonp [7, 11] observed observed observed Phyllocharis undulata (Linnaeus, 1763) Roughly Unclear, mostly abdomens No Not observed No [11, 26] Platyphora selva Daccordi, 1993, P microspina (Bechyne, 1954) Yes Unclear, abdomens Yes Yes No* [15, 27] Platyphora conviva (Stal, 1858), P anastomozans (Perty, 1832), P nigronotata (Stal, 1857), P. nitidissima (Stal, 1857) P fasciatomaculata (Stal, 1857), P vinula (St£, 1858) Yes Yes, heads Yes Yes Yes [7, 9, 10, 28] 4 Psyche Table 1: Continued. Criteria 1 2 3 Aggregation is Is the Organism Best defended Default rest position defensive (not behaviour Reference Circular formation extremity at the periphery for nest protection) cycloalexy? Proseicela vittata (Fabricius, 1781), P. bicruciata Jacoby, 1880, P spectabilis (Baly, 1858) Yes Unclear, abdomens Yes Yes No* (Figure 5(a)) [15] Proseicela crucigera (Sahlberg, 1823) Yes Unclear, abdomens Yes Yes Not enough information [7,9] Pterodunga mirabile Daccordi, 2000 Yes Unclear, abdomens Not reported Not reported Not enough information [11, 12, 19] Coleoptera: Chrysomelidae; Cassidinae Acromis sparsa (Boheman, 1854) Aspidomorpha puncticosta Boheman, Yes Yes, abdomens Yes Yes Yes* [7, 11, 29] 1854, A. miliaris (Fabricius, 1775) Yes Yes, abdomens Yes Yes Yes [7, 30-32] Chelymorpha informis Boheman, 1854, C. alternans Boheman, 1854, Yes Yes, abdomens Yes Yes Yes [7,8] C. cribraria (Fabricius, 1875) Cistudinella foveolata (Champion, 1894) Yes Yes, abdomens Yes Probably Yes (Figure 2(a)) Conchyloctenia punctata (Fabricius, 1787) Yes Yes, abdomens Yes Yes Yes [7, 30, 33] Coptocycla dolosa Boheman, 1855 Yes Yes, abdomens Yes Probably Yes (Figure 4) Eugenysa Columbiana (Boheman, 1850), Yes Yes, abdomens Yes Yes Yes* (Figure 2(b)) E. coscaroni Viana, 1968 [34, 35] Paraselenis flava (Linnaeus, 1758) Yes Yes, abdomens Yes Yes Yes* [8] Nuzonia sp. Yes Yes, abdomens Yes Yes Yes (Figure 2(c)) Ogdoecosta biannularis (Boheman, 1854) Yes Yes, abdomens Yes Yes Yes [7, 11, 36] Omaspides tricolorata (Boheman, 1854), 0. pallidipennis (Boheman, 1854), 0. sobrina (Boheman, 1854), 0. bistriata (Boheman, 1854) and 0. convexicollis Spaeth, 1909 Yes Yes, abdomens Yes Yes Yes* [7, 11, 32, 35, 37-39] (D. Windsors observations) Physonota alutacea Boheman, 1854 Yes Yes, abdomens Yes Probably Yes (Figure 2(d)) Polychalma multicava (Latreille, 1821) Yes Yes, abdomens Yes Probably Yes (Figure 2(e)) Stolas sp., Stolas xanthospila (Champion, 1893) Yes Yes, abdomens Yes Probably Yes (Figure 2(f)) [7] Coleoptera: Curculionidae; Hyperinae Phelypera distigma (Boheman, 1842) Yes Yes, heads Yes Yes Yes [24, 40, 41] Diptera: Ceratopogonidae: Forcipomyiinae Eorcipomyia fuliginosa (Meigen, 1818) Yes Yes, abdomens Yes Probably Yes [1, 11, 42-44] Hemiptera Not specified Not reported Not reported Not reported Not reported Not enough information [11, 12] Ceroplastes sp. (Coccidea), Potnia sp. (Membracidae), Roughly to No, mixed No Unclear No [11] Nephesa rosea (Spinola, 1839) (Flatidae), Derbe sp. (Derbidae) not circular extremities Antiteuchus tripterus (Fabricius, 1787) (Pentatomidae) Yes Unclear, abdomens No No No [45] Parastrachiajaponensis (Scott, 1880) Yes Unclear, No No No [46] (Parastrachiidae) abdomens Psyche 5 Table 1: Continued. Criteria 1 2 3 Aggregation is Is the Organism Best defended Default rest position defensive (not behaviour Reference Circular formation extremity at the periphery for nest protection) cycloalexy? Hymenoptera: Tenthredinoidea Bergiana sp. (Cimbicidae) Yes Not reported Not reported Not reported Not enough information [1] Perga dorsalis Leach, 1817, P. affinis Yes Yes, heads Yes Yes Yes (Figure 3(a)) [7, 11, 12, 47, Kirby, 1882 (Pergidae) 48] Pseudoperga guerini (Westwood, 1880) (Pergidae) Yes Yes, heads Yes Yes Yes [491 Themos olfersii (Klug, 1834) (Argidae) Yes Yes, heads Yes Yes Yes [1, 50] Dielocerus diasi Smith, 1975 (Argidae) Not reported Not reported Not reported Unclear Not enough information [1, 50] Hymenoptera: other superfamilies Trigona sp. (Apidae: Meliponinae) Yes Yes, heads No No No [1, 7, 11, 51] Adult Hymenoptera, bees (Apidae), Some wasps (Vespidae), circular. Yes, usually heads No No No [11] Conomyrma spp. and numerous other ants (Formicidae) some not Apoica sp. (Vespidae: Polistinae) Yes Yes, heads Yes No No [52-54] “Parasitic Hymenoptera larvae and pupae [on] their host” Yes Unclear, abdomens No No No [11] Lepidoptera: Papilionidae: Papilioninae Papilio laglaizei Depuiset, 1877 Yes Unclear, heads Yes Not reported Tentatively [24, 55] Lepidoptera: Saturniidae Hylesia spp. (Hemileucinae) Unclear Not reported Yes Probably Not enough information [7] Lonomia spp. (Hemileucinae) Yes Probably, heads Yes Probably Probably (Figure 3(b)) [11, 56, 57] Arsenura spp. (Arsenurinae) Not circular No, mixed extremities Yes Probably not No [1. 11]. Lepidoptera: other families Noctuidae and Not circular No, mixed Not Probably not No [11] Sphingidae extremities reported Neuroptera: Ascalaphidae Ascaloptynx furciger (McLachlan, 1891) Yes, around No, mixed [1, 11, 12, 58] twig extremities No Yes No Thysanoptera: Phlaeothripidae Anactinothrips nigricornis Hood, 1936 and A. gustaviae Mound & Palmer, 1983 Yes Yes, abdomens Yes Probably Yes (Figure 5(b)) [59] Non-insect arthropods Phronima sedentaria (Forskal, 1775) (Crustacea: Amphipoda: Hyperiidea) Yes Unclear, heads? Yes No No* [24, 60, 61] Platydesmidae, Unidentified sp. (Myriapoda) Yes Unclear, abdomens Not reported Not reported Tentatively analogous [24, 62] Vertebrates Some ungulates, for example. Muskoxen Ovibos moschatus Yes Yes, heads No Yes No [1, 11, 12] (Zimmermann, 1780) Antarctic penguins Yes Unclear, backs Yes No No [12, 63] * These taxa are maternally defended and pose a special challenge to the definitions of cycloalexy (see Section 3.2.5). 6 Psyche Figure 1: Cycloalexywith heads outwards in shining leaf beetle larvae (Criocerinae). (a) Larvae of Lema sp. at rest, photograph in Potrerillos del Guenda, Dept. Santa Cruz, Bolivia, © D. Windsor, (b) Lilioceris nigropectoralis larvae in Taiwan. Seven larvae are distinctly larger and appear to be from a different cohort than the other twelve. Photograph taken in Yangmingshan National Park on 2 August 2011, by (Liu Da Wei), and licensed under the Creative Commons 3.0 Taiwan (CC BY-NC 3.0 TW). 3.2.2. Mixed Head Orientations. Larvae of the owlflj Ascalop- tynx furciger (McLachlan, 1891) (Neuroptera: Ascalaphi- dae) are gregarious. After eclosion and their first meal of abortive eggs, they settle head-downwards on and around the twig on which they were laid [58]. Jolivet et al. [1] deem the behaviour of A. furciger is “not strictly cycloalexy but related to it” since the owlfly larvae all point in the same downward direction: this does not meet the second criterion of the revised definition. We agree with Jolivet and Verma [12] that cycloalexy exists around twigs and is not restricted to flat surfaces. However, even on small branches, cycloalexic larvae collectively orient their heads either outwards or inwards, but not both. This is true for lar- vae of Perga sp. (Hymenoptera: Tenthredinoidea: Pergidae) (Figure 3(a)), Omaspides tricolorata (Boheman, 1854) [39], and this arrangement is retained in the pupae of Omaspides pallidipennis (Boheman, 1854) (Chrysomelidae: Cassidinae) [37]. For owlfly larvae, heads form the periphery at the bottom of the aggregation and abdomens are at the periphery on top, but unlike Cassidinae or Coelomera larvae, their abdomens are more vulnerable. It is more accurate to describe the behaviour as unidirectional defence rather than circular defence; larvae are only protected from predators walking up to the group. Secondly, larvae also feed while, in this position, making it a passive hunting formation and not only a resting position [58]. Because they do not meet the second and third criteria, we question reports of cycloalexy in Neuroptera [7, 11, 12, 64]. 3.2.3. Nonresting Behaviours. As stated in the third criterion, cycloalexy is adopted preemptively by animals at rest. When immature insects are active and feeding, the circular for- mation is normally broken (Figure 4). Larvae of Plagiodera versicolora (Laicharting, 1781) and other Plagiodera species form a loose circle when feeding and at rest, with individual larvae not consistently facing outwards or inwards [22, pers. obs.]. Hence their formation is not an example of cycloalexy. Their formation is not only adopted at rest but also while feeding and is often influenced by the shape of the leaf, with multiple “feeding rings” on larger leaves [22]. Some authors [11] feel that cycloalexy facilitates feeding in P. versicolora as well as in sawflies. Larval aggregations can increase feeding efficiency through synchronized, coordinated, and spatially concentrated feeding [23-25] .The size of P. versicolora groups does not influence survival of larvae, but does help with feeding [25]. Thus, available evidence suggests grouping in P. versicolora is related principally to the process of feeding rather than defence. 3.2.4. Nondefensive Behaviour. Cycloalexy is a defensive behaviour; it protects individuals from predation or para- sitism. Yet, some reported behaviours are not defensive. Such is the case for huddling in Antarctic penguins, where the huddle is a resting behaviour, usually with heads inwards, but it is for heat conservation rather than defence [63]. For these reasons, we disagree with Jolivet and Verma [12] that penguins are cycloalexic. To conclusively prove the defensive value of a behaviour, ecological studies are needed. Yet, for many species, the defensive value of cycloalexy has been inferred from anec- dotal evidence or personal observations or has simply been presumed. For example, the defensive value of cycloalexy in Phelypera distigma larvae is supported by the following statement: “P. distigma larvae are not harvested by polistine wasps, ants, spiders, and other generalist predators that readily harvest caterpillars in dry forest habitats (D. H. Janzen, pers. obs.)” [65]. Rather than rejecting the many reports of cycloalexy on the basis of insufficient ecological studies, we propose that defensive nature of the aggregation can be accepted if the Psyche 7 (e) (f) Figure 2: Cycloalexy in tortoise beetle larvae, (a) Cistudinellafoveolata (Ischyrosonychini) larvae on host Cordia alliodora (Ruiz & Pav.) Oken. Gamboa, Colon Province, Panama; (b) Eugenysa coscaroni (Eugenysini) larvae and mother on host Mikania guaco Bonpl. (Asteraceae), Cerro Campana, Panama Province, Panama; (c) Nuzonia sp. on host Maripa nicaraguensis HemsL, Chiriqui Grande, Bocas del Toro Province, Panama; (d) Physonota alutacea (Ischyrosonychini) larvae on host Cordia spinescens L., Gamboa, Colon Province, Panama; (e) Polychalma multicava (Goniocheniini) larvae on host Helicteres guazumaefolia Kunth. (Sterculiaceae), Gamboa, Colon Province, Panama; (f) Stolas xanthospila (Mesomphaliini) larvae on host Turbina corymbosa (L.) Raf. (Convolvulaceae), Cerro Campana, Panama Province, Panama; all photographs © D. Windsor. 8 Psyche (a) (b) Figure 3: (a) Larvae of Perga sp. (Pergidae) rest aggregated in a cycloalexic formation. Even around a stem of their host plant, Eucalyptus sp., spitfire larvae rest with their heads outwards, Black Mountain, Canberra, ACT, Australia. Photograph by Donald Hobern on 24 May 2010 (CC BY 2.0). (b) Probable cycloalexy with heads pointing outwards in caterpillars of Lonomia sp. (Saturniidae: Hemileucinae) on tree trunk in Peru. Photograph taken near Pongo de Caynarachi, Lamas, San Martin, Peru, and reproduced with the author s permission © Marc Diaz Rengifo (Universidad Nacional Federico Villarreal, Lima, Peru). (a) (b) Figure 4: Coptocycla dolosa larvae, Potrerillos del Guenda, Dept. Santa Cruz, Bolivia, (a) when active, feeding, or moving; (b) at rest. © D. Windsor. animals meet the other criteria: they are in a circle, taken pre- emptively with defensive armature uniform at the periphery. When ecological studies are conducted, if defensiveness is disproved, then the behaviour is another type of aggregation and not cycloalexy. This is the case for larval aggregations of Plagiodera versicolora: ecological studies were conducted and the survival of larvae is not significantly influenced by group size [25]. 3.2.5. Circular Formations That Do Not React to Threats. The original definition of cycloalexy requires coordinated movements in response to threats [1] . We disagree with this requirement: in some larvae with passive protection, like the exuvial or exuvio-fecal shields of tortoise beetles, the circular groups do not always use coordinated movements when threatened by predators. For example, the larvae of Conchyloctenia punctata (Fabricius, 1787) (Cassidinae) are passively protected by their shields but do not have coor- dinated reactions to threats [30]. In our opinion, larvae of C. punctata meet the basic criteria of cycloalexy. Although coordinated group reactions to threats are an indication of the defensive nature of the group, we propose it is not an essential criterion for cycloalexy. The removal of this criterion is also important for several taxa in which the larvae receive maternal care. Cassidinae larvae in maternal care species (e.g., species of genera Acromis, Omaspides, Paraselenis, and Eugenysa) generally have reduced fecal shields and do not always react defensively Psyche 9 when threatened. Larval grouping in these species can be considered as increasing the efficiency of maternal guarding. In these cases, all criteria of the revised definition are met: larvae are in a circle, the best defended extremity is always at the periphery, and the circle is the default resting position. Thus, we consider larval aggregations in these maternal care species as further examples of cycloalexy (Table 1). Similarly, larvae of several chrysomelines rest in tight circular groups with the heads pointing inwards: Doryphora paykulli (Stal, 1859), D. reticulata Fabricius, 1787, Platyphora microspina (Bechyne, 1954), P. selva Daccordi, 1993, Proseicela vittata (Fabricius, 1781), P spectabilis (Baly, 1858), P. bicruciata Jacoby, 1880 and Pr. sp. nov. “Yasuni” [15] . All these species also have maternal care, and when disturbed, larvae do not have coordinated defensive reactions. Instead, the mother acts as the defensive element of the formation (Figure 5(a)) [15]. Is this behaviour still cycloalexy? In other words, should the defensive element obligatorily be found, at least in part, in the larvae? To this question, our answer is yes, through the second criterion. In Cassidinae larvae, the furca and shield are obvious defensive attributes positioned at the periphery. In Chrysomelinae, the best defended extremity is less obvious. Cycloalexic larvae of nonmaternal care Chrysomelinae face outwards. Their best defended extremity is the head and thorax, through regurgitation and biting [7]. We hypothesize that, in species with maternal care, the individuals face inwards not because the best defended extremity is the abdomen but because of herding by the mother, and thus, these species do not meet the second criterion of the revised definition. Ultimately, only ecological and evolutionary stud- ies will provide a clear answer. 3.2.6. Adult Insects. We use Apoica as an example even though cycloalexy was not explicitly reported in this genus. During the day, these nocturnal wasps rest on the circular or nearly circular lower surface of their nests [52-54]. The wasps rest facing outwards, resulting in a circular formation that could loosely be termed cycloalexy. When disturbed, the formation breaks up as wasps fly off the nest. Even though this behaviour meets several criteria of the revised definition, we argue it is not cycloalexic because the shape of the nest or nest entrance explains the circular formation. In a similar fashion, stingless bees of genus Trigona (Apidae: Meliponinae) are not cycloalexic as suggested by Vasconcellos-Neto and Jolivet [7]. In this case, fully developed individuals are not even at rest: in most Meliponinae, the nest entrance is protected by bees positioned in or around the entrance tube and, at night, the entrance is closed [51] . The bees are not resting but are actively guarding and the ring formation is an artefact of the nest entrance shape. These examples motivate limiting and specifying cycloalexy as a formation taken by individuals, whether immature or adult, for increased individual and mutual defences, thus excluding formations taken for defence of a nest, brood, or food stores. We argue that evolution of circular nests and resource guarding may have little to do with the evolution of cycloalexy. 3.2.7. Circular Defence in Vertebrates and the Selfish Herd. Several authors compare cycloalexy to the “circle-the- wagons” formation employed by American pioneers to defend themselves against Native Americans [1, 11, 12, 24] . In Jolivet et al. [1] and Jolivet and Verma [12], the authors discuss behaviours analogous to cycloalexy in vertebrates: muskoxen {Ovibos moschatus), eland {Taurotragus oryx (Pallas, 1766)), elk {Cervus canadensis (Erxleben, 1777)), and penguins. The authors do not provide citations for the behaviour in eland or elk and cite Wilson [4] for descriptions of this behaviour in muskoxen and penguins. Wilson [4] does not mention penguins in this manner but does mention similar behaviours in several terrestrial ungulates and killer whales {Orcinus orca (Linnaeus, 1758)) ([3, 66-69]; all page 45 in [4]). Wilson [4] describes elk grazing in a “windrow” formation but does not mention circular defence [70] and [4, page 45]. We agree that several vertebrates employ defensive circular formations analogous to cycloalexy. However, we would not broaden the definition to include these behaviours. Unlike invertebrates, mammals do not use circular defence when resting but take the formation when threatened. This does not meet the third criterion of the revised definition. In cycloalexic species, the circular formation is the main resting position. The circular defence of vertebrates is reactive, while cycloalexy in invertebrates is largely preemptive. Hamilton used herding animals as an example of how individuals may form a group to lessen individual chances of falling to a predator without reducing overall predation [71]. Hamilton then cited the circular defence of muskoxen as a potential exception to the selfish herd theory but attributes it to selfish reasons: “they are probably connected on the one hand with the smallness of the risk taken and, on the other, with the closeness of the genetical relationship of the animals benefited” [71]. Because cycloalexy may lessen both overall and individual predation risk, it can also be considered selfish. Cycloalexy can be explained by animals exploiting the best defended extremity of nearby individuals. The preemptive aspect of arthropod cycloalexy also distinguishes it from muskoxen circular defence and Hamilton’s selfish herds and may therefore provide interesting systems for study of group defence. 3.2.8. Cycloalexy in Immature Hemimetabolous Insects. We report cycloalexy in Anactinothrips nigricornis Hood, 1936 (Thysanoptera). We observed a group of 14 thrips, in their pupal instar, forming a tight circle with abdomens outwards on a leaf of the woody vine Maripa panamensis Hemsl. (Con- volvulaceae) (Eigure 5(b)). When disturbed, the threatened individuals and those beside them waved their abdomen. When disturbance continued, a brown liquid was exuded and formed a droplet at the end of the abdomen. The group was then further disturbed and the individuals dispersed. Approximately an hour later, the thrips had reassembled in a circular resting formation. In the lab, after the final moult, the adult thrips dispersed in the container in which they were kept. Similar observations were made in another species of the same genus: the thrips A. gustaviae, Mound and Palmer, 10 Psyche (a) (b) Figure 5: (a) Larvae of Proseicela spectabilis Baly (Chrysomelidae: Chrysomelinae) at rest encircling the stem of their host plant with tachinid fly at the bottom left of the cluster and the adult female beetle on the opposite side. Photograph taken in Reventador, Napo Province, Ecuador, © G. Dury. (b) Circular resting position in Panamanian thrips (Anactinothrips sp.) on Maripa panamensis Hemsl. (Convolvulaceae). Photograph taken 24 April 2013, on Cerro Campana, Panama. © G. Dury. 1983, rest in bivouacs and exude a defensive liquid from their abdomen when disturbed [59, 72]. The behaviour was observed in the mobile pupal stage rather than in the larvae. This goes against the original definition but meets all other criteria and assuming it is defensive, we consider the behaviour is cycloalexy. Thus, we propose to remove the taxonomical restriction of the original definition. 3.3. Common Traits of All Cycloalexic Species. When the revised definition of cycloalexy is strictly applied, a set of traits common to all species becomes apparent. Foremost, all cycloalexic species are insects with gregarious immature stages. Gregarious lifestyles have implications in terms of cooperative feeding and continued group cohesion through chemical, tactile, or acoustic communication [24] . To date, all cycloalexic species appear to use chemical defences of one sort or another. The cycloalexic larvae in genera Lema (Criocerinae) and Platyphora (Chrysomelinae) regurgitate when threatened [7, 9]. The larvae of Forcipomyia have paired setae on the head, thorax, and abdomen that exude hygroscopic substances that repel ants [44]. The chemical defences of gregarious Lonomia caterpillars are so potent that the resulting trauma caused by venom injected from their setae can be lethal to humans [73] . Most tortoise beetle larvae carry an exuvial or exuvio-fecal shield on the furca of their eighth abdominal segment which serves as a mechanical or chemical barrier against predators [74-76]. In all cases, the best protected extremity faces outwards. Furthermore, all the species that exhibit cycloalexic behaviour are miniature grazers, and most feed on leaves. This is the case for cycloalexic caterpillars, and larvae of sawflies [47], weevils, and leaf beetles [7, 40]. Some feed on fungal hyphae, such as Forcipomyia fuliginosa (Meigen, 1818) midge larvae [42], and the rest graze on lichen, like Anactinothrips gustaviae thrips [59] . Gregarious lifestyles, chemical defence, and grazing groups of immature insects are all traits of Gostas [24] “larval herd” syndrome of group living. Like cycloalexy, parental care is only present in some of these larval herds [24] . Possibly, the slow-moving and exposed lifestyle of these immature insects makes them more vulnerable to predators and parasitoids [24, 77]. Increased threats probably explain the multiple defences of insect herbivores, including chemical defence whose evolution generally precedes that of aggregation [78]. 4. Conclusion Several immature insects exhibit cycloalexy, a behaviour whose definition we have amended to: “A preemptive defence employed at rest, where individuals form a circle with their best defended extremity exposed at the periphery. Sometimes remaining individuals rest at the centre of the circle.” In leaf beetles (Ghrysomelidae), cycloalexy with abdo- mens oriented outwards is found in one genus of skeletoniz- ing leaf beetles (Galerucinae: Coelomera spp.), at least fifteen tortoise beetle genera (Cassidinae), two genera of shining leaf beetles (Griocerinae: Lema and probably Lz/zoccns), and several genera of broad-shouldered leaf beetles (Chrysomeli- nae: Platyphora, probably Chrysophtharta and tentatively Eugonycha and Pterodunga). Cycloalexy with heads outwards is found in some sawflies (Tenthredinoidea: Pergidae: Perga spp. and Argidae: Themos olfersii (Klug, 1834)) of Australia and Brazil. Social caterpillars often form aggregations, but Psyche 11 these aggregations are rarely cycloalexic. However, caterpil- lars of Lonomia spp. (Saturniidae: Hemileucinae) are prob- ably cycloalexic and Papilio laglaizei Depuiset, 1877 (Papil- ionidae) are tentatively cycloalexic. One weevil Phelypera distigma (Boheman, 1842) (Curculionidae) is cycloalexic and one midge Forcipomyia fuliginosa (Ceratopogonidae) exhibits cycloalexy We propose that some immature thrips are also probably cycloalexic and suggest formally changing the definition of cycloalexy to remove taxonomical restric- tions so that any animals that meet all other criteria of the definition can be included. New instances of cycloalexy will undoubtedly be discovered. For example, Platydesmid millipedes sometimes aggregate in a tentatively analogous fashion. Several reports of cycloalexy do not meet one or more of the revised definition criteria, including reports of cycloalexy in feeding aggregations of Hemiptera and larvae of Hymenopteran parasitoids. The behaviour has also been mistakenly attributed to adult Hymenoptera, for example, stingless bees (Apidae: Meliponinae), ants (Formicidae), and wasps (Vespidae), guarding their nest. This is active protec- tion of a nest and not cycloalexy. Similarly, the term has been applied to the circular assembly of an amphipod crustacean which helps the mother herd the larvae. Owlfly larvae (Neu- roptera: Ascalaphidae: Ascaloptynx furciger) form unidirec- tional defensive groups which are not cycloalexic, allowing larvae to feed without changing position. Defensive circles are sometimes observed in mammals: muskoxen, eland, water buffalo, red deer, and killer whales. Contrary to cycloalexy, the defensive formations in these mammals are a reaction to imminent threat. Other vertebrates, like penguins, huddle to reduce heat loss. Application of a more precise definition of cycloalexy, as provided by Jolivet et al. [1] and revised here, may make unravelling the evolution of cycloalexic behaviour more tractable. Much remains to be learned about whether larval aggregation, cycloalexy, sequestration of plant metabo- lites, and maternal care are alternative defensive strategies or are honed evolutionary responses to particular threats. Chrysomeline leaf beetles are an ideal group for using phylogenetic reconstruction and character analysis of these behaviours to unravel the number of independent evolution- ary origins of cycloalexy and larval aggregation. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work was funded by the Natural Sciences and Engi- neering Research Council of Canada (NSERC), the Fonds de recherche du Quebec Nature et technologies (FQRNT), McGill University, the Smithsonian Tropical Research Insti- tute, and the Entomological Society of Quebec. The authors thank Dr. Laurence Mound (CSIRO) for identifying the thrips from Panama. References [1] P. Jolivet, J. Vasconcellos-Neto, and P. Weinstein, “Cycloalexy: a new concept in the larval defense of insects,” Insecta Mundi, vol. 4, pp. 133-142, 1990. [2] C. von Clausewitz and H. W. Gatzke, Principles of War, vol. 82, Military Service Publishing Company, Harrisburg, Pa, USA, 1942. [3] J. Tener, A Preliminary Study of the Musk-Oxen of Fosheim Peninsula, Ellesmere Island, NWT, vol. 9 of Wildlife Manage- ment Bulletin, Canadian Wildlife Service, 1954. [4] E. O. Wilson, Sociobiology: The New Synthesis, Belknap Press of Harvard University Press, Cambridge, Mass, USA, 1975. [5] J. VasconceUos-Neto and P. Jolivet, “Une nouvelle strategie de defense: la strategie de defense annulaire (cycloalexie) chez quelques larves de Chrysomelides bresiliens (Col.)y,” Bulletin de La Societe Entomologique de Erance, vol. 92, no. 9-10, pp. 291- 299, 1988. [6] J. Vasconcellos-Neto and P. Jolivet, “Ring defense strategy (cycloalexy) among Brazilian chrysomelid larvae (Coleoptera),” Entomography, vol. 67, pp. 347-354, 1989. [7] J. Vasconcellos-Neto and P. Jolivet, “Cycloalexy among chrys- omelid larvae,” in Novel Aspects of the Biology of Chrysomelidae, P. Jolivet, M. L. Cox, and E. Petitpierre, Eds., pp. 303-309, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1994. [8] J. Vasconcellos-Neto and P. Jolivet, “Ring defense strategy among Brazilian Chrysomelid larvae (Col.),” in Proceedings of the 18th International Congress of Entomology, Vancouver, Canada, 1988. [9] L. Medeiros, D. Eerro, and A. Mafra-Neto, “Association of Chrysomelid beetles with solanaceous plants in the south of Brazil,” in Chrysomelidae Biology, P. Jolivet and M. L. Cox, Eds., pp. 339-363, SPC Academic Publishing, Amsterdam, The Netherlands, 1996. [10] L. Medeiros, Aspectos da Interagdo Entre Especies de Chrys- omeliNae (Coleoptera: Chrysomelidae) e Plantas da Eamilia Solanaceae Na Serra Do Japi, Jundiai, SP, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil, 1991. [11] J. A. Santiago -Blay, P. Jolivet, and K. K. Verma, “A natural his- tory of conspecific aggregations in terrestrial arthropods, with emphasis on cycloalexy in leaf beetles (Coleoptera: Chrysomeli- dae),” Terrestrial Arthropod Reviews, vol. 5, no. 3-4, pp. 289-355, 2012. [12] P. Jolivet and K. K. Verma, “Reflexions on cycloalexy among Chrysomelidae (Coleoptera),” Nouvelle Revue D’Entomologie, vol. 27, no. 4, pp. 311-329, 2011. [13] C. Bontems and C.-E. Lee, “A new case of viviparity among Chrysomelinae,” in Research on Chrysomelidae, P. Jolivet, J. A. Santiago-Blay, and M. Schmitt, Eds., pp. 260-264, Brill Academic Publishers, Leiden, The Netherlands, 2008. [14] T. L. Simmul and D. W. de Little, “Biology of the paropsini (Chrysomelidae: Chrysomelinae),” in Advances in Chrysomeli- dae Biology 1, M. L. Cox, Ed., pp. 463-477, Backhuys Publishers, Leiden, The Netherlands, 1999. [15] D. M. Windsor, C. J. Dury, E. A. Erieiro-Costa, S. Lanck- owsky, and J. M. Pasteels, “Subsocial Neotropical Doryphorini 12 Psyche (Chrysomelidae, Chrysomelinae): new observations on behav- ior, host plants and systematics,” Research on Chrysomelidae, vol. 4, no. 332, pp. 71-93, 2013. [16] S.-I. Kudo and E. Hasegawa, “Diversified reproductive strategies in Gonioctena (Chrysomelinae) leaf beetles,” in New Contribu- tions to the Biology of Chrysomelidae, P. Jolivet, J. A. Santiago- Blay, and M. Schmitt, Eds., pp. 727-738, SPB Academic Pub- lishing, The Hague, The Netherlands, 2004. [17] M. Daccordi, L. LeSage, and M. L. Cox, “Revision of the genus Labidomera Dejean with a description of two new species (Coleoptera: Chrysomelidae: Chrysomelinae),” in Advances in Chrysomelidae Biology 1, M. L. Cox, Ed., pp. 437-461, Backhuys Publishers, Leiden, The Netherlands, 1999. [18] M. Daccordi, “Nuove specie di Platyphora della regione neotropicale (Coleoptera: Chrysomelidae, Chrysomelinae),” Memorie della Societd Entomologica Italiana, vol. 72, pp. 221- 232, 1993. [19] C. A. M. Reid, M. Beatson, and J. Hasenpusch, “The morphol- ogy and biology of Pterodunga mirabile Daccordi, an unusual subsocial Chrysomeline (Coleoptera: Chrysomelidae),” Journal of Natural History, vol. 43, no. 7-8, pp. 373-398, 2009. [20] B. J. Selman, “The biology of the paropsine eucalyptus beetles of Australia,” in Novel Aspects of the Biology of Chrysomelidae, P. Jolivet, M. L. Cox, and E. Petitpierre, Eds., pp. 555-565, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1994. [21] P. Came, “Ecological characteristics of the eucalypt-defoliating chrysomelid Paropsis atomaria Ol,” Australian Journal of Zool- ogy, vol. 14, no. 4, pp. 647-672, 1966. [22] M. }. Wade, “The biology of the imported willow leaf beetle, Pla- giodera versicolora (Laicharting),” in Novel Aspects of the Biology of Chrysomelidae, P. Jolivet, M. L. Cox, and E. Petitpierre, Eds., pp. 541-547, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1994. [23] M. J. Wade and E Breden, “Life history of natural popula- tions of the imported willow leaf beetle, Plagiodera versicolora (Coleoptera: Chrysomelidae),” Annals of the Entomological Society of America, vol. 79, no. 1, pp. 73-79, 1986. [24] J. T. Costa, The Other Insect Societies, Belknap Press of Harvard University Press, Cambridge, Mass, USA, 2006. [25] E Breden and M. J. Wade, “An experimental study of the effect of group size on larval growth and survivorship in the imported willow leaf beetle, Plagiodera versicolora (Coleoptera: Chrysomelidae),” Environmental Entomology, vol. 16, pp. 1082- 1086, 1987. [26] M. S. Mohamedsaid, “A simple type of cycloalexy in larvae of Phyllocharis undulata (Linnaeus) (Chrysomelidae: Chrysome- linae),” Chrysomela, vol. 50-51, pp. 9-10, 2008. [27] J. C. Choe, “Maternal care in Labidomera suturella Chevro- lat (Coleoptera: Chrysomelidae: Chrysomelinae) from Costa Rica,” Psyche, vol. 96, pp. 63-68, 1989. [28] L. Medeiros and J. VasconceUos-Neto, “Host plants and seasonal abundance patterns of some Brazilian Chrysomelidae,” in Novel Aspects of the Biology of Chrysomelidae, pp. 184-189, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1994. [29] D. M. Windsor, “Natural history of a subsocial tortoise bee- tle, Acromis sparsa Boheman (Chrysomelidae, Cassidinae) in Panama,” Psyche, vol. 94, no. 1-2, pp. 127-150, 1987. [30] H. D. C. Heron, “Cycloalexy in two South African tortoise beetles (Chrysomelidae: Cassidinae),” Chrysomela, vol. 27, pp. 3-4, 1992. [31] K. K. Verma, “Cycloalexy in the tortoise beetle, Aspidomorpha miliaris E (Col. Chrys. Cass.),” Chrysomela, vol. 26, p. 6, 1992. [32] J. Swi^tojanska, The Immatures of Tortoise Beetles With Biblio- graphic Catalogue of All Taxa (Coleoptera: Chrysomelidae: Cas- sidinae)”, vol. 16 of Polish Taxonomical Monographs, Biologica Silesiae, Wroclaw, Poland, 2009. [33] H. D. C. Heron, “The biology of Conchyloctenia punctata (Eabri- cius). A cycloalexic cassid (Chrys. Cassidinae),” in Advances in Chrysomelidae Biology 1, M. L. Cox, Ed., pp. 565-580, Backhuys Publishers, Leiden, The Netherlands, 1999. [34] C. S. Chaboo, “Eirst report of immatures, genitalia and mater- nal care in Eugenysa columbiana (Boheman) (Coleoptera): Chrysomelidae: Cassidinae: Eugenysini),” Coleopterists Bulletin, vol. 56, no. 1, pp. 50-67, 2002. [35] D. M. Windsor and J. C. Choe, “Origins of parental care in Chrysomelid beetles,” in Novel Aspects of the Biology of Chrysomelidae, P. Jolivet, M. L. Cox, and E. Petitpierre, Eds., pp. 111-117, Kluwer Academic Publishers, Dordrecht, The Nether- lands, 1994. [36] J. Romero-Napoles, “Morfologia y biologia de Ogdoecosta bian- nularis (Coleoptera: Chrysomelidae) en su huesped silvestre Ipomoea murucoides (Convolvulaceae) en al Estado de Morelos, Mexico,” Polia Entomologica Mexicana, vol. 78, pp. 85-93, 1990. [37] P. A. A. Gomes, E Prezoto, and E. A. Erieiro-Costa, “Biol- ogy of Omaspides pallidipennis Boheman, 1854 (Coleoptera: Chrysomelidae: Cassidinae),” Psyche, vol. 2012, pp. 1-8, 2012. [38] V. Rodriguez, “Sexual behavior in Omaspides convexicollis Spaeth and O. bistriata Boheman (Coleoptera: Chrysomelidae: Cassidinae), with notes on maternal care of eggs and young,” The Coleopterists’ Bulletin, vol. 48, no. 2, pp. 140-144, 1994. [39] E. A. Erieiro-Costa, “Biologia de popula^oes e etologia de Omaspides tricolorata (Boheman, 1854) (Coleoptera: Chrysom- elidae: Cassidinae) na Serra do Japi-Jundiai-SP,” in Institute de Biologia, p. 175, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil, 1995. [40] P Jolivet and J. -M. Maes, “Un cas de cycloalexie chez un Curculiondae: Phelypera distigma (Boheman) (Hyperinae) au Nicaragua,” L’Entomologiste, vol. 52, pp. 97-100, 1996. [41] T. D. Eitzgerald, A. Pescador-Rubio, M. T. Turna, and J. T. Costa, “Trail marking and processionary behavior of the larvae of the weevil Phelypera distigma (Coleoptera: Curculionidae),” Journal of Insect Behavior, vol. 17, no. 5, pp. 627-646, 2004. [42] A. M. Young, “Ecological notes on cacao associated midges (Dipt. Ceratopogonidae) in the “Catongo” cacao plantation at Turrialba,” Proceedings of the Entomological Society of Washing- ton, vol. 86, no. 1, pp. 185-194, 1984. [43] L. G. Saunders, “On the life history and the anatomy of the early stages of Porcipomyia (Diptera, Nemat., Ceratopogoninae),” Parasitology, vol. 16, no. 02, pp. 164-213, 1924. [44] H. E. Hinton, “Protective devices of Endopterygote pupae,” Transactions of the Society Por British Entomology, vol. 12, pp. 49-92, 1955. [45] W. G. Eberhard, The Ecology and Behavior of a subsocial pentato- mid Bug and Two Scelionid wasps: strategy and counterstrategy in a Host and Its parasites, vol. 205 of Smithsonian Contributions to Zoology, Smithsonian Institution Press, 1975. [46] L. Eilippi, M. Hironaka, and S. Nomakuchi, “A review of the ecological parameters and implications of subsociality in Parastrachia japonensis (Hemiptera: Cydnidae), a semelparous species that specializes on a poor resource,” Population Ecology, vol. 43, no. 1, pp. 41-50, 2001. [47] P. Weinstein, “Cycloalexy in an Australian pergid sawfly (Hymenoptera, Pergidae),” BullEtin et Annales de La Societe Entomologique de Belgique, vol. 125, pp. 53-60, 1989. Psyche 13 [48] P. B. Came, “The characteristics and behaviour of the saw- fly Perga affinis affinis (Hymenoptera),” Australian Journal of Zoology, vol. 10, no. 1, pp. 1-34, 1962. [49] P. A. Morrow, T. E. Bellas, and T. Eisner, “Eucalyptus oils in the defensive oral discharge of Australian sawfly larvae (Hymenoptera: Pergidae),” Oecologia, vol. 24, no. 3, pp. 193-206, 1976. [50] B. E de Souza Dias, “Comportamento pre-social de Sinfltas do Brasil Central. 1. Themos olfersii (Klug) (Hymenoptera, Argidae),” Studia Entomologica, vol. 18, no. 1-4, pp. 401-422, 1975. [51] D. Wittmann, “Aerial defense of the nest by workers of the stingless bee Trigona (Tetragonisca) angustula (Latreille) (Hymenoptera: Apidae),” Behavioral Ecology and Sociobiology, vol. 16, no. 2, pp. 111-114, 1985. [52] J. van der Vecht, “The social wasps (Vespidae) collected in Erench Guiana by the Mission du Museum national d’Histoire naturelle, with notes on the genus Apoica Lepeletier’,’ Annales de La Societe Entomologique de Erance, vol. 8, pp. 735-743, 1972. [53] K. M. Pickett, J. M. Carpenter, and A. Dejean, ““Basal” but not primitive: the nest of Apoica arborea de Saussure, 1854 (Insecta, Hymenoptera, Vespidae, Polistinae),” Zoosystema, vol. 31, no. 4, pp. 945-948, 2009. [54] A. M. S. Neto and S. R. Andena, “New records of Apoica pallida (Olivier, 1792)(Hymenoptera: Vespidae, Epiponini) in Bahia State,” EntomoBrasilis, vol. 4, no. 3, pp. 152-153, 2011. [55] R. Straatman, “Notes on the biologies of Papilio laglaizei and P toboroi (Papilionidae),” Journal of the Lepidopterists’ Society, vol. 29, pp. 180-187, 1975. [56] L. M. Lorini, P. H. G. Zarbin, and C. D. Tedesco, “Biology of laboratory-reared Lonomia Obliqua (Lepidoptera: Saturni- idae),” Elorida Entomologist, vol. 90, no. 4, pp. 770-771, 2007. [57] M. da Saiide, “V-Acidentes por Lepidopteros,” in Man- ual de Diagnostico d Tratamdnto de Acidentes Por Animais Pegonhentos, pp. 75-84, Punda^ao Nacional de Saiide, Brasilia, Brazil, 1998. [58] C. S. Henry, “Eggs and rapagula of Ululodes and Ascaloptynx (Neuroptera: Ascalaphidae): a comparative study,” Psyche, vol. 79, no. 12, pp. 1-22, 1972. [59] A. R. Kiester and E. Strates, “Social behaviour in a thrips from Panama,” Journal of Natural History, vol. 18, no. 2, pp. 303-314, 1984. [60] P. Laval, “The barrel of the pelagic amphipod Phronima seden- taria (Porsk.) (Crustacea: hyperiidea),” Journal of Experimental Marine Biology and Ecology, vol. 33, no. 3, pp. 187-211, 1978. [61] P. Laval, “Hyperiid amphipods as crustacean parasitoids asso- ciated with gelatinous zooplankton,” Oceanography and Marine Biology: Annual Review, vol. 18, pp. 11-56, 1980. [62] G. Hoffmann, “Diplopoda,” in Synopsis and Classification of Living Organisms, S. P. Parker, Ed., vol. 2, pp. 689-724, McGraw- Hill, New York, NY, USA, 1982. [63] C. Gilbert, G. Robertson, Y. Le Maho, Y. Naito, and A. Ancel, “Huddling behavior in emperor penguins: dynamics of hud- dling,” Physiology and Behavior, vol. 88, no. 4-5, pp. 479-488, 2006. [64] P. Jolivet, “Cycloalexy,” in Encyclopedia of Entomology, J. L. Capinera, Ed., pp. 1139-1140, Springer, New York, NY, USA, 2008. [65] J. T. Costa, T. D. Pitzgerald, A. Pescador-Rubio, J. Mays, and D. H. Janzen, “Social behavior of larvae of the neotropical pro- cessionary weevil Phelypera distigma (Boheman) (Coleoptera: Curculionidae: Hyperinae),” Ethology, vol. 110, no. 7, pp. 515- 530, 2004. [66] H. Kruuk, The Spotted Hyena: A Study of predation and social behavior. University of Chicago Press, Chicago, IE, USA, 1972. [67] J. P. Eisenberg and M. Lockhart, “An ecological reconnaissance of Wilpattu National Park, Ceylon,” Smithsonian Contributions to Zoology, vol. 101, pp. 1-118, 1972. [68] P. P. Darling, A Herd of Red Deer, Oxford University Press, London, UK, 1937. [69] D. R. Martinez and E. Klinghammer, “The behavior of the whale Orcinus orca: a review of the literature,” Zeitschrift Eur Tierpsychologie, vol. 27, no. 7, pp. 828-839, 1970. [70] M. Altmann, “Patterns of herd behavior in freeranging elk of Wyoming, Cervus canadensis nelsoni,” Zoologica, vol. 41, pp. 65-71, 1956. [71] W. D. Hamilton, “Geometry for the selfish herd,” Journal of Theoretical Biology, vol. 31, no. 2, pp. 295-311, 1971. [72] L. Mound and J. Palmer, “Spore-feeding Thysanoptera of the genus Anactinothrips with a new sub-social species from Panama,” Journal of Natural History, vol. 17, no. 5, pp. 789-797, 1983. [73] A. B. G. Veiga, B. Blochtein, and J. A. Guimaraes, “Structures involved in production, secretion and injection of the venom produced by the caterpillar Lonomia obliqua (Lepidoptera, Saturniidae),” Toxicon, vol. 39, no. 9, pp. 1343-1351, 2001. [74] N. E. Gomez, L. Witte, and T. Hartmann, “Chemical defense in larval tortoise beetles: essential oil composition of fecal shields of Eurypedus nigrosignata and foliage of its host plant, Cordia curassavicaj Journal of Chemical Ecology, vol. 25, no. 5, pp. 1007-1027, 1999. [75] K. L. Olmstead and R. P. Denno, “Effectiveness of tortoise beetle larval shields against different predator species,” Ecology, vol. 74, no. 5, pp. 1394-1405, 1993. [76] P. V. Vend, T. C. Morton, R. O. Mumma, and J. C. Schultz, “Shield defense of a larval tortoise beetle,” Journal of Chemical Ecology, vol. 25, no. 3, pp. 549-566, 1999. [77] H. V. Cornell and B. A. Hawkins, “Survival patterns and mortality sources of herbivorous insects: some demographic trends,” The American Naturalist, vol. 145, no. 4, pp. 563-593, 1995. [78] G. D. Ruxton and T. N. Sherratt, “Aggregation, defence and warning signals: the evolutionary relationship,” Proceedings of the Royal Society B, vol. 273, no. 1600, pp. 2417-2424, 2006. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 705397, 5 pages http://dx.doi.org/10.1155/2014/705397 Research Article Prospects for the Use of Pongamia pinnata Oil-Based Products against the Green Peach Aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) Elena A. Stepanycheva, Maria O. Petrova, Taisiya D. Chermenskaya, and Roman Pavela ^ All-Russian Institute of Plant Protection, Podbelsky Shosse 3, St. Petersburg, Pushkin 196608, Russia ^ Crop Research Institute, Drnovska 507, Ruzyne, 161 06 Prague 6, Czech Republic Correspondence should be addressed to Taisiya D. Chermenskaya; tchermenskaya@yandex.ru Received 31 January 2014; Accepted 14 March 2014; Published 7 April 2014 Academic Editor: Nawal Kishore Dubey Copyright © 2014 Elena A. Stepanycheva 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 is devoted to an estimation of the action of preparations based on Pongamia pinnata oil on the life cycle (survival, fecundity) of green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae). The M. persicae is a widespread pest and damages more than 100 species of plants. All test formulations had aphicidal activity for M. persicae adults and larvae. Moreover, they possess prolonged action, exerting a negative influence on the offspring. The preparations differed in speed of onset of mortality. The single treatment with these formulations provides significant reduction in the number of aphids during the observation period, because of the efficiency rising in time. 1. Introduction A wide range of chemicals are used to protect plants from pests in modern agriculture. Most of them are not destroyed by enzyme systems of plants, or by external natural impacts, that is cause their accumulation in the crop, and as a result — in humans and animals. Regular treatments cause the occurrence of resistance in harmful objects and, at the same time, are dangerous for beneficial arthropods. One possible solution to these problems is the search for new active substances — secondary plant metabolites. The ability of plants to produce antibiotic substances is well known [1, 2] . Currently, the list of known plant secondary metabolites continues to expand. Enough information to support the use of plant preparations for crop protection is already accumulated [3,4]. Botanical insecticides are natural chemical substances isolated from plants. Such preparations may be considered as an alternative to synthetic chemical compounds, but they are not always less toxic to mammalian. But botanical insecticides are easily decomposed in the soil and will not be stored in the tissues of plants and animals. Among the substances of plant origin, essential oils, which have well-known activity against various pests, occupy a special place. So, essential oils from plants of the Mentha genus. Origanum vulgare L., Ocimum basilicum L. (Lami- aceae), and Carum carvi L. (Umbelliferae) are highly toxic to Trialeurodes vaporariorum, Tetranychus cinnabarinus, Acan- thoscelides obtectus, and Meligethes aeneus [5-8]. One of the most interesting objects of study in recent years is Pongamia oil, the use of which is widespread in various areas of activity. According to the literature and earlier results, this plant has insecticidal activity to some pest and synanthropic insects [9, 10] . Myzus persicae (Sulzer) (Hemiptera: Aphididae) is a widespread polyphagous aphid, which damages more than 100 species of plants. In greenhouses, there are forms with incomplete development cycle that can reproduce contin- uously throughout the year [11, 12]. Pesticide with broad spectrum is not effective enough due to the high resistance of M. persicae to organophosphate and pyrethroid insecticides [13]. With year-round growing season, the problem of the replacement of traditional pesticides with new means of pest 2 Psyche control is actual in greenhouse crop production, where there is a constant accumulation of harmful arthropods. The aim of our work is to evaluate the action of prepa- rations based on Pongamia pinnata (L.) Pierre oil on the life cycle (survival, fecundity) of green peach aphid. 2. Materials and Methods 2.1. Insects. The green peach aphid Myzus persicae (Sulzer) was reared on sprouting broad beans {Viciafaba L.), grown at 22 ± 2°C, 16 : 8 LD, and 50 ± 10 RH. 2.2. Plant Material and Chemicals. Pongam is seed oil from Pongamia pinnata (Parker Group, India) emulsified using Tween 85. The oil was tested by HPLC to determine the Karanjan content that is over 22 000 ppm. Cinnamomum verum oil was obtained from the bark of Cinnamomum verum J. Presl, which were collected from plants growing in distinct areas of commercial plantations in southern India. The essential oil was extracted by hydrodis- tillation using a modified Clevenger apparatus. The bark was pulverized and 20 g was distilled in 300 mL DH20 in a 500 mL flask for 60 min. Oil samples were stored at 4“C until bioassays. Extracts from Sapindus saponaria L. and Thymus vul- garis L. were prepared from the seeds of S. saponaria or flowering plants of T. vulgaris, respectively, by pulverization and extracted using 100% pure methanol during 48 h at the laboratory temperature (ratio plants : methanol; 1:10). Thymol (pure 99.9%) was obtained from Sigma-Aldrich, Czech Republic. 2.3. Formulations RE: Pongam (emulsified using Tween 85 — ratio Tween : Oil = 1:9). REP: Pongam -i- thymol (emulsified using Tween 85 — ratio Tween : Oil : thymol = 1:8:1). REP3: Pongam -i- Thymus vulgaris extract (emulsified using Tween 85 — ratio Tween : Oil : extract = 1:8:1). REP4: Pongam + Cinnamomum verum oil (emulsified using Tween 85 — ratio Tween : Oil : EO = 1:8:1). REP5: Pongam -i- Sapindus saponaria extract (emul- sified using Tween 85 — ratio Tween : Oil : extract = 1 : 8 : 1 ). NA: NeemAzal TS — The commercial insecticide NeemAzal-U (a.i. azadirachtin A 10 g/kg) (Trifolio-M GmbH, Lah-nau, Germany) was used for treatment. 2.4. Bioassays. To estimate insecticidal activity of the formu- lations in laboratory bioassays, filter paper discs impregnated with test solutions (0.25 mL/disc) were placed in the bottom and the lid of small Petri dishes (36 mm in diameter) (Corning Inc., USA); then a bean leaf treated with the same test solutions and 10 adult aphids were added. The control was treated with water. Twenty- four hours later, live and dead aphids and their offspring were counted. There were 5 replicates for each treatment. Biological efficacy, inhibition of oviposition, and mortality of subsequent nymphs were corrected by Abbott’s formula [14] . The effect of the treatments on aphid larvae mortality was determined as follows. The pepper plants {Capsicum annuum var. annuum L.) were grown particularly in the flowerpots at 23 ± 2°C, 16 : 8 LD. The 15 aphid’s first instar larvae were placed on each plant with two true leaves freshly treated with formulations or water (control). The number of live and dead aphids was counted 1, 2, 3, and 7 days after. This procedure was replicated 10 times for each formulation and the control. Efficacy was corrected by Abbott’s formula [14]. Data were examined using analysis of variance (ANOVA), and means were separated using the Tukey honestly signifi- cant difference (HSD) multiple comparison test (P < 0.05). The LG50 95% confidence limit of upper and lower confidence levels were calculated by using probit analysis [15] . 3. Result and Discussion At the maximum concentration (3%), almost all formu- lations, except REP5, resulted in 90% or above mortality of treated females (Table 1). With such a high mortality rate in tests NA, REP, REP3, and RE only a few larvae hatch and immediately died. On the other hand, after REP4 treatment, the fertility did not change significantly compared to the control, but the emergence of the next generation of individuals was not viable. The gradual decrease in the concentration of working solutions in 2 times allowed us to determine the samples that retain their activity. In particular, even at a concentration of O. 75%, RE caused the death of more than 60% of females and inhibited the development of subsequent nymphs on 80%. After dilution to 0.375%, the efficacy (mortality of females) was 50%. The NA and REP4 somewhat inferior to RE in imago cidal activity. When studying the influence of preparations on mortality of larvae of green peach aphid on vegetative plants, all sam- ples showed high aphicidal activity at 3 and 1.5% (Table 2). There is a clearly expressed dynamic of aphid’s death, which indicates the accumulation of toxins in the body of insects. The preparations differed in speed of onset of mortality. Thus, the larval mortality on day 3 was over 60% in the tests with REP and REP3, while in tests with other formulations — below 50%. After dilution of the working solution to 0.75%, the activity of all samples sharply reduced, except REP (40%). The test samples are inferior to standard NeemAzal TS (NA) in the speed of appearance of the effect and retention of activity at reducing the concentration of the working solution, but nevertheless have certain larvicidal action to this insect. The main component in our experimental formulations — P. pinnata oil — was not chosen by chance. So, Pongam oil treatments reduced the number of whiteflies on the chrysan- themum plants [16]. Also, the Pongamia oil caused high mortality of Spodopfera littoralis, M. persicae, and Tetranychus urticae on greenhouse plants [10]. Psyche 3 Table 1: Activity of plant preparations to green peach aphid female and to its offspring. Treatment Concentration Biological efficacy hCso Inhibition oviposition Corrected mortality of hCso % %* (CI95) % subsequent nymphs %* (CI95) 3.0 90.3" 93.5 1 — ‘ 0 0 0 REP 1.5 84.3"’' 0.86 81.5 92 . 9 ’' 0.59 0.75 27.2"^’ (0.72-0.96) 60.9 54.4" (0.42-0.68) 0.375 11.6" 56.5 20.8 3.0 90.6" 89.8 0 0 0 REP3 1.5 97.9" 0.93 100.0 — N.D. 0.75 28.6‘^ (0.85-1.11) 64.1 0.0" 0.375 16.7^^" 47.8 0.0" 3.0 97.8" 28.6 0 0 0 REP4 1.5 71.5’' 0.95 11.7 0 0 0 1.25 0.75 42.8’'" (0.78-1.05) 18.2 26.6‘^ (1.18-1.35) 0.375 35.3" 158.6 24.ff 3.0 73.5’' 79.7 95 . 0 ’' REPS 1.5 24.6‘^ 2.18 60.1 66.0" 0.89 0.75 27.7^’ (1.98-2.25) 75.7 42.7^^ (0.78-0.99) 0.375 3.ft 48.6 0.0" 3.0 95.9" 97.4 0 0 0 RE 1.5 97.8" 0.36 94.8 0 0 0 0.58 0.75 60.4’' (0.28-0.45) 67.5 78.9’'" (0.39-0.65) 0.375 50.0’'" 58.4 5.5" 3.0 88.6’' 97.3 0 0 0 NA 1.5 83.3’' 1.25 78.4 91 . 7 ’' 0.72 0.75 36.3" (0.96-1.38) 53.8 63.4" (0.67-0.95) 0.375 29.3"^^ 36.5 45.3"^^ *The values within columns with the same lowercase letter do not differ significantly (Turkeys HSD test, P < 0.05). There is not enough information about insecticidal prop- erties of P. pinnata oil. Considering that, it is similar to well- known neem oil by a number of properties. Additional components of our experimental formulations have insecticidal activity per se. The thymol is one of the main substances of T. vulgaris. The extract and oil of this plant were toxic for stored pests Tribolium castaneum (Herbst), Callosobruchus macu- latus, and Sitophilus granarius [17, 18], Sitotroga granarius, Acanthoscelides obtectus [19], and phytophage Trialeurodes vaporariorum [20]. A methylene chloride extract of the C. verum was shown to be insecticidal to T. castaneum and Sitophilus zeamais Motsch [21]. Cinnamon oil provided >90% mortality of citrus mealybug Planococcus citri (Risso), but did not provide sufficient control of sweetpotato whitefly Bemisia tabaci (Gennadius) or green peach aphid M. persicae 7, 14, and 21 d after application [22]. A vast number of species showing great potential as anti- insect agents belong to Sapindus genus. These plants are mostly known for being rich in saponins, which provide plant extracts with biological activities in medicine as well as in pest control [23]. Comparative evaluation of the activity of the tested for- mulations showed that the addition of various components to P pinnata oil did not result in synergistic effects. Nevertheless, they all had aphicidal activity for M. persicae adults and larvae. Moreover, they possess prolonged action, exerting a negative influence on the offspring. When preparations are recommended for pest manage- ment programs, besides the biological efficacy, the absence of side effects related to beneficial species (pollinators and insect predators) and protected plants plays a significant role. In practice, the field treatment with 1% Pongamia oil did not have a negative influence on insect pollina- tors: Hymenopterans — Apis florea. Apis dorsata, and so forth, Dipterans — Muscidae, Syrphidae, and so forth, other orders — Lepidoptera, Hemiptera, and Coleoptera [24]. A high concentration of formulations we applied, (maximum 3%, at practical application, the most commonly used con- centrations for formulations of plant insecticides are 0.5- 1%) did not cause burns of plants (beans and peppers). In addition, the single treatment with these formulations provides significant reduction in the number of aphids during the observation period, because of the efficiency rising in time. 4 Psyche C4> c o -M rt rt Dh (U C cu • ^ S' +1 rt a t5 cu ;h -vP ph O o rt o 3 (U 3 ^ o • ^ bJD 3 • ^ m (N CS * -M vO Ph O^ o o rt o 3 cu 3 ^ u • ^ bO 3 • ^ m * S— I o^ O d o * »-H -M rt Ph "a . vq oo 3 3 3 00 ^ XI 00 00 Tfi VO LO CO 'Co ^ xi ^ CO oo ^33 +1 +1 +1 3 (N f-; 3 3 ^ Lo m m i>^ ^ o (N m 3 3 ^ tC m ^ ^ Ph W Pi !>, i-H O) 3 3 <7v 0^ (N rt rt u m ^ (N (N 3 +1 +1 +1 ^ VO On 3 3 3 Ov Ov ^ *3 o 3 VO " m 00 3 3 +1 +1 +1 ^ (N Ov 3 3 o 00 ^ u u 00 OV 3 3 +1 +1 +i CO rq 3 3 3 T:t< rn Ov o LD (N 3 -O -Q 35 cd CO »— •o 3 3*^ +1 +1 +1 3 3 3 3 3"^ LO LO 0 OV o oo 3 3 00 i>^ 3 m i>. 3 3 o r<) ' — ^ ' — I o vj -d 3^ 3 3 3 CO ^ q 3 N CO ^ ^ W Pi o 3 3 O LO VO '— I <7v Cyv O o iq (N ^ (N +1 +1 VO 00 3 3 0> Ov ■o 00 In o\ ' — j 3 3 3^ IN IN IN 3 3 3 3 +1 +1 q 3 3 00 3 +1 00 3 ■o 00 N 00 N c7v q Tfi f— I LO CO CO y-yq CO ^ 00 3^3 +1 +1 +1 3 rq 3 3 3 LO ^ m 3 <7v m LO 00 q 4-» cr> G a 3 u p 3 aj 3 > Psyche 5 Thus, the high activity of the formulations with Pongamia pinnata oil against the green peach aphid, absence of negative effects on pollinators, and phytotoxicity may be used as a basis for the study of their effects on complex arthropods, damag- ing crops in greenhouses, for inclusion in the integrated pest management program. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgment This study was supported by Grants from the Czech Republic Ministry of Education, Youth and Sports (LH11133). References [1] A. Prakash and J. Rao, Botanical Pesticides in Agriculture, CRC Press, Boca Raton, Fla, USA, 1997. [2] S. K. Okwute, “Plants as potential sources of pesticidal agents: a review,” in Pesticides — Advances in Chemical and Botanical Pesticides, chapter 9, pp. 207-232, InTech, Rijeka, Croatia, 2012. [3] M. B. Isman, “Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world,” Annual Review of Entomology, vol. 51, pp. 45-66, 2006. [4] K. Tiilikkala, I. Lindqvist, M. Hagner, H. Setala, and D. Perdikis, “Use of botanical pesticides in modern plant protection,” in Pes- ticides in the Modern World — Pesticides Use and Management, pp. 259-272, InTech, Rijeka, Croatia, 2011. [5] C. Regnault-Roger, “The potential of botanical essential oils for insect pest control,” Integrated Pest Management Reviews, vol. 2, no. 1, pp. 25-34, 1997 [6] W.-I. Choi, E.-H. Lee, B.-R. Choi, H.-M. Park, and Y.-J. Ahn, “Toxicity of plant essential oils to Trialeurodes vaporariorum (Homoptera; Aleyrodidae),” Journal of Economic Entomology, vol. 96, no. 5, pp. 1479-1484, 2003. [7] E. Sertkaya, K. Kaya, and S. Soylu, “Acaricidal activities of the essential oils from several medicinal plants against the carmine spider mite (Tetranychus cinnabarinus Boisd.) (Aca- rina: Tetranychidae),” Industrial Crops and Products, vol. 31, no. 1, pp. 107-112, 2010. [8] R. Pavela, “Insecticidal and repellent activity of selected essen- tial oils against of the pollen beetle, Meligethes aeneus (E abri- cius) adults,” Industrial Crops and Products, vol. 34, no. 1, pp. 888-892, 2011. [9] M. Kumar and R. Singh, “Potential of Pongamia glabra vent as an insecticide of plant origin,” Biological Agriculture & Horticulture, vol. 20, no. 1, pp. 29-50, 2002. [10] R. Pavela, “Effectiveness of some botanical insecticides against Spodoptera littoralis Boisduvala (Lepidoptera: Noctudiae), Myzus persicae Sulzer (Hemiptera: Aphididae) and Tetranychus urticae Koch (Acari: Tetranychidae),” Plant Protection Science, vol. 45, no. 4, pp. 161-167, 2009. [11] A. J. Troncoso, R. R. Vargas, D. H. Tapia, R. Olivares-Donoso, and H. M. Niemeyer, “Host selection by the generalist aphid Myzus persicae (Hemiptera: Aphididae) and its subspecies specialized on tobacco, after being reared on the same host,” Bulletin of Entomological Research, vol. 95, no. 1, pp. 23-28, 2005. [12] L. M. Eericean, I. Palagesiu, R. Palicica, A. M. Varteiu, and S. Prunar, “The behaviour, life cycle and biometrical mea- surements of Myzus persicaej Research Journal of Agricultural Science, vol. 43, no. 1, pp. 34-39, 2011. [13] A. X. Silva, L. D. Bacigalupe, M. Luna-Rudloff, and C. C. Eigueroa, “Insecticide resistance mechanisms in the green peach aphid Myzus persicae (Hemiptera: Aphididae) II: costs and benefits,” PLoS ONE, vol. 7, no. 6, Article ID e36810, 2012. [14] W. S. Abbott, “A method of computing the effectiveness of an insecticide,” Journal of Economic Entomology, vol. 18, pp. 265- 267, 1925. [15] D. J. Einney, Probit Analysis, Cambridge University Press, Cambridge, UK, 3rd edition, 1977. [16] R. Pavela and G. Herda, “Effect of pongam oil on adults of the greenhouse whitefly Trialeurodes vaporariorum (Homoptera: Trialeurodidae),” Entomologia Generalis, vol. 30, no. 3, pp. 193- 201, 2007. [17] S. Clemente, G. Mareggiani, A. Broussalis, V. Martino, and G. E erraro, “Insecticidal effects of Lamiaceae species against stored products insects,” Boletm de Sanidad Vegetal. Plagas, vol. 29, no. 3, pp. 421-426, 2003. [18] E. Dezfouli, S. Moharramipour, and S. H. Goldasteh, “Ovicidal, larvicidal and oviposition deterrency effects of essential oil from Thymus vulgaris L. (Lamiaceae) on Callosobruchus maculatus (E.) (Col, Bruchidae),” Journal of Entomological Research, vol. 2, no. 2, pp. 73-84, 2010. [19] 1. Kalinovic, J. Martincic, V. Rozman, and V. Guberac, “Insecti- cidal activity of substances of plant origin against stored product insects,” Ochrana Rostlin, vol. 33, no. 2, pp. 135-142, 1997 [20] H. Aroiee, S. Mosapoor, and H. Karimzadeh, “Control of greenhouse whitefly (Trialeurodes vaporariorum) by thyme and peppermint,” KMITL Science Journal, vol. 5, no. 2, pp. 511-514, 2005. [21] Y. Huang and S. H. Ho, “Toxicity and antifeedant activities of cinnamaldehyde against the grain storage insects, Tribolium castaneum (Herbst) and Sitophilus zeamais Motsch,” Journal of Stored Products Research, vol. 34, no. 1, pp. 11-17, 1998. [22] R. A. Cloyd, C. L. Galle, S. R. Keith, N. A. Kalscheur And, and K. E. Kemp, “Effect of commercially available plant-derived essential oil products on arthropod pests,” Journal of Economic Entomology, vol. 102, no. 4, pp. 1567-1579, 2009. [23] M. Diaz and C. Rossini, “Bioactive natural products from Sapindaceae deterrent and toxic metabolites against insects,” in Insecticides — Pest Engineering, pp. 287-308, InTech, 2012. [24] H. Singh, R. Swaminathan, and T. Hussain, “Influence of certain plant products on the insect pollinators of coriander,” Journal of Biopesticides, vol. 3, no. 1, pp. 208-211, 2010. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 871605, 5 pages http://dx.doi.org/10.1155/2014/871605 Research Article A New Species of Dikrella Oman, 1949 (Hemiptera: Cicadellidae: Typhlocybinae) Found on Caryocar brasiliense Cambess. (Caryocaraceae) in Minas Gerais State, BrazU Luci Boa Nova Coelho, ’ Germano Leao Demolin Leite, and Elidiomar Ribeiro Da-Silva^ ^ Laboratorio de Entomologia, Departamento de Zoologia, Instituto de Biologia, Universidade Federal do Estado do Rio de Janeiro, Caixa Postal 68044, 21944-970 Rio de Janeiro, RJ, Brazil ^ Programa de P6s-Gradua^do em Biodiversidade Neotropical, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil ^ Universidade Federal de Minas Gerais, Instituto de Ciencias Agrdrias, Caixa Postal 135, 39404-547 Montes Claros, MG, Brazil '^Laboratorio de Insetos Aqudticos, Departamento de Zoologia, Instituto de Biociencias, Universidade Federal do Estado do Rio de Janeiro, 22290-240 Rio de Janeiro, RJ, Brazil Correspondence should be addressed to Luci Boa Nova Coelho; lucibncoelho@gmaiLcom Received 11 November 2013; Accepted 18 February 2014; Published 9 April 2014 Academic Editor: Jacques Hubert Charles Delabie Copyright © 2014 Luci Boa Nova Coelho 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. A new species of Dikrella is described and figured based on specimens from Minas Gerais, Southeastern Brazil. The new species is diagnosed by the process of pygofer and the general form of aedeagus. Adult males, females, and also nymphs were found on pequi tree, suggesting that Dikrella caryocar n. sp. has its full life cycle in this plant. 1. Introduction The genus Dikrella Oman (Dikraneurini) includes 40 species and occurs in the United States, Mexico, Costa Rica, Cuba, Puerto Rico, Panama, Ecuador, Colombia, Bolivia, and Brazil [1-6]. In Brazil there are records of Dikrella fumida (Osborn) from Santa Catarina, D. albonasa (McAtee) from Mato Grosso do Sul, D. aculeata Coelho & Nessimian, D. reticulata Coelho & Nessimian, and D. spinifera Coelho & Nessimian from Minas Gerais [1, 6-9]. Studying the relationships of insects with pequi tree or souari nut {Caryocar brasiliense Cambess., Caryocaraceae), a new species of Dikrella was found [10] . According to Leite et al. [11] the species is classified as constant, occurring throughout the year, and more abun- dant in summer and autumn. The species is found sucking seedlings with potential to become a pest in commercial crop of C. brasiliense. 2. Material and Methods The study was developed in a savannah ecosystem (“Cer- rado”) in Montes Claros municipality, north of Minas Gerais State, Brazil {43° 55' 7.3" W; 16°44^55.6^^S; altitude 943 m a.s.L). The climate and vegetation characteristics are considered in Leite et al. [10, 11]. To study the morphology of the genital apparatus it was necessary to remove the abdomen and dip it in a warmed sol- ution of 10% KOH (modified from Oman [12]). The genitalia structures were sunk in glycerin jelly to make the illustrations [13]. The terminology was based on Young [14], except for the wings [15] and female genitalia [16, 17]. The type-specimens are deposited in the Cole^ao Entomologica Professor Jose Alfredo Pinheiro Dutra, Departamento de Zoologia, Instituto de Biologia, Universidade Eederal do Rio de Janeiro (DZRJ), Rio de Janeiro, RJ, Brazil. 2 Psyche Figure 1: Dikrella caryocar n. sp, male: habitus (latero dorsal). 3. Results and Discussion Dikrella caryocar n. sp. (Figures 1-3). Total length 2.7-2.9 mm. General color light green (Figure 1). Crown longer medially than next to eyes; distinct suture until just beyond half the length of crown, marked with brown at base; small smoky brown spot on each side of median line, near anterior margin. Face with small brown spot near anterior margin, between midline and inner margin of each eye. Pronotum about twice wider than long, slightly longer than median length of crown; margin of laterobasal angles not exceeding width of head; anterior margin with three irregular dark spots; lateral mar- gin with an elongated irregular light brown spot linked to a yellowish brown spot in central region on each side of median line; a dark brown macula near each lateral angle and a black spot at middle of posterior margin. Mesonotum brown with dark brown spot on each side of median line. Scutellum pale yellow with brown longitudinal band on each side of the median line; apex with black spot. Forewing (Figure 2(a)) translucent green with big yellow spots; oval spots finely outlined with brown in apical and subapical cells; small red spots concentrated near CuP and two larger, also red, in claval area; apex of CuP and end external SmR with small brown spot. Hind wing (Figure 2(c)) with vein CuA 2 originating from point slightly more basal than MP 2 . Abdom- inal apodeme (Figure 2(d)) long, parallel margins and apex rounded, reaching fifth segment. Male genitalia (Figures 2(e) -2(m)): subgenital plate rather broad and triangular in ventral view (Figure 2(e)), and long in lateral view (Figure 2(f)), with apex upturned and rounded, exceeding pygofer apex; outer margin with a med- ian dark brown lobe, from this a fold (towards inner margin of preapical region) also marked with dark brown till half width of plate; four macrosetae in outer margin, one more basal and three most apical to lobe, continued by small and robust setae till apex; microsetae present throughout surface of api- cal curvature. Pygofer (Figure 2(g)) elongated with five robust setae in ventroapical region; process (Figure 2(h)) elon- gated, dorsal in origin, with median curvature forming two branches; basal branch extending to posterior margin till ventral curve, marked by a large lateroventral tooth; apical branch free, directed posteriorly, thinner, with three small teeth on apical region, apex acute. Style (Figures 2(i) and 2(j)) elongated, preapical region curved, apex acute; preapical lobe well developed, broadly rounded, with group of five setae. Connective “Y” shape (Figure 2(k)), main stem shorter than lateral arms. Aedeagus (Figures 2(1) and 2(m)) with atrial complex developed, stem robust and laterally compressed to well-developed dorsal apodeme; tubular, slender, membra- nous dorsal extension (about 1/3 of stem size) coating gonoduct; pair of robust processes proceeding the main stem, with bases fused next base of membranous extension; processes apex thin and curved dorsally. Female genitalia (Figure 3): posterior margin of sev- enth sternite (Figure 3(a)) with median globular promi- nence and each side folds forming an embossed “V” shape. Pygofer (Figure 3(b)) with nine macrosetae in ventral mar- gin, three smaller macrosetae in posterior margin. Valvulae I (Figure 3(c)) with dorsal margin crenulated, curved towards ventral margin, apical region abruptly tapered, apex acute. Valvulae II (Figures 3(d) and 3(e)) with apical region con- spicuously curved, apex narrow and rounded; right and left valvulae II asymmetrical; left valvulae (Figure 3(d)) smaller, with small rounded teeth in dorsal margin; right valvulae (Figure 3(e)) with strong teeth in dorsal margin decreasing in size towards apex. Valvulae III (Figure 3(f)) covered by short setae; three longer setae regularly spaced in ventral margin. 3.1. Studied Specimens. Holotype (male): Montes Claros, Minas Gerais State, Brazil (43“55^7.3^^W; 16°44^55.6^^S; alti- tude 943ma.s.l.), 16/xi/2007, G.L.D. Leite leg. (DZRJ); para- types (4 males, 10 females), same data of holotype (DZRJ). 3.2. Etymology. Karuon, Ancient Greek for “nut,” “kernel”; kard. Ancient Greek for “head,” referring to the generic epithet of pequi tree. 3.3. Comments. The color of the specimens (Figure 1) can be changed with time of conservation to a pale green or yellow. Brown spots on head and thorax may be more reddish or yellowish. No differences were found between male and female in body size. Fore wings show a variation in the studied specimens of Dikrella caryocar n. sp.; the base of apical cell 3 should be sessile or pedunculated (Figures 2(a) and 2(b)). This variation was found in males and females. In male specimens the process of pygofer is robust and has a characteristic shape (Figure 2(g)) differentiated from its congeners. Aedeagus (Figures 2(1) and 2(m)) is quite unique in general shape and not similar to any other species of Dikrella. In lateral view the subgenital plate (Figure 2(f)) of males of D. caryocar n. sp. resembles that of D. angustella Ruppel and DeLong and D. venella Ruppel and DeLong [3] by its elongated shape and rounded curved apices. The presence of a rounded lobe can differentiate the new species from Psyche 3 Figure 2; Dikrella caryocar n. sp., male: (a) fore wing; (b) fore wing showing a variation in base of third apical cell; (c) hind wing; (d) abdominal apodeme; (e) subgenital plate (ventrolateral); (f) subgenital plate (lateral); (g) pygofer (lateral); (h) process of pygofer (dorsoposterior); (i) style (ventral); (j) style (lateral); (k) connective (ventral); (1) aedeagus (ventroposterior); and (m) aedeagus (lateral). D. angustella and D. venella. Also in subgenital plate the lobe in outer margin should superficially resembles structures present in D. venella Ruppel and DeLong, D. bimaculata Ruppel and DeLong, D. mella Ruppel and DeLong, and D. nigrinota Ruppel and DeLong [3], but in those species the prominence is in shape of one or two spines. Adult males and females were found, as well as different nymphal instars, demonstrating that pequi tree is an ideal host for development and maintenance of the species, further suggesting that D. caryocar n. sp. has its full cycle in this plant. Disclosure The species was recorded in ZooBank under the num- ber urn: lsid:zoobank.org:pub:0A2D5238-8998-48E7-A884- 34DB31281385. The new names included in this paper are available under the International Code of Zoological Nomen- clature. This work and the nomenclatural acts it contains have been registered in ZooBank. ZooBank Life Science Identifier (LSID) for this publication is urn:lsid:zoobank. org:pub: XXXXXXX. The LSID registration and any associated infor- mation can be viewed in a web browser by adding the LSID to the portal “http://zoobank.org/.” 4 Psyche Figure 3: Dikrella caryocar n. sp. (female genitalia): (a) seventh sternite (ventral); (b) pygofer (lateral); (c) valvulae I; (d) valvulae II (left); (e) valvulae II (right); (f) valvulae III. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. References [1] W. L. McAtee, “Notes on Neotropical Eupteryginae, with a key to the varieties of Alebra albostriella (Homoptera: Jassidae),” Journal of the New York Entomological Society, vol. 34, pp. 141- 175, 1926. [2] D. M. DeLong and H. H. Ross, “A new species of Dikraneura from Witch-Hazel (Homoptera: Cicadellidae),” The Ohio Jour- nal of Science, vol. 50, no. 2, pp. 86-87, 1950. [3] R. F. Ruppel and D. M. DeLong, “Some new species of mexican Dikrella (Homoptera: Cicadellidae),” The Ohio Journal of Sci- ences, vol. 52, no. 2, pp. 89-95, 1952. [4] R. F. Ruppel and D. M. DeLong, “Ten new species of mexican DikrellaJ Bulletin of the Brooklyn Entomological Society, vol. 48, no. 1, pp. 1-9, 1953. [5] D. A. Young, “Three new neotropical Typhlocybinae leafbop- pers from economic plants,” Bulletin of the Brooklin Entomolog- ical Society, vol. 51, pp. 72-74, 1956. [6] L. B. N. Coelho and }. L. Nessimian, “Three new species of Dik- rella Oman (Hemiptera: Cicadellidae: Typhlocybinae) from Minas Gerais state, Brazil,” Zootaxa, no. 2142, pp. 20-28, 2009. [7] D. A. Young, “Description of the Osborn types of Typhlocyb- inae (Homoptera, Cicadellidae) in the Carnegie Museum col- lection,” Revista Brasileira de Entomologia, vol. 7, pp. 183-226, 1957. [8] Z. R Metcalf, “Pt 17. Cicadellidae,” in General Catalogue of the Homoptera. Easc.VI Cicadelloidea, United States Departament of Agriculture, Washington, DC, USA, 1968. [9] K. M. R. Zanol and M. Menezes, “Lista preliminar dos cica- delideos (Homoptera, Cicadellidae) do Brasil,” Iheringia Serie Zoologia, vol. 61, pp. 9-65, 1982. [10] G. L. D. Leite, R. V. S. Veloso, A. C. Redoan, P. S. N. Lopes, and M. M. L. Machado, “Artropodes associados a mudas de pequiz- eiro,” Arquivos do Instituto Biologico, vol. 73, no. 3, pp. 365-370, 2006. Psyche 5 [11] G. L. D. Leite, R. V. S. Veloso, J. C. Zanuncio et al, “Seasonal abundance of hemipterans on Caryocar brasiliense (Malp- ighiales: Caryocaraceae) trees in the cerrado,” Florida Entomol- ogist, vol. 95, no. 4, pp. 862-872, 2012. [12] P. W. Oman, The Neartic Leafhoppers (Homoptera: Cicadellidae). A Generic Classification and Check List, vol. 3, Memoirs of the Entomological Society of Washington, 1949. [13] R. W. Pennak, “Fresh-water invertebrates of the United States,” Hydrobiologia, vol. 7, no. 1-2, p. 126, 1955. [14] D. A. Young, “A reclassification of Western Hemisphere Typhlo- cybinae (Homoptera: Cicadellidae),” Kansas University Scien- tific Bulletin, vol. 35, pp. 1-217, 1952. [15] I. Dworakowska, “Remarks on Alebra Fieb. and Eastern Hemi- sphere Alebrini (Auchenorrhyncha: Cicadellidae: Typhlocybi- nae),” Entomotaxonomia, vol. 15, no. 2, pp. 91-121, 1993. [16] H. D. Blocker and B. W. Triplehorn, “External morphology of leafboppers,” in The Leafhoppers and Planthoppers, L. R. Nault and J. G. Rodriguez, Eds., John Wiley & Sons, New York, NY, USA, 1985. [17] C. A. Viraktamath and C. H. Dietrich, “A remarkable new genus of Dikraneurini (Hemiptera: Cicadomorpha: Cicadellidae: Typhlocybinae) from southeast Asia,” Zootaxa, no. 2931, pp. 1-7, 2011. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 164271, 9 pages http://dx.doi.org/ 10 . 1 155/2014/16427 1 Research Article Age Stage Two-Sex Life Table Reveals Sublethal Effects of Some Herbal and Chemical Insecticides on Adults of Bemisia tabaci (Hem.: Aleyrodidae) Fatemeh Jafarbeigi,^ Mohammad Amin Samih/ Mehdi Zarabi,^ and Saeideh Esmaeily^ ^ Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University, PO. Box 518, Rafsanjan, Iran ^Department of Life Sciences Engineering, Faculty of New Sciences & Technologies, University of Tehran, PO. Box 14174, Tehran, Iran Correspondence should be addressed to Fatemeh Jafarbeigi; fatemeh.jafarbeigi@yahoo.com Received 6 January 2014; Revised 6 March 2014; Accepted 11 March 2014; Published 27 April 2014 Academic Editor: Taya Chermenskaya Copyright © 2014 Fatemeh Jafarbeigi 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 sweetpotato whitefly, Bemisia tabaci (Genn.) (Hem.: Aleyrodidae), is an important pest of agriculture in subtropical and tropical areas. In this study, we used the age-stage two-sex life table to evaluate the sublethal effects of the herbal extracts taken from Fumaria parviflora Lam. (Fumariaceae), Teucrium polium L. (Lamiaceae), Calotropis procera (Willd.) R. Br. (Asclepiadaceae), and Thymus vulgaris L. (Lamiaceae) as well as the two commercial synthetic insecticides, pymetrozin and neemarin. The whiteflies were exposed to each insecticide using leaf-dip method. Analysis of life table parameters revealed significant differences (P < 0.05) in the net reproductive rate {Rq, NRR), intrinsic rate of increase (r^), and finite rate of increase (A) among different insecticides. The lowest values of the three population parameters, Rq, r, and A, were observed on whiteflies treated with pymetrozin (2.455, 0.036, and 1.036), T polium (2.828, 0.044, and 1.045), and neemarin (2.998, 0.046, and 1.047), respectively. Results of this study highlights the satisfactory insecticidal effects of the extract taken from T polium on B. tabaci, which is comparable to the two commonly used synthetic insecticides. 1. Introduction The sweetpotato whitefly, Bemisia tabaci (Genn.) (Hem.: Aleyrodidae) is one of the most important pests of agriculture in subtropical and tropical regions as well as in greenhouse production systems across the world [1-4]. Both adult and nymphal stages cause economic damages through a com- bination of direct feeding on sap phloem [5], excretion of honeydew, which serves as a substrate for growth of black sooty molds [6], and transmission of a large number of plant pathogenic viruses [7-10]. The use of chemical insecticides has long been considered as the primary strategy for con- trol of B. tabaci [10, 11]. However, the rapid and frequent development of resistance against these compounds as well as the unwanted effects of synthetic pesticides on nontarget organisms and environment makes them a nonsatisfactory tool in integrated management programs of B. tabaci. For several decades, the plant-derived insecticides (botanicals) have been considered as potential alternatives to synthetic pesticides due to their safety to human health as well as their low detrimental effects on nontarget organisms and environment. Plants produce a variety of chemicals that are predom- inantly used to defend themselves against herbivores [12]. Although, a large number of these substances have been already extracted and identified in different plant species, the availability of botanical insecticides has been limited to a few products [13]. Although, in the immediate future biopesticides may continue to be limited mainly to niche and specialty markets, there is great potential for long- term development and application in different areas of pest management science [14, 15]. A large volume of studies have been conducted to evaluate the insecticidal properties of plant-derived substances on some important pests [16-23]. 2 Psyche Traditionally, measurement of the acute toxicity of pesticides to beneficial arthropods has relied largely on the determination of an acute median lethal dose or con- centration. However, the estimated lethal dose during acute toxicity tests may only be a partial measure of the deleterious effects. In addition to direct mortality induced by pesticides, their sublethal effects on arthropod physiology and behavior must be considered for a complete analysis of their impact [24, 25]. Therefore, accurate assessment of sublethal effects is crucial to acquire knowledge on the overall insecticide efficacy in controlling insect pest populations, as well as on their selectivity towards nontarget organisms [26]. On the other hand, it has been shown that the efficacy of insecticide may be different in relation to the times they are used [27, 28] . Therefore, awareness of pest life tables and plant phenology are very important as the two consequential aspects in pest ecology. Because pest susceptibility to control agents alters between life stages, knowledge on the stage structure of a pest population is necessary for establishing the most effective pesticide application schedule [28] . Since, simulations based on the age- stage, two -sex life table explain the stage structure of a pest population at each time period, the population simulations according to this method can be efficiently used to select the best control strategy based on the stage structure [28]. In this research, we used the age-stage two-sex life table model to analyze the life table parameters of B. tabaci, as a new method to evaluate the sublethal effects of some botanical and synthetic insecticides. They include the extracts taken from Fumaria parviflora, Teucrium polium, Calotropis procera, and Thymus Vulgaris as well as the two commercially available synthetic pesticides, pymetrozin and neemarin. 2. Materials and Methods 2.1. Host Plants. We used cotton plants, Gossypium hirsutum var. Varamin, as the main host for mass rearing of B. tabaci and tomato plants, Lycopersicum esculentum var. Bakker brothers, as host in experiments. The cotton and tomato seeds were cultured in transplant trays at greenhouse conditions. Some stand plants were separately transferred in plastic pots (15 X 15 X 20 cm) which filled with a commercial Sterile Plant Growth Media, BAGA (Bastare Amade Giah Arganic, manufactured by Dashte Sabz Atie Go., UTSTP, Iran), while others were planted in hydroponic pots. All pots were kept in 60 X 50 X 80 cm cages covered by fine cloth mesh. Old pots were replaced with new one monthly. 2.2. Study Insects. Adult B. tabaci, collected from native colonies of Rafsanjan cotton fields (Kerman province. South east of Iran), was released on cotton plants in greenhouse of Gollege of Agriculture (Vali-e-Asr University of Rafsanjan). The pupae were analyzed taxonomically and biotype A specimens were isolated and reared as original experimental source. The stock colony was reared in controlled conditions (27 ± 2“G, 50 ± 5 RH and 16 : 8 h L : D). Young fully expanded tomato leaves were put in small plastic pots (10 cm diameter, height: 15 cm) containing dis- tilled water. The pots were then covered by similar transpar- ent pots to make a transparent glass cages. Adult whiteflies were released into the cages through a small pore located at the upper pot. In order to produce the same age 24 h adults, the eye-red pupa was checked daily and new born adults were collected and released into glass cages. 2.3. Pesticides. Gommercial formulations of the two com- monly used insecticides, pymetrozin (Ghess 25% WP, Sin- genata Gompany), and azadirachtin (Neemarin EG1500), were used in this study. 2.4. Plant Extract Preparation. Four medicinal plants includ- ing fumitory, F. parviflora (Fumariaceae), germander, T. polium (Famiaceae), swallowwort, C. procera (Asclepi- adaceae), and thyme, Th. Vulgaris (Famiaceae) were used in this study. Herbal parts of plants (leaves and flowers) were collected from natural habitats in Kerman province, southeastern Iran, during their flowering stages (May and June 2010). Plant materials of F. parviflora, T. polium,a.nd Th. vulgaris were collected from Rafsanjan (30°23^46^^E, 55°56^25^^N), while those of C. procera were gathered from Jiroft (28°40^37^^E, 57°43^52^^N). The collected plants were identified at species level by Dr. M.A. Vakili at the Plant Taxonomy Department of the Islamic Azad University of Jiroft. Sampling was carried out from an average number of 50 plant stalks and three samples were taken from each individual plant. The plant materials were air dried for 4- 5 days and coarsely powdered using an auto mixer. Twenty grams of dried materials were placed on filter paper and steeped in ethanol (90 mL) and water (210 mL) for 12 hours. These extractions were prepared according to the Soxhlet extraction method [29, 30]. Afterwards, rotation was used to reach an extract amount of one-third. 2.5. Concentration-Mortality Response. The effects of five concentrations of the six used pesticides on B. tabaci were assayed using the leaf-dip method. Two-leave tomato plants were dipped in pesticide dilutions for 5 s [31] and then put separately in glass cages. After drying the treated leaflets, fifteen adult B. tabaci were released into each cage. The cages were maintained under aforementioned controlled condi- tions. A solution of 3% ethanol/distilled water was used as control. The total numbers of dead adults were counted after 24 h. Adults were considered died when they were not able to move properly as a result of stimulation with a soft brush. This assay was conducted in a completely randomized design with three replicates being considered for each insecticide. 2.6. Sublethal Effects on Life History Traits. Adult whiteflies (24 hours old) were released into glass cages containing tomato stalks and allowed to oviposit for 24 h. After ovipo- sition, adults were removed from the cages. Fifty eggs were randomly controlled every 24 h and the egg duration was recorded. After that all immature stages (nymph and pupa) growth periods were studied daily. Tomato leaflets were Psyche 3 dipped in the lethal concentration 25% (LC25) of each insecticide for 5 s [31]. After air drying, the plants were transferred to the glass cages. Thirty new emerged adults were randomly captured from the stock colony and released into cages. After 24 h, all adults were removed from the cages and released into new ones. The numbers of eggs laid by each female were recorded by digital microscope Dino Capture and Stereomicroscope and continued until the death of the last female. After adult emergence, the sex ratio of FI generation was determined based on the method of Gerling [4]. 2. 7. Data Analysis. Probit analysis was performed to estimate the LC50 and LC25 values using Polo-Plus 2.00 software. The population parameters data were analyzed using SPSS soft- ware (version 16). One-way analysis of variance (ANOVA) and Duncans multiple range tests were conducted to compare the effects of different insecticides on life table parameters of B. tahaci {P < 0.05). The life table parameters were analyzed using age-stage two-sex life table and female age-specific life table methods. In age-stage two-sex life table, developmental time of all individuals, including males, females, and those dying before adult stage, as well as female daily fecundity was analyzed according to the age-stage, two-sex life table [32, 33]. Processing of raw data analysis was facilitated by a computer program; TWOSEX-MSChart [34]. No standard error was calculated for the data which was analyzed by the TWOSEX-MSChart program [34]. Various life table parameters including the age- stage specific survival rate {s^j, where x = age and j - stage), the age-stage specific life expectancy {e^j, where x = age and j = stage; it gave the expected time that an individual of age x and stage j will live), the age-stage specific fecundity (/^j), the mean fecundity (F), which explained the contribution of an individual of age x and stage j to the future population, the age-specific survival rate (/^), the age- specific fecundity (m^), and the population parameters (r, the intrinsic rate of increase; A, the finite rate of increase, A = e^; Rq, the net reproductive rate; and T, the mean generation time) were calculated according to Chi method [33]. The intrinsic rate of increase was appraised using iterative bisection method from the following equation using age indexed from 0 [35]: CX3 = 1 . ( 1 ) x=0 The mean generation time was explained as the time length that a population requires increasing to RQ-times of its size as the firmly fixed age distribution to obtain the stable increase rate of population. In other words, it means = Rq or A = Rq- The mean generation time (T) and the gross reproductive rate (GRR) were calculated by equations the following, respectively: ^ LnRo GRR = ^ m^. Table 1: The LC 25 (mg/mL), slop ± SE, lower and upper 95% confidence intervals, and of some synthetic and botanical insecticides on B. tahaci. Insecticides Slop ± SE LC 25 Limits 95% a' Pymetrozin 2.236 ± 0.357 0.089 53.99-120.47 1.936 Neemarin 1.073 ± 0.194 0.070 0.023-0.129 0.171 Th. vulgaris 1.991 ± 0.432 69.087 36.78-95.09 4.122 T. polium 2.011 ± 0.366 90.948 50.76-125.97 0.463 F. parviflora 2.936 ± 0.654 314.082 183.35-406.95 3.553 C. procera 2.100 ± 0.498 250.532 130.3 7-338.92 0.762 Chi [33] demonstrated that the relationship among the mean female fecundity (F) and the net reproductive rate (Rq) can be explained as (3) where N is the total number of individuals used at the start of the life table study and N j: is the number of the emerged female adults from these N eggs. This also means Nj: x F - Rq X N. On the other hand, the total number of offspring produced by all females is equal to the net reproductive rate multiply the cohort size. This relationship shows the accuracy in the age-stage, two-sex life table analysis [33]. In the age-specific female life table, the data were calcu- lated according to Carey method [36]. Jackknife resampling methods were used to calculate the mean and standard error of population parameters [37]. 3. Results 3.1. Concentration-Mortality Response. Log-probit regression analyses of concentration-mortality data showed that, 24 h after exposure of adult B. tahaci to the six studied insecticides, the LC 25 values for different concentration of pymetrozin (106, 150, 210, 298, 420, and 593|Wg/mL), neemarin, (0.060, 0.135, 0.330, 0.780, and 1.860 mg/mL), Th. vulgaris, (44, 58, 76, 100, 132, 173, and 228 mg/mL), T. polium (58, 76, 100, 132, 173, 223, 300, and 395 mg/mL), F.parviflora (100, 153, 234, 359, 550, and 842 mg/mL), and C. procera (200, 283, 400, 566, and 800 mg/mL) were 89.95 and 0.070 pg/mL and 69, 90.9, 314, and 250.5 mg/mL, respectively (Table 1). 3.2. Sublethal Effects. The age-stage survival rate (S^j) (Figure 1) shows the probability that a new born pupa will survive to age x and stage j. In addition to survival, this curve also illustrates the stages difference, stages overlapping, and the variable developmental rate between individuals [33, 38]. As the figure shows that all adults emerged on the same day in all treatments, but the females survived longer than the males (Figure 1). The curve shows the probability that a newborn pupa will survive to age x. Results showed that the survival duration adult whiteflies (from birth to death) were 36, 36, 35, 33, 30, 33, and 35 days in control, pymetrozin, neemarin, T. polium, C. procera, Th. Vulgaris, and F. parviflora treatments. Survival rate Survival rate Survival rate 4 Psyche — Egg Female — o— Nymph — Male —w— Pupa Figure 1: Effects of some synthetic and botanical insecticides on age-stage specific survival rate (s^j) of B. tabaci. Psyche 5 respectively. The highest rate of longevity was observed in control and pymetrozin treatments (36 days), while the lowest longevity was recorded for C. procera extract (30 days). Our results also showed that the survival rate of females was 15, 15, 14, 12, 9, 12, and 14 days for control, pymetrozin, neemarin, T. polium, C. procera, Th. Vulgaris, and F. parviflora, respectively. The life expectancies (e^j) of adult B. tabaci calculated using the life duration of female and male whiteflies after the appearance of newborn adults were 4.40, 3.16, 3.21, 3.46, 3.37, 3.52, and 3.83 days for adult females and 3.94, 2.47, 2.75, 2.73, 2.85, 3.07, and 2.65 days for adult males in control, pymetrozin, neemarin, T. polium, C. procera, Th. Vulgaris, and F. parviflora treatments, respectively. The lowest life expectancy of adult females was observed in whiteflies treated by pymetrozin, neemarin, and C. procera, respectively. 3.3. Population Parameters. The results of statistical analysis revealed signiflcant differences in sublethal effects of the synthetic and botanical insecticides on the net reproductive rate (F = 8.271; df = 6, 37; P < 0.001), the intrinsic rate of increase (F - 8.619; df = 6,37; P < 0.001), and the finite rate of increase {F - 8.688; df = 6, 37; P < 0.001). The average values for Rq, A, r, and T parameters have been shown in Table 2. The highest and the lowest values of gross reproductive rate were observed in control (39.61) and C. procera (17.16), respectively. The lowest value of net reproductive rate was detected in pymetrozin (2.45), while the whiteflies in control showed the highest net reproductive rate. The highest amount of intrinsic rate of increase and finite rate of increase was observed in the control (0.076 and 1.079, resp.), while the lowest values were recorded for whiteflies treated by pymetrozin (0.036 and 1.036, resp.). The lowest value of mean generation time was recorded in whiteflies treated by pymetrozin (25.28) and C. procera (25.43). while the longest generation time was observed in those treated by T. polium (30.76) (Table 2). The net reproductive rate (Rq) and the mean female fecundity (F) of control, pymetrozin, neemarin, T. polium, C. procera, Th. Vulgaris, and F. parviflora were 7.32, 2.45, 2.99, 2.82, 3.18, 3.67, and 4.91 offspring and 14.15, 5.38, 5.97, 5.58, 6.68, 6.45, and 9.07 egg/adults, respectively. 3.4. Comparison of Age-Stage Two-Sex Life Table and the Female Age-Specific Life Table. To make a comparison between the efficiency of two commonly used models, the population parameters of whiteflies treated by all insecti- cides were simultaneously calculated using both age -specific female life table and age-stage two-sex life table (Table 3). Statistical analyses revealed no significant difference among parameters calculated by these models. 4. Discussion In the present study, we conducted some bioassays to assess the sublethal effects of pymetrozin and neemarin as well as the extracts taken from F parviflora, T. polium, C. procera, and Th. vulgaris on demographical parameters of B. tabaci. Meanwhile, we compared the age-stage two-sex life table and the female age-specific life table to clarify the differences between the two methods. The life-table studies under dif- ferent environmental conditions and on different host plants have been proposed to be a relatively time-consuming pro- cess; thus the use of life tables in pest management programs seems not to be appropriate. However, the life table studies provide us with some basic knowledge about the biological properties of studied pests, without them development of an appropriate control strategy is impractical [38]. The traditional age-specific life tables [39-41] concentrate only on the survival and the fecundity of the female popula- tion, thus ignoring the male population, the stage differences, and stage overlapping leading to a miscalculation in the survival and fecundity curves [33, 42]. To overcome these problems, the age-stage two-sex life table has been developed by Chi and Liu [32] and Chi [33]. The age-stage two-sex life table has been widely used to study the population dynamics of insect [38, 43-45]. As far as we know, this study is the first one that uses the life table parameters as a suitable index for evaluation of sublethal effects of insecticides on B. tabaci. Our results showed that the female whiteflies survived longer than the males and the lowest survival rate of females was observed in T. Polium followed by neemarin. In the usual condition without application of pesticides, female whiteflies have been shown to survive longer than the males [43] . In our study, the overlapping in curves of S^j shows the potential of the age-stage two-sex life table in displaying the stage dissociation of B. tabaci due to variable developmental rate between individuals. Similarly, stage differentiation can be perceived in curves of e^j. In addition to stage overlapping, a correct relationship between Rq and F can be received. In our research, the total number of offspring produced by all females was nearly equal to the net reproductive rate multiplied by the cohort size and the minor difference was attributed to rounding- off. This equality shows the accuracy of the age-stage two-sex life table analysis. These results in all treatments were consistent with the relationship obtained by Chi [33], and Yang and Chi [43]. According to our results, the survival of a population can be predicted at each condition. Our results are in line with those of Chi and Su [42], Yang and Chi [43], and Hu et al. [38] who showed the variable developmental rate and overlapping among different stages using age-stage two- sex life table. Results of our current study clearly showed that the susceptibility to pesticides and botanical compounds varied significantly among different developmental stages of B. tabaci. These findings are in accordance with those results of Liu and Stansly [46] on B. argentifolii. Yang and Chi [43] proved that Rq < F meaning that the net reproductive rate is lower than the mean female fecundity. If there was preadult mortality, Rq is expected to be lower than F, a condition that was shown by our results as well. In contrast to our results, Liu and Stansly [47] reported that the net reproductive rate was higher than the mean fecundity (i.e., Rq > F). This repugnance maybe related to the methods they used for calculation of Rq and F. The results of comparison between the age-stage two-sex life table and the traditional age specific life table indicated that consideration of male whiteflies in calculation of life table 6 Psyche o C/5 C/5 i-i cu cu a> Jh ,1> >< a> c/5 I O ■I-* cu bO rt (U bO rt cu ■!-• 'T 3 C CS rt o) ■I— > — ^ +1 +1 CO +1 CO +1 CO +1 X m bO VO m m 00 • ^ CU CO fN (N vq 3 VO IN •H— * 3 Lci in CO in d vd d 0 (N (N m cn m m CN o • ^ H Uh D d cu !dX) c •M (N 00 00 C 73 C 7 \ 00 CN ; »-H (N d vq 1— 1 0 K G cu pb| CN m m m (N m CN cu 3 C 3 1— H C 3 V 0 00 m m o fli (N rn m (N cn q C-l— i • d 0 0 d CO cd o; CU +1 +1 +1 +1 +1 +1 +1 m Ln o\ CN 00 IN 1— H c/5 (N O) esq »— 1 vq <03 •u CD K (N (N ni rn rn -M eS u G XJ O Ph cu Zo VO u a\ 'Cf +-» -M 00 0 1— H cq CN F^ •u d C-l— 1 1-H 0 0 CO CO CO cd a> • ^ +1 +1 +1 +1 +1 +1 +1 eS LD < 3 v 00 VO m 0 3 c 3 q C0> i>. vq Ov IN a 06 ni 3 (N in cu P-t cu > < VO VO (N m 0 0 0 0 0 0 0 R f ^ C-l— ( « ^ 0 0 0 0 cq 0 q 0 d 0 CO CO CO cd cu X +1 +1 +1 +1 +1 +1 +1 c/5 d S < 3 V VO m 0 o\ m a> c/5 m m m VO Uh 0 0 0 cO> cq cq q q cu G 1-H 1— H 1— H 1-H t-H 1— H • ^ C4-1 H 0 a> cu •U ■ 4 _» 1 < +3 00 0 0 C 3 V CN VO • ^ 0 1— H 1— H 0 1— H 0 C f C-l— 1 • »-H 0 0 0 cq cq 0 q cjq 0 0 0 0 0 CO cd cu e 5 CU +1 +1 +1 +1 +1 +1 +1 3 00 VO VO IN 0 0 0 cq cq q q G cu pb| 1-H 1-H 1— H 1— H 1— H t-H cu 3 VO m (N m rt 0 0 0 0 0 0 0 C-l— 1 • 0 0 CO 0 cq 0 q CU 0 d CO CO CO CO cd c/5 G > G •u !h •M G 0 u 1 Ph a lU cu 3 T. poli 0 ex, u ;s p <3 Cu te Psyche 7 Table 3: Results of t-test analyses showing differences between the age-specific female life table and the age-stage two-sex life table in estimation of life table parameters of B. tabaci. The intrinsic rate of increase The finite rate of increase The net reproductive rate The mean generation time Treatments (r) (A) (Ko) (D P t P t P t P t Control 0.661 0.449 0.655 0.457 0.492 0.707 0.774 0.293 Pymetrozin 0.670 0.434 0.633 0.486 0.048 2.126 0.317 1.040 Neemarin 0.773 -0.293 0.807 -0.249 0.353 -0.958 0.798 0.262 T polium 0.679 0.425 0.266 1.162 0.011 3.019 0.905 0.122 C. procera 0.556 -0.602 0.590 -0.550 0.016 -2.700 0.919 0.103 Th. vulgaris 0.643 0.475 0.604 0.533 0.008 3.183 0.491 0.710 P. parviflora 0.526 0.652 0.499 0.696 0.124 1.644 0.936 0.082 indices and variable developmental rates had little effects on population parameters of B. tabaci. The suitability of age stage two-sex life table for calculation of population param- eters has been also approved by several previous studies [44]. In recent decades, with the increasing knowledge on detrimental effects of chemical pesticides on human, environ- ment, and nontarget organisms (e.g., development of resis- tance by key pests, environmental pollution, human health dangers, and pest resurgence), the use of environmentally friendly compounds with the least side effects on non- target organisms and environment have received relatively great attention. Among these compounds, the plant-derived insecticides have been traditionally considered as efficient candidate alternative to synthetic insecticides [15, 48]. A large numbers of studies have clarified that the extracts taken from the four plant species, T. polium, R parviflora, Th. Vulgaris, and C. procera, have insecticidal properties [16-22, 49]. Our current study, however, showed that in addition to the direct mortality caused by these compounds they may also impose some sublethal effects on target pests. These effects included decreased growth, fecundity, and survival rate, increased development time, and probably increased susceptibility to natural enemies. In our study, the lowest values of were observed in whiteflies treated by pymetrozin, T. polium, and C. procera. Therefore, the two mentioned botanical insecticides seem to be suitable for integrated management programs of B. tabaci. Altogether, results of the current study showed that some biological characteristics of the cotton whiteflies are affected at sublethal concentrations of the studied insecticides. These effects are easily distinguishable using the age stage two-sex life table method used in this study However, before decision making for establishment of a control strategy using these insecticides, their effects on non- target organisms, especially natural enemies, should be also evaluated. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors are most grateful to Professor Hsin Chi (Lab- oratory of Theoretical and Applied Ecology, Department of Entomology, National Chung Hsing University Taichung, Taiwan) for his valuable contributions to this paper. The authors thank Dr. Rouhollah Rahmani for generously helping with editing. References [1] A. H. Greathead, “Host plants,” in Bemisia Tabaci a Literature Survey on the Cotton Whitefly with an Annotated Bibliography, M. J. W. Cock, Ed., pp. 17-26, International Institute of Biologi- cal Control, Berkshire, UK, 1986. [2] f H. Martin, “An identification guide to common whitefly pest species of the world (Homoptera: Aleyrodidae),” in Tropical Pest Management, vol. 33, no. 4, pp. 298-322, 1987. [3] D. N. Byrne and M. A. Houk, “Morphometric identification of wing polymorphism in Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae),” in Annals of the Entomological Society of Amer- ica, vol. 83, no. 3, pp. 487-493, 1990. [4] D. Gerling, Whiteflies: Their Bionomics, Pest Status and Manage- ment, Intercept Ltd, Wimborne, UK, 1990. [5] M. A. Husain and K. N. Trehan, “Final report on the scheme of investigation on the whitefly on cotton in the Punjab,” The Indian Journal of Agricultural Sciences, vol. 10, pp. 101-109, 1940. [6] M. f Berlinger, “Host plant resistance to Bemisia tabacij Agriculture, Ecosystems and Environment, vol. 17, no. 1-2, pp. 69- 82, 1986. [7] R. C. Dickson, M. fohnson, and E. F. Laird, “Leaf crumples a virus disease of cotton,” Phytopathology, vol. 44, pp. 479-480, 1956. [8] f E. Duffus, “Whitefly transmission of plant viruses,” in Current Topics in Vector Research, K. H. Harris, Ed., vol. 4, pp. 73-91, Springer, New York, NY, USA, 1987. [9] 1. D. Bedford, R. W. Briddon, J. K. Brown, R. C. Rosell, and P. G. Markham, “Geminivirus-transmission and biological characterisation of Bermisia tabaci (Gennadius) biotypes from different geographic regions,” Annals of Applied Biology, vol. 125, no. 2, pp. 311-325, 1994. [10] D. G. Riley and W. Tan, “Host plant effects on resistance to bifenthrin in silverleaf whitefly (Homoptera: Aleyrodidae),” 8 Psyche Journal of Economic Entomology, vol. 96, no. 4, pp. 1315-1321, 2003. [11] Y. O. H. Assad, N. H. H. Bashir, and E. M. A. Eltoum, “Evaluation of various insecticides on the cotton whitefly, Bemisia tabaci (Genn.), population control and development of resistance in Sudan Gezira,” Resistant Pest Management Newsletter, vol. 15, pp. 7-12, 2006. [12] W. Dermauwa, N. Wybouwa, S. Rombautsb et al, “A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticaej Proceedings of the National Academy of Sciences of the United States of America, vol. no, no. 2, pp. E113-E122, 2012. [13] M. B. Isman, “Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world,” Annual Review of Entomology, vol. 51, pp. 45-66, 2006. [14] L. G. Gopping and J. J. Menn, “Biopesticides: a review of their mode of action and efficacy,” Pest Management Science, vol. 56, no. 8, pp. 651-676, 2000. [15] A. Ali, E. Ahmad, A. Biondi, Y. Wang, and N. Desneux, “Potential for using Datura alba leaf extracts against two major stored grain pests, the khapra beetle Trogoderma granarium and the rice weevil Sitophillus oryzaej Journal of Pest Science, vol. 85, no. 3, pp. 359-366, 2012. [16] L. A. Hummelbrunner and M. B. Isman, “Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuidae),” Journal of Agricultural and Pood Chemistry, vol. 49, no. 2, pp. 715-720, 2001. [17] E. H. Koschier and K. A. Sedy, “Labiate essential oils affecting host selection and acceptance of Thrips tabaci lindeman,” Crop Protection, vol. 22, no. 7, pp. 929-934, 2003. [18] A. M. El-Shazly and K. T. Hussein, “Ghemical analysis and biological activities of the essential oil of Teucrium leucocladum Boiss. (Lamiaceae),” Biochemical Systematics and Ecology, vol. 32, no. 7, pp. 665-674, 2004. [19] N. M. Arab, R. Ebadi, B. Hatami, and Kh. }. Talebi, “Insecticidal effects of some plant extracts on Callosobruchus maculatus E under laboratory condition and Laphigma exigua H. in greenhouse,” Journal of Science and Technology of Agriculture and Natural Resources, vol. 11, pp. 221-234, 2008. [20] M. S. Al-mazraawi and M. Ateyyat, “Insecticidal and repellent activities of medicinal plant extracts against the sweet potato whitefly, Bemisia tabaci (Horn.: Aleyrodidae) and its parasitoid Eretmocerus mundus (Hym.: Aphelinidae),” Journal of Pest Science, vol. 82, no. 2, pp. 149-154, 2009. [21] E. Sertkaya, K. Kaya, and S. Soylu, “Chemical compositions and insecticidal activities of the essential oils from several medicinal plants against the cotton whitefly, Bemisia tabaci’,’ Asian Journal of Chemistry, vol. 22, no. 4, pp. 2982-2990, 2010. [22] A. Taghizadeh-Sarokalaii and S. Moharramipour, “Eumi- gant toxicity of essenssial oil. Thymus persicus (Lamiaceae) and Prangos acaulis (Apiaceae) on Callosobruchus maculatus (Coleoptera: Bruchidae),” Iranian Journal of Plant Protection Science, vol. 33, pp. 55-68, 2010. [23] E. Jafarbeigi, M. A. Samih, M. Zarabi, and S. Esmaeily, “Effect of some herbal compounds on biological parameters of Bemisia tabaci (Genn.) (Hem.: Aleyrodidae) on tomato under con- trolled condition,” Journal of Plant Protection Research, vol. 52, no. 4, pp. 375-380, 2012. [24] N. Desneux, A. Decourtye, and J.-M. Delpuech, “The sublethal effects of pesticideson beneficial arthropods,” Annual Review of Entomology, vol. 52, pp. 81-106, 2007. [25] N. Desneux, X. Eauvergue, E.-X. Dechaume-Moncharmont, L. Kerhoas, Y. Ballanger, and L. Kaiser, “Diaeretiella rapae limits Myzus persicae populations after applications of deltamethrin in oilseed rape,” Journal of Economic Entomology, vol. 98, no. 1, pp. 9-17, 2005. [26] A. Biondi, N. Desneux, G. Siscaro, and L. Zappala, “Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: selectivity and side effects of 14 pesticides on the predator Orius laevigatas’,’ Chemosphere, vol. 87, no. 7, pp. 803-812, 2012. [27] B. H. Stanley, W. H. W. L. Reissig, M. Roelofs, R. Schwarz, and C. A. Shoemaker, “Timing treatments for apple maggot (Diptera: Tephritidae) control using sticky sphere traps baited with synthetic apple volatiles,” Journal of Economic Entomology, vol. 80, no. 5, pp. 1057-1063, 1987. [28] H. Chi, “Timing of control based on the stage structure of pest populations: a simulation approach,” Journal of Economic Entomology, vol. 83, no. 4, pp. 1143-1150, 1990. [29] A. L. Vogel, Text Book of Practical- Organic Chemistry, Long- man, Harlow, UK, 1978. [30] M. J. Pascual-Villalobos and A. Robledo, “Screening for anti- insect activity in Mediterranean plants,” Industrial Crops and Products, vol. 8, no. 3, pp. 183-194, 1998. [31] A. Heydari, S. Moharrami pour, A. A. pour mirza, and A. A. Talebi, “Effects of buprofezin, pymetrozin and fenpropathrin on reproductive parameters of Trialeurodes vaporariorum West- wood (Horn: Aleyrodidae),” Applied Entomology and Phy- topathology, vol. 71, pp. 29-46, 2003. [32] H. Chi and H. Liu, “Two new method for the study of insect population ecology,” Bulletin of the Institute of Zoology, Academia Sinica, vol. 24, pp. 225-240, 1985. [33] H. Chi, “Life-table analysis incorporating both sexes and variable development rates among individuals,” Environmental Entomology, vol. 17, no. 1, pp. 26-34, 1988. [34] H. Chi, “TWOSEX-MSChart: a computer program for the age-stage, two-sex life table analysis,” 2010, http://140.120 .197.173/Ecology/. [35] D. Goodman, “Optimal life histories, optimal notation and the value of reproductive value,” American Naturalist, vol. 119, no. 6, pp. 803-823, 1982. [36] J. R. Carey, Applied Demography for Biologists with Special Emphasis on Insects, Oxford University Press, New York, NY, USA, 1993. [37] A. D. H. N. Maia, A. J. B. Luiz, and C. Campanhola, “Statistical inference on associated fertility life table parameters using jack- knife technique: computational aspects,” Journal of Economic Entomology, vol. 93, no. 2, pp. 511-518, 2000. [38] L.-X. Hu, H. Chi, J. Zhang, Q. Zhou, and R.-J. Zhang, “Life-table analysis of the performance of Nilaparvata lugens (Hemiptera: Delphacidae) on two wild rice species,” Journal of Economic Entomology, vol. 103, no. 5, pp. 1628-1635, 2010. [39] E. G. Lewis, “On the generation and growth on the population,” Sankhya, vol. 6, pp. 93-96, 1942. [40] P. H. Leslie, “On the use of matrices in certain population mathematics,” Biometrika, vol. 33, no. 3, pp. 183-212, 1945. [41] L. C. Birch, “The intrinsic rate of natural increase of an insect population,” Journal of Animal Ecology, vol. 17, no. 1, pp. 15-26, 1948. [42] H. Chi and H.-Y. Su, “Age-stage, two-sex life tables of Aphidius gifuensis (Ashmead) (Hymenoptera: Braconidae) and its host Psyche 9 Myzus persicae (Sulzer) (Homoptera: Aphididae) with mathe- matical proof of the relationship between female fecundity and the net reproductive rate,” Environmental Entomology, vol. 35, no. 1, pp. 10-21, 2006. [43] T.-C. Yang and H. Chi, “Life tables and development of Bemisia argentifolii (Homoptera: Aleyrodidae) at different tempera- tures,” Journal of Economic Entomology, vol. 99, no. 3, pp. 691- 698, 2006. [44] R. Farhadi, H. Allahyari, and H. Chi, “Life table and predation capacity of Hippodamia variegata (Coleoptera: Coccinellidae) feeding on Aphis /abac (Hemiptera: Aphididae),” Biological Control, vol. 59, no. 2, pp. 83-89, 2011. [451 Y.-B. Huang and H. Chi, “Age-stage, two-sex life tables of Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) with a discussion on the problem of applying female age-specific life tables to insect populations,” Insect Science, vol. 19, no. 2, pp. 263-273, 2012. [46] T.-X. Liu and R A. Stansly, “Toxicity of biorational insecticides to Bemisia argentifolii (Homoptera: Aleyrodidae) on tomato leaves,” Journal of Economic Entomology, vol. 88, no. 3, pp. 564- 568, 1995. [47] T.-X. Liu and P. A. Stansly, “Life history of Bemisia argen- tifolii (Homoptera: Aleyrodidae) on Hibiscus rosasinensis (mal- vaceae),” Elorida Entomologist, vol. 81, no. 3, pp. 437-445, 1998. [48] H. Izadi and M. A. Samih, Biopesticides and Compounds with Novel Mode of Action, Jahad Daneshgahi, Tehran, Iran, 2006. [49] M. K. Irannejad, The Side-effects of several insecticides and plant extracts on green lacewing Chrysoperla carnea (Neuroptera: Chrysopidae) under laboratory conditions [M.S. thesis], Vali-e- Asr University of Rafsanjan, Rafsanjan, Iran, 2010. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 762704, 5 pages http://dx.doi.org/10.1155/2014/762704 Research Article A Survey of Bedbug {Cimex lectularius) Infestation in Some Homes and Hostels in Gboko, Benue State, Nigeria Onah Isegbe Emmanuel, Alu Cyprian, and Omudu Edward Agbo Department of Biological Sciences, Benue State University, AGO, RO. Box 5, Wadata Makurdi, Nigeria Correspondence should be addressed to Onah Isegbe Emmanuel; isegbeonah@gmail.com Received 15 February 2014; Revised 11 April 2014; Accepted 14 April 2014 Academic Editor: Cleber Galvao Copyright © 2014 Onah Isegbe Emmanuel 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. A Survey of bed bug infestation in some homes and hostels, in Gboko, Benue State, Nigeria, was conducted from January to April, 2011. Bed frames, bunks, mattresses, pillows, chairs, and clothes were inspected. A total of 2,642 bed bugs were collected. 73.3% were from hostels while 26.7% were from homes. There was a significant difference between in the number of homes infested and those not infested = 61.44, df = 4, P < 0.05). Nymphs were the most populated, with 292 (41.4%), followed by males 223 (31.6%), and females 190 (27.0%). There was no significant difference in the number of infested hostels and those not infested = 0.8, df = 4, P < 0.05). The nymphs being the most populated with 901 (64.1%), followed by males 538 (36.1%), and then females 496 (35.3%). The greater number of infestation recorded in the hostels was as a result of poor hygiene, lack of adequate knowledge of the best control practices and the high population density. In homes, lack of the awareness of the resurgence of the emerging pest and lack of proper health education is responsible for the high infestation. Proactive approach should be taken towards public health education against bed bug infestation. Government and NGOs should take critical steps in preventing spread and stigma. 1. Introduction Bedbugs are small parasitic insects of the family Cimicidae (most commonly Cimex lectularius). Two species are associ- ated with humans, Cimex lectularius and Cimex hemipterus, which are cosmopolitan or found in tropical and subtropical regions, respectively [1] . Bedbugs are blood sucking ectopara- sites that infest human habitations and usually feed during the night when the host is sleeping [2] . Under optimal conditions, the adult bedbug feeds once a week. The major attractants appear to be human body temperature and carbon dioxide production and also by certain chemicals [2]. Bedbug infestation associated problems include lack of sleep and psychological and social distress from society’s stigma concerning pests [2]. Although bedbugs have not been linked to disease transmission, they have been shown to harbor the causative organisms of plague, relapsing fever tularemia, Q fever, and Wolbachia. Symptoms from their bites include severe irritation, itching, inflammation, and swelling of the skin [3]. Special nocturnal search is often required as the definitive diagnosis depends upon collection and identification of bedbugs [1]. In the 1980’s bedbugs were considered relatively uncom- mon in many developed countries, such as the UK proba- bly due to better building practices, better education, and emphasis on wide use of insecticides [4]. In developing countries, bedbug infestation is at a high level [5]. Recent observations suggest that urban settings have experienced increased infestation over the past 10 years [6-8]. There is also recent evidence for insecticide resistance in bed bugs in addition to effective application techniques [9-13]. An increasing poor attitude towards housekeeping and poor hygiene is responsible for a high infestation in Makurdi and Otukpo [14]. This survey was carried out to determine the status of bed bug infestation in Gboko, with a view to investigate control measures adopted by residents against the pest; as well as their knowledge on the hazards associated with harboring bedbugs. 2 Psyche Table 1: Sex and growth stage of bedbugs (Cimex lectularius) collected from homes in Gboko. Location Gboko Number of homes inspected Number of homes infested (%) Males (%) Females (%) Nymphs (%) Total number of bedbugs (%) Eggs (%) Central 107 12 (11.2) 42 (31.8) 30 (22.7) 60 (45.5) 132 (18.7) 313 (16.6) East 101 7 (6.9) 2 (24.2) 18 (19.8) 51 (56.0) 91 (12.9) 273 (14.5) West 118 20 (16.9) 50 (27.5) 60 (33.0) 72 (40.0) 182 (25.8) 401 (21.3) North 132 38 (28.8) 73 (33.0) 67 (30.3) 81 (36.7) 221 (31.3) 701 (37.2) South 142 9 (6.3) 36 (45.6) 15 (19.0) 28 (35.4) 79 (11.2) 196 (10.4) Total 600 86 223 190 292 705 1884 (100) ^Alcalculated ~ 61.44, df = 4, ^Tabulated ^ = 4.278 at 95% level of significance). Table 2: Sex and growth stage of bedbugs (Cimex lectularius) collected from Hostels in Gboko. Location Gboko Number of Hostels Inspected Number of homes Infested (%) Males (%) Females (%) Nymphs (%) Total number of bedbugs (%) Eggs (%) Central 2 1 (50.0) 59 (19.0) 48 (15.5) 203 (65.5) 310 (16.0) 307 (21.8) East 2 1 (50.0) 78 (25.0) 81 (26.0) 153 (49.0) 312 (16.1) 178 (12.7) West 1 1 (100.0) 111 (27.8) 107 (26.8) 182 (25.5) 400 (20.7) 341 (24.3) North 2 2 (100.0) 198 (32.9) 201 (33.4) 201 (33.4) 602 (31.1) 301 (21.4) South 2 1 (50.0) 92 (29.4) 59 (18.4) 162 (51.8) 313 (16.2) 279 (19.8) Total 9 6 538 496 901 1937 1406 (100) (xJalculated = df = 4, y^^bulated = 4-278 at 95% level of significance). 2. Materials and Method The survey was conducted in Gboko, Local Government area of Benue state, Nigeria, from January to April, 2011. Gboko is located on 7°19 30^^N 9°0^18^^E and lies in the Savanna region of North-Gentral Nigeria, with a temperature range from 29°C to 33°G. It has a population of over 500,000 people who are predominantly farmers and civil servants. They speak Tiv and English language. Benue State covers an area of about 34,059 km with a population of over 4.2 million people. Written informed consent was obtained from the Gboko Local Government Health Authority and verbal consent from heads of household heads and owners of hostels. Randomly selected homes and hostels were visited and bed frames, mattresses, bamboo beds, carpets, mosquito nets, benches, walls, cushioned chairs, pillows, and bed sheets were thor- oughly inspected for bedbugs infestation. Gollecting bottles, paint-like brushes, and 70% alcohol were the materials used. Where bedbugs were seen, they were brushed into the collecting bottles. The material from which the bedbug was collected was noted and the number of bedbugs collected per material was noted. Residents were interviewed on the control measures adopted against bedbugs. The specimens transferred to the General Zoology Laboratory of the Benue State University for identification according to published keys by Pratt and Smith [15]. 3. Result Out of the 600 homes and 9 hostels surveyed, 86 (14.3%) homes and 6 (66.7%) hostels were infested with bedbugs. A total of 705 bedbugs were collected from homes, com- prising 223 (31.6%) males, 190 (27.0%) females, and 292 (41.4%) nymphs. 1884 eggs were also collected. A total of 1937 beg bugs were collected from hostels. This comprises 538 (27.8%) males, 496 (25.6%) females, and 901 (46.5%) nymphs. A total of 2,642 bedbugs were collected from all the hostels and homes, comprising 761 (28.8%) males and 686 (26.0%) females and 1,193 (45.2%) nymphs alongside 3290 eggs. 73.3% of the total collected bedbugs were from hostels while 26.7% of the total bedbugs were from residential homes. Gboko North had the highest number of infestation rate of 221 (31.3%) in residential homes and 602 (31.1%) in hostels, while Gboko South had the least infestation rate of 79 (11.2%) in residential homes and 313 (16.2%) in hostel (Tables 1 and 2). There was a significant difference between the number of infested and uninfested homes. In the residential homes (^calculated ^ 61.44, df = 4, ^Tabulated ^ 4.278 at 95% level of significance). In the hostels there was no significant difference between the number of infested and uninfested hostels (^calculated = 0'8> ‘*f = 4. xlabulated = 4-278 at 95% level of significance). Psyche 3 A total of 3,602 items were inspected, out of which 760 (21.1%) items were infested. Bed frames had highest infestation; out of 700 inspected bed frames, 314 (44.9%) were infested with a total number of 1,356 bedbugs. 11 bamboo beds were inspected and only 9 (81.8%) were infested with a total number 28 bedbugs (Table 3). The survey of different control measures used at different sites revealed a total of 8 different control measures. A total of 600 homes use one or more of the measures. Those who use Nuvan, dichlorvos (2,2-dichlorovinyl dimethyl phosphate) had the highest frequency of 196 (164.0%) while those who use Omo (detergent) and soap had the least frequency of 27 (22.2%) (Table 4). Insect powder is mostly used outdoors, and furniture assumed to be infested is taken away from the main building and the powder is applied to harborages. Snipper and Nuvan are applied inside houses mostly at night. Similarly, syringes are used to spray the insecticide in corners of the house where the pest is likely to be hiding. The same insecticide is also used with the aim at controlling other insects in the houses visited. 4. Discussion This survey recorded a high rate of infestation of bedbug. This agrees with the other findings in Australia [7, 8] , in Canada [16], and in Benue State, Nigeria [14]. This is as a result of the increase in the movement of people (including students from holidays and prisoners) from where infestation is very common via infested luggage, clothing, and other personal belongings and the increasing movement of bedding, furniture, and other materials by foot, car, and train. Recent increase in incidence of bedbug infestations in Canada, Italy, UK, USA and many other developed western countries has been linked to increase in international travel and the reduction in the use of insecticides to control cockroaches and ants (TerPoorten and Prose, 2005). 85.8% of the very recent cases of bedbug infes- tations recorded in Central Italy were recorded in apartment that housed immigrants and tourists from Mediterranean countries [17]. The data collected indicated high percentage of males than females and yielded greater percentage of nymphs. This agrees with a finding in Gbajimba, a settlement in Benue, where records show a greater percentage of males than females [14]. The greater percentage of nymphs recorded is an indication of the possibility of an increase in infestation where the right control measures are not employed. The greatest percentage of eggs points to a possible persistence of infestation by adult females. This percentage may be due to treatments applied which might have killed some adults without affecting the eggs. The infestations were highest in bed frames; these include single or double bunk (iron beds) frames and wooden bed frames. This is as a result of conducive hiding places the harborage provide and their close proximity to their feeding or host location. This however disagrees with Doggett’s findings, who indicated that iron bed harbor less bedbugs in his survey [7]. Table 3: Distribution of bedbugs in different harborages in homes and hostels in Gboko. Harborages Total number of harborages Number of harborages with bedbugs (%) Total Number of bedbug Bed frames 700 314 (44.9) 1,356 Mattresses 905 192 (21.2) 603 Pillows 800 93 (11.6) 262 Carpets 26 13 (50.0) 62 Walls 600 86 (14.3) 157 Wooden chairs 107 14 (13.1) 53 Wooden benches 36 9 (25.0) 24 Bamboo beds II 9 (81.8) 28 Executive chairs 217 13 (6.0) 36 Other Furniture 200 17 (8.5) 61 Total 3,602 760 2,642 The percentage recorded in hostels is probably due to poor or bad housekeeping and poor hygiene which is very common in Gboko. The World Health Organization suggests that poor housekeeping encourages bedbug breeding [18]. A review of the control measures (treatment) used at different sites indicated a high percentage on the use of Nuvan (2,2-dichloroethenyl phosphate) as it was the chemical method that was easily and readily available and affordable by most residents. Lack of technical knowledge of the control measures, knowledge of their hiding places, knowledge of resistance of eggs to spraying insecticides, and wrong use of the insecticides may have contributed to the high rate of infestation [4, 9-11, 13]. Infestation of bedbugs in homes and students hostels is epidemiologically significant. The nuisance they cause may result into sleeplessness, anxiety, and insomnia which could affect concentration in school and work. The high infestation is likely to arise from greater disper- sal of bed bugs, control strategies that are not fully effective, partial treatments that disrupt but do not eradicate infestation are used which fragment and spread bed bug colonies rather than disintegrating them. The research indicates that most people have little knowledge about bed bug control measures. Conclusively high rate of bedbug infestation recorded in some homes and hostels is no doubt a reflection of poor hygiene, sanitation, and lack of adequate knowledge concerning the pest. It is important that proactive approach should be taken on public education and awareness against bedbug. Critical steps in preventing bedbug spread and stigma should be taken up by the government and nongovernmental organizations. Training is an important component that should be offered and tailored to stakeholders, like property owners, pest man- agement professionals, and staff working in the residential setting. 4 Psyche o O c • ^ C4> -M C/3 o !=1 rt C4> cu a o !=1 (U n3 • ^ C4> cu Sh ns O 'Dh a cu C4> o "S a -M cu a "S cu pq h-l CQ iS bJD !=1 • ^ c o n3 C cS 'T3 fi C cu bC Ph cu CLi4^ o ^ CPO bC • ^ C Sh 13 m Sh CU no P P cu Sh P d C4> O c/5 Dh X w cu !=1 CU c/3 o Ph cu >3 I a ji c« cj CU cu cu c/) C Sh CU n3 O Dh -M CU CU c/3 C Ph CU 'a =i :z: c/5 a ^ K ^ Sh CU up 13 SP 13 c. -M e3 o Xi O sP o • ^ 13 cu o H-l C4> cu a o UP MU un MU in m o 00 C3^ LO 3 VO CM CM o cc3 Sh +-• Pi cu u o LO LD o LD VO CUV o m in MU 00 (vS in vq in 00 CUV CUV o O un CUV K LO CUV O CUV O CM m MU CUV CUV O) 3 00 CO 1-H CM CO 00 LO CO 1-H VO OV MU !>; CO CO CO m CO CO CO m CO CO O m o CM in 00 (vS vq un un 00 CM 00 CO o q MU CM LO CM CM CM CM CM 00 CO CO CM CM (N CO CO m , — ^ CUV 00 CUV MU 00 CO o vd 1-H CM ^ — ' o 00 o^ MU CM m 00 00 CM m CM c/3 rt W C4> Ph O P o CU5 vq (v3 CM 00 CM CM CM CM CM LO (T3 m 00 cn CM in LO MU O 00 MU CUV CM (T3 CM 00 CM cq 00 o o MU rt +-• Psyche 5 Conflict of Interests [17] M. Masetti and E Bruschi, “Bedbug infestations recorded in Central Italy,” Parasitology International, vol. 56, no. 1, pp. 81- The authors declare that there is no conflict of interests regarding the publication of this paper. [18] World Health Organization (WHO), Public Health Significance of Urban Pests, 2009. 83, 2007. References [1] K. Reinhardt and M. T. Siva-Jothy, “Biology of the bed bugs (Cimicidae),” Annual Review of Entomology, vol. 52, pp. 351-374, 2007. [2] I. Thomas, G. C. Kihiczak, and R. A. Schwartz, “Bedbug bites: a review,” International Journal of Dermatology, vol. 43, no. 6, pp. 430-433, 2004. [3] S. Tharakaram, “Bullous eruption due to Cimex lecticularisj Clinical and Experimental Dermatology, vol. 24, no. 3, pp. 241- 242, 1999. [4] M. R Potter, “The business of bedbug,” Pest Management Professional, vol. 76, no. 1, pp. 28-44, 2008. [5] E. Panagiotakopulu and P. C. Buckland, “Cimex lectularius T., the common bed bug from Pharaonic Egypt,” Antiquity, vol. 73, no. 282, pp. 908-911, 1999. [6] C. Boase, J. Small, and R. Naylor, Interim Report on Insecticide Susceptibility Status of UK Bedbugs. Professional Pest Controller, 2004. [7] S. T. Doggett, A Code of Practice for the Control of Bed Bug Infes- tations in Australia, vol. 5, Bed Bug Code of Practice Working Group, Australian Environmental Pest Managers Association, New South Wales, Australia, 2004. [8] N. Ryan, B. Peters, and P. Miller, “A survey of bedbugs in short stay lodges,” New South Wales Public Health Bulletin, vol. 15, no. 11-12, pp. 215-217, 2004. [9] H. Harlan, M. Faulde, and G. Gaumann, Bed Bugs, Health Significance of Urban Pets, 2008. [10] E. A. Temu, J. N. Minjas, C. J. Shiff, and A. Majala, “Bedbug con- trol by permethrin-impregnated bednets in Tanzania,” Medical and Veterinary Entomology, vol. 13, no. 4, pp. 457-459, 1999. [11] J. Myamba, C. A. Maxwell, A. Asidi, and C. E Curtis, “Pyrethroid resistance in tropical bedbugs, Cimex hemipterus, associated with use of treated bednets,” Medical and Veterinary Entomology, vol. 16, no. 4, pp. 448-451, 2002. [12] S. H. P. P. Karunaratne, B. T. Damayanthi, M. H. J. Fareena, V. Imbuldeniya, and J. Hemingway, “Insecticide resistance in the tropical bedbug Cimex hemipterusj Pesticide Biochemistry and Physiology, vol. 88, no. 1, pp. 102-107, 2007. [13] A. Romero, M. F. Potter, D. A. Potter, and K. F. Haynes, “Insecticide resistance in the bed bug: a factor in the pests sudden resurgence?” Journal of Medical Entomology, vol. 44, no. 2, pp. 175-178, 2007. [14] E. A. Omudu, “A survey of bedbug (Hemiptera: Cimicidae infestations in some homes and hostels in Makurdi and Otupkpo, Benue State, Nigeria, with notes on public health implications,” The Nigerian Journal of Pure and Applied Sciences, vol. 1, pp. 84-91, 2008. [15] H. D. Pratt and J. W. Smith, “Arthropods of public health importance,” in Diagnostic Procedure for Mycotic and Parasitic Infections, B. B. Wentworth, Ed., American Public Health Association, Washington, Wash, USA, 2005. [16] S. W. Hwang, T. J. Svoboda, I. J. De Jong, K. J. Kabasele, and E. Gogosis, “Bed bug infestations in an urban environment,” Emerging Infectious Diseases, vol. 11, no. 4, pp. 533-538, 2005. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 839261, 10 pages http://dx.doi.org/ 10 . 1 155/2014/83926 1 Research Article Nothochrysinae (Neuroptera: Chrysopidae): New Larval Description and Generic Synonymy, with a Consideration of Generic Relationships Catherine A. Tauber ^ Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853-2601, USA ^Department of Entomology and Hematology, University of California, Davis, CA 95616, USA Correspondence should be addressed to Catherine A. Tauber; cat6@cornell.edu Received 13 February 2014; Revised 5 April 2014; Accepted 6 April 2014; Published 11 June 2014 Academic Editor: Jacques Hubert Charles Delabie Copyright © 2014 Catherine A. Tauber. 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. Semaphorant B of Kimochrysa africana (Kimmins) expresses all of the larval synapomorphies that characterize the subfamily Nothochrysinae. Except for its head markings, the larva appears identical to that of Hypochrysa elegans (Burmeister). Based on consideration of both larval and adult similarities, Kimochrysa (Tjeder) is designated to be a subjective synonym of Hypochrysa Hagen {New Synonymy). The morphological basis for a previously proposed generic subdivision of Nothochrysinae is evaluated; the results indicate that the subfamily can be organized into two generic groupings each with distinct suites of shared adult characters. As yet, apomorphic support is not forthcoming from adult characters, and, unfortunately, larvae are known from only a few genera in the subfamily. 1. Introduction Chrysopid taxonomists generally agree that the subfamily Nothochrysinae is an archaic, probably monophyletic group- ing [1-7]. Nevertheless, synapomorphic adult features for the subfamily have been elusive, and proposals regarding the monophyly of the subfamily largely rested on the retention of presumed plesiomorphic character states, mostly in wing venation. In contrast, recent investigations have identified several larval features (listed in a later section) that may lend apomorphic support for the monophyly of Nothochrysinae [7-10]. These studies also indicate that larvae within the subfamily express a range of variation in their overall body form — from naked to light debris carrying. To date, larvae are described for only three of the nine genera of Nothochrysinae; thus, the taxonomic breadth of the recently recognized morphological support for the subfamily is limited, and the range and the phylogenetic value of larval variation among genera remain unknown. In the mid 1800s, Brauer [11] provided the first description of a larva from the Nothochrysinae; his article illustrated and described the monotypic European Hypochrysa Hagen (Semaphorant B — second or third instar, as Hypochrysa nohilis Heyd.). More recently, modern descriptions of the first and third instars of this species appeared [8, as Hypochrysa elegans Esben- Petersen] . All instars of the lone North American and the two European species of Nothochrysa McLachlan are described (see [7, 8, 12]). Einally, the first instars of Dictyochrysa fulva Esben- Petersen were described and compared with those of Nothochrysa [13]. In 2010, Duelli et al. [14] published images and biological notes on the larvae of Kimochrysa africana (Kimmins). These authors made the specimens available for morphological study and description. Here, I describe and compare the K. africana larvae with those known from other Nothochrysi- nae. As a result of the comparison, the genus Kimochrysa Tjeder is shown to be synonymous with Hypochrysa and questions arise concerning the currently held generic group- ings of Nothochrysinae. These questions are addressed. 2 Psyche 2. Materials and Methods The specimens were collected in the Republic of South Africa, Hoek-se-Berg Pass nr. Bushmans Kloof, 32°07^04.9^^ S, 19°10^29.7^^ E, 650 m, 2-X-2004 (see [14]). Unfortunately an early shipment of larvae was lost in the mail; the second shipment contained second instars {n - 3) preserved in alcohol. Upon arrival, the specimens were photographed and the external gross features were described. One of the specimens was cleared in KOH and transferred to glycerine for examination of fine structures and setation. Two speci- mens are now returned to P. Duelli, Swiss Federal Research Institute WSL, Birmensdorf/Zurich, Switzerland; the cleared specimen is in the Tauber Research Collection. Morphological terminology and chaetotaxy followed the usage of Rousset [15], Tsukaguchi [16], Tauber et al. [17], and Monserrat and Diaz -Aranda [8]. The larval stage of the Chrysopidae typically includes three instars, the first of which (Semaphorant A) is markedly different from the latter two (Semaphorant B), which resemble each other very closely except for size and small differences in chaetotaxy. Thus, it is appropriate to compare the second instars described here with third instars of other species. 3. Second Instar Kimochrysa africana 3.1. Diagnosis. The K. africana larvae express the three fea- tures previously identified as potential synapomorphies for Nothochrysinae [7], including (i) antenna: terminal segment is short (~ten times shorter than remainder of antenna), (ii) antenna: terminus has a group of small apical setae, not an elongate seta, (iii) labial palpus: terminal segment has more than three lateral sensilla. As Duelli et al. [14] noted, the K. africana larvae closely resemble those of the nothochrysine H. elegans in their elongated, naked bodies and their bright green coloration. They also share the following morphological features: (i) head: primary setae are blunt, (ii) thorax: lateral tubercles (LTs) are absent, (iii) abdomen: LTs are absent, (iv) abdomen: laterodorsal tubercles are absent or very small and with only one seta, and (v) types of setae: thoracic notal setae are short, blunt to slightly clavate, and abdominal (A1-A6) submedian setae are short, blunt to clavate, without hooks. The most notable difference between the larvae of the two species is that K. africana lacks the dark, elongated median head marking of H. elegans. 3.2. Description 3.2.1. Body (Figures 1(a) and 1(b)). Length ~5.9-6.9mm (measured in lateral view through spiracles), depth ~0.85- 1.1 mm (thickest section of abdomen). Bright green coloration of living specimens [14] faded in preserved specimens; dorsal surface largely cream-colored to tan, with pronotal sclerites brown, with pair of broad, reddish brown, vertical bands along lateral margins, extending from cervix to tip of A9. All setae short, smooth, pale, and of two types: “blunt/clavate” with blunt or slightly enlarged tip, usually erect and straight (primary cranial setae, dorsal thoracic, and abdominal setae (submedian setae, SMS)); “simple” with acute tip, usually erect and slightly curved (setae on cephalic appendages, some small, usually secondary, cranial setae, some very small setae on thoracic notum, setae on legs, posterior part of A9, AlO, and ventral setae). 3.2.2. Head (Figures 1(d) -1(f), 2(a) and 2(b)). Dorsum cream- colored, with light brown to brown markings as in Figures 1(d) (dorsal), 1(e) (ventral), and 1(f) (lateral); eyes with stem- mata clear, surrounding integument dark brown; cranium tapered posteriorly and roughly triangular, with rounded posterior (dorsal view); width (across eyes) ~0. 60-0. 64 mm, length (dorsum) ~0. 52-0.54 mm, and depth (midregion to top of eye) ~0.13 mm; base fully exposed. Anterior margin of labrum protruding slightly, straight anteriorly, and rounded laterally. All dorsal primary cephalic setae present (Figures 2(a) and 2(b)), blunt to slightly knobbed apically; two Vx setae present; labrum with two or three pairs of setae (one mesally and two laterally); dorsum with several secondary setae: one long, indistinguishable from posterior primary setae, others very short, mesal to S6. Ventral primary setae (S9, SIO) present; S8 absent. 3.2.3. Cephalic Appendages (Figures l(d)-l(f), 2(a) and 2(b)). Mandible long, thin, with length ~1. 0-1.1 mm, width ~ 0.10 mm; ratio of mandible length to head width 1.54-1.63; ratio of mandible length to head length (dorsal) 1.90-1.00. Mandible slightly upturned distally, with single acute baso- lateral seta; terminus sharp, with six teeth. Antennal length 0.90-0.97 mm, ~1.7-1.8x length of cranium; width ~0.03- 0.04 mm (at widest part of pedicel); scape with straight sides, two pairs of distal setae (one lateral, other mesal); pedicel long, about 17-19x length of flagellum, slightly broader than the base of flagellum; flagellum short, stubby, basal flagellom- ere with or without mesal seta, terminus with short basolat- eral seta, several very short setae. Labial palp long, slender, ~0.75x length of mandible; basal segment with one short, dorsal setabasally, three setae distally (two mesal, one lateral); middle segment long, with long, undivided basal subsegment bearing ~ five setae, five shorter mesal subsegments, with one seta mesally, elongated terminal subsegment, with two long setae distally; terminal segment ~one- third length of middle segment, slightly tapered distally, terminus with several very small setae; palpiger erect, with relatively straight sides, with one mesal seta, one lateral seta; mentum with smooth plate mesal to stipes, lateral to palpiger, with three pairs of long setae; cardo and stipes elongate, narrow, longitudinally arranged; cardo behind stipes. Cervix expanded laterally, ventrally, withdrawn from cranium dorsally; with pair of setae dorsolaterally, two pairs laterally, two pairs ventrally. 3.2.4. Thorax (Figures 1(c) and 3(a)). Lateral tubercles absent; dorsum with scattered, short, blunt to slightly clavate setae; primary setae unidentified (except as noted below). Legs (Figure 1(g)) cream-colored; coxae with diffuse, light brown marks anterolaterally; trochanter, femur with dark brown, longitudinal stripes anterodorsally, posterodorsally, with dark vertical stripe at tip of segment; tibia with dark brown vertical Psyche 3 Figure 1: Hypochrysa africana Kimmins, second instar (Hoek-se-Berg Pass, South Africa), (a) Body, dorsal, (b) Body, lateral, (c) Thorax, dorsal, (d) Head, dorsal, (e) Head, ventral, (f) Head, lateral, (g) Legs, dorsal. 4 Psyche Figure 2: Head of Hypochrysa africana Kimmins, second instar, (a) Dorsal, (b) Ventral. Scale applies to both (a) and (b). Abbreviations: co, cardo; c.m., cranial margin; fl, flagellum; l.p.x, labial palpus, number of segment; md, mandible; mx, maxilla; ped, pedicel; pg, palpiger; sc, scape; sen, sensilla; stp, stipes; Sx, primary seta, number; Vx, Vx setae. stripe basally, short, diffuse, light brown, longitudinal marks anterobasally, posterobasally; tarsus, claw, empodium dark brown. Prothorax (Tl) with two well delineated subsegments separated by transverse depression. Scl brown, elongated, extending almost to the full length of anterior subsegment. narrow anteriorly, posteriorly, broad mesally, embedded within lateral stripe, delineated laterally by curved, cream- colored strip. Sc2 elongated, narrow, extending posteriorly to margin of anterior subsegment. Mesothorax (T2) consisting of three well delineated subsegments separated by distinct depressions. Anterior Psyche 5 Figure 3: Thorax and abdomen (dorsal) of Hypochrysa africana Kimmins, second instar, (a) Thorax, (b) First and second abdominal segments, (c) Seventh and eighth abdominal segments, (d) Ninth and tenth abdominal segments. Abbreviations: Ax, number of abdominal segment; dep, smooth-surfaced, intrasegmental depression between subsegments; SlScl, S2Scl, first and second primary setae on Scl, Sc2, and Sc3, the first, second, and third primary sclerites of each segment; sc, abdominal sclerite; sp, spiracle; Tx, number of thoracic segment. subsegment with pair of small setae on anterior margin (probably SlScl, S2Sc), but Scl not distinguished; spiracles simple, sessile, circular, brown, with cylindrical, tapering atrium. Second subsegment with pair of small sclerites (Sc2) on anterior margin, each with single, very small seta (SlSc2); subsegment separated mesally from posterior subsegment by transverse depression with pair of large, bifurcating sclerites (Sc3) laterally. Sc3 with anterior arm extending anterolaterally, posterior arm extending posterolaterally, each toward edge of subsegment; center of Sc3 marked with brown, embedded in lateral stripe, entirely delineated by cream- colored area. Metathorax (T3) consisting of three well delineated subsegments separated by transverse depressions each with pair of transversely elongated sclerites (Sc2, Sc3) laterally. Sc3 large, with bifurcating arms — anterior arm extending anterolaterally, posterior arm extending posterolaterally, each toward edge of segment; center of sclerite marked with brown, embedded in lateral stripe, entirely delineated by cream- colored area. 3.2.5. Abdomen (Figures 3(b)-3(d)). Lateral tubercles (LTs), laterodorsal tubercles (LDTs) absent; spiracles (A1-A8) cir- cular, sessile, with simple, cylindrical, tapering atrium. Each segment (A1-A7) divided into three subsegments separated by transverse depressions; spiracle located laterally on dor- sum of second subsegment; second, third subsegments sepa- rated by pronounced depression bearing pair of clear sublat- eral sclerites. Al: anterior subsegment small, spindle shaped, separated from second subsegment by small depression; dorsum with single pair of SMS; second, third subsegments longer, broader, similar in size and setation to those of A2, A3. A2-A8: subsegments roughly quadrangular, of similar size, extending to margin of segment, separated from adjoining subsegments by pronounced depressions; dorsum of each subsegment with five (four on anterior subsegment of A8) to 14 pairs of SMS. A4-A8: pleural region (dorsal region of second and third subsegments) with cluster of four to seven short setae. A9: cylindrical, with three subsegments (dorsal view); anterior subsegment without setae; posterior two subsegments sclerotized, with setae; mesal subsegment with ~seven pairs of dorsal setae; posterior subsegment with ~12 pairs of dorsal setae, about half of them lateral. AlO: dorsum with transverse brown band, separated mesally by large arrow-shaped, darker brown mark; segment with -five pairs of setae, numerous microsetae. All segments with few small “simple” or “denticulate” setae. Venter with subsegmentation 6 Psyche lightly demarcated (A1-A4) or without demarcation (A5- A9); each segment with numerous “simple” setae, usually longer than dorsal setae. 4. Discussion 4.1. Synonymy Based on Larval Characters. A comparison of the K. africana and H. elegans larvae (Semaphorant B) does not reveal any significant, generic-level differences; for comparison, see [8]. Indeed, the larvae of the two species appear to differ largely in their head markings. Because of their similarity, I consider them congeneric, and hereby, Kimochrysa Tjeder becomes a junior (subjective) synonym of Hypochrysa Hagen (New Synonymy). The type species of the genus is Chrysopa nobilis Schneider, and the species under study here reverts to its original name, Hypochrysa africana Kimmins. Given this generic synonymy, the questions now are as follows, (i) Is this synonymy, which is based on larval charac- ters, also supported by adult morphological characters? And, (ii) how does the synonymy affect current understandings of relationships among the genera within Nothochrysinae? 4.2. Support from Adult Morphology? Of the several adult characters purported to differentiate Kimochrysa and Hypochrysa, two tend to support the synonymy; one weakly contradicts it, and three are neutral (either variable or without sufficient comparative information) as follows: consistent: wing venation [21]; spiracle of eighth abdominal segment (female) — opening on the eighth tergite versus on membrane (see [4]; also see Table 1). contradictory: ninth tergite and ectoproct (male and female) — fused versus separate (see [1, 4, 21]; also see Table 1). neutral: ectoproct (male) — with versus without a long, slender “appendage” (apodeme) [21]; subgeni- tale (female) — sclerotized versus unsclerotized [21]; microtholi (domelike cuticular glands) on male abdomen present versus absent (see [4], also see Table 1). Below I discuss the perspectives of two sets of authors who provided evidence for separating the genera: (i) Tjeder [21] and (ii) Brooks and Barnard [2] and Brooks [4] . 4.2.1. Tjeder s Perspective. Tjeder [21] described Kimochrysa on the basis of adult specimens of three South African species. His generic description noted similarities in the wing venation of Kimochrysa and Hypochrysa, but he felt that sig- nificant differences in their male terminalia required placing them in separate genera. Specifically, he mentioned (i) tergite 9-i-ectoproct — separate in Kimochrysa (as opposed to fused in Hypochrysa, both sexes), (ii) male ectoproct in Kimochrysa — lacking a long, slender “appendage” (probably the apodeme) that occurs in Hypochrysa, and (iii) subgenitale (female) — unsclerotized in Kimochrysa (as opposed to sclerotized in Hypochrysa). Of Tjeder’s above three characters, subsequent studies have shown that the first continues to provide the strongest (albeit weak) support for the separation of Kimochrysa from Hypochrysa. In males of H. elegans the T9-i-ect is fused, and in Kimochrysa impar (Tjeder) they are separate (males are undescribed for the other two Kimochrysa species) (Table 1). In females, the structures are partially fused in H. elegans and separate in all three species of Kimochrysa. It should be noted that, within at least two genera of Nothochrysinae {Pimachrysa Adams (males) and Nothochrysa (females)), this character is known to express interspecific variation. Consequently, given the small number of studied species within Hypochrysa and Kimochrysa (males: n - 1 for each genus; females: n = 4, with one showing an intermediate condition) (Table 1), this character lends only weak support for separating the two genera. The second character (an elongate male ectoproct) is reported from H. elegans [1, 23] but not from any other species in Nothochrysinae [2, 4]. Thus, although this character distinguishes H. elegans males from the single species of Kimochrysa whose males are studied, its phylogenetic value at the generic-level (versus species-level) remains open. The third character (sclerotized subgenitale) also is of questionable value at this time. Tjeder illustrated the tips of the subgenitale of the three Kimochrysa species. However, he was not specific about what he meant by the character and he did not include equivalent drawings of the Hypochrysa sub- genitale for comparison. Subsequent drawings and descrip- tions by other authors neither provide comparative informa- tion, nor mention any differences between the subgenitale of Hypochrysa and Kimochrysa [2, 23]. Second, the character state of the genitale has not been reported for species in other genera of Nothochrysinae for meaningful comparison. In sum, the generic-level value of the character needs further evaluation. 4.2.2. Brooks and Barnards Perspective. In subsequent studies. Brooks and Barnard [2] and Brooks [4] retained Kimochrysa s distinction as a genus, apparently based on three characters. First, like Tjeder [21], they noted that the condition of the ninth tergite and ectoproct differentiated Hypochrysa (fused) from Kimochrysa (whose structures they considered partially fused). As stated above, this character currently has little informative value regarding the synonymy. The second character was the presence or absence of microtholi on the male abdomen. This character differs between the single Kimochrysa species with known males (microtholi absent) and H. elegans (microtholi present) (Table 1). However, the condition is highly variable among the genera of Nothochrysinae both within and outside the group that contains Hypochrysa (Table 1). Thus, the character does not provide strong support either for or against the synonymy. The third character concerns the placement of the spirac- ular opening on the female eighth abdominal segment — on Psyche 7 Table 1: Summary of adult characters currently used for classifying Nothochrysinae. All known species are included. Species name Male T9 + ect* Female Character Micro tholi** c • 1 * * * Spiracle Nothochrysa grouping Asthenochrysa viridula (Adams) + [3] - [3, 18] -[3] -[3] Dictyochrysa fulva Esben-Petersen^ + [2, 19] + [19] ? -[19] latifascia Kimmins + [20] ? ? + [19] peterseni Kimmins + [19] + [2] ? +/- [19] Hypochrysa africana Kimmins^ ? - [2, 21, 22] ? + [2, 21] elegans (Burmeister)^ + [2, 23] +/- [2, 23] + [2, 23] + [2, 23] impar (Tjeder) - [2, 21] -[21] -[2] + [21] raphidiodes (Tjeder) ? -[21] ? + [21] Nothochrysa californica Banks^ + [1] ?[3] + [1] ? capitata (Fabricius)^ ? + [2] + [2] ? fulviceps (Stephens)^ + [2, 23] -[23] + [2, 23] + [23] indigena Needham + [24] ? ? ? sinica C.-k. Yang ? -[25] ? + [25] turcica Kovanci and Canbulat + [26] -[26] ? + [26] Triplochrysa pallida Kimmins + [2] -[19] -[2] + [2] kimminsi New + [19] - (prob.) [19] ? + [19] Pamochrysa grouping Leptochrysa prisca Adams and Penny ? -[18] ? -[3] Pamochrysa stellata Tjeder -[21] - [2, 21] -[2] -[21] Pimachrysa albocostalis Adams -[1] ? + (prob.) [1] ? fusca Adams - [1. 2] - [1. 2] + [1. 2] - [1. 2] grata Adams ? - (prob.) [1] ? ? intermedia Adams ? - (prob.) [1] ? ? nigra Adams + /- [1] - (prob.) [1] + [1. 2] ? Characters: *tergite 9 and ectoproct: fused (+), unfused (-), partially fused (+/-); * * microtholi on male sternites: present (+), absent (-); ***location of spiracular opening on female eighth abdominal segment: on membranous pleuron below T8 (+), on T8 (-), spanning both T8 and the membrane below (+/-); male or female unknown or character state not reported (?). ^Species with larvae described. Numbers in square brackets: references. the membrane versus on the tergite. For all know species of Hypochrysa and Kimochrysa, the spiracle opens on the membrane (Table 1); thus, it is consistent with the new synonymy. 4.3. Relationships among Genera of Nothochrysinae. The new synonymization of Kimochrysa with Hypochrysa led to a review and reevaluation of the current generic groupings within Nothochrysinae and the characters used to support the groupings. Previously, the subfamily was proposed to contain a derived “monophyletic” group of five genera (the “Nothochrysa group”) that included Hypochrysa and that was supported by characters presumed to be apomorphic. The four remaining genera, including Kimochrysa, were considered less derived and lacked apomorphic support [4]. The present findings favor the notion that the subfamily contains two generic groupings that are similar but not identical to those listed earlier. Moreover, the strength of the “apomorphic” support for the groupings is questioned for two main reasons. First, ancestral states for the presumed apomorphies lack strong supporting evidence. Second, the characters exhibit variation within the generic groupings and sometimes even within genera. Below is a summary of the now tentative groupings within Nothochrysinae followed by a discussion of the underlying 8 Psyche support and a recommendation for fuller comparative stud- ies. 4.3.1. Groupings of Nothochrysinae Genera. Nothochrysinae is now proposed to contain two relatively distinct groupings of genera. (i) Nothochrysa grouping. This category contains five genera: Asthenochrysa Adams, Dictyochrysa Esben- Petersen, Hypochrysa Hagen (including Kimochrysa), Nothochrysa McLachlan, and Triplochrysa Kimmins. Larvae have been described for three of the genera [7] . (ii) Pamochrysa grouping. This category contains three genera: Leptochrysa Adams and Penny, Pamochrysa Adams, and Pimachrysa Adams. Larvae are unde- scribed. 4.3.2. Supporting Characters. To provide perspective and encourage stronger comparative morphological studies, the three presumed “apomorphic characters” for these groupings are discussed below. (i) Character number 1 — ninth tergite (T9) and ectoproct of both sexes fused (versus separate). The ancestral state of this character is not determined, but an unfused condition has been considered plesiomorphic for Chrysopidae [2]. Among three neuropteran families considered to be related to Chrysopidae [5, 6, 27, 28], the T9 and ectoproct are separate in Polystoechotidae and variable (fused or unfused) in Hemerobiidae and Osmylidae [27, 29-32]. The fused character state occurs in various taxa of all three chrysopid subfamilies, including the two that are considered basal (Nothochrysinae and Apochrysinae) (for phylogenetic rela- tionships among chrysopid subfamilies, see [5, 6]; for the distribution of morphological features, see [2, 33, 34]). Prom the available literature, a fused condition occurs in males of all genera in the Nothochrysa grouping and in females of most genera. The condition is largely absent from both males and females of the Pamochrysa grouping (Table 1). The most parsimonious conclusion from the distribution of the features is that the fused T9 and ectoproct began to evolve within males of the Pamochrysa grouping, but that the presumed plesiomorphic state (separate T9 and ectoproct) was largely retained by both males and females. Apparently, full fusion appeared in males and fusion began in females during or soon after the differentiation of the Nothochrysa grouping. The variability (especially partial fusion) in the expres- sion of the trait may reflect differences in the degree of integumental sclerotization, rather than in the actual fusing of segments. It is well known that chrysopid adults become more sclerotized as they mature (often requiring a period of several weeks after emergence) and that their patterns of sclerotization vary considerably, individually and with age [1, 35-37] . Thus, caution is necessary in scoring and interpreting this character. (ii) Character number 2 — male sternites with microtholi being present (versus absent). Microtholi are not known from Neuroptera other than the Chrysopidae, and their absence is probably the basal state for the family. Within the Chrysopidae, microtholi are usually, but not always, absent in Apochrysinae males [2, 34], and their occurrence is highly variable among the Chrysopinae and Nothochrysinae (see [2]; also see Table 1). It is not clear why Brooks [4] considered this feature as an apomorphy for the Nothochrysa group, when earlier Brooks and Barnard [2] reported several genera in the group as being without microtholi and it was known that the structures occur within Pimachrysa. Thus, the feature’s pattern of occurrence is not consistent with its iden- tification as an apomorphy for the subfamily Nothochrysinae or for either of its proposed generic groupings. (iii) Character number 3 — female with spiracle on eighth abdominal segment opening on membrane (versus on ter- gite). The spiracular opening is consistently on the eighth tergite in Osmylidae [25, 27, 29, 38] and Polystoechotidae [30, 32] but its placement shows significant variation within Hemerobiidae [27, 31]. Among the Chrysopidae, the opening is on the membrane throughout the Apochrysinae and in most Chrysopinae and its placement is variable within Nothochrysinae see [4, 23, 35]; also (Table 1). The presumed, but as yet unconfirmed, plesiomorphic state for the Chrysop- idae is for the eighth abdominal spiracle to open on the tergite [4]. In Nothochrysinae, a spiracular opening on the eighth female tergite typifies the Pamochrysa grouping; however the numbers of exemplars/genus are very small {n - 1 /genus). In the Nothochrysa grouping, a spiracular opening on the pleural membrane appears to typify three of the five genera {Hypochrysa, Nothochrysa, and Triplochrysa), but in Dictyochrysa it is variable, and it is absent from the single species of Asthenochrysa (Table 1). An interesting interme- diate situation occurs in Dictyochrysa peterseni Kimmins; in this species the spiracular opening appears to span the membrane and the tergite [19, Pigure 66]. Thus, although Brooks may be correct in his proposal [4] that the spiracular opening on the membrane is a “stem apomorphic character” for the “Nothochrysa group,” it appears more likely that the character evolved within the Nothochrysa grouping after its differentiation. It is noteworthy that all species of the Chrysopinae subgenus Chrysopodes {Neosuarius) Adams and Penny have their spiracular opening on Tergite 8 [35] — a presumed reversal to the pleisiomorphic state. In this group, where large series of specimens are available for comparative study, there appears to be developmental and interspecific variation in the extent and intensity of the tergite’s lateral sclerotization, so that, in some specimens, especially those that are teneral, the spiracle appears to open on an unsclerotized portion of the pleuron membrane. If a similar situation occurs in the Nothochrysinae, it could present a confounding factor similar to that in Character number 1 above. 4.3.3. Future Studies. Prom the above, it is evident that the few adult characters currently used to explain the phylogeny of Nothochrysinae (the presumed basal chrysopid group) offer interesting but extremely limited information on the evolutionary history of the group and of the subfamily. Efforts to improve this situation have been hampered, in large part. Psyche 9 by the lack of specimens (both adult and larval) for many of the rare, crucial taxa. It is hoped that the discussion here helps stimulate efforts to collect and study this ancient group so that future studies will be based on a broad ranging set of characters (from comparative adult and larval morphology, molecular studies, and comparative biology) and the full range of taxa. Conflict of Interests The author declares that there is no conflict of interests regarding the publication of this paper. Acknowledgments The author thanks Peter Duelli (Swiss Federal Research Insti- tute WSL, Birmensdorf/Zurich, Switzerland) and Mervyn W. Mansell (University of Pretoria, Pretoria, South Africa) for generously providing the specimens of K. africana and Maurice J. Tauber for his helpful comments on the paper. The website “Lacewing Digital Library” (J. D. Oswald, chief editor) was helpful during this research [http://lacewing.tamu.edu/LDL/lacewingcitation.html] . The research benefitted from funding by the National Science Foundation, the USDA Competitive Grants Program, the National Geographic Society, and Cornell University (to Maurice J. Tauber and Catherine A. Tauber) and is part of Regional Project W-3185. References [1] P. A. Adams, “A review of the Mesochrysinae and Nothochrysi- nae (Neuroptera: Chrysopidae,” Bulletin of the Museum of Comparative Zoology, vol. 135, no. 4, pp. 215-238, 1967. [2] S. J. Brooks and P. C. Barnard, “The green lacewings of the world; a generic review (Neuroptera; Chrysopidae),” Bulletin of the British Museum of Natural History, Entomology, vol. 59, pp. 117-286, 1990. [3] P. A. Adams and N. D. Penny, “Review of the South American genera of Nothochrysinae (Insecta: Neuroptera: Chrysopidae,” in Current Research in Neuropterology: Proceedings of the 4th International Symposium on Neuropterology (24-27 June 1991, Bagneres-De-Luchon, Haute-Garonne, France), M. Canard, H. Aspock, and M. W. Mansell, Eds., pp. 35-41, M. Canard (Privately Printed), Toulouse, France, 1992. [4] S. J. Brooks, “An overview of the current status of Chrysopidae (Neuroptera) systematics,” Deutsche Entomologische Zeitschrift, vol. 44, pp. 267-275, 1997 [5] S. Winterton and S. de Freitas, “Molecular phylogeny of the green lacewings (Neuroptera: Chrysopidae),” Australian Jour- nal of Entomology, vol. 45, no. 3, pp. 235-243, 2006. [6] N. Haruyama, A. Mochizuki, P. Duelli, H. Naka, and M. Nomura, “Green lacewing phylogeny, based on three nuclear genes (Chrysopidae, Neuroptera),” Systematic Entomology, vol. 33, no. 2, pp. 275-288, 2008. [7] C. A. Tauber, M. J. Tauber, and G. S. Albuquerque, “Debris- carrying in larval Chrysopidae: unraveling its evolutionary history,” Annals of the Entomological Society of America, vol. 107, no. 2, pp. 295-314, 2014. [8] V. J. Monserrat and L. M. Diaz-Aranda, “Los estadios larvarios de los crisopidos ibericos (Insecta, Neuroptera, Chrysopidae), nuevos elementos sobre la morfologia larvaria aplicables a la sistematica de la familia,” Graellsia, vol. 68, no. 1, pp. 31-158, 2012. [9] L. M. Diaz-Aranda and V. J. Monserrat, “Aphidophagous preda- tor diagnosis: key to genera of European chrysopid larvae (Neur.: Chrysopidae),” Entomophaga, vol. 40, no. 2, pp. 169-181, 1995. [10] L. M. Diaz-Aranda, V. J. Monserrat, and C. A. Tauber, “Chapter 4.3. Recognition of early stages of Chrysopidae,” in Lacewings in the Crop Environment, P. K. McEwen, T. R. New, and A. E. Whittington, Eds., Cambridge University Press, Cambridge, UK, 2001. [11] F. Brauer, “Larve von Hypochrysa nobilis Heyd,” in Ver- handlungen der Kaiserlich-Koniglichen Zoologisch-Botanischen Gesellschaft in Wien, vol. 17, pp. 27-30, 1867. [12] C. A. Toschi, “The taxonomy, life histories, and mating behavior of the green lacewings of Strawberry Canyon (Neuroptera, Chrysopidae),” Hilgardia, vol. 36, no. 11, pp. 391-433, 1965. [13] T. R. New, “Some early stages of Dictyochrysa Esben-Petersen (Neuroptera, Chrysopidae),” Neuroptera International, vol. 1, no. 3, pp. 136-140, 1981. [14] P. Duelli, H. Holzel, and M. W. Mansell, “Habitat and lar- vae of the enigmatic genus Kimochrysa Tjeder (Neuroptera: Chrysopidae) in South Africa,” in Proceedings of the 10th International Symposium on Neuropterology (22-25 June 2008, Piran, Slovenia), D. Devetak, S. Lipovsek, and A. E. Arnett, Eds., pp. 153-158, University of Maribor, Maribor, Slovenia, 2010. [15] A. Rousset, Morphologic Cephalique des Larves de Planipennes (Insectes Nevropteroides), vol. 42 of Memoires du Museum Nationale d’Histoire Naturelle, Paris, Series A, Zoology, Editions du Museum, 1966. [16] S. Tsukaguchi, Chrysopidae of Japan (Insecta, Neuroptera), S. Tsukaguchi, Aioi-cho 6-14-102, Nishinomiya-shi, Hyogo, 662 Japan (Privately published), 1995. [17] C. A. Tauber, M. J. Tauber, and G. S. Albuquerque, “Berch- mansus elegans (Neuroptera: Chrysopidae): larval and adult characteristics and new tribal affiliation,” European Journal of Entomology, vol. 103, no. 1, pp. 221-231, 2006. [18] P. A. Adams, “A new species of Hypochrysa and a new subgenus and species ofMallada (Neuroptera: Chrysopidae),” Pan-Pacific Entomologist, vol. 54, no. 4, pp. 292-296, 1978. [19] T. R. New, “A revision of the Australian Chrysopidae (Insecta: Neuroptera),” Australian Journal of Zoology, Supplementary Series, vol. 28, no. 77, pp. 1-143, 1980. [20] D. E. Kimmins, “A revision of the genera of the Apochrysinae (Earn. Chrysopidae),” Annals and Magazine of Natural History: Series 12, vol. 5, no. 58, pp. 929-944, 1952. [21] B. Tjeder, “Neuroptera- Planipennia, The Lace-wings of South- ern Africa. 5. Family Chrysopidae,” in South African Animal Life, B. Hanstrom, P. Brinck, and G. Rudebec, Eds., vol. 12, pp. 228-534, Swedish Natural Science Research Council, Stockholm, Sweden, 1966. [22] D. E. Kimmins, “A new African Hypochrysa (Neuroptera),” Annals and Magazine of Natural History: Series 10, vol. 19, no. 110, pp. 307-308, 1937. [23] H. Aspock, U. Aspock, and H. Holzel, Die Neuropteren Europas, vol. 2, Goecke and Evers, Krefeld, West Germany, 1980. [24] S. K. Ghosh, “Contribution to the taxonomical studies of Neuroptera (suborder Planipennia) from eastern India. III. 10 Psyche Family Chrysopidae,” Records of the Zoological Survey of India, vol. 86, pp. 329-354, 1990. [25] C. Yang -K, “Neuroptera,” in Agricultural Insects, Spiders, Plant Diseases and Weeds ofXizang, S. Zhang, Ed., vol. 1, pp. 191-222, Xizang Renmin Press House, Xizang, China, 1st edition, 1987. [26] B. Kovanci and S. Canbulat, “A new species of the genus Nothochrysa McLachlan 1868 from northwestern Turkey (Neu- roptera: Chrysopidae) with a key to western Palaearctic species,” Annales de la Societe Entomologique de France, vol. 43, no. 2, pp. 165-168, 2007. [27] U. Aspock and H. Aspock, “Phylogenetic relevance of genital sclerites of Neuropterida (Insecta; Holometabola),” Systematic Entomology, vol. 33, no. 1, pp. 97-127, 2008. [28] S. L. Winter ton, N. B. Hardy, and B. M. Wiegmann, “On wings of lace: phylogeny and Bayesian divergence time estimates of Neuropterida (Insecta) based on morphological and molecular data,” Systematic Entomology, vol. 35, no. 3, pp. 349-378. [29] P. A. Adams, “A new genus and species of Osmylidae (Neu- roptera) from Chile and Argentina, with a discussion of Pla- nipennian genitalic homologies,” Postilla, vol. 141, pp. 1-11, 1969. [30] R M. Carpenter, “A revision of the Nearctic Hemerobiidae, Berothidae, Sisyridae, Polystoechotidae and Dilaridae (Neu- roptera),” Proceedings of the American Academy of Arts and Sciences, vol. 74, no. 7, pp. 193-280, 1940. [31] J. D. Oswald, “Revision and cladistic analysis of the world genera of the family Hemerobiidae (Insecta: Neuroptera),” Journal of the New York Entomological Society, vol. 101, no. 2, pp. 143-299, 1993. [32] J. D. Oswald, “Rediscovery of Polystoechotes gazullai Navas (Neuroptera: Polystoechotidae),” Proceedings of the Entomolog- ical Society of Washington, vol. 100, no. 3, pp. 389-394, 1998. [33] S. L. Winterton and S. J. Brooks, “Phylogeny of the Apochrysine green lacewings (Neuroptera: Chrysopidae: Apochrysinae),” Annals of the Entomological Society of America, vol. 95, no. 1, pp. 16-28, 2002. [34] C. A. Tauber, G. S. Albuquerque, and M. J. Tauber, “Charac- teristics of the Loyola Navas male (Neuroptera: Chrysopidae: Apochrysinae),” Proceedings of the Entomological Society of Washington, vol. 107, no. 3, pp. 543-547, 2005. [35] C. A. Tauber, “Revision of Neosuarius, a subgenus of Chrysopodes (Neuroptera, Chrysopidae),” ZooKeys, vol. 44, pp. 1-104, 2010. [36] C. A. Tauber, F. Sosa, and G. S. Albuquerque, “Two common and problematic leucochrysine species — Leucochrysa (Leucochrysa) varia (Schneider) and L. (L.) pretiosa (Banks) (Neuroptera: Chrysopidae): redescriptions and synonymies,” Zookeys, vol. 301, pp. 57-101, 2013. [37] C. A. Tauber, F. Sosa, G. S. Albuquerque, and M. J. Tauber, “Adults and larvae of two Leucochrysa (Leucochrysa) species (Neuroptera: Chrysopidae): descriptions, biological notes, and relationships,” Zootaxa, vol. 3750, no. 2, pp. 101-129, 2013. [38] J. D. Oswald, “Two new South American species of the genus Kempynus Navas (Neuroptera: Osmylidae: Kempyninae,” Pro- ceedings of the Entomological Society of Washington, vol. 96, no. 2, pp. 367-372, 1994. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 232057, 7 pages http://dx.doi.org/10.1155/2014/232057 Research Article Evidence for the Absence of Worker Behavioral Subcastes in the Sociobiologically Primitive Australian Ant Nothomyrmecia macrops Clark (Hymenoptera: Formicidae: Myrmeciinae) Robert W. Taylor ^ ARC Center of Excellence in Vision Science, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia ^ CSIRO Ecosystem Sciences, Canberra, ACT 0200, Australia Correspondence should be addressed to Robert W. Taylor; bob.taylor@homemaiLcom.au Received 27 November 2013; Revised 2 March 2014; Accepted 20 May 2014; Published 18 June 2014 Academic Editor: Jacques Hubert Charles Delabie Copyright © 2014 Robert W. Taylor. 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. Activity in three colonies of the nocturnally foraging Australian ant Nothomyrmecia macrops is investigated. Workers apprehended while foraging were marked, released, and later recaptured within nests following excavation. Every forager in each nest was encountered and marked. It was expected that unmarked, nonforaging, domestic-specialist workers would be discovered in the nests. This was unexpectedly not the case as all workers, apart from one or two in each colony, had been marked, and therefore had foraged at least once during the three-night experiment. The few unmarked individuals are considered to have been temporarily residential nest-entrance guards. Behavioral subcastes comprising “domestic” versus “foraging” workers were thus not indicated, evidencing absence of worker caste polyethism in Nothomyrmecia. The experiment predated emergence in the nests of adult workers from cocoon-enclosed pupae at a season when large feeding larvae of the current annual brood were still being provisioned by foragers. Because Nothomyrmecia is univoltine and emergence of current -brood adults had not yet occurred, all workers present were from preceding annual broods and defined as “postjuvenile.” A previous laboratory study separately evidenced absence of polyethism in Nothomyrmecia. Relevance of the apparent absence of food sharing in N. macrops is discussed. Dedicated to the memory of Roger Bartell (1940-1985) esteemed friend and colleague in much Nothomyrmecia field work 1. Introduction Following evolution of the gyne/queen and worker castes in ants, task specialization (division of labor or polyethism) among workers would likely early have involved distinction between (1) intranidal domestic functions and (2) extranidal food-collecting activities. This could encourage situations where workers operated either as domestics or as foragers, (1) intermittently, (2) in age-based succession, or (3) in either role essentially for life (thus comprising two behavioral subcastes exemplifying caste polyethism) (see [1-5]). The experiment reviewed here investigated the relativities of intranidal versus extranidal activity among workers in three colonies of the Australian endemic Dinosaur Ant Nothomyrmecia macrops Clark (subfamily Myrmeciinae), seeking to determine whether or not dedicated domestic versus specialist foraging subcastes were differentiated among Nothomyrmecia workers. The negative alternative was clearly evidenced. Phylogenetically N. macrops is a member of the myrme- ciomorph group of subfamilies in the formicoid clade [6, 7]. It appears to have retained many behavioural and sociobio- logical traits considered ancestral for ants as a whole [8, 9], and it lacks many of the sociobiologically advanced features known in other ants [8, 9]. The absence of caste polyethism has been previously reported in only one other ant species, the “sociobiologically 2 Psyche primitive” Stigmatomma pallipes (Haldeman) (Amblyoponi- nae) [10] (discussed there as Amblyopone pallipes, nomencla- ture here follows Yoshimura and Fisher [11]). Alternatively, the Nothomyrmecia-related, evidentially more sociobiologi- cally derived, myrmeciines Myrmecia brevinoda Forel [12], and M. gulosa (Fabricius) have bimodally size-ranged poly- morphic workers. In M. gulosa colonies the smallest workers associate with the queen, eggs and small larvae [13], and apparently never leave the nests [14 and pers. obs]. They appear to constitute a nest-bound non-foraging domestic worker sub -caste. Most ants, including Nothomyrmecia, Myrmecia, and Stigmatomma, maintain perennial univoltine colonies [9]. In general, a single annual brood is reared from eggs laid by queens during a few weeks in spring. Development to adulthood takes around 12 months. The growing larvae are eventually overwintered and begin to pupate the following spring, about a year after oviposition, at around the time eggs of the next generation are being laid. Adult workers eclose from pupation (prior to escaping pupal cocoons in the above genera) during the late spring and summer, concluding at around the autumn equinox. For this reason the presence or absence and relative proportions of eggs, hatchling, and other small larvae, large larvae, and pupae vary seasonally, and there is a long autumn/winter period during which larvae of a single generation alone are present as brood in nests (and during which there is very little extranidal activity by workers, apparently because the larvae do not feed in winter, and prey collection is suspended [9]). Colonies in spring contain eggs and recently hatched small larvae of the latest generation, along with large previously overwintered old larvae and pupae of the preceding generation, with egg numbers declining as hatching proceeds and large-larval numbers reducing as pupation progresses. As the season advances workers begin to eclose and escape from their pupal cocoons, and all emerge by late summer or early autumn, leaving only part-grown larvae of the latest generation which are subsequently overwintered. These factors dictated the seasonal timing of the experiment described here. Univoltinism results in recruitment of a single cohort of newly emerged workers each year during summer and early autumn, covering the time from beginning to end of eclosion of adults from pupation. Because of this, workers in mature colonies fall into discrete, usually morphologically unidentifiable, age-cohorts, each on average approximately one year younger than its predecessor. The number of worker generations present in any particular nest is determined by individual longevity and the age of the resident colony. Individuals of the earliest broods, including the first (initially laid down by queens at colony foundation), would often probably not survive to be represented in older colonies. “Juvenile” Nothomyrmecia workers are defined here as adults under several months of posteclosion age, which are too young to have experienced the winter following their emergence versus “postjuvenile” adult workers, compris- ing older, previously overwintered, individuals surviving in colonies from former annual brood cohorts. Significance of the first overwintering experience involves the theoretical possibility that any (if any) polyethism among juvenile workers would possibly be lost during their first experience of winter colony dormancy, when foraging for prey by workers and behavioral acts related to eggs, actively feeding larvae or pupae are suspended. The Nothomyrmecia colonies studied here were consid- ered to be at least 3 or 4 years old (based on their worker numbers, and that their experimentally marked active nest entrances had been noted approximately one and two years previously). Whether their oldest workers were survivors from the initial founding brood is not known. All workers present, however, would have been postjuvenile and more than 10 or 11 months old, given that the current nearly mature ca 1-year- old annual brood comprised large larvae and cocoon-enclosed pupae and had not yet produced (prospec- tively juvenile) adults. Two, three, or more annual age cohorts are estimated to have been represented. The experiment was conducted in mid-November, 48-54 days after the vernal equinox, in Mallee Eucalyptus wood- land at 85 m elevation, just south of Poochera, South Aus- tralia, about 600 m ESE of the Eyre Highway/Streaky Bay Road junction, at estimated coordinates 32°43^26.43^^S and 134“50'21.78"E. 2. Experimental Assumptions The following characteristics of Nothomyrmecia were assumed when designing the experiment or relate to dis- cussion of its results. All have been repeatedly observed or experimentally demonstrated. (See also references [8, 9, 15-17].) (1) Nests function independently of each other and there is no morphological polymorphism among workers. (2) Each nest has a single, obscure entrance hole about 4-5 mm in diameter. (3) At all times, night and day, single guard workers may be observed inside nest entrances. They emerge briefly following insertion of a disturbing filament or a live ant of another species. (4) Eoraging activity is strictly nocturnal. With the exception of assumption 11 below, Nothomyrmecia workers have never been observed naturally abroad in day- light. Departing foragers cross the ground promptly at dusk to ascend host trees on which they remain until returning directly across the ground at dawn to home nests. (5) Eoragers depart nests en masse at dusk, emerging from the entrance, often partly queuing briefly in close single files, over a period of 10 to 20 minutes beginning at about the time they cease to be discernable by naked eye. (6) Each forager “head- scans” on leaving the nest entrance, apparently confirming or recording visual navigational landmarks. The head, which is normally held parallel to the substrate, is pivoted slowly from side to side over an angle of about 80-100 degrees for up to 20 seconds while the ant stands otherwise motionless (often still partly within the nest entrance) with the antennal scapes held symmetrically, enclosing an angle of about 120 degrees. (7) Departing foragers disturbed experimentally near nest entrances after initial head scanning move away, rarely retreating back into the nest. (8) Prolonged uninter- rupted liquid feeding for personal nutrition (terminated and presumably satiated by full crop distension) by foragers at Psyche 3 interceptive diluted honey baits smeared experimentally on host tree trunks (Figure 2) does not suppress their behavioral drive to continue hunting for prey. Unless they eventually capture prey, these ants do not return to the nests until dawn. (9) Successful huntresses return to home nests during the night carrying the undissected prey in their jaws (Figure 3). (10) Unsuccessful huntress foragers return to nests at dawn. Their numbers usually greatly exceed those of previously returned successful foragers. The appearance of first-light apparently cancels their prey-getting drive, initiating nest return. Arrival at home nests is concentrated over 25-40 minutes from first light to approximately when the ants are faintly visible without artificial illumination. (11) Returnees failing to reach home nests before being overtaken by daylight secrete themselves in leaf litter near tree bases or ground- level bark crevices. And presumably return eventually to their nests. (12) Nothomyrmecia queens leave nests to forage during colony foundation, probably up to the time when their first daughter workers appear. They are not known to exit the nests of worker-right colonies. (13) All adult ants are liquid feeders [9, pages 592-593] . Nothomyrmecia workers seek and imbibe appropriate nourishment while foraging outside the nests. Captured prey is returned to nests without dissection, largely as food for the carnivorous larvae (with some in- nest liquid feeding on hemolymph labiated from the remains of larval- dissected prey by workers and by the queen). (14) Nothomyrmecia workers do not lay chemical scent trails, and navigation is entirely visual. 3. Experimental Procedure (1) Foraging workers departing an active Nothomyrmecia field nest were intercepted by covering the nest entrance with a large (ca. 190 mm dia., 20 mm deep) inverted clear glass evap- oration dish (Figure 1). This was placed during the day and revisited after dark following accumulation under its cover of prospective foragers which had emerged from the nest entrance at dusk. (2) All detained ants were collected, carried together to a nearby field caravan, and promptly marked with single mesosomal or gastral paint spots of a single color (using diluted Tipp-Ex typists correction fluid) applied with a fine artists brush under a low-power stereomicroscope. Specimens were collected by aspiration and handled using soft spring-steel forceps. They were not anaesthetized or chilled. The marked ants were then promptly released as a group within 10 cm of the uncovered nest entrance. (3) After all had dispersed the cover dish was replaced over the nest entrance and checked regularly during the night and at dawn for returned foragers standing on its upturned base or at its periphery. Any previously marked returnees were replaced under the dish. The cover was removed in the morning after the last returnees had accumulated. Returnees without color spots were intercepted and color marked. (4) These procedures continued nightly until all ants seen on two successive dusk-to-dawn periods were observed to have been previously color marked. (5) The nest was then fully excavated and all resident ants collected. (6) The numbers of marked and unmarked ants were recorded. (7) The experiment was Figure 1: Nothomyrmecia macrops Clark: prospective foragers impounded at dusk under experimental cover dish prior to colour marking. The nest entrance is at the top of the picture (Poochera, SA, nest “yellow,” R. W. Taylor). simultaneously replicated on three adjacent colonies situated in a triangle with sides approximately 3 M long; workers from each colony were separately distinguished by white, yellow, or green paint marks. 4. Experimental Results (1) All ants replaced near nest entrances after marking promptly performed head scans and moved off to forage. None were observed to return immediately to their nests. (2) Morning- marking was required for only one forager return- ing at dawn on day 2 (colony “green”). It was presumably overlooked the previous evening or had secreted itself during the previous day after failed colony return. (3) The numbers of successful foragers returning with prey during the night were low: 2 for colonies “white” and “green” on night 1, 1 for colony “yellow” on night 1, and 2 on night 2 (about 5.1% of the total worker count). The great majority of returnees accumulated outside the covering dishes during the dawn return period without prey. (4) Single nest-entrance guards were observed in all colonies both by day and at night while the foragers were abroad. None were collected for color marking for fear of disrupting their behavior. (5) The marking 4 Psyche Figure 2; Nothomyrmecia macrops Clark: foraging workers feeding nocturnally at experimental honey bait on host Eucalyptus tree trunk. These ants accumulated as individuals, without collaborative interaction. Note the progressively distended gasters from right to left. (Poochera, SA, R. W. Taylor). Figure 3: Nothomyrmecia macrops Clark: foraging worker with prey (male psyllid bug, Hemiptera: Psyllidae) (nocturnal, Poochera, SA, Ajay Narendra). of workers was completed on night 3 for all three colonies. All those accumulated under the cover dishes at dusk or on their surfaces or surrounds at dawn on nights 4 and 5 were observed to have been previously marked, terminating the experiment. (6) The colonies were excavated and tallied on day 6. (7) The frequency of marked versus unmarked workers collected from each colony is given in Table 1.(8) All recovered colonies contained only one queen. All contained the appropriate seasonal brood of old, near fully grown larvae and congenerational cocoon-enclosed pupae of the current annual generation, with small, young larvae of the following years cohort, destined to complete development to adulthood about 10-12 months later. There were no recognizable newly emerged callow workers or evidence (such as freshly cast pupal cocoons in nest middens) that eclosion had recently occurred. (9) No marked workers were recovered from nests other than those at which they were marked. (10) Queens were not observed to leave the nests. If hard-wired caste polyethism was present in the sub- ject colonies, dedicated domestic workers would not have departed the nests as foragers and would have been retrieved unmarked at excavation. This was not the case. The experi- mental results instead convincingly demonstrate that postju- venile workers in mature, actively foraging field colonies of Nothomyrmecia macrops during the peak annual prey-getting season regularly engage in foraging and that any intranidal activity by individuals generally does not persist continuously for more than a few days before being interrupted by foraging. In the three nests (1) 41-50% of workers foraged on the first night; (2) 28-37% of workers first foraged after at least 1 night spent in the nest; (3) 13-25% of workers first foraged after at least 2 nights spent in the nest. The consistency of results suggests that workers generally do not spend more than two nights in nests between foraging expeditions. During the three-night observation period 96-98% left the nests to forage. The interception of previously marked workers on nights 2 and 3 indicates departures by individuals on successive nights. Four unmarked workers (2.9% of the 137 in all 3 colonies) evidently did not leave the nests during 5 nights prior to excavation. The unmarked workers were probably nest-entrance guards. The observed presence of guards during the experi- ment supports this conclusion. The assumption that guards are temporarily excused from foraging is tenable. Supporting evidence is provided by Jaisson et al. [16] . It is unlikely that these ants were foragers previously overlooked for marking or newly emerged juvenile workers not yet behaviorally programmed to forage. They might have been members of a queen retinue. The numbers indicate, however, that all but one or two workers of any such group present on day 1 must have foraged during the experiment (even assuming that the stayers were not guards or juveniles). Experimental procedure prevented successful foragers from departing on a second same-night foray (they were inserted under the cover dish when apprehended in its vicin- ity with prey and so restricted to the nest for the remainder of the night). It is therefore not known if repeat foraging is practiced by Nothomyrmecia. By comparison, about 10% of individually numbered foragers of the phylogenetically related night-active bulldog ant Myrmecia pyriformis Smith have been reported to depart their nest repeatedly (usually twice, but up to 4 times in one individual) in a 1- night observation period [17]. In Nothomyrmecia (and Myrmecia pyriformis [17]) visual navigation across the ground between nest and host tree in dusk and dawn light is essentially crepuscular, and vision is probably less important than geotaxis for navigation on host trees during full darkness [15]. Nonetheless, nest return across the ground by prey-laden foragers in full nocturnal darkness clearly demonstrates capacity for visual navigation under such conditions. The Nothomyrmecia field program did not routinely record lunar phase, though for at least some nights of peak activity the Poochera site is clearly recalled to have been brightly moonlit. Future researchers should note this detail (see [18, 19]). The experiment would have been more informative if different sets of color marks had been used each night and foragers marked or remarked so as to record individual 5- night foraging histories. Also, the entrance guards should perhaps have been extracted and marked with separate identifying colors. Repetition of the experiment later in the season after emergence of juvenile workers would indicate whether there is a delay preceding foraging activity by juveniles after eclosion, because many more unmarked workers would then Psyche 5 Table 1: The numbers of previously unmarked foraging workers departing 3 Nothomyrmecia macrops nests on 3 consecutive experimental nights, with the total numbers present in each nest as determined by a later excavation. Percentages are those of the full tallies for day 6. Colony White Yellow Green Night 1 19 (41.3%) 26 (50.0%) 18 (46.1%) Night 2 17 (37.9%) 18 (34.6%) 11 (28.9%) Night 3 8 (17.3%) 7 (13.4%) 10 (25.2%) Totals 44 (95.6%) 51 (98.0%) 38 (97.4%) Excavated (day 6) 46 (44 + 2) 52 (51 + 1) 39 (38+ 1) (100%) (100%) (100%) be expected to remain in the nests during the experimental period. If colonies were marked as described here prior to the eclosion season and excavated weeks later following the emergence of juveniles, marked postjuvenile and unmarked juvenile workers would be distinguishable. Similar use of a cover dish to intercept returning foragers could provide frequency estimates of successful versus unsuccessful prey- collection departures and facilitate identification of prey organisms. 5. Observation Nest Studies Social organization in two laboratory-based queen-right observation colonies of N. macrops with individually num- bered workers was reported by Jaisson et al. [16]. The colonies were at approximately the same stage of annual brood cycle as those used in the field experiment discussed above, with a cohort of large larvae and pupae but no congenerational recently emerged workers, along with eggs and small larvae of a younger cohort. All workers must have been more than 10-11 months old. Results demonstrated that (1) there is a strong tendency by the ants not to engage in social interaction (interac- tive acts were less than 1% of the 4,002 recorded) and (2) very few workers performed queen-directed acts. Some temporarily located near the queen more often than others, but were seldom in direct contact. “Their high co-presence indices. . .indicate. . .that they constitute a functional “royal retinue”;” (3) worker polyethism was otherwise not apparent. Some individuals showed a propensity to guard the nest entrance. This activity “evidenced a high degree of special- ization among its executors.” Three workers performed as guards as well as foraging and caring for larvae in Colony 1, while one was involved at high frequency, and seven at very low frequency in Colony 2. None of the latter engaged in foraging, which in any case was relatively infrequent in colony 2; (4) exchange of food directly by trophallaxis or as trophic eggs was not observed between workers, workers and queen, or adults and larvae, apart from worker placement of intact, undissected prey near the larvae, which tore intersegmental membranes and dismembered prey carcasses by their own feeding activity; (5) the queen was observed to feed on hemolymph without assistance from workers, by licking larval-predissected prey; (6) there was no evidence of a dichotomy between dedicated domestic and foraging worker subcastes nor was there evidence in this study that workers specialized long term in performing particular in- nest activities, so it is reasonable to assume that this was also the case within the nests investigated in the field study reported here. 6. Discussion and Conclusions All available evidence indicates that division of labor is likely absent in postjuvenile Nothomyrmecia macrops workers, apart from slight tendencies for one or two individuals at any one time to act as nest-entrance guards or several as members of a royal retinue. These acts are temporary and do not divert participants for long from other tasks including foraging and brood tending. Individual service in the queen retinue is short-lived judging from the laboratory study and field results in which so few workers remained in nests over the 5 subject nights. Because their brood cycles antedated emergence of work- ers from cocoons, neither the field nor laboratory colonies discussed contained juvenile adult workers (as defined above). Future observation-nest investigation of juvenile- worker-right colonies could facilitate recognition (or not) of behavioral differences among juveniles or between juveniles and postjuveniles and the possibility (or not) of identifying such behavior as hard-wired age-based temporal polyethism. Until this is investigated it cannot be affirmed that inherently programmed polyethism is absent among Nothomyrmecia workers, despite the evidence of this account and Jaisson et al. [16]. That is the reason why juvenile workers have been distinguished and defined here. Future researchers will need carefully to consider the findings of Traniello and associates [ 1 , 2 ]. There is no evidence for intergenerational polyethism between workers of different annual brood cohorts, or among similarly aged workers of any single postjuvenile generational cohort nor, in the absence of anatomical polymorphism, is there any other evidence of caste polyethism among Nothomyrmecia workers. The Stigmatomma pallipes colonies studied by Traniello [10] contained both juvenile and postjuvenile workers. The former, recognizable as relatively recently emerged callows, engaged in both brood-tending and foraging similarly to older (postjuvenile) noncallow individuals, except that they were not involved in egg care. Some individuals showed a degree of attachment to particular tasks, but this did not exclude them from other actions, and none of the acts 6 Psyche under observation were significantly correlated. Traniello and Rosengaus [20] rightly point out that in these “primitive” ants “foraging is part of brood-care activity because larvae are directly provisioned with prey, and the same worker performs both tasks,” and so it is with Nothomyrmecia. Nothomyrmecia workers operate almost entirely “in the manner of solitary insects” (to use Wilsons expression [4]). Alleged behav- iorally programmed (as opposed to chance) interactions or cooperation between noncallow workers is extremely rare [15, 16]. Interactions between Nothomyrmecia individuals are predominantly those between workers and brood [16]. It would be difficult to envisage less -collegial adult ants! Exchange of food between workers, workers and queen, or adults and larvae by trophallaxis or as trophic eggs (as in Myrmecia [21, 22]) was not observed in Nothomyrmecia by Jaisson et al. [16] or by the author and others during many hours of focused or casual observation of labora- tory colonies. Neither has larval hemolymph feeding been observed, as in Stigmatomma oregonense Wheeler [23] (cited there as Amblyopone oregonensis), a species close to S. pallipes. The ability secondarily to distribute nourishment therefore appears to be lacking in N. macrops, which presumably might never have evolutionarily acquired this capacity (secondary loss of such valuable behavior seems unlikely). This is evi- dently yet another “primitive” sociobiological characteristic of Nothomyrmecia. Workers, like queens, lick hemolymph from prey in nests following its dismemberment by feeding larvae. When foraging, they feed at experimental honey baits for up to 45 minutes while their gasters expand visibly due to crop distension (Figure 2). They normally lick carbohydrate- rich psyllid lerps, honeydew, and other sugary materials on leaves. The specific need for workers to depart nests seeking liq- uid or soluble food for personal nourishment is exacerbated by the apparent absence of food-sharing behavior. Workers are known also to drink nocturnally at rainwater droplets accumulated in concave dry leaves lying on the ground (pers. obs.). It could be argued that the prospect for further evolu- tionary sociobiological progression in N. macrops is com- promised because food is not secondarily distributed within its colonies. For example, support of a domestic worker subcaste confined more-or-less permanently within nests might be functionally untenable, since its members might not receive adequate nourishment without personal foraging (though nest-bound queens clearly do survive and function effectively, apparently on prey hemolymph, perhaps on that alone in the apparent absence of food sharing). It is of interest that foraging workers, whatever their motivation for nest departure (whether personal hunger or stimulation by hungry larvae), engage in prey-hunting following personal satiation at experimental liquid-food baits. Thus, in terms of both personal feeding, and obtaining food for larvae, all departures are maximally utilized. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments Philip Ward, James Traniello, Keiichi Masuko, Ajay Narendra, Jochen Zeil, David Yeates, and anonymous referees gave helpful advice. References [1] M. L. Muscedere, T. A. Willey, and J. R A. Traniello, “Age and task efficiency in the ant Pheidole dentata: young minor workers are not specialist nurses,” Animal Behaviour, vol. 77, no. 4, pp. 911-918, 2009. [2] M. A. Seid and J. R A. Traniello, “Age-related repertoire expan- sion and division of labor in Pheidole dentata (Hymenoptera: Formicidae): a new perspective on temporal polyethism and behavioral plasticity in ants,” Behavioral Ecology and Sociobiol- ogy, vol. 60, no. 5, pp. 631-644, 2006. [3] R. O. Wilson, “Behavioral discretization and the number of castes in an ant species,” Behavioral Ecology and Sociobiology, vol. 1, no. 2, pp. 141-154, 1976. [4] R. O. Wilson, “The sociogenesis of insect colonies,” Science, vol. 228, no. 4707, pp. 1489-1495, 1985. [5] R. O. Wilson, “The Principles of caste evolution,” in Experimen- tal Behavioral Ecology, B. Holl-dobler and M. Lindauer, Eds., pp. 307-324, Gustav Fischer, Stuttgart, Germany, 1985. [6] P. S. Ward, “Phylogeny, classification, and species-level taxon- omy of ants (Hymenoptera: Formicidae),” Zootaxa, no. 1668, pp. 549-563, 2007. [7] G. S. Moreau, G. 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. [8] R. W. Taylor, “Nothomyrmecia macrops: a living-fossil ant rediscovered,” Science, vol. 201, no. 4360, pp. 979-985, 1978. [9] R. W. Taylor, “Bloody funny wasps: speculations on the evo- lution of eusociality in ants,” in Advances in Ant Systematics (Hymenoptera: Eormicidae) Homage to E. O. Wilson: 50 Years of Contributions, R. R. Snelling, B. L. Fisher, and P. S. Ward, Eds., vol. 80, pp. 580-609, Memoirs of the American Entomological Institute, 2007. [10] J. F. A. Traniello, “Gaste in a primitive ant: absence of age polyethism in Amblyopone” Science, vol. 202, no. 4369, pp. 770- 772, 1978. [11] M. Yoshimura and B. L. Fisher, “A revision of male ants of the malagasy amblyoponinae (hymenoptera: Formicidae) with resurrections of the genera Stigmatomma and Xymmer” PLoS ONE, vol. 7, no. 3, Article ID e33325, 2012. [12] S. Higashi and G. Peelers, “Worker polymorphism and nest structure in Myrmecia brevinoda Forel (Hymenoptera: Formi- cidae),” Journal of the Australian Entomological Society, vol. 29, pp. 327-331, 1990. [13] V. Dietemann, B. Holldobler, and G. Peelers, “Caste specializa- tion and differentiation in reproductive potential in the phylo- genetically primitive eeni Myrmecia gulosaj Insectes Sociaux, vol. 49, no. 3, pp. 289-298, 2002. [14] C. P. Haskins and E. F. Haskins, “Notes on the biology and social behaviour of the archaic ponerine ants of the genera Myrmecia and PromyrmeciaJ Annals of the Entomological Society of America, vol. 43, pp. 461-491, 1951. [15] B. Holldobler and R. W. Taylor, “A behavioral study of the primitive ant Nothomyrmecia macrops Clark,” Insectes Sociaux, vol. 30, no. 4, pp. 384-401, 1983. Psyche 7 [16] P. Jaisson, D. Fresneau, R. W. Taylor, and A. Lenoir, “Social orga- nization in some primitive Australian ants. I. Nothomyrmecia macrops Clark,” Insectes Sociaux, vol. 39, no. 4, pp. 425-438, 1992. [17] S. F. Reid, A. Narendra, R. W. Taylor, and J. Zeil, “Foraging ecology of the night-active bull ant Myrmecia pyriformis” Australian Journal of Zoology, vol. 61, no. 2, pp. 170-177, 2013. [18] A. Narendra, S. F. Reid, and J. M. Hemmi, “The twilight zone: ambient light levels trigger activity in primitive ants,” Proceedings of the Royal Society B: Biological Sciences, vol. Ill, no. 1687, pp. 1531-1538, 2010. [19] N. Kronfeld-Schor, D. Dominoni, H. de la Iglesia et al., “Chronobiology by moonlight,” Proceedings of the Royal Society B: Biological Sciences, vol. 280, no. 1765, 2013. [20] J. F. A. Traniello and R. B. Rosengaus, “Ecology, evolution and division of labour in social insects,” Animal Behaviour, vol. 52, pp. 209-213, 1997. [21] C. P. Haskins and R. M. Whelden, “Note on the exchange of ingluvial food in the genus MyrmeciaJ Insectes Sociaux, vol. 1, no. 1, pp. 33-37, 1954. [22] J. Freeland, “Biological and social patterns in the Australian bulldog ants of the genus MyrmeciaJ Australian Journal of Zoology, vol. 6, pp. 1-18, 1958. [23] A. Wild, “Observations on larval cannibalism and other behav- iors in a captive colony of Amblyopone oregonensisj Notes from the Underground, vol. 11, pp. 27-38, 2005. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 328030, 6 pages http://dx.doi.org/ 10 . 1 155/2014/328030 Research Article Immature Stages and Life Cycle of the Wasp Moth, Cosmosoma auge (Lepidoptera: Erebidae: Arctiinae) under Laboratory Conditions Gunnary Leon-Finale and Alejandro Barro Department of Animal and Human Biology, Faculty of Biology, University of Havana, Calle 25 No. 455 Entre I y J, Vedado, Municipio Plaza, 10400 Ciudad de la Habana, Cuba Correspondence should be addressed to Alejandro Barro; abarro@fbio.uh.cu Received 24 February 2014; Revised 20 May 2014; Accepted 29 May 2014; Published 9 July 2014 Academic Editor: Kent S. Shelby Copyright © 2014 G. Leon-Finale and A. Barro. 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. Cosmosoma auge (Linnaeus 1767) (Lepidoptera: Erebidae) is a Neotropical arctiid moth common in Cuban mountainous areas; however, its life cycle remains unknown. In this work, C. auge life cycle is described for the first time; also, immature stages are described using a Cuban population. Larvae were obtained from gravid wild females caught in Vihales National Park and were fed with fresh leaves of its host plant, the climbing hempweed Mikania micrantha Kunth (Asterales: Asteraceae), which is a new host plant record. Eggs are hemispherical and hatching occurred five days after laying. Larval period had six instars and lasted between 20 and 22 days. First and last larval stages are easily distinguishable from others. First stage has body covered by chalazae and last stage has body covered by verrucae as other stages but has a tuft on each side of A1 and A7. Eggs and larvae features agree with Arctiinae pattern. Pupal stage lasted eight days, and, in general, females emerge before males as a result of pupal stage duration differences between sexes. 1. Introduction Cosmosoma is a large Neotropical moth genus with approx- imately 155 species [1], and some of them are broadly distributed. Wasp moth Cosmosoma auge L. (Lepidoptera: Erebidae) occurs in Central America, South America, and the Caribbean islands. In Cuba, it occurs throughout the main island with big populations in the main mountainous areas. As in other moth taxa, knowledge about immature stages of this genus is lacking and there are no descriptions of their life cycle. Dyar [2] described C. myrodora immature stages from a Florida population and Castillo [3] described C. myrodora larval and pupal stages duration which were fed with three Mikania species, but no instar duration or larval morphology were taken into account. Wild larval ecological traits are also unknown for Cosmosoma species. In this paper we describe life cycle and larval stages for Cosmosoma auge from a Cuban population. We also report a new host plant for this species and some immature ecological traits. Other host plants reported for C. auge in HOSTS databases are Cecropia peltata (Cecropiaceae), Ipomoea sp. (Convolvulaceae), Lage- naria siceraria (Cucurbitaceae), Mikania pachyphylla, M. parviflora, and M. scandens (Asterales: Asteraceae) [4] . 2. Materials and Methods 2.1. Collecting and Rearing. Larvae were obtained from seven wild Cosmosoma auge females collected at Vinales National Park in September 2011. As Peterson [5] suggested, females were kept confined together in a plastic jar until they laid eggs. Newly hatched larvae were placed in plastic Petri dishes (100 X 10 mm) and they were provided with fresh host plant leaves daily. Larvae that hatched the same day were placed together in groups of 30 individuals. When growth desynchronization occurred, larvae were sorted in other Petri dishes in order to keep the same age groups to detect individual variations in stages length. Larvae were reared in captivity at Cojimar, Eastern Havana, under natural photoperiod, humidity, and temperature between September 29th and October 21st 2011. 2 Psyche Daily temperature fluctuated between 24 and 28° C with average of 26° C, and the relative humidity varied from 54 to 93% with average of 79%. Both abiotic variables were provided by Casablanca Meteorological Station, Havana. Host plant identification and ecological traits descriptions were made with botanical samples and animals from Sierra del Rosario Biosphere Reserve. Some individuals {n - 3) were fed with Cecropia peltata to prove its suitability as host plant. 2.2. Life Cycle and Description of Immature Stages. Egg laying patterns were described from wild clutches found on the host plant leaves. Duration of the egg stage was measured and proportion of infertile eggs was also recorded. Egg shape and dimension were described using eggs obtained from wild females caught in Sierra del Rosario Biosphere Reserve. Two base diameters and height were measured using an ocular micrometrer (0.05 mm precision) attached to a stereomi- croscope Olympus. Egg volume {N - 36) was calculated following Garcia- Barros [6]. Surface relief and micropilar details were observed with a SEM (250x magnification). Petri dishes were checked daily for head capsule exuviae to establish larval instar duration. The head capsule width was measured (using the same ocular micrometer) directly on the larvae while resting and was defined as the distance between outermost ocelli. Two body lengths per stage were measured. One length was taken after molt (minimum length) and another in the premolt phase (maximum length), when larvae have emptied their gut and became light yellow. Eirst and second instar larvae length were measured using the ocular micrometer mentioned above, and third to sixth instar larvae length were measured using a Vernier caliper with 0.05 mm precision. Larvae that pupated the same day were placed together to measure the duration of the pupal stage. Pupae maximal length and width were measured using the same Vernier caliper. At emergence, adults’ sex was recorded in order to detect differences in pupal stage duration among sex. Stehr [7] and Scoble [8] were followed for morphological terminology. Photographs of larvae, pupae, and adults were taken using a macro mode of a 10.1 megapixels digital camera. Eggs, at least three individuals of each larval stage, two pupae, and cocoons were preserved in 75% alcohol and housed in Eelipe Poey Natural History Museum, University of Havana. Eourth to sixth larval instars were killed in very hot water to prevent gut content decomposition. 2.3. Statistical Analysis. Mean and standard deviation of larval stages duration were calculated. Stage duration and pupal period length between sexes were compared using a randomization test from Monte Carlo algorithm, using the PopTools complement of Microsoft Excel; 10000 iterations were made. 3. Results 3.1. Host Plant and Ecological Considerations. Wild cater- pillars were found feeding on climbing hempweed Mika- nia micrantha Kunth (Asterales: Asteraceae) and laboratory Table 1: Larval stages duration of Cosmosoma auge raised at Cojimar, Havana, under laboratory conditions (September-October 2011 ). Instar Duration (days) Mean ± SD 1st (N = 40) 3 (N = 15) 4 (N = 25) 3.6 ± 0.49 2nd (N = 40) 2(N = 21) 3 (N = 19) 2.5 ± 0.50 3rd (N = 40) 2 (N = 13) 3 (N = 27) 2.7 ± 0.47 4th (N = 39) 2{N = 8) 3 (N = 31) 2.8 ± 0.44 5th (N = 35) 3 (N = 22) 4 (N = 13) 3.4 ± 0.59 6th (N = 24) 6 (N = 7) 7 {N = 17) 6.7 ± 0.46 cohorts were successfully raised with it. Larvae fed with Cecropia peltata spent most of the time walking all over the Petri dish; they did not eat the leaves and died of starvation. Cosmosoma auge is a multivoltine species with multiple generations along the year; adults were caught almost every month. In the wild, eggs were laid singly {n - 9), in pairs {n - 5), and in trios {n = 3) on the underside of mature leaves of its host plant. Only two clusters with five eggs and two clusters with eight and ten eggs, respectively, were found. Eggs were always found in the basal portion of host plant leaves, near to veins. Eirst and second instar larvae fed on the leaf epidermis while other instar larvae fed on all the laminae but not on the 1st and 2nd order veins. Second instar larvae fed on shed skin as all the other larval instars did. In the wild, 4th and 6th instar larvae were found feeding on leaves underside at night. Always one larva per leaf was found. In the wild, larvae were apparently hidden during the day but in laboratory conditions larvae of all instar fed at day also. When larvae were disturbed they fell down holding themselves with a silk thread. In the wild, we found empty cocoons on the upperface and the underface of host plant leaves. 3.2. Life Cycle. A total number of 134 eggs were obtained and 47 of these (35%) did not hatch; but they were not infertile because in all cases a well-formed embryo could be seen through eggshell. Eggs {n - 87) hatched days after oviposition at any time of day five. Larval stage lasted 20 to 22 (21.5 ± 0.66) days and pupae ended their development 8 to 10 (8.6 ± 0.56) days later. Instar’s duration was between two and four days for immature larvae and six or seven days for last instar larvae (Table 1). Only one larva had a four-day 4th instar period, and two larvae had two-day and five-day 5th instar, respectively. There were differences in larval stage duration: 1st and 5th instar lasted longer than 2nd, 3rd, and 4th instar (P < 0.001), and the 6th instar lasted significantly longer than all the previous instars (P < 0.003). Most of the 40 larvae that completed their life cycle reached the 6th instar; only three of them reached the 7th instar and were not included in this analysis. At the end of the larval phase, the larvae have a quiescent prepupal phase, which lasted about two days, and afterward they entered in the pupal stage. Pupal mortality was 15% of the 43 pupae considered. Adult emersion (n - 35) occurred at any time of the day, although most of it occurred in Psyche Emersion day ■ Females ■ Males Figure 1: Number of Cosmosoma auge imagoes (both sexes) that emerged in each emersion day. Specimens were raised under labo- ratory conditions at Cojimar, Havana (September-October 2011). the afternoon hours (n - 29). Pupal stage lasted eight to ten days. Females {n - 16) had a one day shorter pupal period than males {n - 19) (P < 0.0032). There were differences in emersion dynamics between sexes as a consequence of their differences in pupal period. The 68% of total females emerged in the first emersion day while males didn’t reach the 50% of adults emerged in the second emersion day (Figure 1). These results suggest that C. auge presents protogyny. Adult lifespan was 15 days maximum {n - 1). 3.3. Immature Stages. Eggs are hemispherical and upright, with 0.87 ± 0.05 mm diameter and 0.62 ± 0.05 mm height. Eggs volume was 0.25 ± 0.028 mm . The base was not flat but concave. Egg color is ivory due to its content; all thought the transparent and shiny eggshell gives a pearly appearance (Eigure 2(a)). There is no color change during development, though on eclosion day, the larva mandibles can be seen beneath the eggshell as two pale brown spots that are close together and mobile. Chorion was hexagonally and pentagonally reticulated (Eigure 2(b)) except for the micropilar area, which was rosette-like, composed by five petaloid cells (Figure 2(c)). In the eclosion day, the only difference was the presence of two tiny, mobile, and pale brown spots (larva mandibulae) on the egg surface. First instar larvae: minimum length 2.65 ± 0.25 mm {n - 62) and maximum length 3.75 ± 0.20 mm {n - 34), and head capsule width 0.40 ± 0.05 mm {n - 53). Body covered by chalazae and dorsal chalazae longer than lateral ones. Six stemmata in two rows, four dorsal stemmata forming a semicircle, and two ventral stemmata separated from dorsal ones by a distance equal to stemmata diameter. Prolegs with heteroideus crochets in a mesoseries, distal lobe with 10 big hooks in the middle and many little hooks in both sides. Body color was ivory, almost white, but A8, which was light yellow dorsally. Thorax and abdomen turn green when larvae feed because of their translucent body wall (Figure 3(a)). Head is 3 yellow, but stemmata are black and distal edge of mandibles is reddish brown. Heads have white and tiny setae. Larvae had some two-tone dorsal setae (black base and white tip) interspersed with white regular setae. Rarely, these bicolored setae were predominant on larva’s body, which looks grayish. All larval stages have bare venter. Second instar larvae: minimum length 4.10 ± 0.40 mm {n - 43) and maximum length 5.80 ± 0.65 mm {n - 48). Head capsule width was 0.50 ± 0.05 mm {n - 45). The main change in this instar is that chalazae are replaced by verrucae except the chalazae in the anterior edge of T1 which were oriented forward. Thorax segments T2 and T3 and abdominal segments were covered by verrucae. Verrucae with many white setae and two or three black and long setae. Legs and prolegs had white tiny setae. Setae in T2, T3, and A1 are longer than the other body’s setae. There were no significant color changes from first instar (figure 3(b)) in this or next stages. Third instar larvae: minimum length 7.80 ± 0.60 mm {n - 8) and maximum length 8.35 ± 0.45 mm {n - 18). Head capsule width was 0.75 ± 0.05 mm {n - 13). There are five black chalazae in the anterior edge of T1 oriented forward. Body covered by verrucae, included Tl. Verrucae had several black setae in T2, T3, and A1-A8. fourth instar larvae: minimum length 9.30 ± 1.45 mm {n - 23) and maximum length 11.65 ± 0.50 mm {n - 26). Head capsule width was 1.05 ± 0.05 mm {n - 15). Body covered by verrucae with white and black setae. Dorsal verrucae with some long setae and lateral verrucae with setae shorter than dorsal ones. Legs and prolegs covered by short, white, and thin setae. Oval spiracle in Tl and A1-A8. Head with some tiny setae. Fifth instar larvae: maximum length 15.60 ± 0.90 mm {n - 28). Head capsule width was 1.45 ± 0.05 mm {n - 21). Body covered by verrucae with white and black setae. Legs and prolegs have white and tiny setae. Spiracles in Tl and A1 had medium size, A8 spiracle is big and conspicuous, the rest are tiny. Sixth instar larvae: minimum length 15.40± 1.30 mm {n - 35) and maximum length 21.60 ± 2.00 mm {n - 22). Head capsule width was 1.95 ± 0.05 mm {n - 16). At the end of the larval stage they reached eight times their initial length at hatch and about 30% of this growth occurs during the last instar because of an increased growth rate (figure 4). Mature larva’s body was covered by verrucae with white setae, T2, T3, and A1 verrucae with some black setae. A1 and A7 had a pair of white tuft located laterally perpendicular to longitudinal body axis (figure 3(c)). Most of the tuft setae are white but it had some black plumed setae. There are gray setae in A8 and A9. Also, in A8 there are two bright yellow spots dorsally, one in each side of heart. In the prepupal phase, the mature larvae stopped feeding and searched for a place to pupate. Then, they remained quiet, emptied their gut, and turned bright yellow, especially setae (figure 3(d)). Only white setae changed color, black setae remained the same. Afterward they shrank and detached setae from their bodies to construct cocoons mixing them with silk. The denuded larvae are light yellow and they keep that way until early pupal stage. 4 Psyche Figure 2: Cosmosoma auge eggs. General view (a), chorion surface detail (b), micropilar area (c). Scale bar represents 1mm (a) and 500 f^m ((b) and (c)). (g) (h) Figure 3: Some immature stages and cocoon of Cosmosoma auge. First instar larva (a), second instar larva (b), last instar larva (c), prepupa (d), cocoon with pupa inside (e), recently molted pupa, dorsal view (f), pupa 12 hours before emersion, lateral view (g), and pupa just before emersion, dorsal view (h). Pupae: obtect and enclosed in a thin, walled, and bright yellow ellipsoidal cocoon (Figure 3(e)). Pupae length is 12.45 ± 0.65 mm {n - 43) and width is 4.60 ± 0.25 mm (n - 42). Abdominal segments with punctures moderately dense on dorsal part. Few setae dispersed on the lateral part close to the spiracles and also on dorsal part. Ventrally, the wing tips reach the A4 segment. The cremaster is weak and consisting of three rows of translucent hook-like setae only visible with a magnification of 40x. Pupae color is shiny pale yellow, head and thorax are translucent yellow but abdomen was whitish yellow. Newly formed pupae have two little dark spots on anterior mesonotum, which correspond with thoracic spiracles (Figure 3(f)). Eyes (only a line) and spiracles are brown, which quickly became black. After four days, eyes became brown oval areas and in the 5th day, they became black. In the 7th day, joints are light brown. In the 8th day, head and thorax as well as two stripes on the abdominal dorsum from A1 to A4 turn orange red (Figure 3(g)); wings and the four rear segments as well as a middorsal stripe on the thorax and abdomen between the red stripes are dark 5 Psyche Instar □ Mean n Mean ± 2*SE I Mean ± SD Figure 4; Growth curve of Cosmosoma auge larvae raised at Cojimar, Havana, under laboratory conditions (September-October 2011). In each stage lengths were taken in the premolt phase. gray. Just before emersion these gray zones turn black with dark blue iridescence (Figure 3(h)). Pupae show adult’s color and looked shiny at that point. After that, it turned opaque because pupae skin separates from imago within it. 4. Discussion Mikania micrantha is the first host plant reported for C. auge in Cuba and belongs to the same genus of M. scandens, host plant reported for North American C. myrodora [9]. It is known that Lepidoptera phytophagous larvae had a strong specificity for a host plant family or even genus [10]. As Thompson and Pellmyr [11] state, females are capable of dis- crimination among plant species, host plant genotypes, and host plants in different microhabitats. The record of Cecropia peltata as C. auges host plant could be a misidentification of larvae feeding on it. Also, it could be a result of taxonomic problems in C. auge, in case of being a complex group cryptic species. The finding of wild small clutches and solitary eggs, as in Florida’s C. myrodora [2], suggests that C. auge has a solitary larval strategy in concordance with Uvarov [12] definition. Wild larvae feeding pattern also reinforces this hypothesis. Larval growth under different density conditions was studied for this species (Leon-Finale and Barro unpublished data) and results agree with the hypothesis of larva solitary strategy in C. auge. According to Stamp [13], species with solitary larva reduced the probabilities of been found by parasitoids and predators, and also reduced food and pupation sites competition. Matsumoto [14] states that grouped larvae are at risk of suffering starvation due to host plant defoliation if plant is small or isolated. Mikania micrantha is a vine with limited leaves number and C. auge larvae are voracious in later stages. A large number of individuals could potentially defoliate a single plant quickly. Occasionally, M. micrantha grows grouped and there is potentially more food available for larvae. Females could detect this unusual availability of larval food [11] and laid larger clusters of eggs (eight or ten), as we detect in Sierra del Rosario population. All these arguments make a study to define C. auge larval strategy necessary. Eggs shape, surface relief, and color were similar to Florida’s C. myrodora eggs [2, 5] . Eggs are fairly uniform in structure throughout the subfamily [7]. Dyar [2] found that C. myrodora had an egg period length of eight days but we found an egg period of five days. This difference could a result of differences in abiotic parameters, as temperature. Time and size traits are affected by temperature, photoperiod, humidity, diet, density, and host quality [15] . Also this could be a prod- uct of inherent differences between C. auge and C. myrodora. As a whole, morphology of immature stages of C. auge is similar to general arctiid pattern [7, 8] and is very constant along non- first instar larvae in this species, without major changes in color or morphology. As in Phoenicoprocta capistrata [16], the first instar is only covered by chalazae bearing one long filiform seta and the intermediate instars are covered by verrucae. The last instar (6th) was morpholog- ically recognizable from the previous ones for the presence of one pair of tufts in A1 and A7. The drastic color change in the prepupal period could be a result of accumulation of colored substances in setae, in case of C. auge having transparent and hollow setae. The larvae features are basically the same of C. myrodora [2] but we observed six instars in almost all the cases instead of seven, also the head widths of all instars were larger in this work, but the seventh stage mature larvae in Dyar [2] have a wider head capsule than sixth stage mature larvae of C. auge. In C. auge, as in other Lepidoptera species [17-20], last instar is where maximal growth and reserve accumulation occurred, because it was the only instar that lasted more than four days and the larvae grew around 30% of their total length. In this case, the last instar could be a critical period determinant of adult size, fecundity, and lifespan. The adult size could be determinant in sexual selection [21], mate frequency [22], and number of eggs laid by females [23]. Life cycle of C. auge was similar to but more dynamic than other Arctiinae species, probably as a result of an intense feeding behaviour [24, 25], because larvae were feeding during day and night [7] . Besides C. auge, there are differences in pupal period between sexes in other Arctiinae species as Empyreuma pugione, Dyauxes ancilla, and Phoenicoprocta capistrata [16, 26, 27]. In those, as in C. auge, males emerge after females. Differences in emersion rate could be a result of sex specific needs in gonadal maturation or a strategy to avoid inbreeding [28]. Also, C. auge time lapse between the start of females and males’ sexual receptivity could be even longer if males consume pyrrolizidine alkaloids after the emersion as C. myrodora males do [9]. 5. Conclusions Cosmosoma auge life cycle was quite similar to that described for other Arctiinae species but was much more dynamic probably as a result of intense feeding behavior. Larvae are very constant in color and shape but pupae, on the other 6 Psyche hand, are brightly colored and variable, on the contrary of most other arctiid moths. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors would like to thank Elier Fonseca and Dania Saladrigas (Universidad de La Habana) for their help in maintenance of Cosmosoma auge cultures in laboratory, to Jorge L. Guerra and Sierra del Rosario Biosphere Reserve staff for their help in the field collect, to Ramona Oviedo for host plant identification, to Dr. William Conner for answering some questions about taxonomic status of C. auge and finally to Casablanca Meteorological Station for providing meteorological data. References [1] R. B. Simmons, S. J. Weller, and S. J. Johnson, “The evolution of androconia in mimetic tiger moths (noctuoidea; Erebidae: Arctiinae: Ctenuchina and euchromiina),” Annals of the Ento- mological Society of America, vol. 105, no. 6, pp. 804-816, 2012. [2] G. Dyar, “Preparatory stages of Cosmosoma auge Linn,” Psyche, vol. 7, no. 244, pp. 414-415, 1896. [3] J. A. Castillo, Desarrollo de Cosmosoma myrodora y Estigmene acrea (Lepidoptera: Arctiidae) en la maleza Mikania micrantha y las plantas nativas Mikania cordifolia y Mikania scandens (Asteraceae) en Elorida [Bachelor Thesis], Departamento de Ciencia y Produccion Agropecuaria, Zamorano, Honduras, 2012. [4] G. S. Robinson, P. R. Ackery, I. J. Kitching, J. W. Beccaloni, and L. M. Hernandez, HOSTS — A Database of the World's Lep- idopteran Hostplants, The Natural History Museum, London, UK, 2013, http://www.nhm.ac.uk/research-curation/projects/ hostplants/. [5] A. Peterson, “Some of the eggs moths among the Amatidae, Arctiidae and Notodontidae,” The Elorida Entomologist, vol. 46, pp. 169-182, 1963. [6] E. Garcia-Barros, “Egg size in butterflies (Lepidoptera: Papil- ionoidea and Hesperiodea): a summary of data,” Journal of Research on the Lepidoptera, vol. 35, pp. 90-136, 2000. [7] E W. Stehr, Immature Insects, Kendall/Hunt Publishing Com- pany, Dubuque, Iowa, USA, 1987. [8] M. J. Scoble, The Lepidoptera: Eorm, Eunction and Diversity, Oxford University Press, New York, NY, USA, 1992. [9] W. E. Conner, R. Boada, E C. Schroeder, A. Gonzalez, J. Mein- wald, and T. Eisner, “Chemical defense: bestowal of a nuptial alkaloidal garment by a male moth on its mate,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 26, pp. 14406-14411, 2000. [10] T. Gotoh, P. W. Schaefer, and N. Doi, “Pood plants and life cycle of Lymantria bantaizana Matsumura (Lepidoptera: Lymantri- idae) in northern Honshu, Japan,” Entomological Science, vol. 7, pp. 125-131, 2004. [11] J. N. Thompson and O. Pellmyr, “Evolution of oviposition behavior and host preference in Lepidoptera,” Annual Review of Entomology, vol. 36, pp. 65-89, 1991. [12] B. P Uvarov, Grasshoppers and Locusts, vol. 1, Cambridge University Press, London, UK, 1966. [13] N. E. Stamp, “Egg deposition patterns in butterflies: why do some species cluster their eggs rather than deposit them singly?” The American Naturalist, vol. 115, no. 3, pp. 367-380, 1980. [14] K. Matsumoto, “Population dynamics of Luehdorfia japonica Leech (Lepidoptera: Papilionidae) — II. Patterns of mortality in immatures in relation to egg cluster size,” Researches on Population Ecology, vol. 32, no. 1, pp. 173-188, 1990. [15] S. Nylin and K. Gotthard, “Plasticity in life-history traits,” Annual Review of Entomology, vol. 43, pp. 63-83, 1998. [16] L. Rodriguez-Loeches and A. Barro, “Life cycle and immature stages of the arctiid moth, Phoenicoprocta capistrataj Journal of Insect Science, vol. 8, article 5, 2008. [17] H. E. Bratley, “The oleander caterpillar Syntomeida epilais. Walker,” The Elorida Entomologist, vol. 15, no. 4, pp. 57-64, 1932. [18] D. H. Hebeck, “Life cycle of Neoerastria caduca (Lepidoptera: Noctuidae),” The Elorida Entomologist, vol. 59, no. 1, pp. 101-102, 1976. [19] R. C. Lederhouse, M. D. Einke, and J. M. Scriber, “The contributions of larval growth and pupal duration to protandry in the black swallowtail butterfly, Papilio polyxenesj Oecologia, vol. 53, no. 3, pp. 296-300, 1982. [20] R. C. Stillwell and G. Davidowitz, “Sex differences in phenotypic plasticity of a mechanism that controls body size: implications for sexual size dimorphism,” Proceedings of the Royal Society B: Biological Sciences, vol. Ill, no. 1701, pp. 3819-3826, 2010. [21] M. C. Singer, “Sexual selection for small size in male butterflies,” The American Naturalist, vol. 119, no. 3, pp. 440-443, 1982. [22] N. Wedell and P. A. Cook, “Butterflies tailor their ejaculate in response to sperm competition risk and intensity,” Proceedings of the Royal Society B: Biological Sciences, vol. 266, no. 1423, pp. 1033-1039, 1999. [23] T. Tammaru, K. Ruohomaki, and K. Saikkonen, “Components of male fitness in relation to body size in Epirrita autumnata (Lepidoptera, Geometridae),” Ecological Entomology, vol. 21, no. 2, pp. 185-192, 1996. [24] C. V. Covell Jr., A Eield Guide to Moths Eastern North America, vol. 30 of The Peterson Eield Guide Series, 1984. [25] S. W. Applebaum and Y. Heifetz, “Density-dependent physio- logical phase in insects,” Annual Review of Entomology, vol. 44, pp. 317-341, 1999. [26] A. Otazo, N. Portilla, E. Coro, and P. Barro, “Biologia y conducta de Empyreuma pugione (Lepidoptera: Ctenuchidae),” Ciencias Biologicas, vol. 11, pp. 37-48, 1984. [27] P. Betzholtz, “The discrepancy between food plant preference and suitability in the moth Dysauxes ancillaj Web Ecology, vol. 4, pp. 7-13, 2003. [28] C. Wiklund and T. Eagerstrom, “Why do males emerge before females? A hypothesis to explain the incidence of protandry in butterflies,” Oecologia, vol. 31, no. 2, pp. 153-158, 1977. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 530757, 6 pages http://dx.doi.org/10.1155/2014/530757 Research Article Report on a Large Collection of Merope tuber Newman, 1838 (Mecoptera: Meropeidae), from Arkansas, with Notes on Collection Technique, Sex Ratio, and Male Clasper Size Michael J. Skvarla, Jessica A. Hartshorn, and Ashley P. G. Dowling Department of Entomology, University of Arkansas, 319 Agriculture Building, Fayetteville, AR 72701, USA Correspondence should be addressed to Michael J. Skvarla; mskvarla36@gmaiLcom Received 1 July 2014; Accepted 21 August 2014; Published 31 August 2014 Academic Editor: Russell Jurenka Copyright © 2014 Michael J. Skvarla 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. A large collection of earwigflies, Merope tuber, is reported from Arkansas, and flight period and sex ratio are discussed. In contrast to previous studies, earwigflies were caught more frequently in pan traps than in Malaise traps and male clasper size was found not to be bimodal. 1. Introduction Merope tuber Newman, 1838, known as earwigflies or for- cepflies, are uncommonly collected and have fascinated entomologists since their discovery in 1837 (Figure 1). This fascination was initially due to their presumed rarity — only 16 specimens were collected between their discovery and 1904 [1]. Since then, they have continued to receive attention due to their previously assumed basal phylogenetic position within Mecoptera, relatively unknown life history, undescribed larvae, and odd appearance relative to other Mecoptera (e.g., flattened body, opisthognathous head, and broad wings folded over the abdomen) [2, 3] . Only two other extant meropeids exist: Austromerope poultoni Killington, 1933 [4], from Western Australia and Austromerope brasiliensis Machado et al, 2013 [3], from Brazil. One extinct species, Boreomerope antiqua Novok- schonov, 1995 [5] , is known from Middle Jurassic lacustrine claystone near Kubekovo Village in Siberia. Four extinct species of Thaumatomerope (i.e., T. madygenica Rasnitsyn, 1974, T. minuta Rasnitsyn, 1974, T. oligoneura Rasnitsyn, 1974, and T. sogdiana Rasnitsyn, 1974) were originally assigned to Meropeidae but were later reassigned to Thaumatomeropidae [6, 7]. Collections of M. tuber continue to be infrequent. Prior to 1954, it was reported only from areas in or east of the Appalachian Mountains. Since then, the known range has been extended north to southern Ontario [8-10], west to Minnesota [11, 12], Iowa [13], Missouri [14-16], Arkansas [13, 16, 17], and Kansas [13], and south to Alabama [18], Georgia [17], and Florida [19, 20]. Rather than true emigration, this range expansion is best explained by the increased use of various passive trapping techniques [14]. Merope tuber have been collected using Malaise traps, picric acid traps, European chafer traps, carbon dioxide traps, molasses traps, and glue traps [2, 12, 21] , with the most effective being Malaise traps [22]. Little is known about the life history of M. tuber. Adults are nocturnal and attracted to light at night and spend daylight hours under logs and stones [1, 21]. They seem to be associated with moist deciduous woodlands near water [21, 23], although they are occasionally caught in dry grasslands far from any stream or creek [10]. Feeding preferences are unknown, although they may be attracted to carrion [2] similar to another mecopteran, Notiothauma reedi McLachlan, 1877, which has been reported from vertebrate carrion [24]. Adults stridulate by rubbing the jugum of the forewing against the metanotum [25]. The larvae of all meropeids, including M. tuber, remain undescribed [26] and their discovery “is certainly the most exciting thing left to be done in the study of North American Mecoptera” [14]. 2 Psyche Figure 1: Merope tuber, male. Figure 2: Overhead view of the field site at Steel Creek, with approximate limits of the site and blocks and acre/hectare scales in yellow. Base image taken from Google Earth [29]. The flight period of M. tuber lasts throughout the summer with some variation depending on latitude. They have been reported to occur in June through October in Connecticut [27], June through September in Maryland [28], July through September in Ohio [26] , May through September in Alabama [18], and April through December in Florida [19, 20]. Few studies have reported M. tuber in significant num- bers, but, in those that do, the sex ratio appears to be female biased. Scarbrough [30] collected 8 males and 18 females (1 male: 2.25 females) in two Malaise traps over a period of three years. Maier [27] collected 26 males and 43 females (1 male : 1.65 females) in a single Malaise trap over three years. Barrows and Flint [28], in six Malaise traps over the course of seven months, caught no males and 35 females. Johnson [26], in a single Malaise trap over two years, caught 61 males and 102 females (1 male : 1.67 females), the largest number of earwigflies yet reported from a single site. It is not known whether the sex ratio is truly skewed or if sampling bias is the cause. Unlike life history, much is known about the morphology of M. tuber, with both internal and external anatomy of both sexes being well documented [31-34] . Males have elongated genital styli (= claspers) that are thought to be used in mating Basistylus Dististylus Figure 3: Clasper of male Merope tuber with basistylus and dististylus labeled. Date Figure 4: Number of Merope tuber collected across all traps per date. as in other Mecoptera, either holding the female during copulation, fighting rival males, or both [26]. A bimodal distribution in clasper size has been demonstrated for at least one population with differential mating strategies being suggested as a possible cause [26]. 2. Materials and Methods As part of a more extensive arthropod sampling project, five blocks were established at a 4 ha plot located at Steel Creek along the Buffalo National River in Arkansas (Figure 2). In each block, five pan traps (one of each color: blue, red, green, yellow, and white) were randomly arranged under a terrestrial Malaise trap (MegaView Science Co., Ltd., Taichung, Taiwan), which was placed in perceived flight paths. In addition, three Lindgren funnel traps (ChemTica Internacional, S.A., Heredia, Costa Rica) (one of each color: green, purple, and black) were suspended nonrandomly from large trees 4-10 meters from the ground in the lower canopy. Psyche 3 Table 1: Total number of Merope tuber collected per trap type per block, with subtotals of trap type and block. Block Number of females caught Number of males caught Total caught Trap type Malaise trap 1 0 0 0 Pan trap (purple) 1 1 1 2 Pan trap (yellow) 1 1 0 1 Pan trap (blue) 1 0 0 0 Pan trap (white) 1 1 0 1 Pan trap (red) 1 0 0 0 Malaise trap 2 0 1 1 Pan trap (purple) 2 2 0 2 Pan trap (yellow) 2 1 0 1 Pan trap (blue) 2 2 1 3 Pan trap (white) 2 2 1 3 Pan trap (red) 2 4 1 5 Malaise trap 3 0 0 0 Pan trap (purple) 3 2 0 2 Pan trap (yellow) 3 0 0 0 Pan trap (blue) 3 0 1 1 Pan trap (white) 3 1 0 1 Pan trap (red) 3 1 1 2 Malaise trap 4 0 0 0 Pan trap (purple) 4 5 3 8 Pan trap (yellow) 4 8 2 10 Pan trap (blue) 4 7 3 10 Pan trap (white) 4 2 2 4 Pan trap (red) 4 2 1 3 Malaise trap 5 1 0 1 Pan trap (purple) 5 2 3 5 Pan trap (yellow) 5 5 1 6 Pan trap (blue) 5 2 1 3 Pan trap (white) 5 4 0 4 Pan trap (red) 5 2 1 3 Trap subtotal Malaise trap — 1 1 2 Pan trap (purple) — 12 7 19 Pan trap (yellow) — 15 3 18 Pan trap (blue) — 11 6 17 Pan trap (white) — 10 3 13 Pan trap (red) — 9 4 13 Block subtotal — 1 3 1 4 — 2 11 4 15 — 3 4 2 6 — 4 24 11 35 — 5 16 6 22 Total — 58 24 82 4 Psyche Table 2: Minimum, maximum, and mean measurements of various body parts and results of Shapiro-Wilk goodness-of-fit tests on the same. P < 0.05 is considered significant. Significant values are indicated by an asterisk (*). Measurement Sex Minimum (mm) Maximum (mm) Mean (mm) SD (mm) W Prob. < W Head width Female 0.8 1.32 1.1 0.12 0.97 0.247 Pronotum width Female 1.06 1.69 1.41 0.16 0.97 0.196 Forewing length Female 8.86 13.28 11.66 0.9 0.98 0.337 Abdomen length Female 4.1 8.96 6.44 1.3 0.97 0.153 Head width Male 0.77 1.39 1.11 0.15 0.96 0.534 Pronotum width Male 0.95 1.63 1.31 0.17 0.97 0.756 Forewing length Male 9.52 13.39 11.82 1.04 0.971 0.695 Abdomen length Male 4.07 7.61 5.8 0.78 0.95 0.206 Basistylus length Male 2.21 5.09 4.05 0.77 0.95 0.265 Dististylus length Male 1.47 2.91 2.34 0.43 0.91 0.036* Clasper total length Male 3.68 7.97 6.38 1.17 0.94 0.138 Four blocks contained a SLAM (Sea, Land, and Air Malaise, MegaView Science Co., Ltd., Taichung, Taiwan) trap (with top and bottom collectors counted as separate traps). Three blocks contained pitfall trap sets placed every five meters along a transect centered on a Malaise trap. Two of these blocks contained eight pitfall trap sets and one block contained a single set. Pitfall traps were modified from a design proposed by Nordlander [35], which Lemieux and Lindgren [36] demon- strated that it catches carabids in similar numbers but is more efficient at excluding small vertebrate bycatch. Rather than cutting circular entrances in the sides of pitfall traps, we cut three slots, 2 cm tall x 9.3 cm wide and 2 cm under the rim in the sides of plastic soup containers leaving three 1.5 cm posts, equidistant apart, resulting in a 28 cm collecting surface. Diameter at the base of slots is approximately 10.5 cm and the cups are 10.5 cm deep below these slots, resulting in a collecting volume of 2,988 cm . This allowed the matching lid to be secured to the cup instead of using a separate cover. A single cup was placed on either side of a 30.5 cm x 15.5 cm aluminum fence to make a pitfall trap set and the catch from both cups was combined and treated as a single sample. Propylene glycol (Peak RV & Marine Antifreeze) (Old World Industries, LLC, Northbrook, IL) was used as a preservative in all trap types. Traps were placed on March 13, 2013, taken down on December 4, 2013, and collected approximately every two weeks. Trap catch was sieved in the field and stored in Whirl-Pak bags (Nasco, Fort Atkinson, WI) in 90% ethanol until sorting. After sorting, specimens were stored individually in 2 mL microtubes (VWR Interna- tional, LLC, Randor, PA) in 70% ethanol. Voucher specimens have been submitted to the University of Arkansas Arthropod Museum. Head width, pronotum width, wing length, and abdomen length were measured for both sexes. The lengths of the basistylus and dististylus (Figure 3) were measured on the right side of males and combined to measure total clasper length. Measurements were made in the following manner: photographs of a millimeter ruler and dorsal and ventral aspect of each specimen were taken through the eye piece of a Leica MZ 16 stereomicroscope with the camera on an HTC Droid Incredible 4 G LTE; zoom was not adjusted between photographs to ensure they were to the same scale. All photographs were exported onto a desktop computer, opened in Image} [37], and measurements were taken by tracing the structures. Measurements were recorded in Microsoft Excel (Redmond, WA). Shapiro-Wilk goodness-of-fit tests {a - 0.05) were performed in JMP (SAS Institute, Cary, NC) to test normality of previously described measurements. An F-test for signifi- cance was performed by creating a generalized linear model (GLM) with a Gaussian distribution {a = 0.05). Count data were not normally distributed and required transformation. Because the data contained many zeroes, one was added to each count and before a natural log transformation. Because five pan traps were placed with a single Malaise trap, trap types could not be compared due to extremely skewed sample sizes. Instead, Malaise traps were considered a “color” in analyses and tested against each pan trap color. This simultaneously allowed for comparisons among variables of equal sample sizes for both trap type and pan color. 3. Results and Conclusions All totaled eighty-two earwigflies — 24 males and 58 females (1 male : 2.42 females) — were collected (Table 1). This female- biased collection is in line with previous studies [26-28, 30]. Earwigflies were first collected in late June, with the largest collection occurring in July, followed by low, but consistent, numbers caught until late October (Eigure 4). The beginning and end of the flight period were consistent with other areas at similar latitudes [19, 26-28] . Only a single body measurement, the dististylus, differed significantly from a normal distribution, but not in a bimodal manner (Table 2). These results are in contrast to previous studies, which found a bimodal distribution in the size of male basistyli, dististyli, and total clasper length [26]. As the use of the claspers is unknown, the significance of this is also unknown. Earwigflies were not caught in SLAM traps, Lindgren funnel traps, or pitfall trap sets; therefore, these traps were excluded from analyses. Significantly fewer M. tuber were caught in Malaise traps compared to pan traps (t - -2.455, Psyche 5 d.f. - I, P - 0.0145), although pan trap colors were not significantly diflferent from each other. This is the first report of earwigflies being collected in pan traps; however, previous studies which reported large collections of M. tuber traditionally used Malaise traps alone. It should be noted that, because pan traps were directly under Malaise traps, it is unknown whether those pan trap -collected individuals would have been captured in the Malaise trap collecting head, had pan traps not been present. Significantly more earwigflies were caught in blocks 4 {t = 4.307, d.f. = 1, P = 0.00002) and 5 (f = 2.479, d.f. = 1, P - 0.0136) than in blocks 1, 2, and 3. This suggests that trap placement and microhabitat, even within a relatively small area of a few hectares, are important factors when collecting earwigflies. If earwigflies are specifically targeted, we suggest placing multiple traps in an area of known occurrence in order to maximize the microhabitats sampled and increase the chance of collecting these enigmatic insects. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The authors thank Danielle Fisher for her assistance in sorting samples. This project and the preparation of this publication were funded in part by the State Wildlife Grants Program (Grant no. T39-05) of the U.S. Fish and Wildlife Service through an agreement with the Arkansas Game and Fish Gommission. References [1] H. S. Barber, “The occurrence of the earwig-fly, Merope tuber Newman,” Proceedings of the Entomological Society of Washing- ton, vol. 1, no. 1, pp. 50-51, 1904. [2] J. L. Pechal, M. E. Benbow, and J. K. Tomberlin, “Merope tuber newman (mecoptera; meropeidae) collected in association with carrion in greene county, Ohio, USA; an infrequent collection of an elusive species,” The American Midland Naturalist, vol. 166, no. 2, pp. 453-457, 2011. [3] R. J. P. Machado, R. Kawada, and J. A. Rafael, “New continental record and new species of Austromerope (Mecoptera, Meropei- dae) from Brazil,” ZooKeys, vol. 269, pp. 51-65, 2013. [4] R J. Killington, “A new genus and species of Meropeidae (Mecoptera) from Australia,” The Entomologists Monthly Mag- azine, vol. 69, pp. 1-4, 1933. [5] V. Novokschonov, “Der alteste vertreter der meropeidae (Mecoptera, Insecta),” Palaeontologische Zeitschrift, vol. 69, pp. 149-152, 1995. [6] A. P. Rasnitsyn, “Taxonomic names, in new Mesozoic and Cenozoic Protomecoptera,” Paleontological Journal, vol. 8, pp. 493-507, 1974. [7] V. G. Novokshonov, “Scorpion flies of the family Permocho- ristidae, the closest common ancestors of extant scorpion flies (Insecta, Panorpida-Mecopera),” Zoologischeskii Zhurnal, vol. 73, no. 7-8, pp. 58-70, 1994. [81 H. Weidner, “Merope tuber Newman in Canada,” in Entomol- ogische Mitteilungen aus dem Zoologischen Staatsinstitut und Zoologischen Museum Hamburg, vol. 3, pp. 45-46, 1964. [91 D. K. B. Cheung, S. A. Marshall, and D. W. Webb, “Mecoptera of Ontario,” Canadian Journal of Arthropod Identification, vol. 1, 2006. [101 S. M. Paiero, S. A. Marshall, P. D. Pratt, and M. Buck, “Insects of Ojibway Prairie, a southern Ontario tallgrass prairie,” in Arthropods of Canadian Grasslands. Volume 1: Ecology and Interactions in Grassland Habitats, J. D. Shorthouse and K. D. Floate, Eds., pp. 199-225, Biological Survey of Canada, Ontario, Canada, 2010. [Ill J- W. Barnes, “Notes on Minnesota Mecoptera,” Entomological News, vol. 67, pp. 191-192, 1956. [12] D. W. Webb, N. D. Penny, and J. C. Marlin, “The Mecoptera, or scorpionflies, of Illinois,” Illinois Natural History Survey Bulletin, vol. 31, no. 7, pp. 250-316, 1975. [131 G. W. Byers, “Autumnal Mecoptera of Southeastern United States,” University of Kansas Science Bulletin, vol. 55, pp. 57-96, 1993. [141 G. W. Byers, “Descriptions and distributional records of Ameri- can Mecoptera. Ill,” Journal of the Kansas Entomological Society, vol. 46, pp. 362-375, 1973. [15] C. C. Coffman, “Merope tuber Newman (Mecoptera: Meropei- dae) records from West Virginia collections,” Proceedings of the West Virginia Academy of Science, vol. 54, pp. 48-53, 1982. [16] D. E. Bowles and R. W. Sites, “Merope tuber (Mecoptera: Meropeidae) from the Interior Highlands of the United States,” Entomological News, vol. 123, no. 2, pp. 155-160, 2013. [17] H. W. Robison, G. W. Byers, and C. A. Carlton, “Annotated checklist of the Mecoptera (Scorpionflies) of Arkansas,” Ento- mological News, vol. 108, no. 4, pp. 313-317, 1997. [18] T. L. Schiefer and J. C. Dunford, “New state record for Merope tuber Newman (Mecoptera: Meropeidae) in Alabama,” Journal of Entomological Science, vol. 40, no. 4, pp. 471-473, 2005. [191 J- C. Dunford, R W. Kovarik, L. Somma, and D. Serrano, “First state records for merope tuber (Mecoptera: Meropeidae) in Florida and biogeographical implications,” Elorida Entomolo- gist, vol. 90, no. 3, pp. 581-584, 2007. [201 L. A. Somma, “New collections and record for earwigflies and scorpionflies (Mecoptera: Meropeidae and Panorpidae) in Florida,” Insecta Mundi, vol. 165, pp. 1-4, 2011. [21] G. W. Byers, “Zoogeography of the Meropeidae (Mecoptera),” Journal of the Kansas Entomological Society, vol. 46, pp. 511-516, 1973. [221 G. W. Byers, “Order Mecoptera. Scorptionflies and hang- ingflies,” in Borror and DeLong’s Introduction to the Study of Insects, C. A. Triplehorn and N. F. Johnson, Eds., pp. 662-668, Thomson Brooks/Cole, Belmont, Calif, USA, 7th edition, 2005. [23] G. W Byers and R. Thornhill, “Biology of the Mecoptera,” Annual Review of Entomology, vol. 28, pp. 203-228, 1983. [24] E. Jara-Soto, C. Mu, V. Jerez, and C. Munoz-Escobar, “Registro de Notiothauma reedi McLachlan, 1877 (Mecoptera; Eomerop- idae) en cadavers de vertebrados en la Comuna de Concepcion, Chile,” Revista Chilena de Entomologia, vol. 33, pp. 35-40, 2007. [25] P. M. Sanbourne, “Stridulation in Merope tuber (Mecoptera: Meropeidae),” The Canadian Entomologist, vol. 114, no. 3, pp. 177-180, 1982. [261 N. F. Johnson, “Variation in male genitalia of Merope tuber Newman (Mecoptera; Meropeidae),” Journal of the Kansas Entomological Society, vol. 68, no. 2, pp. 224-233, 1995. 6 Psyche [27] C. T. Maier, “Habitats, distributional records, seasonal activ- ity, abundance, and sex ratios of Boreidae and Meropeidae (Mecoptera) collected in New England,” Proceedings of the Entomological Society of Washington, vol. 86, no. 3, pp. 608-613, 1984. [28] E. M. Barrows and O. S. Elint Jr., “Mecopteran (mecoptera: Bittacidae, meropeidae, panorpidae) flight periods, sex ratios, and habitat frequencies in a united states mid-atlantic fresh- water tidal marsh, low forest, and their ecotone,” Journal of the Kansas Entomological Society, vol. 82, no. 3, pp. 223-230, 2009. [29] “Steel Creek,” 36°02’18.11“N, 93o20’21.73”W, Google Earth, 2012. [30] A. G. Scarbrough, “Recent collection records of Merope tuber Newman (Mecoptera: Meropeidae) in Maryland,” Proceedings of the Entomological Society of Washington, vol. 82, no. 1, pp. 153- 154, 1980. [31] E. Potter, “Internal anatomy of the order Mecoptera,” Transac- tions of the Entomological Society of London, vol. 87, pp. 467-501, 1938. [32] G. W. Byers, “Type specimens of Nearctic Mecoptera in Euro- pean museums, including descriptions of new species,” Annals of the Entomological Society of America, vol. 55, pp. 466-476, 1962. [33] A. Kaltenbach, “Mecoptera (Schnabelhafte, Schnabelfliegen),” Handbuch der Zoologie, vol. 4, no. 2, pp. 1-111, 1978. [34] E. Eriedrich, H. Pohl, P. Beckmann, and R. G. Beutel, “The head of Merope tuber (Meropeidae) and the phylogeny of Mecoptera (Hexapoda),” Arthropod Structure and Development, vol. 42, no. 1, pp. 69-88, 2013. [35] G. Nordlander, “A method for trapping Hylobius abietis (L.) with a standardized bair and its potential for forecasting seedling damage,” Scandinavian Journal of Eorest Research, vol. 2, pp. 199-213, 1987. [36] J. P. Lemieux and B. S. Lindgren, “A pitfall trap for large-scale trapping of Carabidae: comparison against conventional design, using two different preservatives,” Pedobiologia, vol. 43, no. 3, pp. 245-253, 1999. [37] G. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ : 25 years of image analysis,” Nature Methods vol. 9, pp. 671-675, 2012, http://www.nature.com/nmeth/journal/v9/ n7/full/nmeth.2089.html. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 897596, 21 pages http://dx.doi.org/ 10 . 1 155/2014/897596 Research Article Molecular Population Structure of Junonia Butterflies from French Guiana, Guadeloupe, and Martinique Amber P. Gemmell, Tanja E. Borchers, and Jeffrey M. Marcus Department of Biological Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Correspondence should be addressed to Jeffrey M. Marcus; marcus@cc.umanitoba.ca Received 29 May 2014; Revised 20 August 2014; Accepted 15 September 2014; Published 12 October 2014 Academic Editor: Martin H. Villet Copyright © 2014 Amber P. Gemmell 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. Up to 9 described species of Junonia butterflies occur in the Americas, but authorities disagree due to species similarities, geographical and seasonal variability, and possible hybridization. In dispute is whether Caribbean Junonia are conspecific with South American species. Cytochrome oxidase I (COI) barcodes, wingless {wg) sequences, and Randomly Amplified Fingerprints (RAF) were studied to reveal Junonia population structure in French Guiana, Guadeloupe, Martinique, and Argentina. Phylogenetic analysis of COI recovered 2 haplotype groups, but most Junonia species can have either haplotype, so COI barcodes are ambiguous. Analysis of nuclear wingless alleles revealed geographic patterns but did not identify Junonia species. Nuclear RAF genotyping distinguished 11 populations of Junonia arranged into 3 clusters. Gene flow occurs within clusters but is limited between clusters. One cluster included all Argentinian samples. Two clusters included samples from French Guiana, Martinique, and Guadeloupe and appear to be divided by larval host plant use (Lamiales versus Scrophulariales). Many Junonia taxa were distributed across populations, possibly reflecting patterns of genetic exchange. We had difficulty distinguishing between the Caribbean forms /. zonalis and /. neildi, but we demonstrate that Caribbean Junonia are genetically distinct from South American /. evarete and /. genoveva, supporting the taxonomic hypothesis that they are heterospecific. I. Introduction Buckeye butterflies, genus Junonia (Nymphalidae), are an important model system for experimental research in the Lepidoptera [1, 2]. Junonia species have been widely used to study the evolution and development of butterfly wing colour patterns [2-9]. Experimental tools to manipulate gene expression developed in Junonia are broadly applicable across the Lepidoptera [10-14] . Junonia has also been used in studies of insect endocrinology [15-17] and has been an important system for examining the evolution of larval host plant preference and tolerance to host plant toxins [18-21]. Junonia butterflies are found throughout the Old and New World tropics. In the Western Hemisphere, forms of Junonia occur from southern Canada to Tierra del Fuego [22-24] and have a complicated taxonomic history. In 1775, Cramer [25] identified and described two similar species of Junonia, J. evarete and /. genoveva, from Suriname, a Dutch colony on the north coast of South America. The species were described according to the standards of the time (without designated type specimens) and the descriptions were accompanied by hand-tinted plates that reproduced the colours from the original watercolour drawings of specimens of each form (republished in [26]). In the 20th century, there was considerable disagreement in the scientific community about whether Cramers two species were truly distinct [27, 28] or whether all of the specimens belonged to /. evarete [29-33]. This is a result of the geographical [29, 34] and seasonal [35] variability of Junonia and the fact that some Junonia forms closely resemble one another [36, 37]. In addition, different Junonia forms share identical karyotypes {N - 31) [26, 38] and are capable of hybridization and the production of fer- tile offspring [39-41], further complicating the process of assigning names to Junonia specimens. These features make applying the biological species concept [42], phylogenetic species concept [43], or morphospecies concept [44] very diflficult in Junonia. Operationally, we use the isolation species 2 Psyche concept that defines species as systems of populations such that genetic exchange between these systems is limited or prevented by one or more reproductive isolating mechanisms [45, 46]. Identifying and understanding the reproductive isolating mechanisms operating in Junonia will be of great importance in clarifying Junonia taxonomy. Authorities who favoured the two-species hypothesis in Junonia called the larger form /. genoveva and the smaller form /. evarete. In 1985, Turner and Parnell [26], using spec- imens from Jamaica and Florida, USA, verified the existence of two Junonia species in both regions. However, after consulting Cramer s hand-tinted plates and comparing them to specimens from Jamaica and Florida, Turner and Parnell [26] switched the names so the larger species was now /. evarete and the smaller species was /. genoveva. Neild [22], using specimens from Venezuela (geographically much closer to the type locality of Suriname), also confirmed the existence of two species. However, Neild [22], unsatisfied with Cramer’s [25] published plates (copies of which differ from one another due to variation among the watercolourists who tinted them and differences in how the plates aged), consulted Cramer’s original watercolours and Junonia specimens from many localities in South America. Using this reference material, Neild [22] reversed Turner and Parnell [26] so that the larger species was again /. genoveva and the smaller species was /. evarete. Neild [22] also designated new types for /. evarete and /. genoveva to facilitate future taxonomic work. Recently, L. Brevignon and C. Brevignon [23, 47, 48] identified 5 Junonia species (/. evarete, J. genoveva, J. wahl- bergi, J. litoralis, and /. divaricata) from French Guiana. This represents the most diverse assemblage of Junonia in the New World. There are two forms of Junonia known from the Caribbean Islands, “zonalis’' and “neildij which were initial- ly recognized as subspecies of mainland /. evarete and /. genoveva, respectively [49] and later as two distinct species: /. zonalis and /. neildi [23]. In recognizing /. neildi and /. zonalis as distinct species, L. Brevignon and C. Brevignon [23] restricted the use of the species epithets /. evarete and /. genoveva to mainland Central and South American forms. If it were confirmed that the Caribbean forms are actually dis- tinct species with respect to Junonia from the mainland, this would explain some of the widespread difficulty of assigning appropriate taxonomic names to specimens from Florida, Jamaica, and elsewhere in the West Indies. Finally, there are two additional Junonia species, /. coenia from North America and /. vestina from the Andes mountains of South America, for a current total of up to 9 species of New World Junonia. The first molecular phylogenetic approaches to under- standing the relationships among the species discussed here established that the New World fauna appears to be mono- phyletic and that the various New World forms are indeed in the genus Junonia [50, 51] (some authorities had pre- viously placed these species in the related genus Precis) [52, 53]. Unfortunately, these early studies, which incor- porated data from both mitochondrial and nuclear loci, had limited taxon sampling, including data from only 3 New World species [50, 51]. More recent studies of the molecular phylogeny of New World Junonia [23, 54, 55], which have better taxon sampling, have focused entirely on the mitochondrial cytochrome oxidase I (CO/) locus, which is widely used as a barcoding locus for animal taxa [56, 57]. Based on mitochondrial haplotype sequences, the relation- ships among many New World Junonia species are ambiguous and most species are not reciprocally monophyletic [23, 24, 54]. The degree to which recent divergences, retained poly- morphisms, and/or hybridization events contribute to these patterns in Junonia is unknown because only mitochondrial markers were considered. What is apparent is that there are two very divergent CO/ haplotype groups (4% sequence divergence between them) present in New World Junonia: Group A, which predominates in South America and is also present in the Caribbean, and Group B, which predominates in North and Central America but which also occurs in the Caribbean and South America [24, 54]. Sequences belonging to each of these haplotype groups can occur in different individuals of the same species at the same locality [24]. The most successful study to date to distinguish between New World Junonia taxa using molecular markers employed a combination of mitochondrial and nuclear markers to examine populations of Junonia in Buenos Aires, Argentina. Borchers and Marcus [24] used DNA sequences from the nuclear wingless gene and anonymous nuclear loci identified by Randomly Amplified Fingerprinting (RAF) (a technique used to assess genetic diversity within populations [58-60] and gene flow between populations [61]) in addition to sequences from the mitochondrial COI gene. They identified 3 distinct populations of Junonia from Buenos Aires: one population with dark-coloured wings referred to as /. evarete flirtea [62] which Borchers and Marcus [24] suggested may correspond to /. wahlbergi and 2 light-coloured populations that correspond to /. genoveva hilaris and either a genetically disparate population of /. genoveva hilaris or an undescribed cryptic Junonia species. However, the relationship of these Argentinian forms with /. evarete and /. genoveva from Suriname and French Guiana is not known, so we refer to them as /. “flirtea” and /. “hilaris.” In the current study, we extend the genetic tools that were employed by Borchers and Marcus [24] to Junonia popula- tions from French Guiana and the French Antilles in order to study the distinctiveness of the named taxa within and between these two localities. This will allow an explicit test of the 7-species taxonomic hypothesis (2 species in the French Antilles plus 5 species in French Guiana) of L. Brevignon and G. Brevignon [23] and also detect possible hybridization events between named forms. By using a common set of markers we will also be able to compare these populations to previously studied Junonia from Argentina [24] . 2. Materials and Methods 2.1. Specimens and DNA Preparation. A total of 104 Junonia specimens were collected from the wild as adults, reared from wild-collected larvae, or reared from eggs laid by wild- collected adults and frozen at -20°G (Table 1). DNA was iso- lated from legs removed from each specimen. Some samples (42 specimens) were prepared by the Ganadian Gentre for DNA Barcoding at the University of Guelph as previously described [23]. The remaining samples (62 specimens) were Psyche 3 Table 1: Number of Junonia specimens included in this study either entirely processed in our laboratory or extracted at the University of Guelph and sent to us for further study. Species and locality DNA extracted in our laboratory DNA extracted by Guelph and whole genome amplified by our laboratory J. coenia, Florida, USA 0 2 /. divaricata, French Guiana 0 5 /. evarete, French Guiana 0 6 /. genoveva, French Guiana 32 6 /. litoralis, French Guiana 8 4 J. neildi, Guadeloupe 6 2 /. neildi, Martinique 2 3 /. wahlbergi, French Guiana 0 10 J. zonalis, Guadeloupe 7 2 J. zonalis, Martinique 7 2 processed in our laboratory using the Qiagen DNEasy Blood and Tissue kit (Qiagen, Diisseldorf, Germany) as previously described [24], except that the extractions were performed in a Qiagen QIAcube instrument using the standard instrument protocol for purification of total DNA from animal tissue. Extracted DNA was stored at -20° C. Only 10 |WL aliquots of DNA were available for the 42 Junonia specimens processed at the University of Guelph, which was insufficient for the number of experiments we wished to conduct. To produce additional template, whole genome amplification using Illustra Genomiphi V2 (GE Health Gare Life Sciences, Pittsburgh, PA, USA) protocol was performed as follows: 1 fiL of DNA template and 9 [jL of sample buffer were incubated at 95° C for 3 min, cooled to 4°G, mixed with 9 f/L of reaction buffer and 1 [aL of enzyme, incubated at 30° G for 90 minutes and then 65° C for 10 minutes, and cooled to 4°G. Deionized distilled water was used as the template for a Genomiphi amplification negative control. Genomiphied samples were stored at -20° G. 2.2. Mitochondrial Cytochrome Oxidase I Protocol. Cyto- chrome oxidase I (COI) PGR products were generated using a seminested two-step amplification with LGO1490 and Nancy primers followed by a reamplification with LGO1490 and HC02198 (Table 2) [63, 64]. Quick-Load Taq 2X Mastermix (New England Biolabs, Ipswich, MA, USA) was used in PGR reactions with total volumes of 25 /rL. Amplification protocols were run on a BioRad MyCycler or SIOOO Thermal Gycler (BioRad, Hercules, Galifornia, USA) for these and all other PGR amplifications unless otherwise specified. LG014- 90/Nancy PGR reaction conditions were 95° G for 5 minutes; 40 cycles of 95° G for 1 minute, 46° G for 1 minute, 72° G for 1.5 minutes; and a final 5-minute extension at 72°C before being placed on a 4°G hold. LGO1490/HGO2198 PGR reaction conditions were 95° G for 5 minutes; 35 cycles of 94° G for 1 minute, 46° G for 1 minute, 72° G for 1.5 minutes; and a final 5-minute extension at 72°G before being placed on a 4°G hold. PGR reactions were evaluated by gel electrophoresis (1% agarose in TAE buffer, 78 V for 1 hour, visualized with ethidium bromide). Samples that failed to amplify with LGO1490 and HGO- 2198 were reamplified with M13-uniminibarEl (miniGOIE) and M13-uniminibarRl (miniGOIR) (Table 2) [65]. MiniGOI PGR reaction conditions were 95° G for 2 minutes; 5 cycles of 95° G for 1 minute, 46° G for 1 minute, 72° G for 30 seconds; 35 cycles of 95°G for 1 minute, 53°C for 1 minute, 72°G for 30 seconds; and a final 5-minute extension at 72°G before being placed on a 4°G hold. Eurther reactions were carried out to obtain overlapping PGR products that could be assembled as contigs to obtain additional sequence data. Additional primers were designed to bind to invariant regions of the Junonia COI gene (miniGOIP2 and miniGOIR2 in one reaction and either miniCOIP3 and HC02198 or miniCOIP2 and HG02198 (Table 2) in a second reaction) to selectively amplify required sequences. Reaction conditions for these primers were the same as the miniGOI protocol described above. 2.3. Nuclear Wingless Protocol. Wingless PGR products were generated using lepwgl and lepwg2 primers (Table 2) [66]. Wingless PGR reaction conditions were 94° G for 5 minutes; 40 cycles of 94° G for 1 minute, 46° G for 1 minute, 72° G for 2 minutes; and a final 10-minute extension at 72° G before being placed on a 4°C hold. While these primers typically work well in Junonia [24], the samples analyzed here failed to produce detectable products, likely due to poor preservation of nuclear DNA. These PGR reactions were used as the template for PGR reamplification with miniwgP and miniwgR (Table 2), which we designed to bracket the most informative interval of the Junonia wingless coding sequence (Table 3). Mini-wingless reaction conditions were 95°G for 5 minutes; 40 cycles of 95° G for 1 minute, 57° G for 1 minute, 72°G for 1 minutes; and a final 5-minute extension at 72°G before being placed on a 4°G hold. 2.4. Sequencing. Gorrectly sized PGR products were sequenced as previously described [24]. Products were sequenced in both directions, usually with the same primers that generated the products. When the miniwgR primer produced poor quality sequences samples were reamplified with miniwgP and T7-miniwgR and sequenced using T7 primer (Table 2). Sequencing reactions were analyzed on an ABI 3730x1 automated sequencer and edited using Se- quencher 4.6 software [67]. Sequences were trimmed to the appropriate size (Table 3) and aligned in GLUSTALW [68]. 2.5. Randomly Amplified Fingerprinting Protocol. Randomly Amplified Pingerprinting (RAP) was used to gather a large multilocus data set [60]. Amplifications were carried out using single fluorescently labelled primers that act as both for- ward and reverse primers. A product is produced only if the primers bind in the correct orientation and close enough to one another for amplification. The 3 RAP primers, each cova- lently bound to a 6-PAM fluorescent molecule (Integrated DNA Technologies, Iowa Gity, Iowa, USA), used in these amplifications were RP2 (5^-/6-PAM/ATGAAGGGGTT-3^), 4 Psyche Table 2: Primer sequences used in cytochrome oxidase I (COI) and wingless (wg) PCR reactions. Primer name COI Nancy LCO1490 HC02198 M13-uniminibarFl M13-uniminibarRl miniCOIF2 miniCOIR2 miniCOIF3 wg lepwgl lepwg2 miniwgF miniwgR T7-miniwgR Sequence 5'CCCGGTAAAATTAAAATATAAACTTC3' 5' GGTCAACAAATCATAAAGATATTGG3' 5'TAAACTTCAGGGTGACC AAAAAATCA3' 5'GTAAAACGACGGCCAGTGGAAAATCATAATGAAGGCATGAGC3' 5'GGAAACAGCTATGACCATGTCCACTAATCACAARGATATTGGTAC3' 5'ATACTATTGTTACAGCCTCATGC3' 5'TGTTGTAATAAAATTAATAGCTCC3' 5'CCCCACTTTCATCTAATATTGC3' 5'GARTGYAARTGYCAYGGYATGTCTGG3' 5'ACTNCGCRCACCATGGAATGTRCA3' 5'ATCGCGGGTCATGATGCCTAATACG3' 5'GTTCTTTTCGCAGAAACCCGGTGAAC3' 5'TAATACGACTCACTATAGGGGTTCTTTTCGCAGAAACCCGGTGAAC3' Table 3: Expected sequence length of trimmed PCR products (primers removed) for each primer pair. Primer pair Trimmed sequence length (base pairs) LCO1490/Nancy 725 LCO1490/HCO2198 658 M13-uniminibarFl/M13-uniminibarRl 153 mC01F2/mC01R2 292 mC01F2/HC02198 520 mC01F3/HC02198 295 Lepwgl/ Lepwg2 402 miniwgF / miniwgR 137 RP4 (5'-/6-FAM/TGCTGGTTCCC-3'), and RP6 (5'-/6- FAM/TGGTGGTTTGG-3^) [59]. Amplifications were per- formed in triplicate along with positive and negative (distilled deionized water) controls for a total of 954 RAF amplifica- tions. Reaction volumes of 10 ffL were used. Samples were run in a BioRad MyCycler Thermocycler under the following reaction conditions: 95° G for 5 minutes; 30 cycles of 94° G for 30 seconds, 57° C for 1 minute, 56° C for 1 minute, 55° G for 1 minute, 54° G for 1 minute, 53° G for 1 minute; and a final 5-minute extension at 72°G before being placed on a 4°G hold. Reactions were shipped at room temperature to the Biotechnology Gore Facility at Western Kentucky University (Bowling Green, Kentucky, USA). 10 fiL HiDye formamide and 1 fiL RX-500 GeneScan Size Standard (Applied Biosys- tems, Garlsbad, Galifornia, USA) were added to each PGR tube upon receipt. The solution was then vortexed for 1-2 seconds and placed in a microcentrifuge at 13,000 rpm for 30 seconds at room temperature. Samples were placed into indi- vidual wells on a sequencing plate and incubated at 95° G for 4 minutes in a thermocycler. Following 3-5 minutes on ice, samples were loaded into an ABI 3130 automated sequencer (Applied Biosystems), which was fitted with a 50 cm capillary filled with Pop-7 sequencing polymer for fragment analysis. 2.6. Mitochondrial Cytochrome Oxidase I Analysis. A subset of the samples in the current study was used in a previous barcoding study performed in another laboratory [23]. To ensure that there was no confusion or contamination of DNA samples during transfer we resequenced COI from 17 samples (of 42 transferred) that had been previously sequenced. In all cases, identical sequences were obtained by our laboratory as previously reported [23]. COI sequence alignments were con- verted to NEXUS format for phylogenetic analysis using sev- eral different reconstruction methods (distance, parsimony, and likelihood) that rely on vastly different assumptions about sequence evolution, each of which recovered essentially the same tree. For the sake of brevity, we will only present the maximum likelihood analysis (HKY model, 10 replicate heuristic searches with random number seeds, tree bisection, and reconnection branch swapping algorithm) [69]. Other previously published Junonia COI sequences were included in this phylogenetic analysis [23, 24, 51, 57, 70-74]. We also conducted a maximum likelihood bootstrap analysis of this dataset (500 fast addition replicates, collapsing all notes with frequency less than 50%). The aligned COI FASTA sequences generated by this study along with 22 previously published Argentinian Junonia COI sequences [24] were analyzed using Arlequin 3.5 [75]. We employed an AMOVA analysis with the following settings: 1000 permutations, determining the minimum spanning network (MSN) among haplotypes, computing distance matrix, and pair-wise difference with a gamma value of 0. The minimum spanning tree out- put from AMOVA was put into HapSTAR-0.7 [76], which displays the haplotype network in graphical form. Since analysis in Arlequin requires all sequences to be of the same Psyche 5 length, the analysis was first conducted using all samples that amplified using LCO1490/HCO2198 (Figure 2) and then repeated after trimming all sequences to the length of mini- C0IF2/HC02198 (Figure 3). Additional adjustments to the network were made using Canvas X (ACD Systems, Seattle, Washington, USA) such as scaling the population circles to reflect sample size and adding pie charts to reflect the RAF population assignment or geographical location and species. 2.7. Nuclear Wingless Analysis. For the Junonia species se- quenced in this study and Argentinian Junonia wingless se- quences from a prior study [24], individuals heterozygous for single nucleotide polymorphisms (SNPs) in the coding sequence were identified using sequencing chromatograms and CLUSTALW alignments. For each polymorphism, the genotype of each individual was entered into PHASE 2.1.1 [77] and analyzed using the default settings. PHASE uses the Markov Chain-Monte Carlo method to group coinherited SNPs in order to determine the most probable wingless alleles present in each individual. The most likely alleles identified in PHASE were assigned to each individual and the data was then entered into GENEPOP 4.0.10 [78]. GENEPOP was used to test for genetic differentiation (Exact G test [79]) by determining if the alleles from each subpopulation were drawn from the same distribution. GENEPOP settings used for testing all populations were a demorisation of 10,000, 10,000 batches and 10,000 iterations per batch. Finally, Structure 2.3.3 [80] was used to analyze the wingless data since, unlike GENEPOP [78], Structure does not require the a priori assignment of individuals to specific subpop- ulations. Population structure exhibited by wingless alleles was analyzed using Structure 2.3.3 [80] with settings for codominant alleles, a 10,000 step burn-in and 1 million Markov Ghain-Monte Garlo Method replicates. Ten replicate structure searches tested each of 15 different population models with 1 to 15 subpopulations among the 88 wingless sequences. The maximum log likelihood (InP(D)) for the 10 replicate searches for each population model was used to calculate the posterior probability {P{K - n)) of each population model. Haplotype networks of wingless alleles were constructed in the same manner as COI except that PHASE output identifying the most likely wingless genotypes was formatted for input into Arlequin 3.5 [75] . 2.8. Randomly Amplified Fingerprinting Analysis. Eragment analysis sample runs were combined with previously studied Argentinian Junonia [24] and analyzed using GENEMAP- PER version 3.7 software (Applied Biosystems). An allelic bin size of 3 base pairs was selected in order to detect poly- morphic alleles without introducing excessive noise into the analysis associated with small differences in run time between samples. The resulting GENEMAPPER genotypic classifi- cations were exported to an Excel spreadsheet (Microsoft, Redmond, Washington, USA) for further analysis. Bands that appeared in negative control amplifications (of deionized distilled water with no DNA added) were considered artefacts and removed from further analysis for all samples. Within the 3 replicate RAE fragment runs for each primer from an individual butterfly, allele -calling for the presence or absence of the dominant allele at each RAE locus was based on a majority rule determination (at least 2 of the 3 runs had to show the allele for it to be scored as present). Each locus was coded in binary with 0 indicating the absence of an allele and 1 indicating the presence of the allele. Such binary data was analyzed using Structure 2.3.3 software [80] with the same settings as described previously for the wingless data except that in the case of the RAE data set only dominant alleles could be scored. A total of 50 replicate searches were carried out on each of K = 1-15 populations, first including only the samples genotyped in this study (primarily from Erench Guiana and the Garibbean) and then again including the 22 Argentinian specimens genotyped in a previous study [24]. Allele frequencies for each RAE locus were calculated for each population identified in Structure and formatted for input for the GONTML application of PHYLIP 3.5 [81] as implemented in EMBOSS Explorer [82]. GONTML uses a rigorous maximum likelihood algorithm to estimate phylogenies based on allele frequencies. In this model, all divergence between populations is assumed to be due to genetic drift in the absence of new mutations [83]. GONTML trees were exported in NEXUS format and rendered in EvolView [84] for interpretation. A parallel analysis was conducted in GONTML for the RAF data set and the allele frequency data obtained for COI and wingless. 3. Results 3.1. Mitochondrial Cytochrome Oxidase I Results. New COI DNA sequences generated by this project were deposited in Genbank (67 accessions, numbers KJ469059-KJ469126), with the exception of specimens with only COI minibar- code sequence fragments [65], which were submitted to the DNA Databank of Japan (DDBJ, 5 accessions AB9353- 41-AB935345). Full COI barcode sequences, covering the interval between LGO1490 and HG02198, were recov- ered from 65 specimens (17 reported previously [23] and 48 new sequences), 15 of which required assembling 3 sequence contigs to obtain the 658 bp sequence. Partial bar- code sequences were obtained from miniGOIF/R sequences assembled into contigs with miniCOIF2/R2 sequences (2 specimens), miniGOIF/R sequences assembled into con- tigs with miniG0IF3/HG02198 sequences (2 specimens), and miniC0IF2/HC02198 sequences alone (16 specimens). Overall, some COI sequence was recovered from 90 of the 104 specimens. Analysis of the COI sequences produced a maximum like- lihood phylogenetic tree (Figure 1). As previously reported [24, 54], there are two distinct mitochondrial haplotype groups in New World Junonia. Haplotype group A is found in South American and Garibbean specimens, while haplotype group B includes many North American, Gentral American, and Garibbean specimens, as well as some South American specimens. A few forms of Junonia appear to be associated with only one haplotype group (group A: the South American forms /. “fiirtea” and /. vestina; group B: the North American forms /. coenia and /. “nigrosufihisa”). All other Junonia species 6 Psyche |/. divaricata French Guiana (LCBI3) wahlbergi French Guiana (LCB25) / A^cnihia(ARGl7) BraiiUNW15J3| — I /. French Guiana (LCB9) /. neiVrfi MjrlirUqut (LCB37) ' /. ‘yh'rWir Argtniiiia tARGl6) /. ‘)2irrffl“LiglilPhvnoiyju-Argcnlhia(ARG18) /. ‘^irrfti"Argcnlma(AkG19) / Brazil (NW1552) / iftnir/rirffi French Guiana (LCB15) /. neilli Guadeloupe (UK419J / ri Mariiniquc(LCBJ57) ]. zomlls Mariinique (LCB158) I /.coiifl/ij Martinique (U1B161J /. zoiialls Guadeloupe (LCB165) /, ndWi Guadeloupe (LCB]7'U I. nelldi Guadeloupe (LCB176} / neildi Guadeloupe (LCBlTB) .uiiii/ii Martinique (LCB162) A zonalis swifB Guadeloupe (NW136 17) — }. Guadeloupe (UK4Ja) r /T f'Bl IfW .'CBm /. jenovcviJ French Guiana LCB204 /. "Jlirtea" Argentina (ARG13) ^ ]. ^nweva French Guiana (LCB19]) French Guiana (LCB207) /. ^npvevQ Frendi Guiana (UK414) Jmmiiia sp. French Guiana (UK416) LCB20J ]. divaricata French Guiana(LCB14) ■ /, mirt-re Brazil (NW 12620) /, emrefe Brazil (NWJ2930) I. genoveva French Guiana Haplotype group A south America and Caribbean All /. “flirtea” and /. vestina Haplotype group B entire western hemisphere All /. coenia and /. evarete nigrosuffusa CIAD10B12 C1AD10B13 C1ADI0B16 CIAD10B18 CIAD10B21 CIAD10B19 /. evarete nigrosuffusi J. evarete nigrosuffusi /. evareteSonora, MX(CIAD10 B23) /. evareteSonora, MX(CIADIOBIJ) /. evarete nigrosuffusa Sonora, MX (QADIO B34) /. evarete Sonora, MX a Sonora, MX (QADIO B32) a Sonora, MX(CIAD10 B35) € 05 SRNP 58208 03 SRNP28069 05 SRNP 58001 05 SRNP 57998 05 SRNP 32756 \ , 00 SRNP 3055 05 SRNP 57995 95 SRNP 6024 /. evarete Costa Rici(03 SRNP 28074) /. coenia coenia TN, USA (NW8513I /. evarete Cosla Rica (03 SRNP 28071} evarete Sonora, MX (QADIO B28J evarete Costa Rica (05 SRNP 58241) CIAD10B27 QADIO B17 CIAD10B37 QADIO B20 QADIO BIO QADIO BOl CIAD10B22 /. zonalis Guadeloupe (LCB170) /. neildi Martinique (LCB172) /. evarete Sonora, MX /. neildi Guadeloupe (LCB175) ^ /, neildi Guadeloupe (LCB173) — /.iUMafo Guadeloupe (LCB1691 J. neildi Guaddoupe (LCB177) /. neildi Martinique (NW 13616) /. evarete Motdos, MX ( |M6iO) /.coenia coenia MA, USA ( rL7\VG0126) /. evarete nigivsulf'usa Sonora, MX (C1AD10B25) /. coenia coenia KA'. USA (Biol7517) /. evarete niff’osuffusu Sonora, MX (CL\D10B26) /. evarete nigrosuffusa Sonora. MX (CIAD10B36) /. zonalis Guadeioipe (LCB166) /. evarete ni^iwu/iiini Sonora, MX (C1AD10B30) /. evarete nigivsuff'usa Sonora, MX (C1AD1DB24) /. evarete nigrivuff'uso Sonora, MX (CIAD10B33) — /. evarete nlgnisuffiiso Sonora, MX (QAD10B31) _|/. coenia amia NC. USA (DNAA'FBIOSie) I /, evarete Costa Rka (04SRNP15110) /. coenhi eofiJW FL. USA (LCB27) ” /. doeniii eoeiJW NC, USA (DN-AATBIOSOA J. evarete Morelos, MX(NWI627) /. coenia coenia UT, USA (NW3818) ■ /. evarete Costa Rica (05SRNP58293) 03 SRNP 28076 /, evarete 05 SRNP 58221 Costa Rica 05 SRNP 58009 05 SRNP 58257 /. evarete Costa Rica(05SRNP31215) / ci-arete Costa Rica(05SRNP31188) QADIO B04 CIAD10B05 CIAD10B03 53 /. coeniagrisea California, USA QADiOBOe QADIO B09 / nei/di Guadeloupe (LCB34) ), evarete Panama (YBBCII2765) /. ^cnovera French Guiana (LCB8) /, ivn/i/()tTgiT-rencli Guiana (LCB24) / ivafrtOetp PrenchGuiana(LCB95) /. sp, aflin. “hilaris" Argentina (ARG4) / sp. jflin. 'Viiiaris" Argentina (ARG9) /. sp. alTin.”iiiiin'd "iVrgcntina (ARG12) /. gtntn'ei’uFrwich Guiana (LCB7) /. riiraniiifd French Guiana (LCB16) / lUOralls Frendi Guiana (LCB38) /. ei'eriin lun/irt PL, USA {LQJ28) /. litoralis Frendi Guiana (LCB40) /, neildi Martinique (LCB36) 7, zonalis Guadeloupe (LCB29) /. zofia/is Martinique (LCB32) C _/. uenone Captive Reared (NW681) 91 ^—7 (lierta Zimbabwe (NW88I2) /, iiierta Bangladesh (NW10116) 1 7 iiiertflZiinbabwe (NW8810) 'ertu Indonesia (NWSOS) •/- hierta China (huangshangyanl) ■ithya Qiina(NW8811) -I. f.icH^i China (EF683678) 7 orithya Malaysia (N\V291} 7 orithya InJia(SHC03BP05) • / wviiemnmni Uganda (NW8211) /. weslcrmanni Uganda (NW834) /, Sophia Uganda (NW8310) 7 wstermanni Central African Republic (DQ385855) 7 /emriutii- India (SEC19LP05) ^“/. nata/ica Zimbabwe (NW831 1) /. natalica Captive Reared (NV\'681.h /. H£Jta/jri? Zimbabwe (NW8813) ' Captive Reared (N\V6815) Uganda (NW83h Taiizaniii(NW8212) /. terea 'I'anzania (NW8214) /. almana Malaysia (NW293) i^^SOril Uganda (NW832) /. almana Vietnam (NW1318) /. fl/uiflMfl Vietnam (NWI317) almana almana China (MY)D001) I ^ — /. erieoMe Pam New Guinea (NW812j J7 hedonia Australia (SIW332) 7. tiedoiiia AustraikfNWTSSi 7 ailites Captive Reared (NW784) 7 fUliies Malaysia (NW296) 7 ut/itts Uganda (NWl3 19) 7 027 al8 ARC 19al7 LCB 193 a24 ) ARC 11 al5 LCB 008 ARC 5 ARC 16 a7 LCB 157 LCB LCB 157 LCB LCB 158 LCB LCB 159 LCB LCB 160 LCB LCB 160 LCB LCB 161 LCB LCB 161 LCB LCB 162 LCB LCB 162 LCB LCB 163 LCB LCB 164 LCB LCB 164 LCB LCB 168 LCB LCB 169 LCB LCB 170 LCB LCB 170 LCB LCB 173 LCB LCB 173 LCB LCB 174 LCB LCB 174 LCB 175 LCB 002 175 LCB 002 177 LCB 014 177 LCB 026 178 ARC 1 178 ARC 5 180 ARC 10 181 ARC 20 187 187 188 191 195 196 197 200 209 210 322 322 326 I 189 LCB 208 I 189 LCB 210 ( 190 LCB 326 ( 191 ARC 1 i 192 ARC 3 1 194 ARC 4 1 194 ARC 4 ; 195 ARC 6 1 197 ARC 7 I 198 ARC 8 1 198 ARC 9 I 199 ARC 11 ; 199 ARC 12 i 200 ARC 13 i 201 ARC 16 i 201 ARC 18 i 202 ARC 19 i 202 ARC 20 i 203 ARC 22 5 204 i208 a23 ARC 18 al6 LCB 008 j ' alO , LCB 007 a2 ^ LCB 021 / LCB 019 LCB 027( ) LCB 024 / LCB 025 [_ al2 \ LCB 018 \ a29 a26 ARC 15 LCB 005 Oa35 ARC 17 ARC 22 T CRl 92 LCB 095 / 'a9 a28 ARC 9 ARC 10 ARC 13 ARC 14 ARC 14 ARC 21 , LCB 172 LCB 185 al9 ARC 6 ARC 8 ARC 12 ARC 15 al3 LCB 193 a32 ARC 7 a30 ARC 21 □ RAF population 1 □ RAF population 2 A □ RAF population 2B □ RAF population 3A □ □ RAF population 3B □ □ RAF population 3G □ RAF population 4 RAF population 5 RAF population 6A □ RAF population 6B □ RAF population 6G (b) Figure 4: Haplotype networks generated using wingless alleles. Circles are scaled to represent the number of individuals that contain a specific wingless allele with the exception of alleles a7 and a9 (if scaled proportionately, would be 50 and 61 times larger, resp., than shown). Colours for both (a) and (b) are as described in Figure 2. (a) Divisions and colours of circles reflect geography and species associated with each wingless allele. Allele a9 is much rarer in the Caribbean populations than it is in the mainland populations. 50% of a9 allele is comprised of /. genoveva. The majority of wingless alleles found in Argentinian Junonia are allele a9 or its derivatives, (b) Divisions and colours of circles reflect the RAF populations associated with each wingless allele. All 11 RAF populations carry the most common allele a9. Individuals from 10 of the 11 RAF populations carry the other common allele, aZ Psyche 11 Table 4: Inferring K, the number of populations, testing for 1-15 subpopulations in STRUCTURE, for Argentinian, French Guianan, Guadeloupean, and Martiniquan Junonia wingless data. K (number of populations in model) Median ln[P(D)] PR (K = n) (posterior probability of the model) 1 -42.70 0.972 2 -46.54 0.021 3 -56.03 1.587F - 06 4 -53.68 1.664F - 05 5 -136.83 1.287F - 41 6 -135.59 4.426F - 41 7 -173.16 2.136F-57 8 -130.03 1.156F-38 9 -149.33 4.797F - 47 10 -61.83 4.806F - 09 11 -59.65 4.251F-08 12 -55.59 2.465F - 06 13 -53.93 1.296F - 05 14 -49.28 0.001 15 -47.82 0.006 Curiously, when this analysis was repeated with the inclusion of Junonia samples from Argentina, the model with the high- est posterior probability (P(i6 m high) and the other was deployed 2 m above ground from a tripod in a forest clearing; the distance between edges of forest clearings and traps in tree canopies was >50 m at all sites. The traps were inspected daily, and the captured SBW moths were collected to determine abundance of males and females. The same design was used in Saint- Quentin, except that light traps were deployed at nine plots spaced 3-4 km apart along a logging road [19]. Egg density was estimated on the most prevalent host trees of SBW at different sites by pruning one branch (usually <1 m long) in the midcanopy of three to five codominant trees and estimating the number of egg masses visually; the surface area of branches was estimated (length x width of midpoint branch section) and the density of eggs was expressed in terms of egg masses per 10 m branch area [2] . Statistical analyses were conducted with the SAS package (SAS Institute, Cary, NC). Three parameters related to phe- nology were calculated for each site and trap location: (1) the median date of captures; (2) the duration of flight (interval in days between the 5th and 95th cumulative captures dates); and (3) the proportion of individuals captured at light traps during the modal date of abundance. The cumulative abundance, sex ratio, and phenology of SBW were compared in relation to trap position at different sites using paired t- tests; at Saint- Quentin, pooled values across plots were used in analysis. The relationship between cumulative captures of males and females and the density of eggs per 10 m of foliage at dif- ferent sites was evaluated in relation to sex and trap position using regressions. In addition to the seven aforementioned sites, the regression models also included two sites sampled in 1978 (Saint-Quentin and Amherst, Nova Scotia) for which the cumulative (but not daily) abundance of SBW males and females at light traps was available; therefore, the regression models each included nine data points. Data were subjected to logarithmic (abundance data) and arcsine (proportion data) transformation to reduce hetero- geneity of variance. 3. Results Daily numbers of males and females at TC and EC are plotted for different locations in Figure 1. Captures of adults at TC were 4-400 greater than those at EC (Table 1). The sex ratio of moths at TC was strongly biased toward males, whereas the sex ratio at EC was approximately even. Neither the median date of flight nor the duration of the flight period varied significantly with trap position (Table 1). The proportion of adults captured during the modal date of flight was two times higher at EC than TC (Table 1). The density of eggs at different sites varied from 36 to 992 egg masses per 10 m of foliage. Numbers of males and females captured at TC were positively correlated with the density of eggs, whereas no significant relationship was observed at EC (Figure 2). 4. Discussion With the imminent SBW outbreak in Atlantic Canada, it is important to consolidate and disseminate the information gathered by research scientists during previous outbreaks, in particular as-yet unpublished data. Results obtained through this study are published for the first time and provide a detailed record of daily captures of male and female SBW at light traps deployed in forest clearings or tree canopies at multiple locations. The findings have important implications for the design of monitoring procedures and analysis of time- series data and also provide insight into the dispersal- migration behavior of SBW. The physical position of light traps must be standardized in monitoring procedures for SBW. Deployment of traps in forest clearings should be avoided due to the low numeric abundance of SBW and the insignificant relationship between moth abundance and egg densities (Table 1, Figure 2). Cap- tures of SBW at light traps in tree canopies, in contrast, provide accurate estimates of egg densities (Figure 2). The relationships were similar for males and females; thus, the time-consuming process of sexing SBW could be omitted in operational programs that are purely management oriented. From a research perspective, recording the sex ratio of moth captures is useful to unravel the role of dispersal-migration on SBW population dynamics. Ideally, both light traps and pheromone-baited traps should be emptied daily or at short intervals to reduce the risk of trap saturation and gain information related to flight phenology and immigration events (nightly captures are characterized by high numerical abundance, predominantly females) [3, 9, 11, 12]. Psyche 3 Acadia 72 Acadia 76 Ordinal date Ordinal date Chipman 73 800 Q i\ A / 1 n— n — — n— O J 0 y— o 185 1 189 193 Ordinal date Chipman 74 Chipman 75 C/5 181 189 197 Ordinal date Juniper 75 St-Quentin 77 189 Ordinal date Males clearing Males clearing Males clearing -O- Females clearing - O- Females clearing -O- Females clearing Males canopy Males canopy Males canopy -o- Females canopy Females canopy -o- Females canopy Figure 1: Daily abundance of adult spruce budworms captured at light traps deployed in tree canopies and forest clearings at different sites in New Brunswick, Canada. The numerical values within individual graphs represent the scale of the y-axis for different locations and trap position. Table 1: Descriptive parameters related to captures of adult spruce budworms at light traps deployed in tree canopies and forest clearings at seven locations — years (mean ± SE). For each parameter, values with different superscripts are significantly different (paired t-tests, P < 0.05). Data were subjected to logarithmic (number of moths) and arcsine (proportional data) transformations to reduce heterogeneity of variance. Independent variable Position of traps f-test Tree canopy Forest clearing t P Abundance of moths (1000) 24.5 ± 5.6" 1.9 ± l.l’' 5.02 0.0024 Sex ratio (Pfemaks) 0.193 ± 0.02" 0.444 ± 0.024’' 7.51 0.0003 Median date of flight 192.4 ± 1.5" 192.7 ± 1.6" 0.26 0.8041 Duration of flight (day) 8.7 ± 1.3" 7.2 ± 1.1" 1.20 0.2741 Proportion of adults (modal date) 0.251 ± 0.026" 0.514 ± 0.077’' 3.71 0.0100 4 Psyche ^ Males o Females traps deployed in tree canopies capture local moths [3, 9, 10, 12]. Although this hypothesis cannot be unambiguously tested (because no genotypic-phenotypic traits are available to distinguish migrant from resident adults), its framework can be used to make a priori predictions related to patterns of captures of SBW in traps deployed in tree canopies and forest clearings, specifically: (1) greater captures in tree canopies than forest clearings (due to the greater number of resident moths than dispersers in forest stands with high densities of SBW); (2) greater proportion of females in forest clearings than tree canopies (because females are more likely to disperse than males); (3) shorter durations of moth activity in forest clearings than tree canopies (due to the transient nature of dispersal events relative to patterns of emergence- activity of resident moths); and (4) greater proportion of moths captured during the modal (peak) date of captures for forest clearings than tree canopies (transience of dispersal events). The data provided strong statistical support for three of the four predictions (Table 1), suggesting that light traps in forest clearings are indeed more likely to capture dispersive SBW than light traps in tree canopies. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 2: Relationships between numbers of adult spruce bud- worms captured at light traps deployed in tree canopies and forest clearings (x-axis) and the density of eggs (y-axis) at nine locations in New Brunswick, Canada, in the 1970s. Regressions based on captures in tree canopies were statistically significant for both males (y = l.l4x - 2.30, = 0.796, and P = 0.0012) and females (y = 0.89x - 0.67, = 0.776, andP = 0.0017) (solid lines). Regressions based on captures in forest clearings were not significant for either sex (males: y = 0.27x + 1.67, = 0.119, and P = 0.3626; females; y - 0.34x + 1.56, = 0.205, and P = 0.221) (dotted lines). Data were subjected to logarithmic transformations to reduce heterogeneity of variance. Daily records of moth captures at light traps were kept for multiple locations in Atlantic Canada and Maine in the 1970s [6, 7], which might in theory provide extensive time- series analysis of moth abundance. These data should not be analyzed in this context, however, because the position of traps is unspecified for most sites; thus, sampling artefacts (captures are 4-400 times greater in tree canopies than forest clearings) would systematically bias the outcome of analyses. The data are apparently suitable for evaluating broad-scale trends related to the phenology of flight in SBW, because neither the median date nor the duration of flight periods is influenced by the position of traps (Table 1). Existing phenology models of SBW adult flight need to be validated and calibrated with field data because they do not appear to accurately reflect the timing of SBW flight (Figure 6 in [20]). It has been hypothesized that light traps deployed in forest clearings capture predominantly dispersive SBW, whereas Acknowledgments The authors express their gratitude to A. W. Thomas who was responsible for the collection of data and generously granted them the permission to publish them. C. Simpson, V. Martel, and one anonymous reviewer provided useful comments on earlier versions of the paper. References [1] R. R Morris, The Dynamics of Epidemic Spruce Budworm Populations, vol. 31 of Memoirs of the Entomological Society of Canada, Entomological Society of Canada, 1963. [2] T. Royama, W. E. MacKinnon, E. G. Kettela, N. E. Carter, and L. K. Hartling, “Analysis of spruce budworm outbreak cycles in New Brunswick, Canada, since 1952,” Ecology, vol. 86, no. 5, pp. 1212-1224, 2005. [3] D. O. Greenbank, W. Schaefer, and R. C. Rainey, “Spruce bud- worm (Lepidoptera: Tortricidae) moth flight and dispersal; new understanding from canopy observations, radar, and aircraft,” Memoirs of the Entomological Society of Canada, vol. 110, pp. 1- 49, 1980. [4] J. Regniere, J. Delisle, E. Bauce et al, “Understanding of spruce budworm population dynamics: development of early intervention strategies,” in Proceedings of the North American Eorest Insect Work Conference, Information Report NOR-X-381, pp. 57-68, Edmonton, Canada, 2001. [5] M. Rhainds, E. G. Kettela, and R J. Silk, “Thirty-five years of pheromone -based mating disruption studies with Choris- toneura fumiferana (Clemens) (Lepidoptera: Tortricidae),” The Canadian Entomologist, vol. 144, no. 3, pp. 379-395, 2012. [6] J. E. Hurley and E. A. Titus, “Summary of light trap catches for the Maritimes 1976-1986,” Information Report M-X-163, Psyche 5 Natural Resources Canada, Canadian Forest Service — Atlantic Forestry Centre, Fredericton, Canada, 1987. [7] D. Weed, Spruce Budworm in Maine, 1910-1976: Infestations and Control, Maine Department of Conservation, Maine Forest Service, 1977 [8] R J. Silk and L. P. S. Kuenen, “Sex pheromones and behavioral biology of the coniferophagous Choristoneura” Annual Review of Entomology, vol. 33, pp. 83-101, 1988. [9] C. A. Miller and G. A. McDougall, “Spruce budworm moth trapping data using virgin females,” Canadian Journal of Zool- ogy, vol. 51, no. 8, pp. 853-858, 1973. [10] G. A. Simmons and N. C. Elliott, “Use of moths caught in light traps for predicting outbreaks of the spruce budworm (Lepidoptera: Tortricidae) in Maine,” Journal of Economic Ento- mology, vol. 78, no. 2, pp. 362-365, 1985. [11] D. O. Greenbank, “The role of climate and dispersal in the ini- tiation of outbreaks of the spruce budworm in New Brunswick. II. The role of dispersal,” Canadian Journal of Zoology, vol. 35, no. 3, pp. 385-403, 1957 [12] R. B. B. Dickison, M. J. Haggis, and R. C. Rainey, “Spruce budworm moth flight and storms: case study of a cold front system,” Journal of Climate & Applied Meteorology, vol. 22, no. 2, pp. 278-286, 1983. [13] J. N. McNeil, “Behavioral ecology of pheromone -mediated communication in moths and its importance in the use of pheromone traps,” Annual Rreview of Entomology, vol. 36, pp. 407-430, 1991. [14] D. C. Allen, L. P. Abrahamson, D. A. Eggen, G. N. Lanier, S. R. Swier, and R. Kelly “Monitoring of spruce budworm (Lepidoptera; Tortricidae) populations with pheromone-baited traps,” Environmental Entomology, vol. 15, no. 1, pp. 152-165, 1986. [15] K. Knowles, “Pheromone trapping of adult spruce budworm in the Manitoba Model Eorest,” 1997, http://www.manitobamod- elforest.net/publications/Pheremone%20Trapping%20of%20 Spruce%20Budworm%20in%20MBMP%2096-3-07.pdf. [16] C. J. Sanders, “Monitoring spruce budworm population density with sex pheromone traps,” The Canadian Entomologist, vol. 120, no. 2, pp. 175-183, 1988. [17] R. C. Johns and D. Pureswaran, “The pending storm — eastern Canadian foresters brace for spruce budworm,” Canadian Eorest Industries Magazine, pp. 14-16, 2013. [18] M. Rhainds, “Eield assessment of female mating success based on the presence-absence of spermatophore: a case study with spruce budworm, Choristoneura fumiferanaj Annales Zoologici Eennici, vol. 50, no. 6, pp. 377-384, 2013. [19] M. Rhainds and E. G. Kettela, “Oviposition threshold for flight in an inter-reproductive migrant moth,” Journal of Insect Behavior, vol. 26, no. 6, pp. 850-859, 2013. [20] J. Regniere, R. St-Amant, and P. Duval, “Predicting insect dis- tributions under climate change from physiological responses: spruce budworm as an example,” Biological Invasions, vol. 14, no. 8, pp. 1571-1586, 2012. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 424078, 6 pages http://dx.doi.org/ 10 . 1 155/2014/424078 Research Article Chemical Composition and Acaricidal Effects of Essential Oils of Foeniculum vulgare Mill. (Apiales: Apiaceae) and Lavandula angustifolia Miller (Lamiales: Lamiaceae) against Tetranychus urticae Koch (Acari: Tetranychidae) Asgar Ebadollahi,^ Jalal Jalali Sendi,^ Alireza Aliakbar,^ and Jabraeil Razmjou^ ^Department of Plant Protection, Faculty of Agricultural Sciences, University ofGuilan, Rasht 416351314, Iran ^Department of Chemistry, Faculty of Basic Sciences, University ofGuilan, Rasht, Iran ^Department of Plant Protection, Faculty of Agriculture, University ofMohaghegh Ardabili, Ardabil, Iran Correspondence should be addressed to Asgar Ebadollahi; asgar.ebadollahi@gmaiLcom Received 17 September 2014; Revised 14 November 2014; Accepted 24 November 2014; Published 14 December 2014 Academic Editor: Nguya K. Maniania Copyright © 2014 Asgar Ebadollahi 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. Utilization of synthetic acaricides causes negative side-eifects on nontarget organisms and environment and most of the mite species such as two spotted spider mite, Tetranychus urticae Koch, are becoming resistant to these chemicals. In the present study, essential oils of fennel, Foeniculum vulgare Mill, and lavender, Lavandula angustifolia Miller, were hydrodistilled using Clevenger apparatus and chemical composition of these oils was analyzed by GC-MS. Anethole (46.73%), limonene (13.65%), and a-fenchone (8.27%) in the fennel essential oil and linalool (28.63%), 1,8-cineole (18.65%), and 1-borneol (15.94%) in the lavender essential oil were found as main components. Contact and fumigant toxicity of essential oils was assessed against adult females of T. urticae after 24 h exposure time. The essential oils revealed strong toxicity in both contact and fumigant bioassays and the activity dependeds on essential oil concentrations. Lethal concentration 50% for the population of mite (LCgg) was found as 0.557% (0.445-0.716) and 0.792% (0.598- 1.091) in the contact toxicity and 1.876 pL/L air (1.786-1.982) and 1.971 pL/L air (1.628-2.478) in the fumigant toxicity for fennel and lavender oils, respectively. Results indicated that F vulgare and L. angustifolia essential oils might be useful for managing of two spotted spider mite, T. urticae. 1. Introduction Spider mites belong to the family Tetranychidae and are named because many members of this family produce silk webbing on the host plants. Some 1,200 species of spider mites belonging to over 70 genera are known in the world especially in the Southern Hemisphere [1]. Two-spotted spider mite, Tetranychus urticae Koch, is widely distributed globally and a common pest of many plant species in greenhouses, orchards and field crops. To date, 3877 host species have been reported around the world in both outdoor crops and greenhouses [2] . T. urticae feeding causes graying or yellowing of the leaves and necrotic spots occur in advanced stages of leaf damage. Mite damage to the open flower causes a browning and with- ering of the petals that resembles spray burn. In addition. small chlorate spots can be formed at feeding sites as the mes- ophyll tissue collapses due to the destruction of 18-22 cells per minute [2] . The importance of this mite pest is not only due to direct damage to plants but also due to indirect damage to plants which decreases photosynthesis and transpiration [3]. Because of their high reproductive rates, its management can be difficult. When mites begin to feed on a plant, they produce webbing that can protect both motile and egg stages from the acaricide [3]. Synthetic acaricides have been used as the main strategy for Tetranychus species resulting in an increased cost for production and environmental impacts as well as resistance development to even the newly synthesized molecules such abamectin [2, 4] . In addition, control methods based on the use of synthetic acaricides sometimes fail to keep the number 2 Psyche of spider mites below economic threshold levels [5]. It is therefore necessary to find alternatives that can minimize negative effects of synthetic acaricides. Essential oils obtained by hydrodistillation, steam distilla- tion, dry distillation, or mechanical cold pressing of aromatic plants [6] have long been used as fragrances and flavorings in the perfume and food industries, respectively, and recently for aromatherapy and medicines [2]. The essential oils can play major roles in pollination by attracting insects and in water loss’s prevention due to excessive evaporation. Repel- lence is another property of essential oils, as some contain numerous secondary metabolites that can deter attacks from pests [7]. Most essential oil constituents degrade quickly in the environment or are rapidly lost from plant foliage through volatilization, which minimizes residual contact. They have short residual activities due to temperature and UV light degradation and, with a few exceptions, their mammalian toxicity is low [8, 9] . Therefore, essential oils can be applied to both field and greenhouse crops in the same manner as current synthetic acaricides [2, 9] . The fennel, Foeniculum vulgare Mill. [Apiales: Apiaceae (Umbelliferae)], is indigenous to the Mediterranean and is largely used to impart flavor to a number of foods, such as soups, sauces, pickles, breads, and cakes. It is an annual, biennial, or perennial herbaceous plant, depending on the variety, which grows in good soils from sunny mild climatic regions and is a well-known aromatic plant species. Tradi- tionally in Europe and Mediterranean areas, fennel is used as antispasmodic, diuretic, anti-inflammatory, analgesic, secre- tolytic, galactagogue, eye lotion, and antioxidant remedy [10, 11]. The lavender, Lavandula angustifolia Miller [Lamiales: Lamiaceae (Labiatae)], is an evergreen bushy shrub with straight, woody branches; the lower parts are leafless, putting out numerous herbaceous stems to a height of about 1 m [12] . It is native to southern Europe and the Mediterranean area and is commercially cultivated in Erance, Spain, Portugal, Hungary, UK, Bulgaria, Australia, China, and USA [13]. This paper describes a laboratory study examining the contact and fumigant toxicity of essential oils of F. vulgare and L. angustifolia grown in Iran against of T. urticae fol- lowed by evaluation of their chemical constituents by Gas chromatography- Mass spectrometry (GC-MS). 2. Materials and Methods 2.1. Rearing of Two-Spotted Spider Mite. The two-spotted spider mites were collected from infested leaves of some wildly grown weeds in the yard of University of Mohaghegh Ardabili which did not have any exposure to acaricides. The Tetranychus urticae Koch species after separation, and slide preparation was identified according to introduced keys by Zhang [1]. Spider mites were reared on navy bean {Vigna unguiculata Walp. [Eabales: Fabaceae]) plants for one year. The infested plants were held in cages (120 x 300 x 100 cm) covered with mesh cloth. To synchronize the adult stage of T. urticae for adulticidal bioassays, 50 adult female mites were transferred to the leaves of trifoliate bean plants (held individually in cylindrical glass containers with appropriate aeration) with a hair brush and allowed to lay eggs for 24 h. after which the adults were removed. The infested leaves were held at the above-mentioned conditions to allow the eggs to hatch and the larvae to develop into synchronized adults. All experiments were carried out at 25 ± 2°G, 60 ± 5% relative humidity (RH) and a photoperiod of 16 : 8 (light : dark) in a growth chamber. 2.2. Plant Materials and Essential Oil Extraction. Aerial parts 5 cm from the top of L. angustifolia at flowering stage and seeds of F. vulgare were collected from Ardabil, Ardabil province, Iran from June to August 2013. The specimens were air dried in the shade at room temperature and chopped into small pieces with electric grinder. The essential oils were extracted using a Glevenger-type water steam distillation apparatus within 3h. To carry out the extraction, 100 g of powdered plant material was used along with 1200 mL distil- lated water. Anhydrous sodium sulphate was used to remove excess water after extraction. The essential oils were trans- ferred to dark brown glass vials covered with aluminum foil and stored in refrigerator at 4“ G until used in the experiments. 2.3. Analysis of Essential Oil. One pL of prepared essential oil was injected to GG-MS (HP Agilent 6800N/(61530N) with GPSiBGB column (Ghrompack, 100% dimethyl polysiloxane 60 m, 0.25 mm (ID) film thickness 0.251). The analysis was performed under temperature programming from 100“ G (3 min) to 250°G (5 min) with the rate of 3“G/min. Injector temperature was 230“ G. Identification of spectra was carried out by study of their fragmentation and also by comparison with standard spectra present in the library of the instrument. Area normalization was used for determination of composi- tion percentage. 2.4. Contact Toxicity. Gontact toxicity was conducted in Petri dishes (6 cm diameter). Goncentrations ranged from 0.12% to 2.8% for L. angustifolia and 0.11% to 1.7% for F. vulgare, using a spreader sticker adjuvant (20|WL Tween, 0.02%) diluted in distilled water. Leaf discs (3 cm diameter) were cut from leaves of greenhouse-grown U unguiculata and immersed in solutions of the each essential oil for 20 seconds. After drying at room temperature for 45 min, each disc was individually placed at the bottom of a Petri dish atop a 6 cm diameter disc of filter paper wetted with distilled water. The wet cotton pads were then placed on excised leaves and ten adult females were transferred thereafter. The lids of Petri dishes were pierced (1cm in diameter) and their openings were covered with mesh cloth in order to evade of fumigant toxicity. Gontrol mites were held on leaf discs immersed in dilutions without essential oils. Mortality was noted after 24 h and there were tree replicates for each treatment. Mites were considered dead if appendages did not move when prodded with a fine paintbrush. 2.5. Fumigant Toxicity. For evaluation of fumigant toxicity, 750 mL plastic containers with tight lids were used as the test chambers. Each treatment consisted of five concentrations of the essential oil and a control. Based on preliminary exper- iments, ranges of concentrations tested against the adult Psyche 3 Figure 1: Concentration-mortality response lines for adult females of Tetranychus urticae exposed to different concentrations of Foeniculum vulgare and Lavandula angustifolia essential oils. females were 0.53 to 4.2 and 1.33 to 2.4 fiL/L air for L. angusti- folia and F. vulgare, respectively. For each concentration, three replicates were used. Each replicate consisted of sixty 24 h old adult females on each leaf plant disc. The discs (3 cm in diameter) punched from leaves of bean plants were placed inside a 6 cm diameter plastic Petri dishes without lids lined with water- soaked cotton. The Petri dishes were then put into plastic containers used as fumigation chambers. To achieve the desired concentration of the oil in the fumigation cham- bers, using a micropipette, the appropriate volume of the oil was applied on a 2 x 2 cm strip of Whatman no. 1 filter paper adhered to the inner surface of the fumigation chamber. The exposure period for assessing the adulticidal effect of the essential oils was 24 h. To determine mortality, the mites were touched with the tip of a fine hair brush. If the mite did not move, it was considered dead. The controls consisted of the same number of mites as the treatments; and were kept under the same conditions on leaf disks left untreated. 2.6. Analysis of Data. Experiments were arranged in a com- pletely randomized design and data were analyzed by ANO- VA. The mortality data were subjected to probit analysis using SPSS software to estimate LC50 values of the essential oils against T. urticae. 3. Results Using hydro distillation process, fennel seed yielded 2.15% essential oil while lavender leaf yielded 2.01%. Results of analysis of the essential oils are presented in Table 1. Twenty six compounds were identified in the essential oil of fennel. representing 99.94% of the total essential oil sample while twenty five compounds were found in the lavender essential oil, representing 99.97% of the total essential oil sample. The major components were found to be anethole (46.73%), limonene (13.65%), a-fenchone (8.27%), carvone (6.12%), and estragole (5.26%) for F. vulgare essential oil and Linalool (28.63%), 1,8-Cineole (18.65%), 1-Borneol (15.94%), Camphor (8.20%), and Terpineol-4 (4.27%) for L. angustifolia essential oil. Amounts of monoterpenes hydrocarbon in the essential oils of F. vulgare and L. angustifolia were 20.43% and 8.55%, respectively. Monoterpenoids content of F. vulgare and L. angustifolia essential oils was 20.27% and 83.36% and the sesquiterpenes were 0.44% and 2.38%, respectively. F. vulgare and L. angustifolia essential oils revealed strong significant toxicity on the adult females of T. urticae in both contact and fumigant assays. The activity depended on essen- tial oil concentrations in both contact and fumigant bioassays and increased susceptibility of mite was directly associated with oil concentration (Table 2 and Figure 1). In the contact toxicity, lethal concentration 50% mite mortality (LC50) was 0.557% and 0.792% with F. vulgare and L. angustifolia, respectively, with F. vulgare essential oil being the most toxic the adult females of T. urticae (Table 2). On the other hand, fumigant toxicity was 1.876 and 1.971 fiL/L air for F. vulgare and L. angustifolia essential oils, respectively (Table 2). 4. Discussion In the present study, anethole, limonene, a-fenchone, and carvone were the major compounds of essential oil of 4 Psyche Table 1: Chemical analysis of essential oils of Foeniculum vulgare and Lavandula angustifolia grown in Iran by GC-MS. Compound E RT vulgare Percentage L. angustifolia RT Percentage Eormula Molecular weight (g/mol) Classification a-Pinene 3.93 3.53 4.80 1.46 ^10^16 136.23 Monoterpene Camphene 4.17 0.32 5.08 0.68 Qo^ie 136.23 Monoterpene Sabinene 4.51 0.67 5.54 0.96 ^10^16 136.23 Monoterpene jS-Pinene 4.61 0.20 5.64 2.16 ^10^16 136.23 Monoterpene Myrcene 4.74 1.09 5.84 1.39 ^10^^16 136.23 Monoterpene a-Phellandrene 5.05 0.54 — — ^10^16 136.23 Monoterpene Limonene 5.61 13.65 — — ^10^16 136.23 Monoterpene l-Octen-3-ol — — 5.73 0.46 CgHigO 128.21 Alcohol y-Terpinene 6.06 0.43 — — Qo^ie 136.23 Monoterpene d-3-Carene — — 6.27 0.72 ^10^^16 136.23 Monoterpene a-Fenchone 6.83 8.27 — — Qo^ie^ 152.23 Monoterpenoid 1 , 8 -Cineole — — 6.87 18.65 ^10^18^ 154.25 Monoterpenoid trans-jS-Ocimene — — 7.10 0.98 ^10^16 136.23 Monoterpene Terpineol-4 — — 7.58 4.27 ^10^18^ 154.25 Monoterpenoid Terpinolene — — 7.99 0.50 Qo^ie 136.23 Monoterpene Linalool — — 8.64 28.63 ^loHigO 154.25 Monoterpenoid cis-Sabinenehydrate — — 8.99 0.51 ^0^18^ 154.25 Monoterpenoid Camphor 7.92 0.28 9.58 8.20 Qo^ie^ 152.23 Monoterpenoid Estragole 9.12 5.26 — — 148.20 Aromatic hydrocarbon trans-Dihydrocarvone 9.24 0.47 — — Qo^isO 152.23 Monoterpenoid D-Fenchyl alcohol 9.49 0.18 — — Qo^18^ 154.25 Monoterpenoid Fenchol 9.83 1.78 — — QoHi80 154.25 Monoterpenoid 1-Borneol — — 10.17 15.94 ^10^18^ 154.25 Monoterpenoid Carvone 10.18 6.12 — — C 10 H 14 O 150.22 Monoterpenoid /-Carvone 10.26 3.17 — — C 10 H 14 O 150.22 Monoterpenoid Hexyl butyrate — — 10.43 1.56 Qo^20^2 172.26 Patty ester Cryptone — — 10.51 0.58 C 9 H ^40 138.21 Ketonic chelate a-Terpineol — — 10.61 3.25 ^10^18^ 154.25 Monoterpenoid Anethole 11.23 46.73 — — ^ 10 ^ 12 ^ 148.21 Aromatic hydrocarbon Bornyl formate — — 11.29 0.61 ^1^18 ^2 182.26 Ethyl ester Linalyl acetate — — 11.83 2.18 Q 2 H 20 O 2 196.29 Monoterpenoid Geraniol acetate — — 12.55 1.05 ^ 2^20 ^2 196.29 Monoterpenoid Eugenol 12.72 3.75 — — Q 0 H 12 O 2 164.20 Carboxylic Acid Thymol — — 12.80 0.68 C 10 H 14 O 150.28 Monoterpenoid cis-Jasmone 13.09 0.69 — — CnHigO 164.24 Patty Acid Anisyl acetone 13.17 0.36 — — ^3^16^2 204.27 Ketonic ether - C ar y ophyllene 13.90 0.22 — — Q 5 H 24 204.35 Sesquiterpene Germacrene d 15.11 0.22 — — Q 5 H 24 204.35 Sesquiterpene Eugenyl acetate 15.96 0.91 — — C 12 H 14 O 3 206.24 Carboxylic Acid jS-Earnesene — — 16.05 0.81 Q 5 H 24 204.35 Sesquiterpene Benzeneacetic acid 17.07 0.17 — — ^8 ^8 ^2 136.15 Carboxylic Acid cis-isoapiole 17.85 0.28 — — Q 2 H 14 O 4 222.24 Phenylpropanoids a-Bisabolol — — 20.56 1.57 Q5^^26^ 222.37 Sesquiterpenoid a- ethyl- 4,4- dimethoxy- Stilbene 30.91 0.64 — — ^6^16^2 240.30 Ketonic chelate 1,2-Benzenedicarboxylic acid — — 33.67 2.17 ^8^6 ^4 166.13 Carboxylic Acid Psyche 5 Table 1: Continued. Compound E vulgare RT Percentage L. angustifolia Formula Molecular weight (g/mol) RT Percentage Classification Monoterpene hydrocarbons 20.43 8.85 Oxygenated monoterpenes 20.27 83.36 Sesquiterpene hydrocarbons 0.44 0.81 Oxygenated sesquiterpenes 0 1.57 Others 58.8 5.38 Total 99.94 99.97 Yield 2.15 2.01 RT: retention time (min). Table 2: Contact and fumigant toxicity of the essential oils isolated from Foeniculum vulgare and Lavandula angustifolia against the adult females of Tetranychus urticae. Results of ANOVA Results of probit analysis Bioassay Essential oil F(df=4, 10)" P value 24-h LC5Q with 95% confidence limits’^ Slope ± SE (df=3) Sigf Toxicity index Contact E vulgare 49.75 1.4446 0.557 (0.445-0.716) 1.181 ± 0.144 3.327 0.351 100.00 toxicity L. angustifolia 33.30 9.3842 0.792 (0.598-1.091) 0.936 ± 0.124 1.620 0.655 0.703 Fumigant E vulgare 37.70 0.000005 1.876 (1.786-1.982) 5.335 ± 0.670 3.086 0.379 100.00 toxicity L. angustifolia 18.167 0.0001 1.971 (1.628-2.478) 1.377 ± 0.187 3.975 0.264 0.951 ‘‘Calculated values are greater than values in F table (a = 0.05, F credit = 3.4780). Therefore, they are significant. v/v and gL/L air for contact and fumigant toxicity, respectively. ‘^Since the significance level is greater than 0.150, no heterogeneity factor is used in the calculation of confidence limits. F. vulgare while linalool, 1,8-cineole, 1-borneol and camphor were the main compounds of essential oil of L. angustifolia. In the study of Chowdhury et al. [14] , anethole (58.5% in seed oil and 51.1% in leaf oil) and limonene (22.9% in leaf oil and 19.6% in seed oil) determined as main components of the seeds and leaves of F. vulgare. In the other study, borneol, a-terpinene, linolool, and geranyl proprionate were found as major constituents in the L. angustifolia essential oil. The variations could be due to differences in location, elevation, and genetic makeup of the plant or due to an adaptive process to particular ecological conditions [15]. Acaricidal activity of F. vulgare and L. angustifolia essen- tial oils reported in the present study has been reported ear- lier by other authors. For example, the essential oils from F. vulgare and L. angustifolia were toxic against Varroa destruc- tor Anderson and Trueman (a major pest of honey bees. Apis mellifera L.) [16] . The fumigant toxicity of essential oil extracted from seeds of F. vulgare was tested against adult females of T. urticae by Amizadeh et al. [17]. Essential oils are characterized by two or three major components at fairly high concentrations (20-70%) com- pared to others components present in trace amounts. The components include two groups of distinct bio synthetical origin. The main group is composed of terpenes and terpe- noids and the other of aromatic and aliphatic constituents, all characterized by low molecular weight [6] . They are made from combinations of several 5-carbon-base (C5) units called isoprene. The monoterpenes are formed from the coupling of two isoprene units (C^q)- They are the most representative molecules of the essential oils and allow a great variety of structures. A terpene containing oxygen is called a terpenoid. Hence, monoterpenes are found as two forms; monoterpenes hydrocarbon and oxygenated monoterpenes or monoter- penoids. The sesquiterpenes are formed from the assembly of three isoprene units (0^5) [6]. Monoterpenes hydrocarbon, monoterpenoids, and sesquiterpenes are present in our tested study too. Regarding their biological properties, essential oils are complex mixtures of numerous molecules, and their bio- logical effects are the result of a synergism of all components or reflect only those of the main components present at the highest levels according to gas chromatographical analysis [6, 18] . It is suggested that the variability of biological activities of essential oils extracted from different plant species against T. urticae could be due to chemical components, differences in their chemical composition, and even in synergic and antag- onistic interactions between these components [2, 19, 20] . One of the most attractive features of essential oils is that they are low-risk products and they are relatively well-studied experimentally and clinically because of their use as medici- nal products [11]. In terms of ecotoxicology, essential oils are safe to use but not without potential problems. For example, constituents of essential oils are biodegradable, with short half-lives ranging from 30 to 40 h for a-terpineol [21] and in contrast to some synthetic insecticides; no biomagnification has been reported to date [11] . Their short residual half-lives on plants also enhance their compatibility with biological control agents and indigenous natural enemies of pests and reduce risks to honeybees and other foraging pollinators [2]. 6 Psyche As cost effective commercial problems, large quantities of plant material must be processed to obtain sufficient quan- tities of essential oils for commercial-scale tests, situation which also requires breeding these plants in great quantities. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. References [f] Z. Q. Zhang, Mites of Greenhouses; Identification, Biology and Control, CABI Publishing, Wallingford, UK, 2003. [2] M. B. Isman, S. Miresmailli, and C. MacHial, “Commercial opportunities for pesticides based on plant essential oils in agriculture, industry and consumer products,” Phytochemistry Reviews, vol. fO, no. 2, pp. 197-204, 2011. [3] R. L. Brandenburg and G. G. Kennedy, “Ecological and agricul- tural considerations in the management of two spotted spider mite (Tetranychus urticae Koch),” Agricultural Zoology Reviews, vol. 2, pp. 185-236, 1987. [4] T. Ramasubramanian, K. Ramaraju, and A. Regupathy, “Acar- icide resistance in Tetranychus urticae Koch (Acari: Tetran- ychidae) — global scenario,” Journal of Entomology, vol. 2, pp. 33-39, 2005. [5] P. Tirello, A. Pozzebon, S. CassaneUi, T. van Leeuwen, and C. Duso, “Resistance to acaricides in Italian strains of Tetranychus urticae: toxicological and enzymatic assays,” Experimental and Applied Acarology, vol. 57, no. 1, pp. 53-64, 2012. [6] F. Bakkali, S. Averbeck, D. Averbeck, and M. Idaomar, “Bio- logical effects of essential oils — a review,” Eood and Chemical Toxicology, vol. 46, no. 2, pp. 446-475, 2008. [7] M. B. Isman, “Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world,” Annual Review of Entomology, vol. 51, pp. 45-66, 2006. [8] S. Miresmailli and M. B. Isman, “Efficacy and persistence of rosemary oil as an acaricide against twospotted spider mite (Acari: Tetranychidae) on greenhouse tomato,” Journal of Eco- nomic Entomology, vol. 99, no. 6, pp. 2015-2023, 2006. [9] C. Regnault-Roger, C. Vincent, and J. T. Arnason, “Essential oils in insect control: low-risk products in a high-stakes world,” Annual Review of Entomology, vol. 57, pp. 405-425, 2012. [10] L. S. Moura, R. N. Carvalho Jr., M. B. Stefanini, L. C. Ming, and M. A. A. Meireles, “Supercritical fluid extraction from fennel (Eoeniculum vulgare): global yield, composition and kinetic data,” Journal of Supercritical Eluids, vol. 35, no. 3, pp. 212-219, 2005. [11] G. D. Semiz, A. Unliikara, E. Yurtseven, D. L. Suarez, and I. Telci, “Salinity impact on yield, water use, mineral and essential oil content of fennel (Eoeniculum vulgare MiU.),” Tarim Bilimleri Dergisi, vol. 18, no. 3, pp. 177-186, 2013. [12] M. Wichtl, Herbal Drugs and Phytopharmaceuticals, Medpharm Scientific Publishers, Stuttgart, Germany, 1994. [13] A. S. Shawl and S. Kumar, “Potential of lavender oil industry in Kashmir,” Journal of Medicinal and Aromatic Plant Sciences, vol. 22, pp. 319-321, 2000. [14] J. U. Chowdhury, M. H. Mobarok, M. N. I. Bhuiyan, and N. C. Nandi, “Constituents of essential oils from leaves and seeds of Eoeniculum vulgare Mill, cultivated in Bangladesh,” Bangladesh Journal of Botany, vol. 38, no. 2, pp. 181-183, 2009. [15] V. A. Facundo, A. R. Polli, R. V. Rodrigues, J. S. L. T. Militao, R. G. StabeUi, and C. T. Cardoso, “Constituintes quimicos fixos e volateis dos talos e frutos de Piper tuberculatum Jacq. e das raizes de P hispidum H. B. K,” Acta Amazonica, vol. 38, pp. 733-742, 2008. [16] F. Kutukoglu, A. O. Girisgin, and L. Aydin, “Varroacidal efficacies of essential oils extracted from Lavandula officinalis, Eoeniculum vulgare, and Taurus nobilis in naturally infested honeybee (Apis mellifera L.) Colonies,” Turkish Journal of Vet- erinary and Animal Sciences, vol. 36, no. 5, pp. 554-559, 2012. [17] M. Amizadeh, M. J. Hejazi, and G. A. Saryazdi, “Fumigant toxicity of some essential oils on Tetranychus urticae (Acari: Tetranychidae),” International Journal of Acarology, vol. 39, no. 4, pp. 285-289, 2013. [18] S. Miresmailli, R. Bradbury, and M. B. Isman, “Comparative toxicity of Rosmarinus officinalis L. essential oil and blends of its major constituents against Tetranychus urticae Koch (Acari: Tetranychidae) on two different host plants,” Pest Management Science, vol. 62, no. 4, pp. 366-371, 2006. [19] C. Regnault-Roger and A. Hamraoui, “Fumigant toxic activity and reproductive inhibition induced by monoterpenes on Acan- thoscelides obtectus (Say) (coleoptera), a bruchid of kidney bean (Phaseolus vulgaris F.),” Journal of Stored Products Research, vol. 31, no. 4, pp. 291-299, 1995. [20] S. Attia, K. F. Grissa, G. Fognay, S. Heuskin, A. C. Mailleux, and T. Hance, “Chemical composition and acaricidal properties of Deverra scoparia essential oil (Araliales: Apiaceae) and blends of its major constituents against Tetranychus urticae (Acari: Tetranychidae),” Journal of Economic Entomology, vol. 104, no. 4, pp. 1220-1228, 2011. [21] G. Misra and S. G. Pavlostathis, “Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems,” Applied Micro- biology and Biotechnology, vol. 47, no. 5, pp. 572-577, 1997. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 687979, 5 pages http://dx.doi.org/10.1155/2014/687979 Research Article Application of Asiatic Honey Bees {Apis cerana) and Stingless Bees {Trigona laeviceps) as Pollinator Agents of Hot Pepper {Capsicum annuum L.) at Local Indonesia Farm System 1 1 ^ Ramadhani Eka Putra, Agus Dana Permana, and Ida Kinasih ^School of Life Sciences and Technology, Bandung Institute of Technology, Ganesha Street No. 10, Bandung 40132, Indonesia ^Department of Biology, Islamic State University Sunan Gunung Djati, Bandung 40614, Indonesia Correspondence should be addressed to Ramadhani Eka Putra; ramadhani@sith.itb.ac.id Received 26 September 2014; Accepted 4 December 2014; Published 30 December 2014 Academic Editor: Jan Klimaszewski Copyright © 2014 Ramadhani Eka Putra 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 Indonesia, hot pepper {Gapsicum annuum) is one of the most important spices. Despite the fact that high yield cultivars and fertilizers have been applied to increase the annual production of this spice, local farming is always unable to maintain constant production. Studies to find the explanation of this problem mostly focused on pest attack while possibility of low fruit production due to lack of pollination was neglected. In this study, the effect of pollinator visitation to fruit set and quality was assessed by application of two local domesticated honey bees, Asiatic honey bees {Apis cerana) and stingless bees ( Trigona laeviceps) as potential pollinator agents at hot pepper plantation. This study found that both bees had similar visitation rate while A. cerana spend less time in flowers. Visitation by A. cerana and Trigona laeviceps improved fruit set, fruit production per plant, average fruit weight, and fruit size. This result confirms the importance of cross pollination for hot pepper production and both species could be used as pollination agent for hot pepper. Advantages and disadvantages for each species as pollination agent for local Indonesia farm system are discussed in this paper. 1. Introduction Hot pepper {Capsicum annuum L.) is cultivated and con- sumed around the world. Its major producers are United States, Mexico, Italy, Japan, India, and Brazil, where this crop has economic importance. Best way to cultivate pepper is in greenhouses which allows production all year round, best management practices, better fruit quality control, lesser or no use of pesticides, earlier harvesting, and superior uniformity of fruits [1] . However, in Indonesia, hot pepper chili usually cultivated at open field where local farmer usually apply best seeds, extensive weed and pest control, and monoculture system in order to obtain high yield. Despite all of these efforts local farmer could not maintain sustained productivity since open field cultivation highly depend on climate condition and ecosystem services, namely. pest control, nutrition cycle, and pollination to produce abundant harvest and good quality fruit [2-5] . Among all available ecosystem services, this study focused on pollination service. Pepper flowers, like those of most cultivated Solanaceae, are pendent from leaf axils, showing a white corolla, five to seven stamens containing 1.0 to 1.5 mg of pollen, and one central style with a round sticky stigma on its top. Anthers are tubular, and dehiscence occurs through lateral opening. Both flower anthesis and anther dehiscence take place in the morning [6]. Although pepper flowers are largely self-pollinated, introducing pollinators could produce beneficial effects on fruit production. Among pollination agents available in nature, wild insects had been considered as the best pollinator agents and receive huge attention as important component of agriculture sys- tems [7, 8]. In Indonesia, wild pollinator insects have not 2 Psyche yet received notice as important component of agriculture system even though some studies have shown the importance of insects as pollinator for some Indonesian perennial and annual crops [9-13]. Lack of understanding on function of particular insects as pollinator agents combined with common practices of synthetic insecticides, removal of wild plants, and destruction of nesting area through plowing significantly reduced population of wild pollinator [14]. In many intensive plantations, to ensure pollination of crop, domesticated bees usually applied as pollinator agents. In case of pepper pollination, best pollinator agent is bum- blebees which carried out “buzz pollination,” a mechanism related with behavior of bumblebees in order to release pollen of pepper flowers to pollinate female flowers [15-17]. However, bumblebees are not native species of Indonesia and could potentially cause negative effects on native pollinators and plants [18, 19]. Alternatively, local bee species, such as Asiatic honey bees {Apis cerana) and various species of stingless bees {Trigona laeviceps), which domesticated by bee farmers for their products (honey, propolis, and wax), may apply as pollinator agents for hot pepper. Previous study showed their possible application as pollinator of tomato flowers, plant with similar flower characteristic with hot pepper flowers [20]. In this study we will evaluate performance of Indonesia domesticated bees as pollination agent of hot pepper and its possible use and concern for application at local farm system. 2. Materials and Methods 2.1. Study Area and Research Materials. The pollination experiment was conducted at local farm in North Bandung, West Java, Indonesia. Average daily temperature of study site was 20-25°C with humidity 70-75%. For this purpose, thirty red chili plants, planted in pots, were arranged in 3 rows of 10 plants each, with two-meter-wide aisle between the rows. Four colonies (~500 bees per colony) of Trigona laeviceps and four colonies of A. cerana (=10,000 bees) were introduced into farm. All colonies were kept in bee hive made from wood and acclimatized for 3 months prior to study. 2.2. Bee Visitation Frequency. Frequency of bee visitation was observed during flowering period by method developed by Klein et al. [12] whereas observation was conducted only at sunny day or 60% cloudy day between 0900 and 1400 (local time). Observation was conducted with interval of 15 minutes for three consecutive days at different plant. Total number of flowers observed for three days was 100 flowers. 2.3. Bee Pollination Efficiency. In this study, 10-20 flowers from each plant (depend on the number of available flowers), that still not bloomed were randomly selected and tagged. Total number of plants used for each experiment group, explained below, was 10. Each group of flower was bagged with mesh nylon bag (diameter 1 mm). Glue was applied at the twig where flowers were located to prevent ant from entering flower. Bags were kept until fruit production for self-pollinated group. As for bee pollinated group, bags were removed when flower started to bloom. Observation for bee pollination effi- ciency started from removal of the bag until bee transferred pollen to female flower. After pollination process, flowers were bagged until fruit was produced. This group of treatment was designed as honey bee (HB) and stingless bee (SB) group. Pollination studies of honey bee and stingless bee were conducted in different period. As for control group (NP), bag was not removed from flower until fruit was produced or all flowers has dehiscenced. Pollination efficiency for each group was measured by Pollination efficiency Total numbers of flowers that produce fruits Total numbers of observed flowers ( 1 ) 2.4. Fruit Production. Fruit production was measured for every type of pollination. Fruit production was measured by subtracting total number of broken fruits from total fruit produced. 2.5. Fruit Quality. Fruit quality was identified by measuring weight and size of fruit produced from 50 fruits, as soon as they were red in color. The fruits were weighed to the nearest gram and their size was measured to the nearest centimeter. 2.6. Data Analysis. Data was analyzed by statistic program Statistica 8.0 (StatSoft Corp.). Prior to analysis, normality of data was tested. Difference of bee visitation frequency between honey bees and stingless bees was analyzed by t- test analysis. Difference of fruit quantity and quality among different pollination types was analyzed by ANOVA and LSD as post hoc test. Significant value for both tests was P < 0.05. 3. Results and Discussion 3.1. Bee Visitation. We found that visitation rate of honey bee to hot pepper flowers similar to stingless bee {t-test analysis, P > 0.05) (Figure 1(a)). Detail observation on visitation pattern of both species showed that honey bee preferred to visit flowers in early morning while stingless bee visited flowers with relatively constant rate (Figure 1(b)). Early foraging bout by honey bees is probably related with availability of fresh pollen and nectar and attractiveness of flower signal. Hot pepper flowers bloom early in the morning, which provides access to fresh pollen and nectar which is located near base of petal [21]. During midday, when most of flowers were dehisced and provided less resources to be harvested by forager honeybees, most of foragers foraged on different flowering plants (Putra, personal observation). The shifting of foraging force of honey bees among food sources was well reported [22-24]. Stingless bees which are smaller and less aggressive preferred late foraging as they usually took long exploratory flight to find rich, suitable, and economic resources [25]. These bees probably exploited remaining pollen and nectar Psyche 3 Table 1: Average fruit production, weight, and fruit size pollinated by local honey bee, stingless bee, and self-pollination (N = 50). Group Apis cerana Trigona laeviceps Self-pollinated Fruit Ppoduction per plant 22 ± 3.5^^ 20 ± 3.5" 17 ± 3.5'^ Average fruit weight (g) 12.55 ± 4.17^ 11.16 ± 4.99" 9.16 ± 2.99’' Fruit size (cm) 25.16 ± 9.99" 24.78 ± 2.47" 20.58 ± 3.47’' Different letter indicated statistical difference at P < 0.05. Time I I Honeybees I I Stingless bees (a) (b) Figure 1: (a) Visitation rate and (b) visitation pattern of honey bees and stingless bees to hot pepper flowers. Figure 2: Pollination efficiency of hot pepper by honey bee, stingless bee, and self-pollination. which is inaccessible by honey bees due to their bigger size. Stingless bee also tends to specialize on hot pepper flow- ers, also known as flower constancy, after competition from honey bee was reduced. Flower constancy is widely found on many species of stingless bee [26-28]. 3.2. Pollination Efficiency and Fruit Quality. On average, pollination efficiency of honey bee on hot pepper much higher than stingless bee and pollination assisted by both bee species more efficient than wind pollination {t-test analysis, P < 0.05) (Figure 2). Total number of fruits produced and quality of hot pepper produces was enhanced significantly by bee pollinated {One way Anova, P < 0.05). Both bee species provide similar Figure 3: Handling time of honey bee and stingless bee on hot pepper flowers. quality of pollination to hot pepper in terms of fruit produce and size of the fruit (Table 1). This study confirmed that cross pollination improve pollination efficiency and quality of pepper as also reported by de Oliveira Cruz et al. [1], Roldan Serrano and Guerra- Sanz [16], and Al-Abbadi [17]. Based on this data, honeybee and stingless bee have great potential as pollinator insect for hot pepper. Even though both species are known for not showing capability to carry out buzz pollination mechanism [15], they seem to provide some disturbances which improve pollination [29, 30] . Both species provided pollination in different way which is related to handling time. Honey bees seem more economic species because they spend less time in flower, yet produce better pollination success than stingless bees (Figure 3). 4 Psyche Higher pollination success could be caused by their bigger size, which produced more disturbances to flowers, and they tend to visit more fertile flowers. On the other hand, higher flower handling and flower constancy of stingless bee increased the possibility of pollen deposited in stigma. 4. Conclusion Both species have several advantages and disadvantages when applied on common Indonesia farming. Asiatic honey bees have higher pollination efflciency, commonly domesticated by local bee farmers, and have wider foraging area which made them suitable candidate as pollinator agent of hot pepper. However, their aggres- sive and absconding behavior with high nectar and pollen requirement reduce their value as pollinator agent of small hot pepper plantation farm located nearby human residence and/or plantation with unsustain nectar and pollen resources. On the other hand, stingless bees more likely to be apply as pollinator agent at plantation located nearby human residence without sustain nectar and pollen resources [31, 32]; small foraging area may provide high visitation rate at small and conflned agriculture [30, 33]; their lack of functional sting and less aggressive behavior made them highly suitable for pollination of crops cultivated nearby human dwelling [34, 35]. Furthermore, these bees foraging on varied plants [36-38] made them applicable as pollinator for varied types of local crops, even though further studies are needed for possible mismatch (see [35]). However, great concern should be addressed on application of insecticide, a common procedure on Indonesia agriculture system, as stingless bee is highly sensitive to common pesticide applied on local farm (Putra, unpublished data). Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Authors’ Contribution All authors contributed signiflcantly to this work. Acknowledgment This research was funded by Grant STRANAS of DIKTI Indonesia granted to first author. References [1] D. de Oliveira Cruz, B. M. Freitas, L. A. da Silva, E. M. S. da Silva, and I. G. A. Bomfim, “Pollination efficiency of the stingless bee Melipona subnitida on greenhouse sweet pepper,” Pesquisa Agropecuaria Brasileira, vol. 40, no. 12, pp. 1197-1201, 2005. [2] T. H. Ricketts, “Tropical forest fragments enhance pollinator activity in nearby coffee crops,” Conservation Biology, vol. 18, no. 5, pp. 1262-1271, 2004. [3] T. Tscharntke, A. M. Klein, A. Kruess, I. Steffan-Dewenter, and C. Thies, “Landscape perspectives on agricultural intensi- fication and biodiversity — ecosystem service management,” Ecology Letters, vol. 8, no. 8, pp. 857-874, 2005. [4] C. Kremen and R. Chaplin -Kramer, “Insects as providers of ecosystem services: crop pollination and pest control,” in Insect Conservation Biology, A. J. A. Stewart, T. R. New, and O. T. Lewis, Eds., pp. 349-382, CABI, Wallingford, UK, 2007. [5] R. Winfree, N. M. Williams, H. Gaines, J. S. Ascher, and C. Kremen, “Wild bee pollinators provide the majority of crop visitation across land-use gradients in New Jersey and Pennsylvania, USA,” Journal of Applied Ecology, vol. 45, no. 3, pp. 793-802, 2008. [6] A. Dag and Y. Kammer, “Comparison between the effectiveness of honey bee {Apis mellifera) and bumble bee (Bombus terrestris) as pollinators of greenhouse sweet pepper (Capsicum annuum)J American Bee Journal, vol. 141, pp. 447-448, 2001. [7] K. S. Delaplane and D. P. Mayer, Crop Pollination by Bees, CABI, Wallingford, UK, 2000. [8] S. S. Greenleaf and C. Kremen, “Wild bee species increase tomato production and respond differently to surrounding land use in Northern California,” Biological Conservation, vol. 133, no. 1, pp. 81-87, 2006. [9] A.-M. Klein, S. A. Cunningham, M. Bos, and I. Steffan- Dewenter, “Advances in pollination ecology from tropical plan- tation crops,” Ecology, vol. 89, no. 4, pp. 935-943, 2008. [10] S. Notodimejo, “Influence of growth regulators Dormex, Pro- malin, foliar fertilizer Algifert and release of honey bees on the development and production of mango in East Java,” Agrivita, vol. 18, no. 2, pp. 43-50, 1995. [11] A.-M. Klein, I. Steffan-Dewenter, and T. Tscharntke, “Emit set of highland coffee increases with the diversity of poUinating bees,” Proceedings of the Royal Society B: Biological Sciences, vol. 270, no. 1518, pp. 955-961, 2003. [12] A.-M. Klein, I. Steffan-Dewenter, and T. Tscharntke, “Polli- nation of Coffea canephora in relation to local and regional agroforestry management,” Journal of Applied Ecology, vol. 40, no. 5, pp. 837-845, 2003. [13] R. Olschewski, T. Tscharntke, P. C. Benitez, S. Schwarze, and A.- M. Klein, “Economic evaluation of pollination services compar- ing coffee landscapes in Ecuador and Indonesia,” Ecology and Society, vol. 11, no. 1, article no. 7, 2006. [14] C. Kremen and R. Chaplin -Kramer, “Insects as providers of ecosystem services: crop pollination and pest control,” in Insect Conservation Biology: Proceedings of the Royal Entomological Society’s 23rd Symposium, A. J. A. Stewart, T. R. New, and O. T. Lewis, Eds., pp. 349-382, CABI, Wallington, UK, 2007. [15] S. L. Buchmann, “Buzz pollination in angiosperms,” in Hand- book of Experimental Pollination Biology, C. E. Jones and R. J. Little, Eds., pp. 73-114, Van Nostrand Reinhold, New York, NY, USA, 1983. [16] A. Roldan Serrano and J. M. Guerra-Sanz, “Quality fruit improvement in sweet pepper culture by bumblebee pollina- tion,” Scientia Horticulturae, vol. 110, no. 2, pp. 160-166, 2006. [17] S. Y. A. Al-Abbadi, “Efficiency of different pollination treat- ments on solanaceae yields grown in plastic house,” Journal of Biological Sciences, vol. 9, no. 5, pp. 464-469, 2009. [18] A. B. Kingston and P. B. McQuillan, “Displacement of Tas- manian native megachilid bees by the recently introduced bumblebee Bombus terrestris (Linnaeus, 1758) (Hymenoptera: Apidae),” Australian Journal of Zoology, vol. 47, no. 1, pp. 59-65, 1999. Psyche 5 [19] D. Goulson, “Effects of introduced bees on native ecosystem” Annual Review of Ecology, Evolution, and Systematics, vol. 34, pp. 1-26, 2003. [20] R. E. Putra and I. Kinasih, “Efficiency of local Indonesia honey bees {Apis cerana L.) and stingless bee (Trigona iridipennis) on Tomato (Lycopersicon esculentum Mill.) pollination,” Pakistan Journal of Biological Sciences, vol. 17, no. 1, pp. 86-91, 2013. [21] S. Vogel, “Remarkable nectaries: structure, ecology, organophy- letic perspectives. III. Nectar ducts,” Elora, vol. 193, no. 2, pp. 113-131, 1998. [22] T. D. Seeley, S. Camazine, and J. Sneyd, “Collective decision- making in honey bees: how colonies choose among nectar sources,” Behavioral Ecology and Sociobiology, vol. 28, no. 4, pp. 277-290, 1991. [23] B. Granovskiy, T. Latty, M. Duncan, D. J. T. Sumpter, and M. Beekman, “How dancing honey bees keep track of changes: the role of inspector bees,” Behavioral Ecology, vol. 23, no. 3, pp. 588-596, 2012. [24] A. E. Wagner, B. N. Van Nest, C. N. Hobbs, and D. Moore, “Persistence, reticence and the management of multiple time memories by forager honey bees,” The Journal of Experimental Biology, vol. 216, no. 7, pp. 1131-1141, 2013. [25] C. S. Danaraddi, Studies on stingless bee, Trigona iridipennis Smith with special reference to foraging behaviour and melis- sopalynology at Dhawad, Karnataka [Master of science thesis], College of Agricultural Science, Dhawad, India, 2007. [26] T. Inoue, S. Salmah, I. Abbas, and E. Yusuf, “Eoraging behavior of individual workers and foraging dynamics of colonies of three Sumatran stingless bees,” Researches on Population Ecol- ogy, vol. 27, no. 2, pp. 373-392, 1985. [27] M. Ramalho, A. Kleiner t-Giovannini, and V. Imperatriz- Eonseca, “Important bee plants for stingless bees {Melipona and Trigonini) and Africanized honeybees {Apis mellifera) in neotropical habitats: a review,” Apidologie, vol. 21, no. 5, pp. 469- 488, 1990. [28] O. Cauich, J. J. G. Quezada-Euan, J. O. Macias-Macias, V. Reyes-Oregel, S. Medina-Peralta, and V. Parra-Tabla, “Behav- ior and pollination efficiency of Nannotrigona perilampoides (Hymenoptera: Meliponini) on greenhouse tomatoes {Lycoper- sicon esculentum) in subtropical Mexico,” Journal of Economic Entomology, vol. 97, no. 2, pp. 475-481, 2004. [29] A. Jarlan, D. de Oliveira, and J. Gingras, “Pollination by Eristalis tenax (Diptera: Syrphidae) and seed set of greenhouse sweet pepper,” Horticultural Entomology, vol. 90, pp. 1646-1649, 1997. [30] A. Raw, “foraging behavior of wild bees at hot pepper flowers {Capsicum annuum) and its possible influence on cross pollina- tion,” Annals of Botany, vol. 85, no. 4, pp. 487-492, 2000. [31] D. R. Campbell and A. E. Motten, “The mechanism of competi- tion between two forest herbs,” Ecology, vol. 66, no. 2, pp. 554- 563, 1985. [32] C. T. Ivey, P. Martinez, and R. Wyatt, “Variation in pollinator effectiveness in swamp milkweed, Asclepias incarnata (Apocy- naceae),” American Journal of Botany, vol. 90, no. 2, pp. 214-225, 2003. [33] T. Kakutani, T. Inoue, T. Tezuka, and Y. Maeta, “Pollination of strawberry by the stingless bee, Trigona minangkabau, and the honey bee. Apis mellifera: an experimental study of fertilization efficiency,” Researches on Population Ecology, vol. 35, no. 1, pp. 95-111, 1993. [34] T. A. Heard, “The role of stingless bees in crop pollination,” Annual Review of Entomology, vol. 44, pp. 183-206, 1999. [35] E. J. Slaa, L. A. Sanchez Chaves, K. S. Malagodi-Braga, and f. E. Hofstede, “Stingless bees in applied pollination: practice and perspectives,” Apidologie, vol. 37, no. 2, pp. 293-315, 2006. [36] D. W. Roubik, Ecology and Natural History of Tropical Bees, Cambridge University Press, Cambridge, UK, 1989. [37] M. Ramalho, T. C. Giannini, K. S. Malagodibraga, and V. L. Imperatriz-Eonseca, “Pollen harvest by stingless bee foragers (Hymenoptera, Apidae, Meliponinae),” Grana, vol. 33, pp. 239- 244, 1994. [38] J. C. Biesmeijer, E. J. Slaa, M. S. de Castro, B. E. Viana, A. Kleinert, and V. L. Imperatriz-Eonseca, “Connectance of Brazi- lian social bee — food plant networks is influenced by habitat, but not by latitude, altitude or network size,” Biota Neotropica, vol. 5, no. 1, pp. 85-93, 2005. Hindawi Publishing Corporation Psyche Volume 2014, Article ID 176539, 4 pages http://dx.doi.org/ 10 . 1 155/2014/ 176539 Research Article Cryptocephaline Egg Case Provides Incomplete Protection from Generalist Predators (Coleoptera: Chrysomelidae) Matthias SchoUer Faculty of Life Sciences, Humboldt-Universitdt zu Berlin, Lentzeallee 55/57, 14195 Berlin, Germany Correspondence should be addressed to Matthias Scholler; matthias.schoeller@hu-berhn.de Received 14 September 2014; Accepted 15 December 2014; Published 30 December 2014 Academic Editor: Jan Klimaszewski Copyright © 2014 Matthias Scholler. 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 egg case of Cryptocephalus rufipes (Goeze) is described and illustrated. In laboratory trials, eggs of field-collected C. rufipes were observed for larval emergence (untreated control) or exposed to two species of generalist predators, Chrysoperla carnea (Stephens) or Xylocoris flavipes (Reuter) in no-choice experiments. The behaviour of the predators upon contact with the C. rufipes eggs was observed. The number of hatching larvae was counted and compared. In the presence of each of the two species of predators, larval emergence was significantly reduced. Eggs that were not protected by an egg case were completely consumed by the predators. C. rufipes eggs were therefore incompletely protected from the studied generalist predators. This is the first study showing experimentally the protective function of cryptocephaline egg case. 1. Introduction Leaf beetles in the subfamilies Cryptocephalinae and Lam- prosomatinae cover their eggs with small faecal plates. These faecal plates compose a solid egg case that is completely covering the egg. After hatching, the larva is biting a hole in the egg case, and it is wearing it as a larval case. This larval case is continuously enlarged with larval faeces when the larva is moulting and growing [1-3] . One of the functions of the egg and larval cases is thought to be protection from natural enemies [4, 5]. However, both mammal and insect predators and hymenopterous parasitoids are known to accept cryptocephaline larvae as prey or host, respectively [1, 4-9]. First-instar larvae that are still in their egg cases, but not eggs of Clytra laeviuscula and C. quadripunctata, were observed to be picked up and transported by ants [10]. However, the larvae of these Clytra species are known to live inside the nests of ants [11, 12]; consequently the ants are not expected to prey upon the eggs. While some information on natural enemies of larvae accumulated, nothing seems to be known about natural enemies of cryptocephaline eggs. One of the reasons of this gap in knowledge may be the difficulty to find eggs in nature. Some species attach their egg with the help of a stalk to the host plant [13, 14], where they can be observed in the field. However, the eggs of most species drop to the ground after the female finished building the egg case [3] , and then the eggs are difficult to find and observe among the leaf litter. To overcome this problem, in this study adult Cryptocephalus (Burlinius) rufipes (Goeze, 1777) were held in a rearing container, the eggs collected and exposed to two different species of laboratory- reared generalist predators, the common green lacewing Chrysoperla carnea (Stephens, 1836) (Neuroptera, Chrysopidae) and the warehouse pirate bug Xylocoris flavipes (Reuter, 1875) (Heteroptera, Anthocoridae). Moreover, the egg cases of C. rufipes are redescribed and illustrated. C. rufipes is widely distributed in Europe, from Portugal to Turkey, and in Northern Africa [15]. In urban areas, it was found feeding on its host plant Salix purpurea Linnaeus, 1753, which is planted as a park tree or to form hedges. 2. Materials and Methods Adult males and females of C. rufipes were collected from Salix purpurea in Berlin, Friedrichshain (52°52.3272^N, 13°46.5825 E), in June and July, 2014. The adults were kept in 250 mL glass-jars covered with pieces of clothing held with rubber bands at 23 ± 2°C and 60 ± 5% RH. The bottom of the jar was lined with filter paper. A twig of the host 2 Psyche Ridge Figure 1: Egg of Cryptocephalus rufipes (Goeze, 1777), scale = 0.5 mm. plant S. purpurea was placed in a narrow plastic tube (5 x 1.2 cm) filled with water and closed with a plug of paper towel to prevent the water from loss by leakage. The twig was replaced when necessary. Eggs laid by several females were collected daily from the bottom of the jars. The eggs were transferred to Petri-dishes (diameter: 5 cm) lined with paper. To each Petri- dish, five C. rufipes eggs and either two adult X flavipes, two larvae of C. carnea, or no predators (untreated control) were added. Each treatment had 11 replications. The behaviour of the predators upon first contact with the C. rufipes-eggs was observed. In the first three days after adding the predators, the experiments were controlled for survival of the predators. After seven days, eggs were controlled daily for larval emergence. Additionally, five Petri-dishes were prepared with five eggs of Ephestia kuehniella each and either two adult X. flavipes, two larvae of C. carnea, or no predators (untreated control). Laboratory-reared larvae of C. carnea and adults of X. flavipes were obtained from Biologische Beratung Ltd., Berlin. The results were analysed with the help of SigmaStat 3.1 software. The number of leaf beetle larvae emerged was subjected to a Kruskal-Wallis One Way Analysis of Variance on Ranks followed by All Pairwise Multiple Comparison Procedures, Dunns Method, to separate means. Treatments were considered significantly different at the P = 0.05 level. Percentage natural mortality data of C. rufipes eggs were not corrected for control mortality, because mortality in the untreated control treatment was <5%. The size of 30 eggs, that is, length and width in lateral view, was measured with a measuring ocular mounted on a dissecting microscope. 3. Results 3.1. Field Observations. Beside C. rufipes, several other Chrysomelidae were occurring on Salix purpurea in June and July, namely Cryptocephalus (Burlinius) ocellatus ocellatus Drapiez, 1819, C. androgyne Marseul, 1857, Phratora vitellinae (Linnaeus, 1758), and Clytra laeviuscula (Ratzeburg, 1837). Moreover, the weevil Polydrusus {Polydrusus) picus (Labri- cius, 1792) was found feeding and mating on S. purpurea. 3.2. Morphology of C. rufipes Eggs. The eggs of C. rufipes are blackish to greyish brown with eight narrow, regular ridges as illustrated in Ligure 1. Each individual faecal plate Figure 2: Larva of Chrysoperla carnea (Stephens, 1836) uplifting an egg of Cryptocephalus rufipes (Goeze, 1777). Figure 3: Adult of Xylocoris flavipes (Reuter, 1875) handling an egg of Cryptocephalus rufipes (Goeze, 1777). bears a little crest; the regular arrangement of the faecal plates composes the ridges that have little gaps in case the faecal plates do not perfectly touch. The mean size ± SD was 0.797 mm ± 0.057 mm length and 0.532 mm ± 0.033 mm width; median was 0.80 for length and 0.53 for width. Length was ranging from 0.700 to 0.975 mm, width from 0.475 to 0.600 mm. The eggs are elongate oval; the mean length to width ratio was 1.50 mm ± 0.095, ranging from 1.25 to 1.70 (median 1.50). 3.3. Experiments with Eggs ofE. kuehniella. The experiments with eggs of E. kuehniella resulted in complete predation of these eggs by both C. carnea and X. flavipes. In the untreated control, 92% of the E. kuehniella-eggs emerged. 3.4. Experiments with Eggs of C. rufipes. Behavioural Obser- vations. When encountering the C. rufipes eggs, the larvae of C. carnea showed the typical prey uplifting behaviour. They fixed the C. rufipes eggs and held them in position (Ligure 2). X. flavipes examined the eggs after contacting it (Figure 3). Within the first three days of the experiment, in all trials one predator consumed the second; consequently, cannibalism occurred. 3.5. Experiments with Eggs ofC. rufipes, Larval Hatch. Larvae hatched after 10 to 13 days. The presence of the predators significantly affected the number of hatching C. rufipes- larvae from the eggs (Kruskal-Wallis One Way Analysis of Variance on Ranks, H - 18.473, DF - 2, P < 0.001). In the untreated control, a mean ± SD of 3.23 ± 1.02 larvae hatched. The presence of both C. carnea and X. flavipes significantly reduced the number of hatching C. rufipes-eggs (All Pairwise Psyche 3 4 Untreated Chrysoperla Xylocoris flavipes carnea Figure 4; Number of Cryptocephalus rufipes-hrysLe hatched out of five eggs exposed to Chrysoperla carnea or Xylocoris flavipes, or no predators (untreated). Means followed by the same lowercase letter do not differ significantly at P < 0.05 (Dunn’s Method) (n = 11). Multiple Comparison Procedures, Dunn’s Method, Q = 3.891 and 2.817, P < 0.05). A mean of 0.91 ± 1.38 and 1.55 ± 1.29 hatched in the presence of C. carnea and X. flavipes, respectively. However, there was no statistical difference in reduction between the two predators (Dunns Method, Q = 0.929, P > 0.05) (Figure 4). 4. Discussion The egg of C. rufipes was first described in 1852 [1] , under two synonyms, that is, C. gracilis Fabricius, 1792, and C. minutus Fabricius, 1792. An egg length of 0.75 mm as well as a surface with eight to nine ridges was given. For C. gracilis, regular ridges and a blackish green colour were described, and for C. minutus irregular ridges and a yellowish green colour. The egg of C. minutus was figured [1, Figure 18] : the egg in this figure is less elongate and the ridges are wider compared to the eggs described in the present study. In 1899, again the egg case was described twice under C. gracilis and C. minutus, possibly mixing information from [1] and own observations [16] . For the eggs described under C. minutus [16, page 51], a length ranging from 0.7 to 0.8 was given, data in accordance with those found in the present study. However, the eggs were described as yellowish grey with slightly irregular ridges, contrary to the blackish brown eggs with rather regular ridges described here. For the eggs described under C. gracilis [16, page 56], a short note was given stating a length of 0.8 mm, a surface with nine narrow bend carinae and a greyish colour, fitting better to the eggs described here. However, as the determination of species in the subgenus Burlinius Lopatin, 1965, requires in many cases the study of aedeagus characters [17] , the identity of immatures described in the 19th Century remains sometimes doubtful, and especially the immatures described as C. minutus probably belong to another species. Even though the shape of the eggs is variable as indicated by the range of the length to width ratio, they can be, for example, easily distinguished from those of the related synoekous species C. ocellatus, which are yellowish brown with blunt ridges. Concerning natural enemies of C. rufipes, reports are available about larval parasitoids [1, 16]. No information was traced on natural enemies of eggs of Crypto cephalinae in general. In the laboratory trials, feeding could not be directly observed, as both species of predators have piercing- sucking moth parts. However, the behavioural observations indicated the predators identified the C. rufipes eggs as prey items. The trials with the E. kuehniella eggs showed the ability of the predators to locate the eggs within the experimental arena, and that food was a limiting factor for survival in these no-choice experiments. At least after cannibalism, the predators relied upon the eggs of C. rufipes for survival. The analysis of C. rufipes hatching showed a significant reduction of larval emergence in the presence of both predators, proving indirectly the predator-induced mortality. However, contrary to the experiments with the eggs of E. kuehniella, predation of C. rufipes eggs was not complete. This observation suggests egg cases of C. rufipes provide a protection against predation by generalist predators, although incomplete. Larvae of C. carnea are known to prey on eggs of various insects [18] . Both adults and larvae of X. fiavipes are known to prey on insect eggs, including eggs of Chrysomelidae [19]. Both C. carnea and related species and anthocorid predators commonly occur in Central Europe. However, they may not frequently encounter C. rufipes eggs in the leaf litter because they are typically foraging on leafs. C. carnea and X. fiavipes were used here as model organisms for generalist predators with piercing- sucking mouth parts. Possibly the egg cases provide even better protection against small predators with chewing mouth parts. Natural enemies of cryptocephaline eggs in biotic communities have to be identified and studied in future field studies. Conflict of Interests The author declares that there is no conflict of interests regarding the publication of this paper. References [1] W. G. Rosenhauer, Uber die Entwicklung und Fortpfianzung der Clythren und Cryptocephalen, einer Insektengruppe aus der Ordnung der Coleoptera [Ph.D. thesis], Philosophische Fakultat Universitat Erlangen, J. J. Barfufi’sche Universitats- Buchdruckerei, Erlangen, Germany, 1852. [2] D. Erber, “Beitrag zur Entwicklungsbiologie mitteleuropaischer Clytrinen und Cryptocephalinen (Coleoptera, Chrysomeli- dae),” in Zoologische Jahrbucher, vol. 96, pp. 453-477, Abteilung fur Systematik Okologie und Geographie der Tiere, 1969. [3] D. Erber, “Biology of Camptostomata Clytrinae-Cryptoce- phalinae-Chlamisinae-Lamprostomatinae,” in Biology of Chrysomelidae, P. H. Jolivet, E. Petitpierre, and T. H. Hsiao, Eds., pp. 513-552, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1988. [4] J. B. Wallace, “The defensive function of a case on a chrysomelid larva,” Journal of the Georgia Entomological Society, vol. 5, no. 1, pp. 19-24, 1970. 4 Psyche [5] C. G. Brown and D. J. Funk, “Antipredatory properties of an animal architecture: how complex faecal cases thwart arthropod attack,” Animal Behaviour, vol. 79, no. 1, pp. 127-136, 2010. [6] M. L. Cox, “The hymenoptera and diptera parasitoids of chrysomelidae,” in Novel Aspects of the Biology of Chrysomel- idae, P. H. Jolivet, M. L. Cox, and E. Petitpierre, Eds., pp. 582-590, Kluwer Academic Publishers Series Entomologica, Dordrecht, Netherlands, 1994. [7] M. L. Cox, “Insect predators of Chrysomelidae,” in Chrysomel- idae Biology: Ecological Studies, P. H. A. Jolivet and M. L. Cox, Eds., pp. 23-91, SPB Academic, Amsterdam, Netherlands, 1996. [8] M. SchoUer, “Eield studies of Crypto cephalinae biology,” in Advances in Chrysomelidae Biology, M. L. Cox, Ed., vol. 1, pp. 421-436, Backhuys, Leiden, The Netherlands, 1999. [9] J. A. Owen, “Eield studies on Cryptocephalus coryli (Linnaeus) (Coleoptera: Chrysomelidae),” Entomologists Gazette, vol. 54, no. 1, pp. 39-44, 2003. [10] M. SchoUer, “Larvae of case-bearing leaf beetles (Coleoptera: Chrysomelidae: Cryptocephalinae),” Acta Entomologica Musei Nationalis Pragae, vol. 51, no. 2, pp. 747-748, 2011. [11] W. G. Rosenhauer, “Uber die Larve der Clythra 4punctata” in Stettiner Entomologische Zeitung, vol. 1842 of Entomologische Mittheilungen 6, pp. 50-52, 1842. [12] E. Skwarra, “Uber die Ernahrungsweise der Larven von Clytra quadripunctata” Zoologischer Anzeiger, vol. 50, pp. 83-96, 1927. [13] M. SchoUer and U. Heinig, “The species of Acolastus Ger- staecker, 1855 of Israel and Sinai with identification key, and description of larva and egg of Acolastus hebraeus ( J. Sahlberg) (Coleoptera: Chrysomelidae: Cryptocephalinae),” Entomologis- che Zeitschrift, vol. 116, pp. 83-90, 2006. [14] M. Wa,sowska, “Morphology of the first instar larva and of the egg of Labidostomis longimana (Linnaeus, 1761) and of Labidos- tomis tridentata (Linnaeus, 1758) (Coleoptera, Chrysomelidae, Clytrinae), with a key to clytrine genera with the first instar larva known,” Deutsche Entomologische Zeitschrift, vol. 54, no. 1, pp. 51-67, 2007. [15] I. Lopatin, A. Smetana, and M. SchoUer, “Tribe Cryptocephalini Gyllenhal, 1813, genus Cryptocephalus Geoffroy, 1762,” in Cat- alogue of Palaearctic Coleoptera, Volume 6, Chrysomeloidea, I. Lobl and A. Smetana, Eds., pp. 580-606, Apollo Books, Stenstrup, Denmark, 2010. [16] R J. V. Xambeu, “Moeurs et metamorphoses des Insectes,” Annales de la Societe Linneenne de Lyon, vol. 45, pp. 1-72, 1899. [17] A. Warchalowski, The Palaearctic Chrysomelidae, Identification Keys, vol. 1, Natura Optima dux Eoundation, Warsaw, Poland, 2010 . [18] G. Shrestha and A. Enkegaard, “The green lacewing, Chrysop- erla carnea: preference between lettuce aphids, Nasonovia ribisnigri, and western flower thrips, Erankliniella occidentalism Journal of Insect Science, vol. 13, article 94, 2013. [19] S. E. Sing and R. T. Arbogast, “Predatory response of Xylocoris fiavipes to bruchid pests of stored food legumes,” Entomologia Experimentalis et Applicata, vol. 126, no. 2, pp. 107-114, 2008. Hindawi