rn r- rn o rn LO ON ri ri d o d d in d o oo d" ’ 1 ri o r- rn d" d- m » n in r- ON ON CM On * — < — m . — < m m *— nO o 00 O o o o o o o O o o o o o o o (N 00 q oo q q n NO 00 n q q o ri d" OO q ri q ri d d ON d q in ri m o O d- r- d" m O m On r- oo o ON nO ri On T— C . — d" n o fN i < — i m o o od q .2 o X < H o cd E ; banks caused by thè confluence of thè Cavaiola stream can be noted at station 4 (Tab. Vili); as a matter of fact, we can say that among all thè samples taken on thè I e 11 bank ,, thè Nematoda were thè only ani mais found in mud samples. At station 3 (Tab. IX) and 2 (Tab. X) there are only Turbellarians - with thè species Mìcrostomum lineare, Microstomum sp., Stenostomum sp., Mesostoma lingua, Mesostoma sp., Rotifera - with thè species Rotaria neptunia, Branchionus sp., Nematoda, with Rhabditis limnicola, Oligochaetes - with thè species Tubifex tuhifex, Limnodrilus sp., Stylaria sp. and Dipterans - once again with larva of Chironomus sp., Chironomus thumnii, Eristalis sp.-. At station 1 , besides thè same taxa found at stations 2 and 3 there were also occasionai findings of some Copepods (Tab. XI). Discussion The contemporary presence of ammonium ion and nitrous nitrogen shows that thè pollution of thè Sarno river is partially of organic origin (sewers, preserved food industry drains, urban waste, farm rejects, etc.) even though toxic inorganic substances due to minor tanning products are also present. Despite thè above, thè pH value is slightly basic at all thè stations. It is our opinion that many anthropogenic aspects concur to this, such as H2S-buffered NH3, both originated by thè decomposition of organic matter or by preserved food industry drains, and anion HS , which in favourable condition is balanced with hydrogen sulphide and may thus dissociate itself a second time giving rise to S2“, both originated by thè decomposition of sulphide and thè drains of some tannery processes using Na2S. Anomalous increases of thè Cl~ ion concentration (probably due to thè hide desalting process) were reported during thè sampling both in thè Cavaiola stream and on thè left bank of station 4. The concentration of ammonia also contributes to thè definition of thè pH; in water it reacts as follows: NH3 + H20 ^ NH| + OH- water will be more basic if ammonia increases; this reaction is completely moved to thè left. An increase of thè ammonia ion in time has been observed. Its utmost level was reached in thè sampling carried out on thè 26th of lune, 1991; Table Vili - Composition of thè fauna at station 4 at thè various times of sampling Soppelsa O., Battaglini F. e Battaglini P . 'sf- oo NO oo T— ( NO o NO V“H ON o nO °N O- oo NO NO et ON o nO d d 04 d d d d ’-1 d d d od d d ON 04 LO <— co LD CO 1— 1 i-H O co LO ON ON in oo NO co uo o co On ON o o in NO ON LO co NO CO d d NO d- NO fN d d d fN 04 d 04 CO - nO 04 - ON On 04 O o o o o O o o o O o o O o ON ^r co o o O in On On ON , o m co NO oo nO NO nO NO 00 CO co fN d 1 d Nf” o- _ 04 fN 04 ON ! CO CO o o O o o o o O o O o o o o — co N" 'd- fN ON d- in (N in o o o o- o o NO od d NO of 00 d; 04 co co co co in ih d fN fN co CO •'Cf fN o O o O O o o o o o o o o o _ o co CO NO CO o NO o o o co o o | d in in co in in d in d co fN o fN co fN o o O o o o o o o o o o o o m co i> NO o o o o o o o o o ON cO d 00 LO CO 1 ih _ ON m in fN o O o o o o o o o o o o o o ON CO cd d .2 cd cd d 5 tu cd cd cd cd o D, cd 2 CU o -d o cd 0) S-I -d d o (U "o Ih 0 o cd E cu a o u to cd d o o .5P s 53 2 cd s 'C cd o cu cu o "d jd o cu cd o cf> o a cu a 0 52 cu c 2 a £ cu cd u <0 a H 2 a O X < U O U CQ X Q o o nO m o o CO Of" o o n- o o oo O o fO O o LO LO o o o o ON co o o LO o o nO oo o o ON co < H o H TOTAL (%) 8.55 28.86 4.39 8.55 2.41 12.06 9.43 25.88 1.54 9.43 100 Table IX- Composition of thè fauna at station 3 at thè various times of sampling 42 Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) in nT ON o n o o o o o o o in o o sO i— ' nj- Ni- o in o o o o o o co o o n}- o oo d i— H d d d d d d in ,~ o (N m LO in NO o in o o o o o o o n oo NT nO oo i — i >— i ▼— ! CO o vO nO ON o o o o o o o o o n o n Q no in ni (N ni vO n cd o NO n6 ; T— 1 in co ni NO ni co co in vO o vO* oo O m o o o o o o o o o NO o m (N co o q in no n ni o co in co co co n O nO Nf o n o o o o o o o _ o _ Q m oo ni n- in oo ni o q co in (O NO in ni co LO o oo o ni co 00 o o o o o o o o o n- o Nf co nO d d ni o q *— < ni in *— i +2 •*— < co ON __ n ni co oo n- o o o o o o o o o oo o oo Q On OS Nf- co o n- rn ni Nf NO Nf ON CO On n}- co n ni Nf (N rn of nO o o o o o o o o o _ o sO d s C cd a cu o d jd a cd co a 3 B 0,800-1,000 □ 0.600-0,800 M 0,400-0,600 H 0,200-0,400 B 0,000-0,200 left bank Figure 3 - Graphic representation of thè Dominance index 12/12/90 08/05/91 St. 1 St. 2 St. 3 St. 4 Stations 26/06/91 St. 5 w fi) 3 ■o a 2,000-2,500 □ 1,500-2,000 B 1,000-1,500 H 0,500-1,000 B 0,000-0,500 righi bank Figure 4 - Graphic representation of thè Diversity index of Shannon 48 Boll Soc . Natur. Napoli - Voi. 101 ( 1992-1993 ) 26/06/91 St. 1 St. 2 St. 3 St. 4 St. 5 Stations Figure 5 - Graphic representation of thè Diversity 12/12/90 c n a> 26/02/91 ■§ CD w m 2,000-2 ,500 □ 1 ,500-2 ,000 ■ 1,000-1 ,500 a 0,500-1 ,000 m o o o p ò ,500 left bank index of Shannon Equality 01/10/90 12/12/90 26/02/91 08/05/91 St. 2 St. 3 St. 4 Stations 26/06/91 St. 5 m 0,800-1 ,000 □ 0,600-0 ,800 ■ 0,400-0 ,600 m 0,200-0 ,400 a o o o p ó ,200 righi bank Figure 6 - Graphic representation of thè Equality index 49 Soppelsa O., Battaglini F. e Battaglini P. Equality 12/12/90 26/02/91 08/05/91 St. 2 St. 3 St. 4 Stations 26/06/91 St. 5 co fi) 3 H 0,800-1,000 □ 0,600-0,800 B 0,400-0,600 H 0,200-0,400 Il 0,000-0,200 left bank Figure 7 - Graphic representation of thè Equality index River pollution due to preserved food industries must therefore be added to thè more serious and steady contribution of tanning industries. The different Dominance, Equality and Diversity index values suggest that thè part of river between thè inflow of thè Cavaiola stream and thè mouth of thè river is thè most damaged, even if a light recovery is observed in its terminal part. In fact, according to Odum (1973), thè Diversity index is low in physically controlled ecosystems, i.e. subject to highly restrictive chemical-physical factors, while it is high in biologicallv controlled ecosy¬ stems. Thus, a higher Diversity means more complex feeding networks, more cases of symbiosis and more possibilities of a negative feedback control, which reduces variations and increases stabilitv. The Diversity index of Shannon clearly shows this zonation of popula- tions too. In view of thè above, it can thus be stated that along thè examined part of thè Sarno river it is possible to identify three areas with different species and different quantities of animals collected for each species. The first area can be identified by stations 5 and 4L. It is thè richest in phyla and its faunistic structure can be included among thè mesosaprobic alpha and beta classes of V. Sladecek’s System (1965). The second area is 50 Boll. Soc. Natur. Napoli - Voi 101 (1992-1993) thè most damaged by thè pollution. It includes thè part downstream from thè inflow of thè Cavaiola river and can be defined as metasaprobic. The third and last area can be identifìed by stations 3, 2 & 1. This area has strong polisaprobic features. Having used research methods adopted in some previous studies (Battaglini et al., 1968, 1971, 1979), thè conditions of thè Sarno river in 1979 could be compared with its current conditions. Compared data show a generai worsening in thè conditions of thè river. Polluting elements cannotbe eliminated, thus causingthe accumula- tion of toxic mud on thè bottoni and thè subsequent moving away from normal conditions, i.e. conditions preventing thè growth up of a typical river environment benthofauna. Significant seasonal variations of waste are another obstacle to thè formation of well defined biocoenose (d'Elia, 1988). The pH is almost unchanged, but NH| and nitrous nitrogen concen- trations have increased, confirming a higher inflow rate of organic origin. Even more worrying is thè fall of thè dissolved oxygen concentration from mean values of approximately 3.3 mg/L in 1979 to approximately 0.6 mg/L. This is likely to be thè cause of thè decreased specific Diversity. As regards total hardness, thè concentration doubled at all stations and in all sam- plings. The worsening and thè increase in thè concentration of polluting substances is also caused by thè increased exploitation of thè springs, so that most of thè spring waters are conveyed to be used as drinking water. Considering animai communities as a whole, thè first things to be noticed are thè strong decrease in thè number of taxa and thè lower number of single animals. The disappearance of some taxa, such as Gastropods, is clearly due to thè worsening of all environmental condi¬ tions. Only Dipterans larva were collected among thè Arthropoda. Some particularly resistant species as Turbeilari (Steno stormirvi sp., Mesostoma sp., Microstomnm sp.), Rotifera (Rotaria neptunia) and Nematoda can stili be found in thè river. The species variety is heavily reduced among thè taxa too. Only species typical of metasaprobic or, at thè most, polysaprobic environments can survive (Liebmann, 1951). As a matter of fact, thè only species of Oligochaeta found is thè Tubifex tubifex. The zoocoenose of thè downstream part and up to thè mouth of thè Sarno river consists of few species - all adapted to typical polysaprobic environment conditions - whose distribution is determined by thè inflow of thè Cavaiola stream. The faunistic data are perfectly consistent with thè 51 Soppelsa O., Battaglini F. e Battaglini P. chemical-physical parameters and lead to thè sarne two basic types of polluting reasons: thè massive number of industries and urban waste. Station 5 stili maintains acceptable conditions confirmedby a not very high Dominance vaine and an almost suffìcient Diversity. From thè con- fluence of thè Cavaiola stream onwards there is a “zonation” even in thè case of thè little Sarno river. In fact, thè waters of this tributary are a reai ecological barrier against thè diffusion of animals. Dominance indexes rise substantially while Diversity indexes decrease, thus revealing thè highly stressing condition. The comparison between thè current data and those previously collec- ted by one of thè Authors (Battaglini et al., 1968) shows a generai worsening of thè conditions of thè river, Anthropogenic polluting elements cannot be eliminated, thus causing thè accumulation of toxic mud on thè bottom and thè subsequent moving away from normal conditions. All this prevents thè growth of a typical river environment benthofauna, also because of thè strong seasonal variations in thè type and quantity of waste. Acknowledgements The present study was supported by a contribution of Italian MURST (40 & 60%) REFERENCES AA.VV. (1989) Standard methods for thè examination of water and wastewater. American Public Healt Association. New York. Battaglini, P. (1979) Aspetto ecologico delle comunità animali del Fiume Sarno (Campania). Rend. Ass Se. Fis Mat. Soc. Naz- Se. Leti. Art., Sez. IV, 46: 1-34. Battaglini, P., Percuoco, G. (1967) Osservazioni ecologiche della fauna limnologi¬ ca del lago craterico di Astroni (Campi Flegrei). Boll. Soc. Nat. in Napoli, 76: 695-771. Battaglini, P., Pierantoni, A., Percuoco, G. (1967) Ricerche sulla fauna del Sarno. I) Descrizioni del corso d’acqua e dati popolazionistici sugli invertebrati della sorgente ed alto corso. Boll. Soc . Nat. in Napoli, 76: 695-771. Battaglini, P., Pierantoni, A., Percuoco, G. (1968) Ricerche sulla fauna del Sarno. II) Studio ecologico di una zona del corso inferiore. Boll. Soc. Nat. in Napoli, 77:481-497. Bianucci, G., Bianucci, E. R. (1982) Il trattamento delle acque inquinate. Hoepli, Milano. Bianucci, G., Bianucci, E. R. (1980) L’analisi chimica delle acque naturali ed inquinate. Hoepli, Milano. 52 Boll. Soc . Natur . iVapo/i - Vo/. 101 (1992-1993) cTElia, E. (1988) L’inquinamento delle acque del bacino del Fiume Sarno. Stato di fatto e interventi di risanamento. Università degli Studi di Napoli, Napoli. d’ELiA, E., Mendia, L., Troncone, M.R. (1974) Condizioni di inquinamento del comprensorio del Fiume Sarno. Istituto di Ricerca sulle Acque, Napoli. Liebmann, H. (1951) Handbuch der Frishwasser und Abwasser Biologie. Verlag R. Oldenbourg, Miinchen. Odum, E.P. (1973) Principi di ecologia. Piccin, Bologna. Pielou, E. C. (1966a) Species-diversity and pattern-diversyity in thè study of ecological successions. J. Theoret. Biol., 10: 370-383. Pielou, E. C. (1966b) The measurement of diversity in different types of biological collections. /. Theoret. Biol., 13: 131-144. Pielou, E. C. (1966c) Shannon's formula as a measure of specific diversity: its use and disuse. Amer Nat. , 100: 463-465. Sladecek, V. (1965) The future of saprobity System. Hydrobiologia, 25: 518-537. Boll Soc . Natun Napoli - Voi 101 (1992-1993): 53-63 53 Bionomics of Myopites stylata F. (Diptera, Tephritidae) and its naturai eri em ics in Vivara island (Gulf of Naples) Fimiani P., Digilio M.C.1 Dipartimento di Biologia, Difesa e Biotecnologie agro-forestali, Università degli Studi della Basilicata, Via Nazario Sauro 85, 85100 Potenza, Italy Dipartimento di Entomologiae Zoologiaagraria, Università degli Studi di Napoli «Federico II» Via Università 100, 80055 Portici (Na), Italy Key words: Inula, host alternation, Bactrocera (Dacus) oleae. Abstract. Myopites stylata F. (Diptera, Tephritidae), is a gall-forming fly living on thè composite Inula viscosa Aiton. The bionomy and parasitoid complex of this insect species were studied in Vivara, a small island in thè Gulf of Naples. M. stylata adults always emerged from thè second half of September to thè end of October. The parasitoid species obtained were different Chalcidoids (Hymenoptera): two undetermined species of Habrocytus Thomson, belonging to albiformis Walker group (Pteromalidae), Eupelmus urozonus Dalman (Eupelmidae), Torymus cyani- mus Boheman and Dimeromicrus kiesenwetteri Mayr (Torymidae), Eurytoma tibia- lis Boheman (Eurytomidae). The role and effectiveness of E. urozonus as a possible naturai control agent of thè Olive Fly, Bactrocera (Dacus) oleae (Gmelin) is at present not well defined. However, in this study M. stylata, compared with other Mediterranean areas, resulted an important alternative host for E. urozonus. Riassunto. Bionomia di Myopites stylata F. (Diptera, Tephritidae) e dei suoi nemici naturali nell'isola di Vivara (Golfo di Napoli). Il presente lavoro riporta alcuni dati bionomici su Myopites stylata F. (Diptera, Tephritidae) e sul complesso dei suoi nemici naturali nell'isola di Vivara (Golfo di Napoli). Lo studio è stato effettuato mediante raccolta dei capolini fiorali della Composita Inula viscosa Aiton, trasformati in galle dall’azione del Dittero. Dalle galle sono sfarfallati il Tefritide ed i parassitoidi relativi, tutti appartenenti alla superfamiglia Chalcidoidea (Hymenoptera). Le specie ottenute sono: Torymus cy animus Boheman e Dimeromicrus kiesenwetteri Mayr (Torymidae), Eurytoma tibialis Boheman (Eurytomidae), due specie non determinate del genere Ha¬ brocytus Thomson, appartenenti al gruppo albiformis Walker (Pteromalidae), e Eupelmus urozonus Dalman (Eupelmidae). I parassitoidi sono sfarfallati lungo un arco di tempo piuttosto lungo, mentre gli adulti del Dittero nel periodo settembre- ottobre, coincidente con la fioritura dell’inula a Vivara. Il parassitoide quantitati¬ vamente più importante è risultato Habrocytus spp. Anche E. urozonus si ritrova in quantità notevole, contrariamente ad altre aree mediterranee dove alcuni Autori hanno ottenuto pochi o nessun esemplare di questo parassitoide dalle galle di I. viscosa. A Vivara, quindi, M. stylata costituisce un importante ospite alternativo di E. urozonus, che potrebbe quindi risultare utile, nonostante le complesse intera- Received 24.4.92, accepted 8.2.93 Boll . Soc. Natur. Napoli - Voi. 101 ( 1992-1993 ) zioni con gli altri parassitoidi, nel controllo naturale della Mosca dell'olivo, Bactrocera (Dacns) oleae (Gmelin). Introduction The mediterranean maquis in Vivara island (Gulf of Naples) is a naturai environment free of antropic influence, which offers interesting opportunities for ecological studies (Fimiani, 1977; 1981). The features of this island are reported in a special issue edited by Regione Campania (1981). A list of thè insect species living on Vivara island, collected with sweeping nets, is available (D'Antonio & Fimiani, 1988). Myopites stylata F. (Diptera, Tephritidae) lives exclusively on thè composite Inula viscosa Aiton, where it produces galls from flowers (Fig. 1). /. viscosa is common in thè Mediterranean area, where it is often associated with olive groves. Myopites females typically lay their eggs in flower ovaries, by introducing thè ovipositor through thè corollar tube. The hatching larva penetrates into thè flower receptacle, which gradually becomes hypertrophic, and finally is transformed in a wooden-plurilocular gali, with special tubercles in place of thè achenes. The association between inules and olive trees was investigated in Italy by Martelli (1910), Bua (1938) and Silvestri (1940), then in Greece by Isaakidès (1955; 1957), in Corse (Féron et al., 1961) and Continental France (Delanoue & Arambourg, 1965), and more recently in Crete (Neuen- schwander et al., 1983). The importance of ecological studies on thè relationship between spontaneous vegetation and olive tree is also recently emerged in thè last OILB Working Group on Fruit Flies, held in Sassari in November 1990 (unpubk). The present note aims at defining thè naturai enemy complex of M. stylata in Vivara, trying also to assess in this environment thè role of inule as a naturai reservoir of Bactrocera ( Dacus ) oleae (Gmelin) parasitoids in olive orchards. Materials and methods The galls were collected once a year from 1982 to 1985, in October, January, November and May respectively in thè four years of thè study. The samples were kept in thè laboratory for over one year to recover all thè Fimiani P„ Digilio M.C. 55 emerging adult flies and parasitoids. In 1982, galls were cumulatively stored in plastic boxes (cm 20 x 30 x 10) closed with a fine gauze. For all thè Figure 1 - Myopites stylata F. gali on Inula viscosa Aiton. 1, 2. Gali formation. 3. Gali completely formed. 4. M. stylata mature larva. remaining years of thè study thè galls were individually stored in glass vials (cm 10 x 0.6) plugged with cotton wool. Part of thè collected galls were also dissected. Adults emerging from galls stored in thè boxes were periodically collected for species identifìcation, while those isolated in thè vials were weekly collected to assess thè time distribution of adult emergence trends. Results and discussion The total number of both fly and parasitoid specimens obtained in thè different years of study is reported in Table I. The parasitoid complex is 56 Boll. Soc. Natur. Napoli - Voi 101 (1992-1993) illustrateci in Table IL The relative abundance of parasitoids emerging from thè galls is shown in Table III. For M. stylata a sex ratio nearly equal to 0.5 was recorded (Tab. I). The adults of this species mostly emerged Table I - Myopites stylata and parasitoids emerged from collected galls. collecting vears 1982 1983 1984 1985 collected galls 530 159 80 43 active galls 124 59 43 Number per sex d’c? 99 dcf 99 dd 99 0 99 Myopites stylata F. 176 151 99 92 40 30 26 20 total parasitoids 668 484 64 130 56 31 13 21 Habrocytus spp. 618 421 40 82 45 27 4 7 Eupelmus urozonus Dalman 13 28 6 21 - - 9 13 Torymus ?cy animus Boheman 8 10 11 1 1 4 1 - 1 Dimeromicrus kiese.nwetteri Mayr 8 14 5 13 6 2 - - Eurytoma tibialis Boheman 21 11 2 3 1 1 - - ' For this collection it was not possible to know thè number of active galls, i.e. thè galls yielding adults, both flies or parasitoids. during thè second and third week of September (Figs. 2, 3), apart from a few individuate emerging through October. For isolated galls it was possible to assess that in average several flies and parasitoids were in most cases present for each inhabited gali (Tab. IV). However, thè galls which did not yield any insect, not even thè Tephritid who had caused thè gali formation, were 22% in 1983 and 26% in 1984 (Tab. I). In fact, upon dissection of galls collected in November, we observed that in some cases they were absolutely empty. Pteromalids Two species of Habrocytus Thomson were obtained, differing in marginai vein length and slightly in colour. Habrocytus spp. was usually thè most abundant species (Tab. III). Its relative abundance ranged from 32.3% up to 90.2%, and was always thè highest except in May 1985, when Eupelmus urozonus Dalman exceeded p.. M ( ' 40 30 20 10 0 -Q .Q _Q JQ ffl ® 0 © © © © a a u ii ii u S5S5<<<< « « « « >.>.>. >^ E O. CL Q, Q, CO CO co co 3 - - - - - -2222n^ ^ ▼“ CSJ CO ^ C\J C\l e\i □ Eurytoma Wk Dimeromicrus □ Torymus H Eupelmus ■ Habrocytus N O N ^ O ffl ^ OJ CO T- CM W Figure 2 - Emergences of Myopites stylata parasitoids. Collection of January 1983. e\i oa Torymus Eupelmus Habrocytus Figure 3 - Emergences of Myopites stylata parasitoids. Collection of November 1984 (left) and May 1985 (right). The galls of both these collections are produced by Tephritids emerged in September-October 1984. Boll Soc. Natur. Napoli - Voi 101 (1992-1993) Table II - The parasitoid complex. parasitoid family and subfamilv feeding behaviour host Habrocytus spp. albiformis Walker group Pteromalidae Pteromalinae ectophagous polyphagous Eupelmus urozonus Dal man Eupelmidae Eupelminae ectophagous poliphagous Tory mas ?cy animus Boheman Torymidae Toryminae ectophagous Tefritid galls on Composites Dimeromicrus kiesenwetteri Mavr Torymidae Monodontomerinae ectophagous Tefritid galls on Composites Eurytoma tibialis Boheman Eurytomidae Eurytominae endophagous Tefritids galls on Composites Table III - Relative abundance of parasitoids emerged from Myopites stylata galls collecting vears 1982 1983 1984 1985 Habrocytus spp. 90.19 62.89 82.76 32.35 Eupelmus urozonus Dalman 3.56 13.91 - 64.71 Torymus ?cy animus Boheman 1.56 1 1.34 5.74 2.92 Dimeromicrus kiesenwetteri Mavr 1.91 9.28 9.2 - Eurytoma tibialis Boheman 2.78 2.58 2.3 - Table IV - Average number of Myopites stylata andparasitoids emerged from one gali . collecting vears M. stylata x ± sd parasitoids x ± sd N. 1983 1.54 + 1 .62 v 1.57 + 1.17 124 1984 1.19 ± 1.40 1.47 ± 1.18 59 1985 1.07 + 1.08 0.79 + 0.94 43 Fimiani P., Dìgiììo M.C< 59 thè number of Pteromalids present in our samples (Tab. III). The adult emergence was mainly registered from January until May (Fig. II, III) and, in 1983, to a lower extent also in September-October (Fig. 2). Habrocytus trypetae Thomson is reported both as an ectoparasitoid of Tephritid larvae and as a hyperparasitoid on Enrytoma curta Walker (Varley, 1937). The possible occurrence of similar ecological relationships in thè case of thè Habrocytus spp. that we found in Vivara remains to be evaluated. In Central (Terracina, Latina, Lazio) and Southern Italy (Cala¬ bria) Habrocytus was already reported as thè most common parasitoid found on M. stylata (Martelli, 1910). The high relative abundance of Pteromalids has been reported also by Féron et al. (1961) for Corsica. In this case an undetermined species of Pteromalus accounted for 40% of thè total insects collected. In contras!, Habrocytus sp. was only rarelv reared from Myopites galls collected in thè Marche region (Rivosecchi, 1960a). However, this different situation could be partly due to thè fact that thè host species were Myopites biodi Brébisson and M. frauenfeldi Schiner. Eupelmids In this group, thè only represented species is Eupelmus urozonus Dalman. The relative abundance of this species varies depending on thè collection periods, reaching up to 64.7% (May 1985 collection, Tab. III). E. urozonus emerged at thè same time as Habrocytus , with thè last flights recorded in October (Figs. II, III). No E. urozonus is obtained from thè November 1984 collection (Tab. III). While some authors (Isaakidès, 1955, 1957) consider M. stylata as a valid alternative host for E. urozonus , others did not (Fèron et al., 1961; Delanoue & Arambourg, 1965; Neuenschwander et al., 1983). In fact, in Crete Neuenschwander et al. (1983) reported a low presence of this Chalcidoid (Tab. V). Their data are different from our findings (Tab. V). Fèron et al. (1961) reported that M. stylata galls collected in Corse in September did not yield any E. urozonus ; but, because thè absence of spontaneous vegetation resulted in a higher rate of olive fly infestation, they suggested a possible positive role of wild plants, such as inules, in olive groves, as naturai reservoirs of Eurytoma sp. In Vivara too we found that no or few E. urozonus emerged from thè samples collected in autumn (October 1982 and November 1984), then reports of Corse are not really in disagreement with Vivara ones. 60 Boll. Soc . Natur. Napoli - Voi 101 (1992-1993) Table V - Eupelmus urozonus adults emerged from 100 galls. ° from Neuenschwan- der et al. , 1 983. localities ratios dates of collection Crete 2.98 December ° Vivara 7.74 October 1982 Vivara 16.98 January 1983 Vivara 51.16 May 1985 Delanoue & Arambourg (1965) found that E. urozonus presents a different oviposition behaviour on M. stylata and on D. oleae. The existen- ce of biotypes adapted to different hosts would prevent a shift between thè two Tephritids. Torymids Torymids were always present in low numbers in our samples, with a relative abundance ranging between 1.6% and 11.3% (Tab. 3). Adult emergences of Torymids species started in January, about at thè same time as Habrocytus , and thè highest values were registered in February-March (Figs. 2, 3). In Marche region (Rivosecchi, 1960b) Dimeromicrus kie- senwetteri Mayr early in thè season shows a few generations on Myopites blotii Brébisson and M. frauenfeldi Schiner, and afterwards it develops and overwinters on M. stylata galls on I. viscosa, since these galls are persistent during winter. Varley (1941) observed in Torymus cy animus Boheman a tendency to superparasitism : thè female commonly lays many eggs in one host, leading to a high mortality rate, which might account for thè low presence of T. cyanimus adults recorded in Vivara. Superparasitism occurrence has been reported also for D. kiesenwette- ri : adult females of this species were found to lay up to 10 eggs per host; D. kiesenwetteri successfully developed also as an ectophagous hyperparasite on Eurytoma curta larvae (Rivosecchi, 1960a). On thè Adriatic coast (Rivosecchi, 1960b) D . kiesenwetteri is a common parasitoid of Tephritids, particularly of species of thè genus Myopites Brébisson. Fimiani P., Digilio M.C. 61 Eurytomids Eurytoma tibialis Boheman (= E . curia Auct., nec Walker) was obtai- ned only in a few specimens (Tab. Ili), which scatteredly emerged in different periods of thè year (Figs. 2, 3). The similarity of emergence times observed for parasitoids in thè different years of thè study, in spite of laboratory Storage of galls, and, in most cases, thè significant association with thè life cycle of thè host seem to suggest thè possible existence of diapause. However, Neuenschwander et al. (1983) after recording thè onset of E. urozonus emergence in December, concluded that diapause in Crete is less severe than in Conti¬ nental Greece (Louskas, in Neuenschwander et al., 1983). Rivosecchi (1958) compared hibernating larvae of two parasitoids of Tephritis stictica Loew, Eurytoma tristis Mayr and Ormyrus hungaricus Erdos, kept at room temperature, at 25 °C and at 30°C, and noticed that exposure to higher temperatures fastens thè development rate, causing pupation to occur much in advance in respect to parasitoids stored at lower temperatures. For M. stylata thè diapause regulated developmental arrest is much more evident. In fact Myopites pupae collected in Vivara in November, in galls not yet completely formed, did not develop into adults until thè occurren- ce of /. viscosa flowering, only 10 months later. For isolated galls it was possible to assess that in average several flies and parasitoids were in most cases present for each inhabited gali (Tab. 4). In conclusion, thè results show that in Vivara island /. viscosa can be an effective naturai reservoir for E. urozonus, which might be useful in thè naturai control of thè olive fly, B. oleae. Acknowledgments Thanks are due to Prof. G. Viggiani and Prof. P. Mazzone (Dipartimen¬ to di Entomologia e Zoologia agraria, Università degli Studi di Napoli Federico II) and to Prof. L. Rivosecchi (Istituto Superiore di Sanità, Roma) for advice in parasitoid (G. V. and P. M.) and Tephritid (L. R.) identifica- tion. Prof. E. Tremblay (Dipartimento di Entomologia e Zoologia agraria, Università degli Studi di Napoli Federico II) is gratefully acknowledged for his interest to this research. 62 Boll Soc . /tewr. Napoli - Voi 101 (1992-1993) REFERENCES Bua, G., 1938. Contributo alla conoscenza dei parassiti temporanei della Mosca delle olive ( Dacus oleae GmeL). L’Olivicoltore, 9: 3-5. D'Antonio, C. & P. Fimiani, 1988. Approccio ad un inventario entomofaunistico dell’isola di Vivara (Na). Nota preliminare. Animar. Ist. Museo Zool. Univ. Napoli, 26: 155-170. Delanoue, P. & Y. Arambourg, 1965. Contribution à l’étude en laboratoire d ’Eupel- mus urozonus Daini. (Hym. Chalcidoidea Eupelmidae). Ann. Soc. Ent. Fr., (N.S) I: 817-842. Feron, M., Benard, R. & S. Poitout, 1961. La mouche de l’olive, Dacus oleae Gmel. et ses parasites en Corse en 1959 et 1960. Entomophaga, 6: 173-183. Fimiani, P., 1 977. Aspetti entomologici della biocenosi olivicola dell’isola di Vivara. Trifoglio Natura, 7: 22-28. Fimiani, P., 1981. Aspetti fenologici e biologici negli Insetti dell'isola di Vivara.- In Vivara Oasi di Protezione Naturale. Giunta regionale della Campania: 51-68. Isaakides, C.A., 1955. La lutte biologique contre la mouche des olives en Grèce. Pragm. Akad. Athen., 22: 1-19. Isaakides, C.A., 1957. Sur la lutte biologique contre le Dacus oleae Rossi. Entomo¬ phaga, 2: 245-249. Martelli, G., 1910. Materiali per la conoscenza dei parassiti della Mosca delle olive. IL Myopites limbardae Schiner. Boll. Lab. Zool. gen. agr. Portici, 4: 303-306. Neuenschwander, P., Bigler, F., Delucchi, V. & S. Michelakis, 1983. Naturai enemies of preimaginal stages of Dacus oleae Gmel. (Dipt. Tephritidae) in Western Crete. I. Bionomics and phenologies. Boll. Lab. Ent. agr. Filippo Silvestri, 40: 3-32. Regione Campania, 1981. Vivara Oasi di Protezione Naturale. -Giunta regionale della Campania: 182 pgs. Rivosecchi, L., 1958. Note sui parassiti dei Tripetidi. I. Eurytoma tristis Mayr e Eurytoma hungaricus Erdòs (Hymenoptera Chalcididae). Boll. Lab. Zool. agr. e Bachic., I: 187-208. Rivosecchi, L., 1960a. Note sui parassiti dei Tripetidi: Dimeromicrus kiesenwetteri Mayr (Chalcidoidea, Torymidae) ed altri Calcidoidei del litorale marittimo delle Marche. Parassitologia, 2: 287-293. Rivosecchi, L., 1960b. Note sui parassiti dei Tripetidi. IL Dimeromicrus kiesenwet¬ teri Mayr (Chalcidoidea, Torymidae) parassita di larve del gen. Myopites Brèbisson. Boll. Zool. agr. e Bachic., s. II, 3: 179-200. Silvestri, F., 1940. La lotta contro la mosca delle olive. In Atti del Convegno Nazionale di Olivicoltura, Bari, 21-22 settembre 1938, 2: 56-82. Varley, G.C., 1937. Description of thè eggs and larvae of four species of Chalcidoid Hymenoptera parasitic on thè knapweed gall-fly. Proc. R. Ent. Soc. London (B), 6: 122-130. Varley, G.C., 1941. On thè search for hosts and thè egg distribution of some Chalcid parasites of thè knapweed gall-fly. Parasitology , 33: 47-66. Boll Soc. Natur. Napoli - Voi 101 (1992-1993): 63-72 63 Immunocytochemistry of carbonio anhydrase in thè chick embryo membranous labyrinth during development Avallone B., Balsamo G., Marmo F. Cattedra di Istologia ed Embriologia Dipartimento di Genetica, Biologia generale e Molecolare Università degli Studi «Federico II», Via Mezzocannone 8, 80134 Napoli, Italv Key words: Immunocytochemistry, Carbonic-anhidrase, Embryo, Labyrinth. Abstract. An immunocvtochemical studv with polyclonal antibodies was carried out with thè aim to localize carbonic anhydrase (CA) isozymes CA I and CA II in thè various cells of chick membranous labyrinth during development. Our data indicate that CA I and CA II are localized in thè same cells in thè chick embryos. In fact CA I and CA II are diffuselv localized in thè membranous labirinth bud during thè early developmental stages, successivelv thè sensorial epithelium of thè maculae acusticae and crista ampullaris stronglv stained bv thè immunocvto- chemical procedure. Cochlear and vestibular dark cells consistentlv appeared stained for CA I and CA II. The epithelial cells of thè endolymphatic sac and of thè brain choroid plexus were similarlv stained for both isozymes. In one day and one week chicken CA was visualized immunocvtochemically in thè same sites as at advanced embryo n al stages. It is suggested that these findings are related to otolith formation and maintenance, as well as to fluid and ionie flux in thè endolymph. Riassunto. Studi Immunocitochimici sull’anidrasi carbonica del labirinto membranoso deH’embrione di pollo durante lo sviluppo. È stato condotto uno studio con anticorpi policlonali allo scopo di localizzare gli isoenzimi dell’anidrasi carbonica (CA) CA I e CA 11 nel labirinto membranoso del pollo durante lo sviluppo. I dati indicano che negli embrioni, già dai primi stadi di sviluppo i due isoenzimi sono diffusamente presenti nell’epitelio otocistico e succesivamente nell’epitelio sensoriale delle creste ampollari e delle macule acustiche, come a livello delle cellule scure del vestibolo e del tegmentwn vasculosum. Sono, inoltre, positivi alla reazione immunocitochimica per i due isoenzimi anche l’epitelio del sacco endolinfatico e dei plessi corioidei. Nei pulcini di un giorno e di una settimana la localizzazione dell’enzima è sovrapponibile a quella degli embrioni in cui è presente un buon differenziamento del labirinto membranoso. Gli Autori correlano tali reperti con l’azione dell’enzi¬ ma nella morfogenesi e nel riassorbimento degli otoliti e con l’intervento dello stesso nel flusso di ioni e di fluidi delTendolinfa. Received 27.1 1.92, accepted 16.6.93 64 Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) Introduction Carbonio anhydrase (CA) catalyses thè reversible reaction between carbon dioxide and water to form carbonio acid which spontaneouslv dissociates into bicarbonate and hvdrogen ions. CA is known to be involved in several cellular functions, such as C02 removai from red blood cells, acidifìcation of secreta in thè parietal cells of thè gastric mucosa, selective release of K+ and resorption of NA+ and water in thè kidney tubules. CA is also responsible for thè production of aqueous humor in thè eye ciliarv bodies, of cerebrospinal fluid in thè choroid plexus, and of endolymph in thè stria vascularis of thè inner ear (Watana- be, 1963; Lim et al., 1983; Hsu & Nomura, 1985). In addition, there is convincing evidence that CA may be involved in thè deposition of calcium carbonate in thè shell of molluscs (Wilbur & Jodrey, 1 955) and chick eggs (Hodges & Lòrcher, 1 967), and, as regards thè membranous labyrinth, in otoconia formation in chicks (de Vincentiis & Marmo, 1968; Fermin and Igarashi, 1986), mice (Purichia & Erway, 1972), and Bufo bufo tadpoles (Campantico, 1968). Cytochemical CA localization in inner ear tissues was studied in thè guinea pig and thè rat bv Watanabe (1963), thè chick embryo bv Marmo (1966), thè chinchilla by Lim et al. (1983), thè guinea pig by Hsu and Nomura (1985) and Takumida et al. (1989a). Biochemical investigations by Erulkar and Maren (1961) in thè cat inner ear demonstrated that thè highest CA concentration is in thè co- chlear duct; moreover, Drescher (1977) found CA similar to that from red blood cells in thè membranous labyrinth of mammals. So far 7 isozymes of CA have been separated (Kaunisto et al., 1990). They can be localized individuali by thè immunocytochemical approach, which is much more sensitive and specific than thè cytochemical techni- ques currently used, in particular those aimed at detecting CA, which are constantly subject to criticism. Therefore, we availed ourselves of this technique, using polyclonal antibodies to localize CA I and CA II isozymes in thè chick membranous labyrinth during development, which had alrea- dy been investigated cytochemically by Marmo (1966). We also tried to elucidate their role in cells with different specialized functions, and in thè differentiation of thè inner ear structures mostlv occurring during embryo development. \ ì-.tii, <■'. /■' fu.. ! C. j ?-; ■ *« ■:> : ' I I : : v HIMMì 65 Material and methods Chick embryos at 2, 5, 7, 9, 11, 13 days of incubation (stage 12-39, according to Lilìie, 1952), as well as 1 day and 1 week old chicks were used. The embryos were removed in normal saline, fixed in Carnoy and embed- ded in paraffim The membranous labyrinths were taken from chicks after anaesthesia and decapitation, and subjected to thè sanie procedure. In Figure 1 - Three day chick embryo. Diffuse positivity can be observed in thè otocyst (OT) (small arrow) and in thè epithelium cells of thè acoustic ganglion bud (AG) (large arrow). PAP CAI.X 144 order to localize CA, thè direct, indirect and peroxidase-antiperoxidase (PAP) methods were used. Depara i il ned and rehydrated Spiri sections were incubated in 3% H202 for 5 min in order to inactivate endogenous peroxidase, and then pretreated with normal rabbit serum (1:20) for 30 min to block thè fragment crystallizable (Fc) receptors of ìmmunoglobuli- nes. The polyclonal antibodies (Serotec, Oxford, England) used were: peroxidase labelled and un labe l! ed sheep an ti human CA I and CA II, and 66 Boll Soc . Natur. Napoli - Voi 101 (1992-1993) peroxidase labelled and unlabelled rabbit anti-sheep IgG; thè sera were diluted 1:50 (CA I, CA II, IgG) 1:40 (PAP) in phosphate buffer saline (PBS) 0.1M pH 7.2, with 1% bovine serum albumin (BSA). Each step was carried out in a moist chamber at room temperature for Ih. In order to reveal peroxidase, sections were then incubated in 3,3’-dia- minobenzidine tetrahydrochloride (9 mg DAB in 15 mi PBS with 30% H202), and dehvdrated and mounted in Canada balsam. At thè same time, negative and positive control procedures were performed bv omitting thè primarv antibody on thè sample sections, and by using guineapig kidnev sections, respectivelv. Results Despite thè different methods used, our investigation provided consi- stent data. Thev demonstrated that thè two isozvmes, CA I and CA II, are localized in thè same cells; however, a quantitative studv was not carried out, and hence it was not possible to establish which is thè more abundant in thè various sites. Figure 2 - Five day chick embrvo. It can be seen diffuse positivity of thè otocvst (OT) epithelium. Endolvmphatic sac (ES). PAP CA II.X60. Avallone B., Balsamo G Marmo F. 67 In embrvos at thè otic pit stage, a diffuse positi vitv was observed in thè epithelium, which persisted in thè next stages until thè 9th day (Fig 1 and 2). This positivity was also present in thè celi cvtoplasm of thè acoustic ganglion bud. Figure 3 - One day chick: intenselv stained sensorial epithelium (SE) of thè utricle. The otolith matrix (small arrow) appears positive. Only one otolith is indicated (O). PAP CA II.X560 From thè llth day thè enzvme was mostly localized in thè sensorial areas of thè saccule, thè utricle and thè lagenae, where sensorial, suppor- iting, transitional and dark cells showed positivity to thè immunocvtoche- mical reaction. Positivity had also been observed in thè otoconial matrix since thè early formation of otoconial crystals in thè 7 dav embrvo (Fig. 3). The same distribution of thè enzyme was also evident in thè ampullar crests (Fig. 4). The localization of thè enzyme in thè epithelial cells of thè endolymphatic duct and sac was alreadv possible at early stages of develo- pment (5 days). Intense immunostaining was visualized in thè dark cells of tegmentum vasculosum (Fig. 5) and in thè supporting and sensorial cells of thè Corti organ. 68 Boll. Soc. Natur. Napoli - Voi 101 (1992-1993) Immunocytochemistrv also evidenced abundant carbonic anhydrase in thè neuron cytoplasm of cochlear and vestibular ganglia at each developmental stage (Fig. 6). In addition, a particularlv intense CA reaction Figure 4 - Five day chick embrvo. Intense positivitv can be observed in thè endolvmphatic sac (ES) epithelium. PAP CA II.X225 was also present in thè epithelium of thè choroid plexns, which are alreadv known to show a high CA content (Fig. 7) Discussion and conclusion Our observations showed that CA I and CA II are diffuselv localized in thè membranous labyrinth bud during thè earlv developmental stages; in later stages, instead, thev are restricted to thè areas where thev fulfil diverse and specialized functions. CA is present in thè areas (maculae) implied in otoconia production. The morphogenetic function of thè enzvme is demonstrated by both thè inhibition of otoconia morphogenesis, which has been observed in em- brvos treated with carbonic anhydrase inhibitors (Marmo, 1965; Campanti- 69 Avallone B., Balsamo G ., Marmo F. co, 1968; Purichia & Erwav, 1972; Kido et al., 1991), and bv thè role that thè enzyme plays in crystal resorption (Lim, 1983; Kawamata et al., 1986; Igarashi, 1989). Ultrastructural investigations aimed at localizing CA in thè Figure 5 - One day chick: tegmentum vasculosum. It is possible to distinguish between negative light cells (small arrow) and intenselv positive dark cells (large arrow). PAP CA II. X 144 vestibular organ of thè guinea pig have confirmed this function; in fact, they have demonstrated that thè globular structures (considered otoconia precursors) that are present in thè supporting, transitional and “planum semilunatum” cells are surrounded bv reaction products (Takumida et al., 1989 a,b). The morphogenetic role of CA in these sites is also supported by thè occurrence of thè enzyme in thè otoconial matrix which is alreadv present in thè embryo on thè early formation of otolithic crvstals. It is noteworthy that CA occurs also in cells involved in fluid and ion transportation, such as vestibular dark cells and thè epithelial cells of thè endolympatic sac and of thè coroid plexus ; an intense activitv of Na+ K+ dependent ATPase and adenylate cyclase has also been demonstrated in these cells (Hsu & Nomura, 1985). However, CA is also present in cells not 70 Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) being involved in fluid and ion transportation, such as supporting cells, that, as suggested bv Lim (1983), may play an important role in thè respiratory mechanism of sensorial cells. Figure 6 - Thirteen day chick embrvo: vestibular ganglion (VG). Positivitv is evident in thè neuron cytoplasm (small arrow). PAP CA I.X60 Figure 7 - Thirteen day chick embrvo: choroid plexsuses. Positivitv is clearlv evident in thè epithelium (small arrow). PAP CA 11X225 Interestingly, unlike thè cytochemical data obtained by Lim et al. (1983) and Hsu and Nomura (1985) in thè maculae, thè cristae and thè Corti organ, our investigation evidenced an intense CA reaction not onlv in supporting cells, but also in thè sensorial ones. As regards more specifical- ly thè chick embrvo membranous labyrinth, our data showed that thè enzyme fails to disappear in 15 day embryos, in contrast to what was demonstrated in previous cytochemical studies (Marmo et al., 1966), but persists after hatching showing thè same localization as in thè embrvo at advanced stages of development. In conclusion, thè present study does not provide any clue to thè possible role played by CA in thè inner ear. Here CA activity is different in Avallone B., Balsamo G., Marmo F . 71 thè various celi types (“secretory”, supporting and sensorial cells) , and hence carbonic anhydrase might be a ubiquitous enzyme. CA might be involved in specialized functions only in thè cells showing intense enzyma- tic activity. Therefore, it could be suggested that this enzyme mav play several functions in thè inner ear: i) regulation of thè endolymph ions and/or fluids; ii) removai of C02 from inner ear tissues; iii) otoconia formation and maintenance; iv) influence on thè endolymphatic potential, in agreement with Prazma (1978) and Vozumi et al. (1991), as well as on thè neural stimulus spread, in view of its occurrence in thè cvtoplasm of acoustic ganglion neurons, as already suggested bv other investigators (Giacobini, 1961; Korhonen et al., 1964; Korhonen & Korhonen, 1965). REFERENCES Campantico, E., 1968. L'azione della diclorofenamide sulla deposizione dei sali di Ca del sacco endolinfatico di larve di Bufo bufo. C.N.R., 37: 866-868. Drescher, D.G., 1977. Purification of a carbonic anhydrase from thè inner ear of thè Guinea pig. Proc. Nati. Acad. Sci., 74: 892-896. Erulkar, S.D. & T.H. Maren, 1961. Carbonic anhydrase and thè inner ear. Nature, 189: 459-460. Fermin, C.D., Y. Igarashi, 1986. Review of statoconia formation in birds and originai research in chicks (Gallus domesticus). Scan. Electron. Micr., 4: 1649-1665. Giacobini, E., 1961. Localization of carbonic anhvdrase in thè nervous svstem. Science, 134: 1524-1525. Hodges, R.D. & K. Lorcher, 1967. Possible sources of thè carbonate fraction of egg shell calcium carbonate. Nature, 216: 609-610. Hsu, C.J. & Y. Nomura, 1985. Carbonic anhydrase activity in thè inner ear. Acta Otolaryngol. (Stockh) [suppl.J, 418: 1-42. Igarashi, Y., 1989. Submicroscopic studv of thè vestibular dark celi area in human fetuses. Acta Otolaryngol. (Stock), 107: 29-33. Kaunisto, K., S. Parkkila, T. Tammela, L. Ronnberg & H. Rajanieml, 1990. Immunohistochemical localization of carbonic anhydrase isoenzvmes in thè human male reproductive tract. Misto chemistry , 94: 381-386. Kawamata, J., Y. Harada & N. Tagashira, 1986. Electron microscopie study of thè vestibular dark cells in thè crista ampullaris of thè Guinea pig. Acta Otolarvn- gol. (Stock), 102: 168-174. Kido, T., T. Sekitani, H. Yamashita, S. Endo, Y. Masumitsu & H. Shimogori, 1991. Effects of carbonic anhydrase inhibitor on thè otolithic organs of developing chick embryos. Am. J. Otolaryngol . Head Neck Med. Surg., 12 (4): 191-195. Korhonen, L.K., E. Naatanen & M. Hyyppa, 1964. A histochemical study of carbonic anhydrase in some parts of thè mouse brain. Acta Histochem., 18: 336-347. 72 Boll Soc . Natur. Napoli - Voi 101 (1992-1993) Korhonen, E. & L.K. Korhonen, 1965. Histochemical demonstration of carbonic anhvdrase activitv in thè eyes of rat and mouse. Acta ophthal, 43: 475-481. Lillie, FR., 1952. Development of thè chick. Ed. H. Holt and Company, New York. Lim, D.J., C. Karabinas & D.R. Trune, 1983. Histochemical localization of carbonic anhvdrase in thè inner ear. Am. J. Otolaryngol, 4: 33-42. Marmo, F., 1965. Osservazioni sulla morfogenesi degli otoliti dell'embrione di pollo in presenza di inibitori dell'anidrasi carbonica. Boll. Zool., 32: 231-238. Marmo, F., 1966. L’anidrasi carbonica del labirinto membranoso deH’embrione di pollo durante lo sviluppo. Acta Embryol. et Morphol. Exp., 9: 1 18-126. Prazma, J., 1978. Carbonic anhvdrase in thè generation of cochlear potentials. Am. J. Physiol., 235: 317-320. Purichia, N. & L.C. Erway, 1972. Effects of dichlorphenamide zinc and manganese on otolith development in mice. Devel. Biol., 27: 395-405. Takumida, M., D. Bagger-Sjoback, J. Wersall & J. Harada, 1989 a. Ultrastructural localization of carbonic anhvdrase in thè vestibular end organs of thè guinea pig. Arch. Otorhinolaryngol , 246: 56-60. Takumida, M., Y. Harada, D. Bagger-Sjoback & J. Wersall, 1989 b. Carbohydrates of thè Guinea pig vestibular supporting cells. Anris-Nasns-Larvnx(Tokyo) , 16: 133-142. de Vincentiis, M. & F. Marmo, 1968. Inhibition of thè morphogenesis of thè otoliths in thè chick embrvo in thè presence of carbonic anhvdrase inhibitors. Expe¬ ri entia, 24: 818-820. Vozumi, N., N. Mori & S. Sakai, 1991. The effect of acetazolamide on thè endolym- phatic sac DC potential. Acta Oto-laryngol , 3 (5): 921-925. Watanabe, Y., 1963. Influence of Diamox (acetazolamide sodium) upon inner ear and histochemical studv of carbonic anhvdrase. J. Otorhinolaryngol. Soc. Jpn., 66: 657-670. Wilbur, K.M., L.H. Jodrey, 1955. Studies on shell formation. V. The inhibition of shell formation bv carbonic anhvdrase inhibitors. Biol. Bull., 108: 359-365. 73 Boll. Soc . Natur. Napoli - Voi 101 (1992-1993): 73-87 Ocurrence of (3-endorphin- and enkephalin- like immunoreactivity in thè hypothalamus of thè domestic fowls, Gallus domesticus Esposito V., De Girolamo P., Sammarco M., Gargiulo G. Dipartimento di Strutture, Funzioni e Tecnologie Biologiche, Università degli Studi «Federico II», Via Delpino, 1, 1-80137 Napoli, Italv Key words: Immunocytochemistrv, hypothalamus, enkephalins, P-endorphin, Gallus domesticus. Abstract. The distribution of P-endorphin and enkephalin-like immunoreacti¬ vity in thè hypothalamus of 2-week-old domestic chicks was studied with immu- nocytochemical methods, using antibodies to p-endorphin, met-enkephalin and leu-enkephalin. P-Endorphin-like neurons were observed in thè nucleus infundibu- li whereas p-endorphin-like fibers were detected both in thè infundibular area and in thè external layer of median eminence. Enkephalin-like immunoreactive peri- karya were observed in thè parvocellular division of thè nucleus paraventricularis and in thè nucleus infundibuli. The enkephalin-like immunoreactive fibers were observed in every part of thè hypothalamus, but mainlv concentrated in thè infundibular area and in thè external layer of median eminence. The distribution pattern of both p-endorphin and enkephalins is compared with that other neu- ropeptides present in thè chick hypothalamus. The results are discussed in relation to thè possibility that both P-endorphin and enkephalins may be involved in hypophysial regulation or neuromodulator activity in thè hypothalamus of dome¬ stic chick. Riassunto. Presenza di immunoreattività P-endorfìna ed encefalino-simile nell’ipotalamo di Gallus domesticus. Nel presente studio immunocitochimico eseguito sulla regione ipotalamica di pulcini di Gallus domesticus secondo il metodo di perossidasi antiperossidasi, ha evidenziato la presenza di immunoreattività ad anticorpi per la metionina-encefali- na, leucina-encefalina e p-endorfina. Test di specificità hanno dimostrato “ cross" - reattività tra i due antisieri per le encefaline ed i rispettivi antigeni e tra l’antisiero per la P-endorfina e la p-lipotropina, per cui l 'immunoreattività appresso descritta è da considerarsi relativa a sostanze encefalinosimile e p-endorfina e/o P-lipotropi- nasimile. L'immunoreattività all’anti-P-endorfina è stata messa in evidenza in corpi cellulari presenti nella regione del nucleo infundibulare ed in fibre principalmente frammiste alle cellule suddette; essa risulta anche presente, seppur scarsamente, nella regione ipotalamica anteriore e nella zona esterna dell’eminenza mediana anteriore e posteriore. Received 27.1 1.92, accepted 6.6.93 74 Boll. Soc . Natur, Napoli ~ Voi 101 ( 1992-1993 ) L’immunoreattività encefalino-simile è presente in neuroni appartenenti alla componente parvocellulare del nucleo paraventricolare ed in cellule del nucleo infundibulare; ed inoltre, in fibre sparse in tutto l'ipotalamo e nella zona esterna dell’eminenza mediana anteriore e posteriore. La presente localizzazione rapportata a quella di altri sistemi peptidergici ipotalamici, già caratterizzati in lavori precedenti, evidenzia correlazioni spaziali il cui significato funzionale è da stabilire, ma che suggeriscono complessi rapporti neuromodulatori soprattutto nella regione infundibulare. La presenza di immuno- reattività encefalino- e g-endorfino-simile nella zona esterna dell’eminenza media¬ na depone a favore dell’ipotesi che questi peptidi siano rilasciati nella circolazione portale ipofisaria e quindi, coinvolti nella funzione neuroendocrina del sistema ipotalamoipofisario. Introduction Both methionine-enkephalin (met-ENK) and leucine-enkephaline (leu-ENK) as well as g-endorphin (g-END), are all members of a naturai opioid peptide familv (Hughes et al., 1980) able to link endogenous opiate receptors in thè brain (Terenius & Wahlstrom, 1974; Hughes, 1975) and in thè pituitary gland of various vertebrates (Cox et al., 1975). At first, g-END was isolated and characterized from carnei and porcine pituitaries as a 3 lamino-acid peptide, identical to thè carboxyl terminal sequence of g-lipotropin (nLPH) which is considered its biosynthetic precursor (Brandburv et al., 1976; Li & Chung, 1976; Chretien et ah, 1976; Cox et al., 1976; Akil et al., 1984). Both met-ENK and leu-ENK were isolated and characterized from thè pig brain as pentapeptides (Hughes et al., 1975) and they stem from a large precursor molecole, called proenkephalin (Akil et al., 1984). In mammals thè enkephalin-containing neurons are distributed widely in thè centrai nervous System, both as locai circuit neurons and as projection neurons, whereas g-END-containing neurons are mainly concentrated in thè region of thè arcuate nucleus of thè mediobasal hypothalamus with fibers projecting rostrallv and caudallv to manv limbic, thalamic and lower brainstem targets; although, another celi cluster is present in thè nucleus tractus solitarius (Elde et al., 1976; Hokfelt et ai, 1977 ; Simantov et ai, 1977; Sar et al., 1978, Bloom et ai, 1978; Uhi et ai, 1979; Bugnon et ai, 1979; Bloch et al, 1979; Sofroniew, 1979; Pickel et al. , 1980; Finley et al 1981; Schwartzberg & Nakane, 1981; Khachaturian et al, 1985 a,b). Esposito V., De Girolamo P., Sammarco M., Garginlo G The very few observations on thè distribution of opioid peptide in thè avian brain, include mainly thè enkephalins (Bàlhser & Dubois, 1980; De Lanerolle et al., 1981; Ryan et al., 1981; Mikami et al., 1983; Ball et al., 1988). Further to our previous studies on characterization of hvpothalamic peptidergic systems in thè chick brain (Gargiulo et al. , 1 990; Esposito et al. , 1992 a,b), we have identified in thè present investigation, by immunocyto- chemical methods, enkephalin and g-endorphin-like centers in thè hv- pothalamus of thè chick Gallus domesticus. The probable interaction with other hypothalamic neuropeptides and their involvement in regulation of thè release of pituitary hormones have been discussed. Materials and methods Male chicks of Gallus domesticus were obtained from a locai breeder at thè age of two weeks. Chicks were killed by decapitation and their brains were rapidly removed and immersed in Bouin's fìxative solution. The fixed brains were then routinelv dehvdrated through a series of graded alcohols, placed in xylene, and embedded in paraffin. Sagittal and transverse serial sections of thè brains 5pm thick, were cut and mounted on glycerinealbu- min coated glass slides. Finally, sections were deparaffinized in xylene, rehydrated in a graded ethanol series and washed in 0.0 1 M phosphatebuffe- red saline (PBS), pH 7.4. The immunocytochemical staining was performed using thè peroxida- seantiperoxidase (PAP) technique (Sternberger, 1979). Briefly, after inhibition of endogenous peroxidase activitv with 3% hydrogen peroxide, thè sections were treated with normal goat serum (IgG, 1:5, 30 min.), to avoid possible background reaction; then incubated overnight at 4°C with antisera to met-ENK diluted 1:500/1500, leu-ENK diluted 1:500/1500 and g-END diluted 1:1000/2000. Subsequentlv, thè sections were treated with goat antirabbit immunoglobulin (1:50, 30min.) and PAP-complex (1:100, 30min.). The reaction was visualized with a solution of 3,3’-diaminobenzidine (DAB; 10 mg in 15 mi of 0.5 M Tris-buf¬ fer, pH 7.6 containing 1.5 mi hydrogen peroxide at 0.03%). Between each step, thè sections were thoroughlv rinsed in PBS. Antisera to g-END (Ì456/002), met-ENK (Ì672/002) and leu-ENK (i67 1/ 002) was purchased from UCB as well as g-END (VB056), met-ENK (VB051), leu-ENK (VB050), normal goat serum (Ì200/002), goat anti-rabbit serum (Ì200/003) and PAP-complex (Ì200/001). DAB was purchased from DAKO. 76 Boll Soc. Natur. Napoli - Voi 101 ( 1992-1993 ) Figure 1 - Schematic drawing of thè hvpothalamus of domestic chick in a parasagittal piane, showing thè distribution of thè immunoreactive perikarya and fibers to anti(3endorphin (▲, ▲) and to anti-enkephalins (•, •). AC: anterior commissure; C: cerebellum; IN: nucleus infundiboli; ME: median eminence; N: neostriatum; NC: neostriatum caudale; NL: neural lobe; Nili: nervus oculomo- torius; OC: chiasma opticum; PC: posterior commissure; PD: pars distalis; POA: preoptic area; PVN: nucleus paraventricularis; TSM: tractus septomesencepha- licus; V: third ventricle. Esposito V\, De Gìrotmmo P., Sammarco Al, Gargiulo G. The specificity of staining were verifìed with control experiments as follow: a) replacement of thè metENK antiserum with thè same antisera adsorbed by both enkephalins, b) replacement of thè leu-ENK antiserum with thè same antisera adsorbed by both enkephalins; c) replacement of thè g-END antiserum with thè same antisera adsorbed by g-END, g-LPH and both enkephalins, d) replacement of thè met-ENK, leu-ENK, and g-END- antisera with normal rabbit serum or phosphate-buffered saline. Some sections were Luxol fast bleu-cresyl violet stained to improve thè identifìcation of nervous structures. All thè sections were dehydrated, coverslipped and finally observed and photographed using a Leitz Aristoplan. Results Immunocytochemical reactions with thè g-END, met-ENK and leu- ENK-antisera were observed in different parts of thè hypothalamus of thè domestic chick, Gallus domesticus (Fig. 1). Figure 2 - Photograph showing g-endorphin-immunoreactive cells and fibers in thè n. infundibuli. Parasagittal section.(X350). IN: n. infundibuli. 78 Boll Soc. Natur. Napoli - Voi 101 (1992-1993) Immunocytochemical specificity Controls were carried out on adja- cent serial sections in each area where positive immunoreactivity to p-END, met-ENK and leu-ENK were identified. The absorption tests by both synthetic methionine-enkephalin and leucine-enkephalin showed that each studied enkephalin antisera revealed both homologous antigens and heterologous antigens; indeed, it was observed no immunoreactivity or attenuated staining when preabsorption of met-ENK antiserum was car¬ ried out with synthetic met-ENK or leu-ENK, on thè other hand thè preabsorption of leu-ENK antiserum with synthetic leu-ENK or met-ENK inhibited or attenuated respectivelv thè immunostaining. v ili ME Figure 3 - Photograph showing P-endorphin-immunoreactive fibers in thè zona externa of median eminence. Transverse section. (X300). ME: median eminence, V: third ventricle The P-END immunostaining was inhibited by absorption of thè p-END antiserum with both synthetic P-END and P-LPH; thus, thè employed antiserum can recognize both P-END and P-LPH. The P-END immunostai¬ ning was maintained when it was used thè p-END antiserum adsorbed by both synthetic met-ENK and leu-ENK. 79 Esposito V />• V f * *«.» 4 Figure 4 - Photograph showing P-endorphin-immunoreactive cells (arrows) in thè n. infundibuli. Transverse section. (X400). IN: n. infundibuli: third ventricle A large population of (3-ENDlike immunopositive celi bodies occurred in thè nucleus infundibuli, a neuron group located ventromedial to thè third ventricle (Figures 2 and 4). P-END-like immunopositive fibers were detected in thè mediobasal hypothalamus and in thè nucleus infundibuli intermingled with p-END-im- munopositive perikarya. Only few thin P END immunoreactive-fibers were found scattered in thè anterior hypothalamic area. Thin P-END-like immu¬ nopositive fibers were also distributed in thè zona externa of thè rnedian eminence (Fig. 3). 80 Boll Soc . Natur. Napoli - Voi. 101 (1992-1993) Enkephalin-immunoreactive neurons were found both in thè parvo- cellular division of thè nucleus paraventricularis and in thè nucleus infundibuli; thè later were very few and small in size (Figures 5 and 6). ENK-immunopositive fibers were detected throughout thè whole hy- pothalamus; anyhow they were mainly concentrated in thè anterior hy- Figure 5 - Photograph showing enkephalin-immunoreactive cells (arrow) and fibers in thè median eminence. Transverse section. (X250). IN: n. infundibuli, ME: median eminence pothalamus, ventrally to thè anterior commissure (Fig. 8), and in thè posterior hypothalamic region around thè noimmunoreactive perikarya of thè nucleus mammillaris medialis (Fig. 7). Discussion The findings of present immunocytochemical study indicate that both enkephalinergic and g-endorphinergic or lipotropinergic System are pre- Esposito V., De Girolamo P., Sammarco M.f Gargiulo G. sent in thè chick hypothalamus and that they have a different localization as well as mammals (Bloom et al., 1978; Watson et al, 1978). Specificity tests for immunocytochemical staining revealed a possible cross-reaction between thè anti-MET-ENK antisera and LEU-ENK, and thè anti-LEU-ENK antiserum and MET-ENK; thè present study, therefore, reports thè localization of an enkephalin-related peptide. Figure 6 - Photograph showing enkephalinimmunoreactive cells in thè n. para- ventricularis. Transverse section. (X300). PVN: n. paraventricularis, V: third ventricle On thè other hand, thè anti-human g-endorphin antiserum employed in this study reveal cross-reactivity with g-lipotropin, suggesting that they also contain antigenic determinants common to g-LPH. Similar behavior has been reported previously for antihuman and anti-porcine g-END (Bloch et ah, 1979; Doerr-Schott et al, 1981; Kiss et al, 1985). Other immunocytochemical studies on lower vertebrates (Doerr- Schott et al., 1981; Nozaki & Gorbman, 1984; Vallarino, 1985, 1986) indicate that cross reactions may occur between mammal anti-g-END and thè corresponding antigen from nonmammalian vertebrates, suggesting an immunogenic relationship between g-END molecules among vertebrates. 82 Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) The g-endorphin-like immunoreactivity evidenced in thè present study is mainly concentrated in thè nucleus infundibuli that appears to be thè homologous of thè arcuate nucleus of mammals (Mikami, 1986), thè Principal site of g-END-containing neurons in mammalian species (Akil et al, 1984). Figure 7 - Photograph showing enkephalin-immunoreactive fibers in thè n. mammillaris. To remarke thè network fibers around thè noimmunopositive pe¬ ri karva of MM. Transverse section. (X300). MM: n. mammillaris, PVO: paraven- tricular organ Many kinds of neuropeptides have been immunocytochemically cha- racterized in thè avian nucleus infundibuli (Mikami, 1986; Esposito, 1990; Gargiulo et al. , 1 990; de Girolamo & Esposito, 1 99 1 ; Esposito et al. , 1 992 a, b); thè phvsiological significance of this spadai relationship between g-END and other peptides is unknown, but it is possible to postulate complex neuromodulator interaction. Furthermore, thè g-END-immuno- reactive fibers terminals in thè median eminence coming very likelv from nucleus infundibuli suggest a possible hypophvsiotropic role of g-END. Sakurai et al. (1986) demonstrated that g-END is involved in thè control of LH release in thè hen as an inhibitory agent. Esposito V., De Girolamo P., Sammarco M., Gargiulo G. 83 According to Blàhser and Dubois (1980) we have observed that immunocytochemical reaction for enkephalins reveals more extensive fìber network than immunoreactive perikarya confirming thè possibilitv of a discontinuous peptide synthesis within thè perikarya, or an unmasking of thè peptide molecule only within thè axons and dendrites. • V,' 4 ■ V ■ è i . .* . I:- , * / * \ >r à H ** k 4. ,f f ■ n 4 • V* % a * * ’4 • f * r . jf ^ // 4 > ...À" -fin J é ■ f*f 4 > v ' /;> V , , v ^ i - ■ '«A , * i : ■ ,** '* , À - . * -, « { | : •4 | a. ■ • $ 4 * 1 • 'V F / s jr ; - . 4 k I J y. r ,*» A ’ - • , » f ** «> . ,v t ti- I ' V I -■ , #? ; ; ’! 'G ' ■ v- ; ‘ v - •t / , ** ’ M - .. 8 Figure 8 - Photograph showing enkephalinimmunoreactive hbers and some cells (arrows) in thè anterior hvpothalamus. Parasagittal section. (X350) However thè hypothalamic distribution of immunoreactivitv to enke¬ phalins described in this study is similar to those reported in previous works (for review see Viglietti-Panzica & Panzica, 1991). 84 Boll Soc. Natur. Napoli - Voi 101 ( 1992-1993 ) The enkephalinergic centers observed in thè hypothalamic region, in both paraventricular and infundibular perikarya, may be related to thè presence in this area of a wide neurosecretory System producing hypophy- siotropic hormones (Scharrer & Scharrer, 1963; Blàhser, 1983). Generally, thè ENK appear to inhibit thè neuronal firing rate by inhibiting transmit- ter-induced depolarization of thè postsynaptic celi membrane (Zieglgan- sberger et al , 1976, North & Williams, 1 985), therefore they may modulate different excitatory signals in this area or in other hypothalamic sites. The present reports mainly emphasizes thè rich enkephalinergic in- nervation of thè infundibular area which is consistent with thè various hypophysiotropic effects of enkephalins (Tramu et al, 1981). The presence of perivascular enkephalins fiber terminals in thè me- dian eminence implies that enkephalin may be released from thè median eminence into thè portai circulation and involved in neuroendocrine function of thè hypothalamo-hypophvsial System. Finally, our observation show a spatial relationship between ENK and g-END in thè mediobasal hypothalamic area, which contains thè main accumulation of g-END perykaria, intermingled with ENK-fibers, but thè possible interaction between them will be able to demonstrate only at thè ultrastructural level. In conclusion, these observation indicate thè presence of both enke- phalin-like and g-endorphin-like immunoreactive substance in thè chicken hvpothalamus, which might be involved in thè pituitary regulation and/or in other neuronal functions as neuromodulator. This studv also provide a foundation for further studies to elucidate thè role of g-END in thè brain of thè domestic chick and other birds. Acknowledgments This studv was partiallv supported by a contribution of Italian Murst (40% and 60%). REFERENCES Akil, H., S.J. Watson, E. Young, M.E. Lewis, H. Khachaturian & J.M. Walker, 1984. Endogenous opioids: biologv and function. Ann. Rev. Neurosci., 7: 223-255. Ball, G.F., P.L. Faris, B.K. Hartman & J.C. Wingfield, 1988. Immunohistochemi- cal localization of neuropeptides in thè vocal control regions of thè songbirds species. J. Comp. Neitrol, 268: 171-180. Esposito V., De Girolamo P., Sammarco AL, Gargiulo G. 85 Blahser, S. & M.P. Dubois, 1980. 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Brani Res., 115: 160-164. ' ' Boll. Soc. Natur. Napoli - Voi 101 ( 1992-1993 ): 89-103 89 Action of thè cadmium on thè Carassius auratus living in lentie waters containing catabolic ammonia Battaglini P.\ Soppelsa O.1, Improta C.2, Ferrara L.2 1 Dipartimento di Zoologia, Università degli Studi «Federico II», via Mezzocannone 8, 80126 Napoli, Italy 2 Dipartimento di Chimica, Università degli Studi «Federico II», via Mezzocannone 4, 80126 Napoli, Italy Key words: cadmium, ammonia, water hardness, Carassius auratus. Abstract. In a previous paper (Battaglini et al., 1991) concerning thè Cadmium ion absorption in ditte re nt Carassius auratus organs in “soft” and “hard” waters, thè Authors have shown that thè absorption of thè Cadmium is much more signifteant in softwater than in hard water. In this paper thè Authors have considered a further element characterizing thè habitat of thè Carassius auratus, i.e. thè limited environment of stagnant water in which, besides thè pollution substances like cadmium salts, thè same catabolic materials of thè Carassius auratus exist. The Carassius auratus were kept for 8 days to become acclimatized. We have then carried out a research on thè results obtained by our tested animals to thè Cadmium collecting thè survival and absorption data of this toxic in different organs by adding cadmium chloride CdCh.Z'/.FUO up to reach a 10 ppm concentration of ion Cd++ in experiment tanks where. We have observed that thè Storage in water of catabolic material such as ammonia is an element increasing cadmium toxicity related to thè Carassius auratus. Such toxicity is revealed both by higher values of absorption of this ion in different organs and in thè survival values of thè specimens which are definitely lower. Riassunto. Azione del cadmio su Carassius auratus viventi in acque lentiche contenenti NH, catabolica In un precedente studio relativo all’assorbimento dello ione cadmio nei vari organi di Carassius auratus in acque “dure” e “dolci”, abbiamo dimostrato che l’assorbimento del cadmio è molto più pronunciato in acqua dolce che in quella dura. Inoltre, avendo constatato in acqua dura contrariamente a quanto avviene in acqua dolce una immediata e quasi completa precipitazione dello ione cadmio sottoforma di CdC03, abbiamo ipotizzato nei due casi due differenti meccanismi di assimilazione del cadmio: a) per osmosi con bioaccumulo nel tempo, in acqua dolce; b) per ingestione di CdC03 solido precipitato, nel caso di acqua dura. Nel presente lavoro abbiamo voluto prendere in considerazione un ulteriore elemento che caratterizza l’habitat del Carassius auratus, ovvero l’ambiente limitato di acqua ferma in cui sono presenti, oltre ad eventuali sostanze inquinanti come i sali di cadmio, gli stessi prodotti di rifiuto dei carassi. Abbiamo quindi Received 27.1 1.92, accepted 16.6.93 Boll Soc . Natur. Napoli - Voi . 101 (19 effettuato uno studio sulla risposta fornita dai nostri animali da esperimento al cadmio, raccogliendo dati di sopravvivenza e di assorbimento di tale tossico nei vari organi. In particolare abbiamo aggiunto cadmio cloruro, CdCI2.2,/2H20, fino a raggiungere una concentrazione di 10 ppm di ione Cd2+ in vasche da esperimento ove erano stati tenuti i carassi per acclimatarli nei precedenti 8 giorni. Nell'acqua utilizzata, (“acqua dura“: pH=7,8; durezza 369 mg/L di CaC03; “acqua dolce” pH=7,6; durezza 152 mg/L di CaC03) erano presenti anche i prodotti catabolici dei carassi tra cui l’ammoniaca che, al momento della aggiunta del cadmio, presenta¬ va una concentrazione di 7. 1 0 mg/L. Le misure di assorbimento atomico sono state eseguite sugli organi mineralizzati. I pesci sono stati sacrificati ai seguenti tempi: Ih 30’, 24h, 72h, 7gg. La presenza nell’acqua di NH3, che è un forte complessante dello ione cadmio, ha notevolmente aumentato la solubilità dei suoi sali permet¬ tendo la sua permanenza in soluzione al 100% per oltre 4 giorni. Successivamente lo ione metallico è cominciato a precipitare come carbonato, rimanendo tuttavia per circa il 20% del suo valore iniziale ancora al 7U giorno dall’inizio dell'esperi¬ mento. La sopravvivenza nella “acqua dura” è stata del 100% dopo Ih 30’, del 50% dopo 24h, del 44% dopo 72h e del 12% dopo 7gg. Nella “acqua dolce” la sopravvivenza, molto simile alla precedente, è stata del 100% dopo Ih 30', del 50% dopo 24h, del 40% dopo 72h e del 10% dopo 7gg. I dati di assorbimento confermano che gli organi che presentano maggiore accumulo di cadmio sono, come nel nostro precedente esperimento, fegato, rene ed intestino, mentre si ottengono valori modesti o addirittura nulli per pelle, cervello, cuore e muscolo. Inoltre i valori di assorbimento di Cd2+ sono sempre più elevati rispetto a quelli riscontrati per esemplari posti in acqua senza ammoniaca. Ciò è stato attribuito al fatto che essendo il cadmio, nel caso attuale, presente in massima parte in soluzione, anche se come complesso cadmioammoniaca, esso possa essere più facilmente assimilato per assorbimento dai Ciprinidi (nel nostro caso Carassius auratus ) e accumulato nei vari organi. In conclusione si può dire che l’accumulo nell’acqua di un prodotto catabolico come l’ammoniaca costituisce un elemento di aggravamento della tossicità del cadmio nei confronti dei carassi, che si manifesta sia in più elevati valori di assorbimento di questo ione nei vari organi che nei valori di sopravvivenza degli esemplari che sono drasticamente più bassi. Introduction The pollution of thè waters caused by heavy metals and thè relevant consequences on flora and fauna of these ecosystems has become a very topic question following thè great development of human activities at industriai level. Among thè different heavy metals cadmium and mercury proved to be thè most toxic, (Ravera, 1984). This metal which is present in nature expecially as an impurity of thè zinc minerals (blenda, ZnS) or as sulphur (grenockite CdS), is spread out in thè biosphere through different ways such as: volcanic activity, exudates from vegetation, foresi fires, windblown dust and leaching of rocks and, in thè latest ten years, also 91 Battaglini P., Soppelsa O., Improta C., Ferrara L. through thè use of phosphate fertilizers, incinerator waste coal and oil combustion and thè same industriai production of cadmium minerals (Ravera, 1984; Polprasert, 1982). The cadmium, spread out in thè soil, in discharge waters and in atmosphere, is at last carried by wind and rain into thè streams of water (Absullah & Royle, 1972; Ajrnal et al, 1985; Ajmal et ai , 1987). The studies on cadmium toxicity on freshwater fish have shown a signifìcant dependence of this toxicity from thè Chemical characteristics of thè environment in which thè toxic is spread out, having sometimes increasing results, some other times decreasing results, ofthe toxic action ofthis heavy metal (Sprague, 1987; Calamari et al. , 1980; Enk & Mathis, 1977). In former studies (Battaglini et al., 1991; 1992a; 1992b; Gargiulo et al., 1991) concerning ion cadmium uptake in different organs of Carassius auratus in waters of different hardness, it was shown that thè metal uptake is much more signifìcant in soft than in hard water. In addition, owing to thè observation of a heavy and sometimes almost complete precipitation of ion cadmium as CdC03 in waters of higher hardness, two possible ways of absorption with consequent toxic action of cadmium were assumed: a) due to osmosis with bioaccumulation during thè time (above all in soft water); b) due to intake of CdC03 solid precipitate (above all in hard water) (Battaglini et al., 1991). The high and sudden mortality of fish in rivers and hatcheries is often caused by lethal concentrations of polluting toxicant substances (Tarazona et al., 1987). Anyway such pollution is not always due to thè intake of allochtoneous substances, as thè same catabolic materials in small habitat and without change water can cause an environmental change, being themselves toxic or simplv increasing thè toxicant action of other substan¬ ces already present in thè habitat (Ravera, 1984). To better understand thè environmental changes that are displayed on thè area, some behavioural and echotoxicology tests were carried out on goldfìsh (Carassius auratus ) aimed at focusing thè interrelation, from an ecotoxicology view, of some behavioural parameters. In this paper thè authors have considered an element that characteri- zes thè naturai habitat of Carassius auratus, i.e. thè feature of a limited habitat with stagnant water in which, besides thè possible polluting sub¬ stances, there is a Storage of catabolic materials of Carassius auratus (Mommsen & Walsh, 1992). Among these, ammonia is thè major nitroge- nous and product comprising usually more than 70% of total waste nitrogen in freshwater teleosts (Warde van, 1983). The importance of 92 Boll. Soc. Natur. Napoli - Voi. 101 ( 1992-1993 ) ammonia in this context is due not only to its reai toxicity but also because it is one of thè main chemical complexing substances of ion Cd2+, causing a modification of thè speciation of this metal in solution. Thus thè authors have carried out a study on Carassius auratus response to thè polluting action of cadmium with ammonia as catabolic product, testing such action in waters of different .hardness in order to compare these results with those obtained in former experiments carried out without ammonia. Materials and methods 20 tanks of 20 1 each were prepared, of which 10 tanks were filled up with tap water of Naples and 10 were filled up with thè same water, previously decalcified with ion exchange resins Soft C (line “C”) CARMAR, Naples. Both kinds of waters were utilised only after a period of aeration in a 600 1 glass aquarium to eliminate thè chlorine. The test fish, supplied by CARMAR Co., Naples, weighed 5 -e 7 g. All animals have been held for 10 days to have them acclimatized in two different kinds of waters in two 600 1 separate glass aquaria, equipped with air pump and filter Eheim, in thè Ecology and Ecotoxicology Laboratory of thè Department of Zoology University of Naples Federico II. Then 200 Carassius auratus, randomly chosen, were transferred in experiment tanks (10 fishes in each tank). The tanks had a photoperiod of 12 hrs and were supplied with an air pump, but without filter, to prevent catabolic materials from being eliminated To simulate thè lentie environ- ment, fishes were held for 8 days in thè same water to let catabolic materials store up, and particularly ammonia. During this period, fish were fed as usuai. The experiment began only after these eight days. Cadmium as CdCl2.2ViH20, equal to 10 ppm, was added to 8 tanks with water of higher hardness and to other eight tanks with water of lower hardness. The Carassius auratus which were in other 4 tanks, 2 tanks with soft water and two tanks with hard water, were utilized as control fishes during thè experiment. During thè whole experiment thè temperature was constantly at 16 + 1 °C. The analysis of thè different substances which are in thè two different kinds of waters utilized for thè experiment, was performed at zero time. Measures were taken utilizing thè traditional volumetrie titration, thè standard solution hardness of sodiumtilendiaminotetracetic acid salt for thè hardness, alkalinity with standard solution of HCl using bromocresol as Battaglini P., Soppelsa O., Improta C., Ferrara L. acidbasic indicator for thè alkalinity, ionchloride with AgN03 solution utilizing Mohr (Kolthoff et al., 1973) method for ionchloride. A pHmeter ORMA, model NK 300 was used to determine thè pH and a dissolved oxygenmeter HI 8543 (Hanna Instruments, USA) to determine thè dissol¬ ved oxygen in water. The NH3 produced and present in water was measured with a spectro- photometer utilizing Nessler reagent to obtain thè typical yellow colour. Cadmium concentration in solution in thè single tanks was measured every 24 hrs till thè end of thè experiment by using thè atomic absorption spectrophotometer VARlAN AA275. To get thè correct interpretation of cadmium concentration values present in solution at different phases, a kinetic precipitation of ion Cd2+ as CdC03 was carried out in parallel in thè same waters utilized during thè tests, but without Carassius auratus and its relevant catabolic materials. The survival values were taken up to 7 days from thè beginning of thè experiment, carrying out also thè pathological anatomy dissection of thè dead specimens. To evidence thè metal effects on Carassius auratus behaviour thè ventilation frequency of experimental and control goldfish was determi- ned by counting thè number of opercular movements per minute (Lang et al., 1987) of 5 fishes for each tank. The computation of 5 fishes for each tank was optimized by using thè average value, which was then compared to thè values obtained at different phases. Such computations were made every 15’ for thè first 90 minutes, every 30' for thè following 150 minutes and every 24 hours during 3 more days. A further parameter to understand thè behavioural reactivity in thè presence of toxic was taken counting thè movements of thè caudal fin per minute (Abel & Papoutsoglou, 1987): more beats = more movement. Of course thè same tests were carried out for thè Controls. For each kind of water two specimens were taken randomly in various periods of time (1.5 h, 24 h, 72 h and 168 h). The organs taken out, after being weighed on an analytic balance, were treated with a mixture (weighing as much as five times thè organs) of HN03/HC104 4:1 in a 10 mi pyrex tube plugged at 7080°C for 24 h in order to obtain a complete mineralization of thè tissues. The solution obtained was brought up to thè known volume by adding HN03 0.1 M and then analyzed by an atomic absorption spectrophotometer VARIAN AA275 in order to determi¬ ne thè cadrnium concentration as parts per million (ppm) of fresh organ. 94 Boll Soc . Ator. Napoli - Voi 101 (1992-1993) Results In both experiments thè concentration of NH3 catabolic reach a value (7 -e 10 mg/L) that have a remarkable influence on thè form in which cadmium (II) is present in thè System. Fig. 1 reports two precipitation kinetics of cadmium as carbonate (thè average cadmium concentration present in solution versus time) in water ppm Figure 1 - Storage of ion cadmium in solution with or without catabolic products. of higher hardness with thè Carassius auratus and, in comparison, in thè same kind of water but without thè Carassius auratus. It can be noted that thè precipitation occurs in significantly different amounts. In point of fact, in hard water and with Carassius auratus, we can notine that total cadmium remains in solution longer than four days of thè experiment. The precipitation occurs only at thè final stage of thè experiment, leaving in solution, however, a cadmium concentration of almost 2 ppm. In Kinetic of comparison, always in hard water but without goldfishes, thè cadmium carbonate precipitation is immediate with a residuai quantity in solution reducing itself to 3.5% of thè starting value after 24 hrs. In less hard water, Battagìmi SoppeJsa Gq Imgrota C., Ferrara L. 95 characterized by greatly lower alkalinity and thus with a lower carbonate cadmium precipitation, no carbonate cadmium precipitation at all occurs, with or without Carassius auratus. The survival, in water of higher hardness, was 100% after 1 hr 30', 50% after 24 hrs, 40% after 72 hrs and 10% after 7 days, Sudi values are defìnitely lower, as compared to thè results obtained in our previous experiments carried out in waters without ammonia, when survival was almost 100% after thè same experiment period of time. (Battaglini et al. , data unpublished). The pathologic anatomy analysis of thè dead specimens, has shown haemorrhagic gills, congested liver, haemorrhagic kidneys, and a hyper mucus production all over thè body and particularly at gills. As to thè beh avi our, fig. 2a reports thè average values of beats per min. of thè operculum and caudal fin in hard water, and fig. 3a reports thè movements/msn Figure 2a - Effect of cadmium + NH3 on thè Carassius auratus behaviour in hard water. relative values in soli water More frequent movements of opercular gill are signs of a greater request of Oxygen (Black 1 95 1 ; Hughes, 1 960) . Caudal fin movements are signs of regular activity, decrease or stop are signs of a significant suffering (Bainbridge, 1961). In thè Controls (Figures. 2b and 3b) thè Àuthors have noted that there is an almost continuous trend having 96 Boll Soc . Natur . Afapo/i - Vo/. 101 ( 1992-1993 ) average values very dose to normality (1040 beats per min. of thè oper- culum). As regards thè fishes treated (figures 2a and 3a), thè values of thè operculum beats were noted to be always high but, after a lowering to 60’, they turn to be stronger and stronger after 4 hours from thè beginning of thè experiment. movements/min Figure 2b - Behaviour of control specimens of Carassius auratus in hard water. The data of cadmium stored up in different organs are characterized by a slight measure error, estimated more or less by 2 3%, but subjected to a variability of cadmium values depending on thè different specimens, and such variability value is much more important. Thus thè values reported, which are thè average among couples of measures on different samples, are effected by a total uncertainty reaching sometimes a value of even 10%. Fig. 4a reports cadmium Storage values in some organs tested in our experiment, as compared to thè same values obtained in a previous experiment (Fig. 4b) (Battaglini et al, 1991), in which no catabolic materials were present when cadmium chloride was added. The data of atomic absorption confimi that organs having a higher cadmium Storage are, as in our previous test: liver, kidney, and gut; while low values or even no value were noted for skin, brain, heart and muscle. Battaglini P Soppelsa O., Improta C., Ferrara L. Discussion In this research, thè striking datum for his dramatic and unexpected low vaine, is thè one relative to thè survival. Such a dramatic discrepancy could not be expected indeed. In fact, among thè different softwater fishes (Flis, 1968) Cyprinids are known to be thè least sensitive to NH3 and Figure 3a - Effect of cadmium + NH3 on thè Carassius auratus behaviour in soft water. cadmium; on thè other hand, thè same results obtained in previous experiments suggested definitely different results (Battaglini et al., 1991). This discrepancy is not overcome even if thè Authors consider that ammonia itself develops its own toxic action which is to be added to thè action of cadmium. In fact, thè values of total ammonia measured by thè Authors in experiment waters, at thè pH values on which we are working, imply thè presence of molecular ammonia in hard and soft water of 0.33 and 0.22 mg/L, respectively. These values are definitely lower than thè standard value considered toxic for thè carp, which is 2.0 mg/L (Malacea, 1968), and for other Cyprinids, like Pimephales promelas, which is between 0.75 and 3.4 mg/L of NH3 (Thurston, Russo & Phillips, 1983). As a matter of fact, thè discrepancy is only apparent and is due to thè fact of considering thè toxic action of cadmium and ammonia as two 98 Boll Soc . Natur. Napoli- Voi 101 (1992-1993) additional phenomena, where, on thè contrary, thè simultaneous presence of such two substances deeply modifies thè chemical nature of both, and consequently, their own toxicity. movements/min Figure 3b - Behaviour of control specimens of Carassius auratus in soft water. To better understand thè environment moclified by these substances, we must consider thè main chemical processes obtained in solution. Ammonia, as already mentioned, is a strong complexing agent of ion cadmium making with this one several compounds up to thè tetraminocad- mium: 4 NH3(aq) + Cd2+(aq) = Cd(NH3)42+(aq) The progressive involvement of NH3 in thè complex with cadmium, gradually moves thè acidbase equilibrium of ammonia towards further molecular ammonia, according to such equilibrium: NH4+(aq) + H20 = NH3(aq) + H30 + (aq) and thus, even if at thè beginning there is a low amount of molecular NH3 at thè pH level on which we are working, thè presence of ion Cd2+ is capable of displacing this equilibrium towards thè molecular NH3 just 99 Battaglini P., Soppelsa O., Improta C., Ferrara L . because of thè capture caused by thè complexing of thè molecules NH3 produced. On thè other hand, thè production of thè complexes Cd (NH3)2+n aims at drastically reducing thè concentration of ion Cd2+ in solution, thus highly increasing thè solubility of CdC03 which is produced in calcareous waters with Cd2+: Cd"+(aq) + HCO 3_(aq) + H2O = CdC03(s) + H30+(aq) Thus, one of thè consequences produced, is thè lower and slower precipitation of CdC03 obtained in experiment tanks with calcareous water and Carassius auratus, as compared to thè one obtained in water of thè same kind but without Carassius auratus. PPm + kidney HW liver HW ^ kidney SW ^ liver SW Figure 4a - Cadmium Storage in liver and kidney of Carassius auratus specimens held in hard (HW) and soft (SW) water with ammonia. Furthermore, we assign to thè complexes Cd(NH3)n2+ a toxicity higher than thè one attributed to thè single Cd2+ and NH3; in fact, we have ascertained that even in soft water, where cadmium remains in solution for thè whole period of thè experiment, thè survival is, under thè same conditions, lower when catabolic ammonia is present together with ion cadmium. In view of thè above, also thè other data can be better explained. 100 Boll. Soc . Natur. Napoli - Voi. 101 (1992-1993) In this sense, we have shown that thè first symptom in goldfish exposed to cadmium + ammonia is respiratory alteration, measured by ventilation frequency, as reportedby Maki (1979). Smart (1978) reported hyperexcita- bility and hyperventilation such as acute toxic mechanism in rainbow trout. Both specimens treated in hard water and those treated in soft water suffer stress which shows an increase of thè opercular beats, and a dramatic decrease of thè caudal movement, showing thè stillness condi- tion of goldfish at thè bottom of thè tanks. No particular difference can be seen between thè two cases, which confirms that both in hard and soft water thè Carassius auratus during most of thè experiment time, are PPm Figure 4b - Cadmium Storage in liver and kidney of Carassius auratus specimens held in hard (HW) and soft (SW) water without ammonia. exposed to thè same amount of cadmium in solution. Similarly, thè dead specimens, no matter if placed in hard or in soft water, did shown a mucus hyperproduction which sometimes covered thè whole specimen like a shirt. This is in agreement with thè relevant literature, since it has been demonstrated that mucus production can be influenced by thè presence of harmful environmental factors, such as ammonia pollution (Jakowska, 1963; Dave & Garside, 1976; Zuchelkowski el al., 1981). As concerns thè Storage of cadmium data in different organs, thè comparison of uptake values obtained in two different water kinds, shows 101 Battaglini P., Soppelsa O., Improta C., Ferrara L. : that, while in harder water thè cadmium Storage occurs alreadv in thè first 24 hrs, in softer water thè Storage occurs only after a longer time. On thè other hand, after 7 days, Storage in softer water becomes more important than thè one in hard water. For thè gut only, higher cadmium values are ascertained in hard waters, even after 7 days, because of thè presence in thè intestinal lumen of CdC03 ingested from thè bottom of thè tank and accumulated there after thè fourth experiment day. Finally, it has to be pointed out that cadmium storages in organs, in thè presence of catabolic ammonia, are higher than those obtained without it, both in hard and soft water. In water of higher hardness, this can be easilv explained attributing thè higher cadmium Storage (in thè presence of ammoniaca! products) to a longer stay of cadmium solution as a complex which, therefore can be easily absorbed by fishes from solution. Viceversa, thè greater Storage observed in soft water, brings thè Authors to thè assumption that cadmium has a higher penetrating capacity into fish tissues when it is coordinated with ammonia, rather than with water molecules. In conclusion, it can be stated that Storage of a catabolic product (such as ammonia) in water will definitely increase cadmium toxicitv as regards fish. Acknowledgements: This study was supported by a contribution of Italian MURST (40% & 60%). REFERENCES Abdullah, M.I. & L.G. Royle, 1972. 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Sprague, Eds., New York, I. Wiley & Sons. pp. 139-169. B attagliili P.t Soppelsa O., Improta C., Ferrara L . 103 Tarazona, J.V., MaJ. Munoz, J.A. Ortiz, MaO. Nunez & J.A. Camargo, 1987. Fish mortality due to acute ammonia exposure. Aquacul. Fish Manag., 18: 167-172. Thurston, R.V., R.C. Russo & G.R. Phillips, 1983. Acute toxicity of ammonia to fathead minnows (Pimephales promelas). Trans. Am. Fish Soc., 112: 705-71 1. Waarde van A., 1983: Aerobic and anaerobic ammonia production by fish. A review. Comp. Biochem. Physiol. , 74B: 675-684. Zuchelkowski, E.M., R.C. Lantz & D.E. Hilton, 1981. Effects of acid-stress on epidermal mucous cells of thè brown bullhead Ictalurus nebulosns (Le Seur): a morphometric study. Anat. Ree., 200: 33-39. Boll Soc. Natur. Napoli - Voi. 101 (1992-1993): 105-120 Catabolic NH3 influence on cadmium toxicity to thè gut of Carassius auratus L. Andreozzi G.1, Antonucci R.1, Affatato C.\ De Girolamo P.\ Gargiulo G.1, Ferrara L.2, Sammarco M.\ Battaglini P.3 1 Dipartimento di Strutture, Funzioni e Tecnologie Biologiche, Università degli Studi «Federico II», via Delpino 1, 80137, Napoli; 2 Dipartimento di Chimica, Università degli Studi «Federico II», via Mezzocannone 4, 80126 Napoli; 3 Dipartimento di Zoologia, Università degli Studi «Federico II», via Mezzocannone 8, 80126 Napoli, Italv Key words: Cadmium, toxicity, gut, immunocytochemistry, C arassius auratus . Abstract. We studied thè toxic action that thè ion Cd:~ exerts on thè gut of Carassius auratus when thè metal is introduced into water containing materials of thè fish catabolism including ammonia. The results have shown that in hard water (369 mg/L of CaC03), thè cadmium produces damage to thè epithelium of thè bulb which appears detached from thè connective and an increase of thè cellular turnover evidenced by a higher number of thè cells in mitosis. On thè contrary, in soft water (125 mg/L of CaC03) thè following was noted: a suffering aspect of thè epithelium of thè bulb, an expantion of thè mucous cells and an increase in thè mucus mass contained in thè intestinal lumen. Riassunto. Influenza dell’NH3 catabolica nell’intossicazione da cadmio sull’intestino di Carassius auratus. È stata studiata la reazione tossica che il cadmio esercita su Carassius auratus quando il metallo viene immesso in acqua contenente anche prodotti del catabolismo dei pesci e in particolare tra questi 1NH3. Poiché Fazione tossica del cadmio è correlata alla durezza dell’acqua, sono sta prese in considerazione acque con diversi valori di durezza e precisamente una contenente 369 mg/L di CaC03 “acqua dura” e un’altra contenete 152 mg/L di CaC03 “acqua dolce”. Sono state allestite due serie di vasche contenenti acqua a diversa durezza. In ciascuna della vasche sono stati immessi 10 carassi. Dopo 8 gg., quando l’acqua si era arricchita dei prodotti catabolici dei pesci, in ogni vasca è stato aggiunto CdCL.2'/2FL0 in quantità pari ad una concentrazione di cadmio nell’acqua di 10 ppm. L’accumulo di cadmio nell’intestino è stato determinato mediante l’assorbimen¬ to atomico previa mineralizzazione dell’organo. Sono state inoltre usate le colorazioni con ematossilinaeosina per l’esame morfologico della mucosa intestinale, e, per la localizzazione e tipizzazione dei mucopolisaccaridi, la reazione PAS, la colorazione con Alcian a pH 2,5 e a pH 1 da sola o associata a digestione con neuroaminidasi. La Received 27.1 1.92, accepted 7.5.93 106 Boll Soc. Natur. Napoli - Voi. 101 ( 1992-1993 ) presenza di sostanze Metencefalina like e Leuencefalina like è stata rilevata con metodica PAP. I risultati ottenuti hanno mostrato che in “acqua dura” con NH, la precipitazione del Cadmio inizia solo dopo quattro giorni dall’inizio dell’esperimento. Inoltre, indipendentemente dalla durezza dell’acqua, in presenza di NH3, l’assorbimento del metallo è sempre elevato, e la sopravvivenza non raggiunge il 15%. Tali dati sono un indice di maggiore tossicità rispetto a nostri precedenti studi su Carassius auratus esposti alla stessa quantità di cadmio, ma senza NH3, ove abbiamo riscontrato valori di assorbimento minori e sopravvivenza del 100%. Le indagini istologiche hanno evidenziato in acqua dura alterazioni della mucosa del bulbo con un aumento del turnover cellulare, numerose mitosi e riduzione delle dimensioni delle cellule mucipa¬ re. In acqua dolce, invece, si osservano segni di sofferenza delle cellule epiteliali e dilatazione delle cellule mucipare. Inoltre, sia in “acqua dura” che in “acqua dolce”, sono presenti cellule immunoreattive per la Met e la Leuencefalina. È possibile quindi affermare che, in presenza di NH3, raggiunta di cadmio determina un ambiente molto più tossico. Viene ipotizzato che ciò avviene, non solo perché l’NH3 si lega al cadmio formando dei complessi che aumentano fortemente la solubilità di quest’ultimo, ma anche perché molto probabilmente le varie specie di cadmio presentano una diversa permeabilità attraverso gli organi bersaglio. INTRODUCTION In previous researches on thè cadmium action concerning thè Caras¬ sius auratus gut kept in strongly calcareous waters thè Authors noticed, in shortterm, remarkable histomorphologic changes of thè intestinal mucosa and, in longterm, a good recovery capacity of thè exposed animals (An- dreozzi et al., 1992). Of particular interest was thè resulting disappearance, after a treatment of 7 days, of thè Met and Leuenkephalinelike immuno- reactivity, probably connected to thè increase of thè mucus production induced by thè cadmium (Andreozzi et al, 1992). On thè other hand, modifications in thè intracellular distribution of a peptide metenkephali- nelike have been described in thè intestinal epithelium of Carcinus moenas contaminated by heavy metals (AmiardTriquet et al, 1986). Battaglini et al. (1991) have reported in Carassius auratus , exposed to thè cadmium action a remarkable metal uptake in thè gut and, furthermore this Storage was connected to thè different hardness of thè experiment water. On thè other hand, thè toxic action of thè Cadmium varies in relation to thè chemicalphysic conditions of thè environment (Calamari et al., 1980) and to thè presence of other substances that can develop a chelating action towards thè ion Cd2+ (humic acids, aminoacids, porphyrins, purins) (Ravera, 1984; Sprague, 1987). 107 Andreozzi G., Antonucci R., Affai ato C., De Girolamo P., et al Among these substances thè Authors focused their attention to ammo- nia, produci of thè Carassius auratus catabolism, which Stores up in lentie waters in which these fìsh live. In particular, tke NH3 is important because it is a strong chelating agent of Cd2+ and then it can alter thè toxicity both varying thè speciation of this metal in thè solution and preventing its precipitation as CdC03 in calcareous waters (Battaglini et al., 1992). The purpose of thè present paper is to study thè toxic action of thè cadmium in thè catabolic NH3 presence on thè Carassius auratus gut in waters of different hardness. In particular we will to study thè histological and immunohistochemical changes of thè intestinal mucosa as well as thè changes of some neuropeptides. Materlals and methods Animals The experiment was carried out with waters of different hardness. The first kind, “hard water”, was thè tap water of Naples (hardness 369 mg/L of CaC03) and was utilized after an aeration period in a tank for an appropria¬ te dechlorination. The second kind, “soft water”, (152 mg/L of CaC03), was obtained by treating tap water with ionie exchange resins “Soft C” (line “C”), CARMAR, Napoli. Samples of Carassius auratus L., supplied by CARMAR sas (Naples), weighing 6 + 1 g, have been kepi for ten days to get acclimatized, in thè different kinds of water in two separate 600 L glass aquaria, equipped with an air pump and a filler (EHEIM, Germany). To get thè experiment 8 tanks (30x30x40 cm) were equipped with an air pump, without fìlter. During thè whole experiment thè temperature was constantly at 16 ± 1°C, and thè photoperiod was 12 hrs. 4 tanks were filled up with 20 L of “hard water” and 4 tanks were filled up with 20 L of “soft water”. 4 groups of 10 Carassius auratus, randomly chosen in thè acclimatiza- tion “hard water” glass aquarium, were transferred in thè four “hard water” tanks and fed as usuai with Tetramin Tetramere, Melle. The same was made for thè four “soft water” tanks. After 8 days in 3 “hard water” tanks and in 3 “softwater” tanks, CdCl2.21/2H20 was added in such a quantity to obtain a cadmium concen- tration in water equal to lOppm in each tank. 108 Boll Soc . Natur. Napoli - Voi . 101 ( 1992-1993 ) In such a way thè toxic was added when thè experiment water, after 8 days of animai stay, was enriched with catabolic fish materials and among them thè NH3, as we have already mentioned. The specimens from thè other two tanks were utilized as control. The cadmium treatment ran out for 7 days The survival was calculated as percentage of thè survived animals seven days after thè beginning of thè experiment. Chemical tests The analyses of thè components dissolved in thè water were carried out through thè traditional methods of volumetrie titration (Kolthoff et ai, 1973). To determine hardness, calcium and magnesium a standard solu¬ tion af sodium ethilendiaminotetracetic acid salt was utilized; to get thè alkalinity a 0.0 1 N solution of HC1 as titrating solution and green bromocre- sol as acidbase indicator was used; at last for thè chlorides a dosage with AgN03 according to Mohr method was used. All chemical produets utilized are Carlo Erba RP reagents, used without any further purification. To determine thè pH a pHmeter ORMA, model NK 300 was utilized; to determine thè dissolved oxygen was utilized a dissolved oxygenmeter HI 8543 (Hanna Instruments, USA). The NH ; produced and present in water was measured with a spectro- photometer using thè Nessler reagent to identify thè NH3 (Kolthoffet ai, 1973). The cadmium concentration in solution in thè different tanks was regularly measured by an atomic absorption spectrophotometer VARIAN AA275. A parallel precipitation kinetic of Cd2+ as CdC03 was effected in each kind of water used during thè experiments, but without fish and relative catabolic materials. Determination of cadmium uptake For each group of experimental animals, taken after seven days of exposure, guts ere used to determine thè cadmium uptake. Guts were weighed and mineralized by thè action of a HN03/HC104 mixture (ratio of concentrated acids 4:1) following K. Bull's method (1975). The resulting solution was brought up to a volume of 10 mL in a calibrated flask and analvsed by an atomic absorption spectrophotometer VARIAN AA275 to determine thè amount of cadmium. H ; • .. Andreozzi G.} Antonucci R., Affettato C., De Girolamo P., et al. 109 P ■" . ■ ■ . ■ ; Histology and Histochemistry After a treatment of 7 days for each experiment group, 2 Carassius auratus were taken and decapitated. At thè same time 2 control fishes were sacrificed for each kind of water. The gut, cranially dissected at intestinal sphincter level and caudally dissected at thè rectum end, was completely taken out. All thè specimens, after being Bxed in thè Bouin’s fiuid, were embed- ded in paraffin and serially cut into 7 pm sections. To study thè mucopolysaccharides thè following methods were car- ried out: - Periodic acid Schiff reaction. PAS was used in order to identify neutral mucopolysaccharides. - Alcian blue procedure. At pH 2.5 (A.B. pH 2.5) and at pH 1 .0 (A.B. pH 1.0) thè Alcian blue procedure was used alone or in combination with other procedures for thè Bnding and subsequent localisation of acid mucopolysaccharides. - Enzyme digestion test. Sections immediately following thè ones stained with A.B. pH 2.5 were digested with neuroaminidase (from Clostridium perf rigens type V, Sigma) at a concentration of 0.2% in phosphate buffer for 24 hr at 37°C to put in evidence thè sialomu- cins. The immunocytochemical tests were carried out by PAP method (Sternberger, 1979) to find out thè possible presence of Met and Leuenke- phalinelike material. In both cases thè primary antibody was used at 1:500 dilution. Then thè reaction product was displayed with 3.3’ diamine benzidine (DAB). The reaction specifity was tested both by replacing thè primary antibody with PBS and by thè absorption procedure. The control sections were stained with ematoxylineeosine (E. E.). Results Survival The survival analyses showed that aBer 7 days survival was inferior to 15% both in hard water and in soB water. Chemical characteristics Table I reports Chemical and physicochemical parameters of two diRerent waters utilized for thè experiments. It must be noted that thè 110 Boll Soc . Natur. Napoli - Voi 101 (1992-1993) presence of catabolic ammorba, at zero time of thè treatment, reached a 7 -e 10 ppm concentration. Table I - Chemical and physico-chemical parameters of thè different waters Water properties Hard water Soft water Hardness (mg/1 CaCO, 369 152 Aìcalinity (mg/1 HC03~ 346 9,8 Ammonia (mg/1 NH3) 7-10 7-10 Chloride (mg/1 Cl" 32 30 Sulphate (mg/1 SO,2-) 25 25 pH 7,8 7,6 Dissolved oxygen (mg/1 0,) > 7 > 7 Temperature (°C) 16 ± 1 16 ± 1 Fig. 1 reports thè average concentration of cadmium in solution in thè experiment tanks with water having a higher hardness as a function of time ppm Figure 1 - Concentration of Cd2+ ion in solution in thè presence and in thè absence of catabolic products. Andreozzi G., Antonucci R., Affettato C., De Girolamo P.t et al 111 and, in comparison with thè sanie trend of thè cadmium concentration in thè same kind of water, but without Carassius auratus and thè relative catabolic materials. It is easy to note in thè two cases that thè kinetic of precipitation of CdC03 is completely different. In fact, in thè sample of water for comparison (without Carassus auratus) it is possible to note already in thè first hours, a fast and practically complete cadmium disappearance from thè solution. On thè contrary, with thè Carassius auratus thè precipitation occurs only after thè fourth day from thè begin- ning of thè treatment and, however, in a lower quantity, with a left concentration in solution of almost 2 ppm after 7 days. Vice versa, in water of lower hardness, thè cadmium lasts in solution for thè whole length of thè test, aside from thè Carassius auratus presence. Table II - Concentration of Cd2 in thè Carassius gut treated for seven days with NH., without NH; Hard water 71 1 412 Soft water 336 103 Cadmium uptake Tab. 2 reports thè cadmium Storage values measured with thè atomic absorption spectrophotometry in thè gut at thè seventh day of thè treat¬ ment for thè Carassius auratus treated in two different types of waters. In comparison, thè data of a previous research (Battaglini et al., 1992) are reported in thè same table, a research relative to thè cadmium uptalce in thè Carassius auratus guts treated for seven days in two different waters having characteristics similar to those utilized in this paper, but at thè beginning of thè previous research no catabolic ammonia was noted. It can be noticed that thè quantity of cadmium in thè organ is higher for thè Carassius auratus treated in water of higher hardness, as observed in thè previous experiment. It has also to be underlined that thè uptake is always higher when ammonia is present at thè beginning, no matter what kind of water is utilized. Carassius auratus gut structure The digestive System of thè Carassius auratus as all Cyprinids is rather simple because it has no stomach. 112 Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) Thus, in thè Carassius auratus, thè oesophagus is in direct communi- cation with thè gut through thè oesophageal sphincter. The gut is of uniform size, except for a short cranial part, more dilated, called bulb ofthe gut. (Caceci, 1984). The gut mucosa, made of thè epithelium and thè connective tunic, raises in folds having a connective axes (Fig. 2). The epithelium is a single layer of cylindrical cells and contains several mucous cells (McVay and Kaan, 1940) less numerous in thè bulb area than thè other parts ofthe gut. They have a typical “drumstick” shape and produce a secretion made of neutral PASpositive mucins and AB pH 2.5 and pH 1.0 positive sulphurated acid mucins, neuroaminidase digestion resistant (Fig. 3). Also Metenkepha- line (Fig. 6) and Leuenkephaline (Fig. 9) positive neuroendocrine cells are mixed up with thè epithelial cells (Andreozzi et al., 1992). Such roughlv pyramidal and open cells are located onlv in thè bulb area and in thè most cranial part of thè gut. Figure 2 - Carassius auratus control -intestinal bulb - Alcian pH 2,5240X. Figure 3 - Carassius auratus control -intestinal bulb - Alcian pH 2,5380X. il 3 Apiér'eozjd G\, Antonucci Aifatato C\, De Girolamo P., et ah Figure 4 and 5 - Carassius auratus treated with Cd2' in hard water for seven davs - intestinal bulb - Alcian pH-3,5. Note very thin mucous cells and cells flankìng away in thè top of thè yiiìus - 600X. Boll Soc , Haiur. Napoli ~ Volò! 01- ( 1992-1993 ) ■y. 9 Figure 6 -- Caras- sius auratus con¬ tro! - intestinal bulb - metenke- phalin 800X Figures 7 and 8 - Carassius au¬ ratus treated wi~ th Cd2+ in hard water for seven days - intestinal bulb - metenke- phaìin 800X Figure 9 - Caras¬ sius auratus con¬ trol - intestina! bulb - leuenke- phalin 800X Figures 10 and 1 1 - Carassius auratus treated with Cd2+ in hard water for seven days - intestinal bulb - ìeu-enke- phaiin 800X Andreozzì G., Antonucci R,f Affettato C., De Girolamo P., et ah 115 Hard water experiment In thè animals exposed for seven days to thè cadmium action in hard water at thè presence of catabolic ammonia, thè gut mucosa appears to be rather damaged at thè bulb level and at thè more cranial part of thè gut. In fact, in these areas thè epithelium is often detached from thè inferior connective and it was noticed that some cells were flaking away. (Fig. 4, 5). The mucosa keeps a normal aspect only at thè bottom of thè folds and at thè remaining part of thè gut. The mucous cells, often much more elongated and thinned as compared to those of thè Controls, have positive granules scattered in thè whole cytoplasm up to thè base. In addition there are several mitosis. The histochemical reactions for thè mucopolysaccharides have shown, as for thè Controls, thè presence of positive A.B. pH 2.5 and pH 1 .0 positive acid mucins and neuroaminidase mucins resistant. There are also eells containing PAS positive neutral muans. The immuno reactive Metenkephaline cells seem to be more nume- rous and more significantlv positive as compared to thè Controls (Figures 7, 8). Their shape and distribution result unchanged. The IR (immunoreac- tive) cells for thè Leuenkephaline seem to be more significantlv positive as compared to thè Controls (Figures 10, 11). Their number and shape are similar to those of thè Controls. Soft water experiment In thè gut of thè animals treated for seven days with cadmium in softwater with catabolic ammonia, thè bulb mucosa appears suffering. In fact, thè epithelial cells appear coarted and thè mucous cells located at thè top of thè folds are often so swollen as to look like a “ball” or a “bag” with a highly thickened secretion. Sometimes it seems that thè nearbv cells merge themselves (Figures. 12, 13). Furthermore there is an increase of thè mucus mass contained in thè intestinal lumen. The histochemical reac¬ tions have shown, as in thè Controls, thè presence of PASpositive mucins and A.B. positive mucins, neuroaminidase resistant. Positive Metenkephaline and Leuenkephaline cells were found and their frequency and distribution were similar to those found in thè animals of Controls. 116 Boll. Soc . Natur Napoli - Voi. 101 (1992-1993) * V - • ^ v - ** 1 * _ ^ ■*{ * %4 * l vv -A*' £ 2* • v# -' «./:■''* «f >%•&* ». 44»?^ * * ' " ■:; * ; ; ^ 1 3r .: . r4* ^ ì; ,i ' : -ù «. » , "■'i WùwA • » * *ÀÌz^«! \ ^ ir V-4 - V A Mv> N * ?• g*^% ' /fè> l,; ■ * ' .v??; * a 4f'A/ . ^ Figure 1 2 and 1 3 - C arassi us anratus treated with Cd2+ in soft water for seven davs - intestina! bulb - Alcian pH 2,5 - Note thè mucous cells enlarged and sometimes merged (arrows). 380X. Andreozzi G Antonucci R Affatalo C., De Girolamo P., et Discussion The results obtained on thè Carassius auratus after 7 day treatment, suggest that thè cadmium shows a higher toxicity in thè presence of catabolic NH3. In addition, unlike what was noticed in our previous researches (Battagliai et ai, 1991) this action is not strictlv influenced by thè water hardness. The most signifìcant results obtained in this experiment are those concerning thè survival. In fact, in thè present experiment, after 7 days thè survival had not reached thè value of 1 5%. On thè contrary, in our previous experiments on Carassius auratus, we have reported that with equal cadmium concentration and lack of catabolic NH3, a survival up to 100% after 40 days in hard water (520 mg/L of CaC03) (Battaglini et al. , 1 992) and up to 90% after 40 days in soft water (150 mg/L of CaC03) (not yet published data). The histological researches, after 7 days of exposure to cadmium with catabolic ammonia, have focused signifìcant changes of thè intestinal mucosa, in particular at thè bulb level, with changes of thè mucus Storage and mucous celi shape. In thè specimens treated in “hard water” thè mucous cells appear thinner and elongated, with positive granules up to thè basai area, while in thè specimens treated in soft water they are verv swollen “bag” shaped with a very thickened secretion. In both cases thè immunoreactive Met and Leuenkephaline cells are positive and more numerous in thè specimens treated in hard water. These data differ from those described in our previous researches (Andreozzi et al, 1992; Andreozzi et al. 199 la, 199 lb). In highlv calcareous waters thè AA have observed, after a treatment of 7 days with cadmium, less damage at thè gut mucosa level and thè disappearance of immunoreac- tivity to Met and Leuenkephaline; in a longer period a remarlcable capacitv of restoring thè histological and immunocytochemical features was noted. Thus, a higher toxicity of cadmium in thè presence of catabolic NH 3 is evident and it appears with different characteristics in environments having different hardness. In fact, while in hard water thè numerous mitoses, thè detached epithelium and thè reduced sizes of thè mucous cells induce us to think of a turnover celi increment, on thè contrary, in soft water thè coarted aspect of thè epithelial cells is a sign of a suffering tissue condition, while thè increase of thè mucous celi size and a greater mucus quantity in thè intestinal lumen make us suppose a defensive reaction of thè epithelium itself. 118 Boll Soc . Natur. Napoli - Voi 101 ( 1992-1993 ) These data match with those described in Fnndulus by Gardner and Yevich (1970) and in Salmo gairdneri by Crespo et al ( 1 986) influenced by cadmium alone. In fact, these authors describe thè cellular “suffering” and thè mucus secretion increase. In addition, Crespo et al (1986), always in trout, describe also an increase in thè mitoses number. The presence of immunoreactive cells for thè Leu and Metenkephaline, after 7 day treat¬ ment, in contrast to what reported by Andreozzi et al (1992) could be related to an emergency situation from which thè animai cannot recover any longer. On thè contrary, thè atomic absorption data in thè gut after 7 days of treatment show a higher metal Storage in thè specimens treated in hard water compared with those treated in soft water. This datum is similar to what previouslv reported in goldfish by Battaglini et al (1991) on thè action of thè cadmium in waters of different hardness, without catabolic ammonia. In this case too, thè higher value in hard water is likely to be attribute to thè ingestion, even if in a lower quantity, of CdC03 which begins to precipitate on thè bottom after four days of experiment. In soft water, at thè same cadmium quantity dissolved in solution, thè presence of NH3 deter- mines a clear increase of metal Storage in thè gut as compared to what reported in specimens exposed onlv to thè cadmium (Battaglini et al, 1991). We can assert now that to thè same quantity of cadmium added thè “experimental environment” created results to be definitely more toxic. Th e reasons of such a higher toxicity can be, in our opinion, attributed just to thè presence of NLL expelled by thè fishes as catabolic material. In fact, thè first effe et of thè molecular ammonia is to bind itself to Cd2+ producing several complexes that stronglv increase thè CdCCft solubility developed in calcareous waters with Cd2+. In fact, in test tanks with thè goldfish and their catabolic materials, thè CdCCb precipitation is slower and inferior as compared to that which occurs in waters of thè same kind but without Carassius anratus. Thus, thè presence of catabolic ammonia determines a cadmium permanence in hard water as we observed in soft water. On this matter, a first hvpothesis would lead us to state that, in waters of higher hardness, thè highest toxicity of Cd2+ with NH3 can be connected to thè higher and longer presence of thè toxic in solution than in thè case in which ammoniacal materials. are lacking. But this element, even if important, cannot explain whv even in water of less hardness where thè CdCO , precipitation does not occur it is possible to obtain a definitely Andreozzi G.. Antonucci R., Affatalo C., De Girolamo P., et ah 119 lower survival with respect to that obtained in absence of NH3. As in ibis second case, thè Carassius auratus, with or without NH3, are in contact with thè same total amount of cadmium dissolved in solution (10 ppm) and are in environmental conditions almost similar, thè different survival observed could be ascribed to thè different cadmium speciation in solution and very likely to a different permeability, of different forms in which cadmium is present in solution, through thè organ targets of thè test animals. Acknowledgements This study was supported by a contribution of italian MURST (40% & 60%). REFERENCES Amiard-Triquet, C., J.C. Amiard, R. Ferrand, A.C. Andersen & M.P. Dubois, M.P. 1986. Disturbance of MetEnkephalinlike hormone in thè hepatopancreas of crabs contaminated bv metals. Ecofoxicologv and environmental s afe tv, 11: 198-209. Andreozzi, G., C. Affatato, R. Antonucci, P. Battagli ni, & G. Gargiulo, 199 la. Comportamento di alcuni peptidi nell’intestino di Carassius auratus trattato .con cadmio. Atti Soc. It. Sci. Vet. , 45: 277-280. Andreozzi, G., C. Affatato, R. Antonucci, P. Battagline G. Gargiulo, & P. Russo, 1 99 1 b. Effetti dell'esposizione al cadmio sulla mucosa intestinale di Carassius auratus. Atti 45" Conv. Soc. It. Anat. Sassari, 261. Andreozzi, G., R. Antonucci, C. Affatato, G. Gargiulo, & P. Battagline 1992. Influence du cadmium sur l’intestin de Carassius auratus. Anat. Histol. Embryol. , (in press). Battagline P-, G. Andreozzi, R. Antonucci, L. Ferrara, G. Gargiulo & C. Affatato, 1992. Azione del cadmio in presenza di NH, catabolico sull’intestino di Carassius auratus. Atti 54 Cong. U.Z.I. , 219. Battagline P., L. Ferrara, & O. Soppelsa, O., 1991. Assorbimento di Cd2+ in vari organi di Carassius auratus in acque HdureU e “dolci”. Atti Soc. It. Sci Vet., 4sT 149-153. Bull, K., 1975. Routine heavv metal analvsis of biological samples. Science Chelsea J. Res. Chel, 7: 23-25. Caceci, T. 1984. Scanning electron microscopv of goldfish, Carassius auratus, intestinal mucosa. J. Fish Biol. , 25: 1 12. Calamari, D., R. Marchetti, & G. Vailati, 1980. Influence of water hardness on cadmium toxicitv to Salmogairdneri Rich. WaterRes. , 14: 1421-1426. 120 Boll. Soc . Natur. Napoli - Voi 101 (1992-1993) Crespo, S., G. Nonnotte, D.A. Colin, C. Leray, L. Nonnotte, & A. Aubree, 1986. Morphological and functional alterations induced in trout intestine bv dietary cadmium and lead. J. Fish Biol., 28: 69-80. Gardner, G.R. & P.P. Yevich, 1970. Histological and hematological responses of an estuarine teleost to cadmium. J. Fish Res. Bd. Can., 27: 2185-2196. Kolthoff, I.M., E.B. Sandell, E.J. Meehan & S. Bruckenstein, 1973. Analisi Chimica Quantitativa. Padova, Piccin. Me Vay, J.A. & H.W. Karm, 1940. The digestive tract of Carassius auratus. Biol. Bull., 78: 53-67. Ravera, O. 1984. Cadmium in freshwater ecosystem. Experientia, 40: 214. Sprague, J.B. 1987. Effect of cadmium on freshwater fish. In Cadmium in Aquatic Environment, Nriagu, J.O. & Sprague, J.B. Eds., New York, J.Wiley, pp. 139-169. Sternberger, L.A. 1979. Imm u nocytochem istry , New York J. Wiley & Sons, Eds. CONTRIBUTI IN ITALIANO Boll Soc » Naiur. Napoli - Voi 101 (1992-1993); 1 23-140 123 Mammiferi olocenici provenienti da uno scavo effettuato nell 'isola di Capri (Italia meridionale) Barbera C., Lene! G., Rapuano M., Virgili A. Dipartimento di Scienze della Terra, Università degli Studi «Federico II», L.go San Marcellino 10, 80138 Napoli, Italy Riassunto, In questo lavoro si segnala la presenza di una mammofauna olocenica rinvenuta durante uno scavo recente nell'isola. Associati a reperti ceramici di età romana sono stati rinvenuti resti di Equus, Sus , Capra, Ovis, Bos e gallinacei. Per la prima volta, viene segnalata la presenza del Bos primigenius, specie non facente parte delle faune endemiche dell'isola. Gran parte dei reperti mostra chiari segni di bruciatura che sono riferibili ad avanzi di pasto, appartengono sia ad animali giovani che ad animali adulti ad eccezione di quello riferito al cavallo che sicuramente è appartenuto ad un individuo adulto. Premessa Questa nota si pone come obbiettivo l’identificazione di alcuni resti ossei rinvenuti nell'isola di Capri, in uno scavo effettuato a scopo edilizio in via Truglio 19. Lo studio dì questi reperti ha portato ad identificare la specie Bos primigenius Boi, mai segnalata prima nell'isola. I resti dei mammiferi in oggetto sono depositati presso il Centro documentale A. Ciccogliano di Capri e siglati con la lettera C seguita da numeri progressivi. Precedenti conoscenze Sulla zona esistono pochi dati bibliografici sia dal punto di vista geologico che paleontologico specialmente per i sedimenti della copertura pleistocenica. Received 14.5.92, accepted 27.1 1.92. 124 Boll Soc. Naiur. Napoli - Voi 101 (1992-1993) Rei lini (1910) riconosce, nel giacimento Qu isisan a Certosa ritrovato casualmente durante uno scavo per fondazioni edilizie e segnalatogli da E. Cerio, appassionato collezionista e raccoglitore di tutto il materiale rac¬ chiuso nel museo di Capri che porta il suo nome, una parte superiore di età olocenica con presenza di ceramiche del bronzo ed industrie del Neolitico medio, una intermedia di età pleistocenica-terminale presentante interca¬ lazioni di terra rossa con resti di cervidi e di una parte inferiore (Pleistoce¬ ne medio) con industrie litiche acheuleane e resti di grandi mammiferi. Bassani e Galderi (1911) verificano la stratigrafia dello scavo di Quisisana creando una apposita sezione ad esso adiacente. Azzaroli (1961) nel suo lavoro sul nanismo dei cervidi insulari dopo aver rivisto il materiale del livello intermedio di Cerio, Pigorini e Pelli. ni (1906), descrive una nuova specie di cervide nano dell’isola di Capri come Cervus tyrrenicus. Successivamente Piperno e Segre (1984) propongono una stratigrafia più aggiornata dei giacimenti Qu isisana C e rto sa e identificano cinque livelli, che dall’alto in basso sono: 1) Suoli e detriti non consolidati. 2) Pozzolane stratificate del Pleistocene superiore finale. 3) Terra rossa . 4) Tufo grigio campano , facies cineritica. 5) Argilla rossa con industria acheuleana e grandi mammiferi. Cinque, G bozzi ed Esu (1988) e successivamente Gliozzi (1989) ricono¬ scono in un riempimento della grotta “ Vascio o’ furino ” localizzata sul lato Sud Est dell’isola, una nuova specie di roditore Apodemus silvaticus thyrre- nicus con un cranio di grandi dimensioni e denti arcaici. Barbera e Cimmino (1990) in materiale subattuale di una grotta del versante orientale di Monte Solaro riconoscono resti di Suncus etruscus (SAVI), Rattus rattus (L.), Mus domesticus RUTTY, Apodemus sp.} Eliomys quercinus (L.). Alcune di queste specie, Suncus etruscus ed Eliomys quercinus non erano mai state segnalate nell’isola. Serie stratigrafxca Lo scavo in via Truglio, come da premessa, è stato effettuato quale sbancamento per uso edilizio. Esso ha raggiunto una profondità di circa 3,50 m e la successione dei sedimenti affioranti dall’alto verso il basso è la seguente: Burbera C\, Retici jjj , Rapitane J§|§ Virgili A, 125 m. 2,80 - terreno di copertura agraria m. 0,50 - ceneri vulcaniche m. 0,80 - pozzolane stratificate m. 0,30 - intercalazioni di terra rossa Data la vicinanza del sito di provenienza del materiale allo scavo di Quisisana Certosa descritto da Piperno e Segre (1984) è stato possibile confrontare le colonne stratigrafiche relative ai due giacimenti si è così notato che il materiale proveniente da via Truglio si rinviene in un livello che corrisponde al livello 3 (intercalazione di terra rossa) del giacimento di Quisisana. Probabilmente se lo scavo non fosse stato interrotto sarebbe stato possibile raggiungere il livello 5 con i grandi mammiferi e Pindustria litica di età acheuleana. Descrizione del materiale Per la descrizione del materiale viene usata la classificazione di Grzimek (1974). Tutto il materiale è stato confrontato con quello presente nei Musei Universitari Napoletani. èssendo la quasi totalità dei reperti in frammenti, i parametri delle tabelle sono riferiti alle ossa sulle quali è stato possibile effettuare misura¬ zioni. Caballus caballus (L.) Materiale esaminato: Un solo frammento di tibia di grandi dimensione appartenente ad un individuo adulto (Cl). Numero di esemplari 1. Sus scrofa (L.) Materiale esaminato. 2 canini sinistri, 1 superiore (C2) ed 1 inferiore (C3) 1 canino destro superiore (C4) 1 incisivo mandibolare sinistro (C5) 126 Boll Soc. Natur. Napoli - Voi , 101 (1 992-1 993 ) 2 PM superiore sinistri (C6, C7) 1 M2 superiore sinistro (C8) 2 frammenti mascellari, 1 sinistro (C9) ed 1 destro (CIO) 4 frammenti mandibolari, 3 sinistri (CI 1, CI 2, CI 3) ed uno destro (CI 4) 2 omeri destri frammentari (CI 5, CI 6) 1 omero sinistro frammentario (CI 7) 1 femore destro frammentario (CI 8) Numero minimo di individui: tre. Osservazioni : Due delle mandibole esaminate appartengono ad un individuo di età avanzata (C 11, C 14), mentre una delle mascelle appartiene ad un individuo di giovane età. Anche i canini appartengono ad individui giovani. Le ossa lunghe sono rotte a livello della dialisi. I parametri sono riportati in tab. I e II. Tab. I - Parametri morfometrici dei denti: Sus scrofa Linneo masc. dx clO h diam. meso-dist. diam. vest.-lingu. PM4 0,59 1,09 1 MI 0,33 1,38 1,31 M2 0,67 1,85 1,17 masc. dx c9 h diam. meso-dist. diam. vest.-lingu. PM4 0,83 1,17 0,86 MI 0,66 1,5 1,13 M2 0,99 1,7 1,24 masc. dx cl 1 h diam. meso-dist. diam. vest.-lingu. PM4 0,27 1,59 1,05 MI 0,76 1,54 1,85 M2 0,35 1,82 1,18 denti isolati h diam. meso-dist. diam. vest.-lingu. I C5 0,53 0,3 P M2 inf. dx C7 1,15 1,23 0,77 P M2 inf. sx C6 1,26 1,28 0,45 M 2 sup. sx C8 0,9 1,23 1,12 Barbera C,, Leuci G., Rapuano M., Virgili A. 127 Tab. II ■ ■ Parametri morfometrici delle ossa: Sus scrofa Linneo OMERO: misure longitudinali C 15 C 16 Diametro trasversale max estremità inf. 3,93 3,25 Diametro trasversale inf. troclea (f. post.) 2,77 2,58 Diametro trasversale sup. troclea (f. anter.) 1,5 1,2 Diametro verticale max troclea 3,62 3,12 Diametro trasversale sup. artic. (f. anter.) 2,82 2,29 Lunghezza foro oleocraneo 0,67 0,56 Larghezza foro oleocraneo 0,57 0,54 FEMORE: misure trasversali C 18 Lunghezza collo 1,78 Diametro trasversale della testa 2,47 TIBIA: misure trasversali C 17 Diametro trasversale epifisi inf. 2,88 Diametro antero-post. sup. art. inf. 1,74 Diametro trasversale sup. art. sup. 1,9 Capra hircus (L.) Materiale esaminato. 1 incisivo destro (CI 9) 2 MI inferiori destri (C20, C21) 2 M2 inferiori destri (C22, C23) 1 frammento di atlante (C24) 3 frammenti mandibolari, due destri (C25, C26) ed uno sinistro (C27) 1 frammento di radio destro (C28) 2 frammenti di coxale sinistro (C29, C30) 1 frammento di tibia destra (C31) Numero minimo di esemplari: due. Osservazioni : Tutte le mandibole, per quanto frammentarie presentano la serie dentale completa dai premolari. Alcune mandibole appartengono ad individui giovani (C26, C27), in quanto le cuspidi sono poco usurate. Le ossa lunghe sono rotte all’altezza della diafìsi. I parametri sono in tabella III e IV. 128 Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) Tab. Ili - Parametri morfometri dei denti: Capra hircus Linneo mand. dx C25 h diam. meso-dist. diam. vest.-lingu. PM1 0,94 0,54 0,53 PM2 1,02 0,74 0,67 PM3 1,1 1 1 0,78 MI 1,13 1,25 0,83 M2 1,18 1,61 0,93 M3 1,27 2,37 0,91 mand. sx C26 h diam. meso-dist. diam. vest.-lingu. PM2 1,07 0,82 0,58 PM3 1,16 0,93 0,6 MI 1,55 1,33 0,77 M2 1,46 1,51 0,84 M3 1,36 1,7 0,77 mand. sx CI 1 h diam. meso-dist. diam. vest.-lingu. PM2 1 0,71 0,61 PM3 1,1 0,84 0,62 MI 1,08 1,05 0,72 M2 1,31 1,31 0,87 M3 1,58 2,25 0,93 Ovis sp. vel Capra sp. Materiale esaminato: 1 calcagno sinistro (C32). Numero minimo di esemplari: uno. Osservazioni: Alcune rugosità decorrono dalla destra del corpo delhosso sino alla suspentaculum, ciò fa pensare che esso dovesse appartenere ad un animale selvatico. Barbera C., Leuci G., Rapuano M., Virgili A . 129 Tab. IV — Parametri morfometrici delle ; ossa: Capra hircus Linneo RADIO: misure trasversali C 28 Diametro trasversale della testa 2,57 Diametro trasversale del collo 2,05 Diametro trasversale fossetta radiale 1,83 Diametro antero-posteriore della testa 1,56 RADIO: misure longitudinali C28 Diametro antero-post. oleocraneo 1,05 Diametro antero-post. del corpo 0,52 Diametro antero-post. apofisi stiloide 0,52 BACINO: misure trasversali C 29 C 30 Diametro branca discendente pube 2,41 2,74 Diametro antero-post. cavità cotiloidea 2,54 TIBIA: misure trasversali C 31 Diametro trasversale del corpo 1,43 Diametro trasversale epifisi inf. 2,88 Diametro antero-posteriore sup. art. inf. 1,32 Diametro trasversale sup. art. sup. 1,82 Bos primigenius Boj Materiale esaminato: 1 MI superiore destro (C33) 1 porzione prossimale di ulna destra (C34) 1 metatarso destro rotto longitudinalmente e trasversalmente (C35) 1 II falange interna destra (C36) Numero minimo di individui: almeno due in quanto la porzione prossima¬ le di ulna appartiene ad un individuo di grandi dimensioni, mentre il metatarso appartiene ad un individuo adulto le cui dimensioni rientrano tra le minori della specie. Osservazioni : Sono stati fatti diversi tentativi di ricercare dei caratteri diagnostici nei denti isolati per distinguere il bove dal bisonte. Houle, 1906, indica come Boll Soc. Natur . Napoli « Voi IO 1 (1992-1 993} 130 caratteristica la forma più quadrata dei molari del bisonte. Altri AA. insistono di più sui caratteri del solo M3 della mandibola (Boessneck et al , 1963). Tuttavia i molari del bisonte sono più quadrati di quelli del bove; il lato labiale è meno appuntito, la copertura di smalto più forte. Il dente del bisonte effettivamente, è reso più corto e tozzo dalla diversa morfologia delle pieghe laterali e da una differente forma e posizione del talonide (Corridi, 1987). I confronti effettuati con materiale presente nel Museo di Paleontologia dell'Università di Roma hanno permesso di attribuire ad un MI superiore destro di Bos primigenius BOJ il reperto (C33) da noi esaminato. La porzione prossimale dell'ulna destra (C34) appartiene ad un indivi¬ duo giovane, dato il tipo di ossificazione, ma di grandi dimensioni; manca della tuberosità superiore e della porzione prossimale del becco dell'ole erano. Il reperto C35 è la porzione mediale di metatarso destro che oltre a rotture dovute all'estrazione probabile del midollo, presenta caratteristi- che striature dovute all'azione dell'uomo. I parametri sono in tabella V. Tab. V - Parametri morfometrici delle ossa: Bos primigenius : Boi SECONDA FALANGE int. dx: mis. longitud. C 36 Lunghezza massima 4 Altezza massima 2,08 SECONDA FALANGE int. dx: mis. trasvers. C 36 Diametro trasversale sup. 2,88 Diametro antero-posteriore estr. sup. 2,9 Diametro trasversale troclea 1,93 Diametro trasversale estr. inf. 2,24 Diametro antero-posteriore condilo interno 2,1 Diametro antero-posteriore condilo esterno 2,85 Diametro trasversale 2,44 Diametro antero-posteriore 2,23 Aves sp. et gen. ind. Tra il materiale da noi esaminato sono stati rinvenuti frammenti lunghi di ossa di gallinacei. Particolarmente ben conservati appaiono: 1 cubito destro (C37) 1 tibia sinistra (C38) 1 metatarso sinistro (C39) Numero minimo di esemplari: uno. B Utf pepli C.t Le&pì C , Mapuan® À, m Osservazioni : I gallinacei osservati sono di discrete dimensioni. Il cubito ed il metatarso sono in buone condizioni di fossilizzazione, la tibia si presenta rotta a circa i due terzi del corpo. I parametri sono in tabella VI. Taf*. VI - Parametri morfometrici delle ossa: Gailineacei TIBIA: misure longitudinali C 38 Lunghezza della cresta 2,47 TIBIA: misure trasversali C 38 Lunghezza incavo popliteo 0,7 Diametro trasversale del corpo 0,92 Diametro trasversale max estr. inf. 1,6 METATARSO: misure trasversali C 39 Diametro trasversale estremità sup. 1,45 Diametro anten>posteriore estr. sup. 1,05 Diametro trasversale del corpo 0,78 Diametro trasversale epifisi inf. 1,34 Diametro antero-posteriore epifisi inf. 0,37 Conclusioni L’insieme faunistico, seppure con i limiti dovuti alla tipologia del giacimento ed al metodo alquanto sommario di recupero può dare alcune informazioni sulla popolazione mammaliana dell’isola di Capri nel tardo Olocene. I grandi mammiferi come: Furo, Bos primigenius Boi, erano ivi rappresentati e probabilmente anche utilizzati dall’uomo sia come cibo che per il lavoro nei campi. Come cibo probabilmente venivano utilizzati gli altri animali costituenti la fauna (cinghiale, pecora, capra ecc.). Gran parte dei resti mostrano tracce di bruciature e tagli longitudinali, caratteristici dell’opera dell’uomo, tesi all’estrazione delle parti edibili in essi contenuti. Gli scarsi resti di ceramica, associati alle ossa che abbiamo fatto esaminare da colleghi archeologi della sovrintendenza ai Beni AA. di Napoli e Caserta ci hanno dato un arco temporale compreso tra il 1° secolo 132 Boll Soc . Natur . A/apo/i - FoT 101 {1991 1993} A.C.: ed il 11° D.C., periodo questo della urbanizzazione da parte dei romani dell'isola di Capri. L'interesse di questa nota resta quindi principalmente limitato alla prima segnalazione certa, con resti accertati di Bos primige- nius BOJANUS in epoca romana nell'isola di Capri. Ringraziamenti Ringraziamo il Prof. F. Barattolo del Dipartimento di Paleontologia per averci fornito il materiale dello scavo. I Direttori dei Musei di Zoologia della Facoltà di Scienze e di Anatomia Veterinaria dell'omonima Facoltà per averci permesso di confrontare il materiale. Ringraziamo le Dr. L. Caloi e E. Gliozzi per le proficue discussioni su Capri e per l'aiuto datoci nel determinare alcuni dei reperti di Bos. Ringraziamo la Dottoressa P. Gargiulo della sovrintendenza archeologica di Napoli e Caserta per l'esame del materiale ceramico. LAVORI CONSULTATI Azzaroli, A., 1961 . Il nanismo nei cervi insulari. Palaeont. It., Pisa, 56, n. s.: 1-32, 25 figg. 10 tavv. Barbera, C. &l M.G. Cimmino, 1990. Resti di insettivori e roditori di età recente raccolti in una grotta dell’isola di Capri (Italia). Histrix, Roma n. s. 2: 1 10, 1 tav. Bassani, F. & A. Galdieri, 1911. Scavo geologico eseguito a Capri. Atti Soc. It. Progr. Se., 4: 671-676, 3 figg. Bassani, F. & A. Galdieri, 1911. Strumenti chelleens dell'isola di Capri. Bull. Paleon., Parma, s. 4, 7: 5762, 3 figg. Boessneck, J., J.P. Jequier & H.R. Stampfli, 1963. Seeberg burgaschiseesud. Acta Bernensia, Berna, II Teil 3. Boule, M., 1906. Paleontologie. In: Villeneuve, Boule, Vernan, Cartallhac, Les grottes de Grimaldi (Baousse Rousse) , Monaco 1: 234-236. Cerio, L, L. Pigorni & U. Rellini, 1906. Materiali paleontologici dell'isola di Capri. Bull. Paleont. It., Roma, 23: 1 16. Cinque, A., E. Gliozzi & V. Esu, 1 986. Il riempimento della grotta “ Vascio ‘o funno" a Capri, primi risultati dello studio geomorfologico e paleontologico. Pubbl. Dip. Se. Terra Napoli, 33: 105-1 16, 2 figg., 1 tav. Corridi, C., 1 987. Faune pleistoceniche del Salento 2 La fauna di fondo Cattie, Maglie, Lecce. Ediz. Scient. Mus. comunale di Paleont. Quaderno 3: 730, 18 tav. Gliozzi, E., 1989. Apodemus sylvaticus tyrrenicus n. ssp. ( Muridae , Rodentia) from thè Upper Pleistocene of ndo Cattie, Maglie, Lecce. Ediz. Scient. Mus. comuna¬ le di Paleont. Quaderno 3: 730, 18 tav. Grzimek, A., 1974. La vita degli animali. Voi. 13. Ed. Bramante, Milano. Piperno, M. & A.G. Segre, 1984. Capri. In: I primi abitanti d’Europa. Scheda 77: 146-149. De Leca editore, Roma. Rellini, U., 1910. L'uomo preistorico nell'isola di Capri. Natura, Pavia, 1: 310. Rellini, U., 1923. La grotta delle Felci a Capri. Monum. Ant. Ac. Lincei, Roma, 29: 305-406, 36 figg., 2 tavv. Rellini, U., 1928. Il Paleolitico italiano secondo il Dr. R. Vaufrey. Bull. Palent. It., Roma, pt. 1: 110. 134 ' Boti, Soc . Natur. Napoli - Voi 101 (1992-1993) Tav. 1 - Capra hircus Linneo. 1. Emimandibola sinistra (C 26). Norma labiale. 2. Emimandibola sinistra (C 27). Norma linguale. 3. Emimandibola destra (C 25). Norma labiale. Boll Soc . Natur. Napoli - Voi 101 ( 1992-1993 ) 136 Tav. 2 - a) Bos primigenius Boj. 1. Porzione prossimale di ulna destra (C 34). Circa 1/2. 2. Primo molare superiore destro (C 33). b) Sus scrofa Linneo. 3. Femore destro (C 18). Norma anteriore. 4. Omerotro (C 16). Norma posterodistale. 5. Omero destro (C 15). Norma posterodistale. 6. Omero sinistro (C 38). Norma posterodistale. 7. Emimandibola sinistra (C 10). SKff 138 Boll Soc. Natur. Napoli - Voi 101 (1992-1993) t Tav. 3 - a) Aves gen. et sp. indet . 1. Cubito destro (C 37). 2. Cubito destro (C 37). 3. Tibia (C 38). 4. Tibia (C 38). 5. Metatarso (C 39). b) Ovis vel Capra 6. Calcagno sinistro (C 32). c) Ceramica. 7 Frammento di vaso. Tutte le figure sono in grandezza naturale tranne C 34 che è circa 1 /2 delToriginale. JMJ, Soc.., Nitur. mpbU ' ir/t Wì '{1992-1 9^3 j: 14J-14S • 141 Il Dissesto Idrogeologico Vallarlo A, Professore Ordinario di Geologia Applicata Dipartimento di Scienze della Terra Facoltà di Scienze» Università degli Studi «Federico II» da una Conferenza alla Società dei Naturalisti in Napoli Gli eventi naturali ed antropici succedutisi negli ultimi due secoli sono stati caratterizzati da un crescendo» rapido superamento di fasi storiche e sociali» in relazione alla progressiva affermazione e diffusione della società industriale» alFincremento della popolazione» all 'ampl iarsi di centri urba¬ ni» alla concentrazione delle aree industriali» al veloce progresso dello sviluppo tecnologico e all 'utilizzo indiscriminato delle risorse naturali» trascurando nel contempo la qualità della vita, le condizioni di lavoro e gli effetti alterativi ed inquinanti che le attività antropiche avrebbero potuto determinare all'ambiente nell'immediato e nel futuro (Dì Donna e Valla¬ no» 1992» 1993a.» 1993b; Vallano, 1992a). In tale contesto il rapporto dell'uomo con l'ambiente naturale è stato improntato» prevalentemente, al principio dello sfruttamento. Posizione questa sviluppata dalle filosofie occidentali per le quali l’uomo è l'essere superiore in grado di sottomettere la natura. Va ricordato a tal proposito che anche l'insegnamento cristiano si pone nella stessa posizione come specificato in un versetto della Genesi che esorta l'uomo e la donna a crescere e moltiplicarsi ed ad usare la terra, rendendosela soggetta. (Dorsi.. 1988). Solo negli ultimi decenni l'incalzante ripetersi di catastrofi e di disastrosi fenomeni alterativi dell'ambiente fisico (Catenacci» 1992), tra l'altro conseguenti al modelli socioeconomici prevalenti» ha determinato l'unanime ammissione che l'uomo ha avuto il molo di agente attivo nell'alterazione degli equilibri naturali. Infatti le attività antropiche» sem¬ pre più incisive ed irrispettose dell'ambiente fìsico e delle risorse naturali» hanno agito come fattori della dinamica esogena al pari delle acque superficiali del vento e delle escursioni termiche» con l'aggravante che l'uomo agisce senza soluzione di continuità ritenendo» tra l'altro» di essere l'unico indiscusso padrone e beneficiario dì tali beni» mentre avrebbe avuto interesse ad assumere il molo del rispettoso e raziocinante custode Boll Soc. Natur. Napoli - Voi 101 (1992-1993) di un patrimonio unico, comune a tutti gli esseri viventi, e non rinnovabile in tempi umani Nel vasto panorama di equivoci voluti e determinati per perseguire lo sfruttamento intensivo ed estensivo delle potenzialità naturali ha svolto un ruolo determinante la mancanza di una visione complessiva delle proble¬ matiche ambientali e, soprattutto, di un disegno programmatico di indiriz¬ zo politico e gestionale che ha agevolato, quando non direttamente inne¬ scato, il progressivo depauperamento dell'ambiente fisico e delle risorse naturali (Ministero dell'Ambiente, 1992, Vallario, 1992b). Ambiente fisico e potenzialità naturali Ambiente è quanto comprende ed interagisce, mediante complesse relazioni funzionali dirette ed indirette con l’uomo, gli altri esseri viventi, il mondo inorganico, le condizioni geologiche, biologiche, fìsiche e chimi¬ che che costituiscono e, quindi caratterizzano un determinato spazio geografico. Tutti gli elementi costituenti l'ambiente risultano tra loro interdipendenti in vario modo e con diversa intensità, in tempi a scala geologica e umana, a seconda delle circostanze, dei processi e dei fenome¬ ni considerati. Un ambiente rappresenta un sistema in cui al variare di condizioni interne e/o esterne possono mutare i rapporti tra le sue varie componenti; in tal modo alcune di esse, col procedere del tempo, possono perdere il carattere di prevalenza per essere sostituiti da altri che rappre¬ sentano l'effetto dei nuovi mutamenti. Il sistema ambiente non può, quindi, ritenersi statico ma in continua evoluzione dinamica (Vallario, 1992a). La complessità degli elementi costitutivi e le loro ampie interrelazioni lasciano intendere che l'ambiente va considerato come una realtà unitaria nella sua struttura, nei suoi meccanismi e nei suoi equilibri dinamici e che l’uomo ha assunto, via via nel tempo, un ruolo sempre più determinante quale parte integrante del sistema. Le tematiche inerenti all’ambiente che più direttamente interessano l’uomo devono avere come obiettivi gli strumenti per giungere ad una approfondita analisi dei fenomeni e la conoscenza delle potenzialità naturali, ciò per consentire una corretta pianificazione e gestione antropi¬ ca del territorio. Gli elementi di giudizio necessari a sviluppare un approccio culturale corretto delle problematiche ambientali derivano dalla conoscenza dei Vallano A . 143 fenomeni naturali. Questa, partendo dall'analisi delle componenti dell'am¬ biente, può consentire di giungere alla ricostruzione dei modelli morfoe- volutivi e quindi alle previsioni sia sull’innesco di nuovi fenomeni alterati¬ vi che sulle reazioni agli stessi. Ciò equivale a prevedere e quindi program¬ mare un'adeguata prevenzione ai conseguenti rischi per la vita e per le opere dell’uomo. Le potenzialità naturali sono rappresentate oltre che dalle risorse rinnovabili e non,, quali le acque superficiali e sotterranee, l'aria, i combustibili fossili, i materiali naturali da costruzione, le fonti energeti¬ che, le materie prime, anche dal paesaggio, dal suolo, dalla fauna, dalla flora, dai parchi naturali, dalle risorse alimentari e dalle caratteristiche fisiche dell'ambiente che in diversi casi hanno costituito presupposti indispensabili per lo sviluppo economico e sociale di alcune regioni (Vallario, 1991). L'assetto e l’uso razionale del territorio dovrebbero scaturire dalle conoscenze acquisite, dalle caratteristiche fisiche dell’ambiente e dalla entità e tipologia delle potenzialità naturali disponibili in un certo ambito geografico. Nell'approccio all’ambiente fisico sembra opportuno ricordare che la configurazione geomorfologica attuale di un certo sito rappresenta solo un istante a scala umana della sequenza che ha dato luogo nei tempi geologici al susseguirsi di processi e forme che hanno determinato l'evoluzione a cui è stata sottoposta quella porzione di superficie terrestre sotto l'azione degli agenti esogeni (acque meteoriche, escursioni termiche, ecc ) ed endogeni (eventi tettonici, fenomeni vulcanici, sismi, ecc.). Aspetti questi che hanno costituito, da circa quaranta anni, argomenti di studio da parte di molti ricercatori italiani quali, ad esempio: Gisotti e Bruschi, 1990; Guida et al., 1974, 1979, 1981; Ippolito, 1954, 1962, 1967, 1972a, 1972b, 1973; Vallario, 1973, 1992b, 1993a, 1993b). Il rischio geologico L'uomo vive ed opera, prevalentemente, in corrispondenza della porzione più superficiale della crosta terrestre; proprio in quella parte in cui avvengono, o si avvertono, gli effetti dei fenomeni di modellamento, di trasformazione, di alterazione, di adattamento o di reazione connessi, in modo diretto o indiretto, agli agenti della dinamica endogena ed esogena, (Bolt et al, 1975, Vallario, 1992b). L'insieme dei fenomeni geologici e dei loro effetti sulla superficie terrestre rappresenta quella che possiamo definire la pericolosità geologi¬ ca, la porzione della superficie terrestre dove vivono ed operano comunità antropiche rappresenta, invece, la potenziale vulnerabilità’ antropica di un dato territorio ai fenomeni geologici. Il rischio geologico è la combinazione della pericolosità geologica e della potenziale vulnerabilità antropica di un territorio, espresso in termini di rapporto tra i prevedibili eventi di pericolosità geologica, la loro intensità e frequenza e le relative interferenze con le attività antropiche. Tra gli eventi di pericolosità geologica devono rientrare sia i fenomeni naturali che quelli indotti dalle attività antropiche quali, ad esempio, i fenomeni vulcanici, i terremoti, le alluvioni, i maremoti, le erosioni intense della superficie terrestre, le frane, la subsidenza, il bradisismo, gli effetti delle grandi infrastrutture antropiche sull'evoluzione della superfi¬ cie terrestre, l'inquinamento, ecc. La potenziale vulnerabilità antropica può comprendere l’intensità e il tipo di urbanizzazione, l’uso del territorio, l’industrializzazione, la presen¬ za di infrastrutture viarie, le opere di captazione di importanti sorgenti, opere di adduzione da liquidi o gas, opere di scarico di liquami, la presenza di bacini artificiali, ecc. Da ciò deriva, quindi, che il rischio geologico è strettamente connesso alle attività antropiche e che può essere ridotto agendo non tanto sui fenomeni geologici, peraltro difficilmente controllabili, ma soprattutto, mediante un uso oculato e razionale del territorio da parte dell’uomo (Vallano 1991). Aspetto particolarmente significativo per la definizione del rischio geologico in un determinato sito è la individuazione di tutti gli elementi geologici e delle cause che, di volta in volta, si combinano negativamente concorrendo a turbare gli equilibri, tali conoscenze dovranno applicarsi ai fattori di potenziale vulnerabilità per definire le strategie di intervento che costituiranno oggetto ed obiettivo della programmazione, della pianifica¬ zione e della gestione del territorio, per ben rispondere alle esigenze umane (Benedini e Gisotti, 1985; Greco, 1992; Vallario, 1992b). Boll. Soc. Natur. Napoli - Voi. 101 (1992-1993) Il dissesto idrogeologico Tra i processi evolutivi di maggiore incisività e velocità che sconvolgo¬ no l’ambiente fisico sono da includere le frane, i fenomeni di intensa erosione e le alluvioni; fenomeni questi di primaria importanza per gli effetti catastrofici che possono produrre sulle opere, sulle attività e sulla Vallano A. 145 vita stessa degli uomini. Questi fenomeni che sconvolgono tanto profonda¬ mente l'ambiente fìsico ed il territorio rientrano nelle fenomenologie del così detto dissesto idrogeologico, definizione questa che tende a mettere in rilievo che tali fenomeni alterativi hanno cause determinanti nelle acque superficiali e sotterranee. Le catastrofi naturali che più frequentemente incombono sul territo¬ rio del nostro paese, sono da collegare a fenomeni di intensa erosione, a movimenti in massa o frane o ad alcuni particolari aspetti della dinamica fluviale (alluvioni) in quanto costituenti gli elementi più diffusi e maggior¬ mente incisivi nell’evoluzione rapida dell'ambiente fìsico, eventi questi che vengono comunemente indicati con la denominazione di rischio idrogeologico e comprendono quelle catastrofi, che derivano dalla fre¬ quente combinazione di irrazionali utilizzazioni antropiche dell'ambiente fìsico e da, più o meno sfavorevoli, condizioni geologiche e morfologiche. Sembra opportuno ribadire che l’evoluzione dell'ambiente fìsico è condizionata prevalentemente dai processi di rapido modellamento conse¬ guenti alla dinamica fluviale (alluvioni), alla dinamica dei versanti (feno¬ meni erosionali e frane) e alla dinamica dei litorali (variazioni delle linee di costa e dei fondali). Si definisce alluvione l’insieme dei fenomeni legati all'alta velocità dell’acqua nella rete drenante superficiale, all’erosione, al trasporto e successivo deposito di ingenti quantità di materiale solido fine e grossola¬ no, sia nell’alveo che nelle aree limitrofe, in occasione dello straripamento dei corsi d'acqua. Gli effetti di tali fenomeni sono disastrosi in quanto 1' onda di piena che si propaga da monte a valle aumenta in portata e velocità. Con l’aumento della portata l’acqua, non più contenuta nell’al¬ veo, può invadere le aree circostanti, con l’aumento della velocità essa si carica di detriti e incrementa ancor più la propria energia, provocando intensi fenomeni erosionali con successivo deposito del materiale eroso e trasportato. Tali fenomeni stravolgono interi bacini imbriferi modificando sia le condizioni morfologiche precedenti e sia i modelli morfoevolutivi; i loro effetti disastrosi sono da collegare, oltre che a condizioni Onaturali, anche all’utilizzazione insensata dell'uomo di quelle parti pianeggianti, prospi¬ cienti gli alvei, che vengono invase dalle acque solo nei periodi di eccezionale piovosità. Adeguate opere idrauliche di regimazione delle piene e di regolazione delle sponde e dei profili di fondo, la sistemazione dei bacini montani e il mantenimento di aree di rispetto per lo smaltimento delle piene sono gli 146! Boll Soc. Natur. Napoli - Voi mi (1992-1993) interventi che hanno consentito di ridurre notevolmente gli effetti distrut¬ tivi delle alluvioni. Tra gli interventi antropici particolarmente incidenti sulla dinamica fluviale è indispensabile ricordare l'asportazione di ingenti quantità di materiali alluvionali dagli alvei dei corsi d'acqua; queste azioni hanno avuto incidenza via via crescente a partire dagli anni cinquanta, quando sotto la spinta del forte sviluppo economico sono iniziate le massicce realizzazioni di infrastrutture viarie e di innumerevoli costruzioni di ogni genere e tipo che hanno utilizzato i depositi alluvionali per realizzare rilevati stradali e per la confezione del calcestruzzo. Questo fenomeno, nel complesso rapporto tra i diversi elementi dell'ambiente fisico, ha provoca¬ to anche sostanziali modificazioni, a volte irreversibili, al regime dei litorali; inoltre, le asportazioni hanno generato l'abbassamento progressi¬ vo degli alvei con conseguenti crolli di manufatti, franamento delle difese di sponda e modificazioni, anche profonde, degli articolati sistemi erosio¬ ne-trasporto-sedimentazione. La dinamica dei versanti comprende i fenomeni di intensa erosione ed i movimenti in massa. I fenomeni erosionali sono legati quasi esclusiva- mente all'azione meccanica delle acque dilavanti che, per la forza di gravità, si spostano da monte a valle. I movimenti in massa o frane sono costituiti dalla caduta più o meno rapida o, comunque, dallo spostamento lento e differenziale di masse rocciose o di materiali sciolti, per gravità. In tal senso appare evidente che i fenomeni erosionali e le frane sono agenti modellatori particolarmente incisivi che non solo modificano localmente, ma intervengono in maniera determinante nell'evoluzione dell'ambiente fisico in quanto, alterando la geometria dei versanti e le forme del suolo, agevolano l'insorgere di nuovi meccanismi di trasformazione che si so¬ vrappongono ai precedenti e, in alcuni casi, si sostituiscono ad essi. In molte zone del territorio nazionale le caratteristiche geologiche inducono all' instaurarsi diffuso di fenomeni di tal tipo che spesso raggiungono l'entità di vere e proprie catastrofi naturali (Vallano, 1992a e b). L'agente principale della dinamica dei litorali è costituito dall'azione del moto ondoso; un ruolo del tutto subordinato spetta alle correnti marine. La tendenza evolutiva delle coste basse, quelle di maggiore interesse per le attività antropiche, oltre che dagli elementi naturali, dipende dagli interventi dell'uomo. I tratti di costa che si possono ritenere naturalmente stabili sono estremamente limitati in quanto è difficile che si possa realizzare l'equilibrio duraturo tra apporti, erosioni e trasporto dei materiali. Vallano A. 147 1 Altra azione antropica di alterazione degli equilibri è la costruzione di opere costiere quali moli, pennelli e scogliere che, modificando le modali¬ tà di trasporto del materiale lungo costa, provocano erosioni ed accumuli anomali rispetto agli schemi naturali. Val la pena di ricordare che se non si ripristinano le condizioni naturali alterate dall’escavazione dei materiali dagli alvei dei corsi d'acqua, non si potranno raggiungere condizioni di equilibrio naturali. Unica alternativa concreta a tali situazioni è il ripascimento artificiale delle coste basse. Il ripetersi di eventi catastrofici di ampiezza ed entità sempre maggio¬ re e, in particolare, l'alluvione dell'Arno del 1966 suggerirono l'istituzione di una Commissione Interministeriale per avviare finalmente una ricogni¬ zione conoscitiva sul territorio nazionale. Gli studi ed i rilevamenti effettuati misero in evidenza nel 1970 che era necessaria una spesa di 8.932 miliardi, da investire in un trentennio, per la difesa del suolo e la sistemazione idraulica sul territorio nazionale. In particolare erano previsti 5.300 miliardi per la difesa idraulica del suolo, 429 miliardi per la sistemazione di frane e la prevenzione di valanghe, 2.370 miliardi per la sistemazione idraulicoagraria e per il potenziamento silvo-pastorale e 824 miliardi per la difesa dei litorali (Commissione Interministeriale, 1970). Da allora molto è cambiato soprattutto nello sfruttamento del territo¬ rio e delle risorse naturali, con conseguenze sempre più catastrofiche ed irreversibili. In ciò l'uomo ha avuto un ruolo prioritario e determinante ed è da ritenere l'agente modellatore che ha avuto le maggiori responsabilità rispetto agli eventi naturali. BIBLIOGRAFIA Benedini, M. & G. Gisotti, 1985. Il dissesto idrogeologico. Cause, effetti e interventi a difesa del suolo. La Nuova Italia Scientifica, Roma. Bolt, B.A., W.L. Horn, G.A. Macdonald & R.F. Scott, 1975. Geologie al Hazards, Springer-Verlag, Berlin. Catenacci, V., 1992. Il dissesto idrogeologico e geoambientale in Italia dal dopo¬ guerra al 1990. Serv. Geol. Naz., Mem. Descr. della Carta Geologica d’Italia, voi 47, Istituto Poligrafico e Zecca dello Stato, Roma. Commissione Interministeriale per lo studio della sistemazione idraulica e della difesa del suolo, 1970. Atti della Commissione, Camera dei Deputati, Roma. Di Donna, V. & A. Vallario, 1992. Ambiente fisico, territorio e realtà socio-econo¬ mica. Geologia Tecnica & Ambientale , Ordine Nazionale dei Geologi, n. 2, Roma. 148 Boll Soc . Natur. Napoli - Voi 101 (1992-1993) Di Donna, V. & A. Vallario, 1993a. Uomo e ambiente fìsico. Istituto di Ricerca e di Didattica Ambientale Napoli (IREDA), Bollettino delle Attività, voi 2. Di Donna, V. & A. Vallario, 1993b. Ambiente: risorse e rischi. Liguori Editore. Dorst, J., 1988. Prima che la natura muoia. Verso una riconciliazione dell’uomo e della natura. Muzzio Editore, Padova. Gisotti, G. & S. Bruschi, 1990. Valutare V ambiente. La Nuova Italia Scientifica, Roma. Greco, N., 1992. La pianificazione dell’ambiente fisico in Italia. Quaderni Ambien¬ te, Edistudio, Roma. Guida, D., M. Guida, G. Iaccarino, G. Metcalf, A. Vallario, V. Vecchio & G. Zicari, 1979. Il bacino del Mingardo (Cilento): evoluzione geomorfologica, fenomeni franosi e rischio a franare, Geol. Appi, e Idrogeologia, voi. 14, p. II, Bari. Guida, D. , M. Guida, D. Luise, G. Salzano, A. Vallario, 1981. Geologia e franosità del Fiume Lambro (Cilento). Geologica Romana, voi. 20, Roma. Guida, M., G. Iaccarino, A. 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