Hymenoptera: Braconidae)

MORPHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM OF PHANEROTOMA (PHANEROTOMA) OCULARIS KOHL (HYMENOPTERA: BRACONIDAE)

J. MORENO & R. JIMENEZ Departamento de Biologia Animal (Entomologia), Facultad de Ciencias Biol6gicas, Universitat de Valencia, Dr. Moliner 50, 46100 Burjassot - Valencia, Spain

Received IO Mars 1992; accepted 7 September 1992

REFERENCEMORENO(J,): & JIMENEZ(R.), 1993.- Morphology of the female reproductive system of Phanerotoma (Phanerotoma) ocularis Kohl (Hymenoptera: Braconidae). Research and Reviews in Parasitology, 53 (1-2): 53-56. ABSTRAcr:The female internal reproductive system of the hymenopteran species Phanerotoma (Phanerotoma) ocularis Kohl consists of 2 pairs of elongated ovarioles. The distal portion of the ovariole or germanium is enlarged and contains a number of reproductive cells. The medial portion contains the ovocytes and «nurse» cells. The proximal portion of the egg tube is enlarged and contains 20-30 mature ova. The calyx is also enlarged and filled with a fluid. The venom apparatus is Type II modified; the reservoir is located at the distal end of the only gland filament. KEy WORDS:Hymenoptera, Braconidae, Phanerotoma (Phanerotoma) ocularis, female reproductive system, ovarioles, calyx, reproductive cells, nurse cells, venom apparatus.

INTRODUCTION as a first step to determine the source of the observed effects on the host- In this paper we examine the mor- Phanerotoma (Phanerotoma) ocularis Kohl phology of the internal reproductive system of the female (Hymenoptera, Braconidae) is an important egg-larval of P. (P.) ocularis. 'parasitoid of lepidopterous pests. It has been reported as a parasitoid of Ectomyelois ceratoniae Zeller, Paramyelois transitella (Walker), Ephestia kuehniella Zeller (Lep., MATERIAL ANDMETHODS Pyralidae) and Prays citri Milliere (Lep., Yponomeutidae) (BILlOTTI & DAUMAL, 1969; CALTAGIRONE, SHEA & FIN- The parasites used in this study were reared from Ephestia NEY, 1964; HAWLlTZKY, 1978; MORENO, 1991; MORENO, kuehniella (Lepidoptera, Pyralidae) maintained according to the HAWLlTZKY & JIMENEZ, 1991). methodology described by BILIOTTI& OAUMAL(1969). Female The first and second larval instar of P. (P.) ocularis are parasites were dissected, just after emergence, in Ringer's saline and endoparasitic and feed exclusively on haemolymph; the examined microscopically. The reproductive system was isolated on a slide, washed with Ringer's saline and manipulated into the cor- third larval instar emerges from the host larva and com- rect position for observation. The isolate system was fixed for 10 pletes development as an ectoparasitoid feeding as a sar- minutes in Oubosq Brasil fixer (alcohol 800/0: formol 40%: picrid eo phage (BILlOTTI & DAUMAL, 1969; HAWLlTZKY, 1978; acid; 150 cm:': 60 cm:': I g) to which 10% of acethic acid was add- MORENO, 1991). ed. The fixer was removed and the tissues were dehydrated with a After studies developed in this laboratory to determine decreasing alcohol series. The fixed reproductive system was fixed the effects of parasitization by this parasitoid on the mor- to the slide with collodion (2 minutes). The reproductive system was rehydrated and stained with Feulgen's (Schiff-Light Green) method. phology and physiology of its host, it was not found that Photographs were taken by a Leitz Orthoplan microscope fitted with the number of larval instars or time of development was a Leitz Orthoplan Automat camera. different from controls. However, a reduction of cephalic capsule and in the size and weight of the parasitized larvae did occur. In addition, a compression and displace- ment of the internal organs of the parasitized larvae were RESULlS AND DISCUSSION observed (HAWLlTZKY, 1978; MORENO, 1991; MORENO, HAWLlTZKY & JIMENEZ, 1991). The ovary is of a typical polytrophic meroistic type con- The action of parasitoids on their host larva has been sisting of two pairs of long slender ovarioles (Fig. lA). reported to induce a modification in the immune response The two ovarioles are similar but separate, being joined and, consequently, in the inhibition of the encapsulation at the proximal region, as reported for Cardiochiles process. Several authors have reported the importance of nigriceps Viereck (Hymenoptera: Braconidae) by VINSON the female calyx and venom fluids in the modification (1969) (Fig. lB). Each ovariole becomes progressively of the immune response observed in hosts (Eo SON et al., slender toward the distal portion. The distal portion of 1981; FERRAN & DAUMAL, 1973; FERRAN & LAFORGE, the ovariole is swollen and forms an elliptical germanium, 1974; LE MASURIER, 1987; NORTON, VINSON & STOLTZ, where preovocytes occur (Fig. lB). 1975; VINSON, 1972, 1974, 1977 a, b; SALT, 1973;SHAW, The egg tube is polytrophic and shows a high number 1981; STOLTZ & VINSON, 1977, 1979; STOLTZ, VINSON & of reproductive cells that can be observed with microscope MACKINNON, 1976). Laboratory studies were conducted after Feulgen's staining. These cells are the ovaric VI Fig. 1.- Female reproductive system of P. (P.) ocularis: A) general aspect: ovariole (01), uteruslike chamber (Utc), calyx (Cx), lateral oviduct (Odl), common oviduct (Ode), sperrnatheca .j::o (Spt), venom apparatus (Va), reservoir (R), gland filament (Glf), ovipositor (Ov); B) detail of the ovariole distal portion and germanium (Gr); C). detail of the mature ovocytes (Ovyrn) and the trophic cells (Tcd) in' degeneration process; D) calyx (Cx) and lateral oviduct (Odl); one mature ovum can be observed embedded in the calyx fluid. Scale bars: A, B: 400 !Am; C: 140 !Am; D: 80 !Am.

..,--- Va i

-01

A II B

" fJ__ iT ~ ,_ .....•,.."•.r1:.- Ih.. 'i 1. VOI- , 0 • ;;0 m Z 0 Ro ?' •..... 3: m· ~,','1)~ -,,1 ',-~J.~ji...,-,_. #,' :- z ~~?'C~"'--;J . - r -4.• m 'L.D ~:~ .•/ 4 '\o.._~-:t,,:'" ~ ,"":'\:~.jl:~ e •••••••• • N Female reproductive system of Phanerotoma (Ph.) ocu/aris 55

follicules, ovocytes and «nurse» cells, more or less evolv- found in other genera of Cheloninae (EDSON& VINSON, ed in function of their location along the ovariole (Fig. 1979), which are egg-larval parasitoids. lC). Each ovocyte is found associated with a group of The function of the venom apparatus secretion in small cells, the trophic or «nurse» cells all along the length females which do not paralyze their host is unknown. Pro- of the ovariole (Fig. lC). The nurse cells degenerate as bably this function, like the calyx fluid, is related to the the ovocytes mature. The trophic cells are not associated observed effects on the morphology and physiology of with the ovocytes in the uteruslike chamber. VINSON the host and, particularly, to the reduction of the host (1969) suggests that the nurse cells could be secreted inmune response after parasitization. At present we are together with the calyx fluid. This author reports that the studying the ultrastructural morphology of the internal number of these small calyx secretory cells increases with reproductive system in P. (P.) ocularis in order to obtain the parasitoid female age. further information with regard to the function of the The proximal portion of each ovariole or uteruslike venom apparatus in those female which do not paralyze chamber (VINSON, 1969) is swollen, and is billed with their hosts. mature ovocytes, even in newly emerged females (Fig. lA). BILlOTTI& DAUMAL(1969) indicate that the eggs remain in non-degenerating females. Moreover about 20 to 30 ACKNOWLEDGEMENlS mature ovocytes can be observed in the basal portion of each ovariole of the just emerged females. This work was supported by financial assistance provided by the c.1.c.Y.T. Department of Spanish Government (Ministry of Educa- The uteruslike chambers of the paired ovary open into tion and Science) (Project PB90-0419). We are grateful to Or. C. an enlarged calyx, forming a cup-shaped structure, where Boulay (INRA-CRA Versailles, France) for her aid in the histological generally only one mature egg can be observed embedd- aspects, to Dr. N. Hawlitzky (lNRA-CRA Versailles, France) for her ed in a Schiff's stained fluid (Fig. lD). This fluid seems critical comments, and to Or. C. Frenoy and Ch. Brachet (lNRA- very similar to the fluid suggested by SALT(1973), VIN- CRA Versailles, France) for their technical support and help in the SON(1969, 1972 b) or SHAW(1981) as responsible for the laboratory. protection of the parasite egg against the host's cellular defense. REFEJU: CES Each calyx of P. (P.) ocularis gives rise to a short lateral oviduct that is fused with the other to form a short, broad, BILIOTTI(E.) & DAUMAL (J.), 1969.- Biologie de Phanerotoma common oviduct that opens into the ovipositor (Fig. lA). flavitestacea Fischer (Hym., Braconidae). Mise au point d'un The spermatheca (Fig. lA) is very small and is situated elevage permanent en vue de la lutte biologique contre Ectomyelois ceratoniae Zell. Annales de Zoologie et Ecologie Animale, 1: near the joining of the lateral oviducts to form the com- 379-394. mon oviduct. CALTAGIRONE (L.E.), SHEA (K.P.) & FINNEY (G.L.), 1964.- The ovary of P. (P.) ocularis is similar to the ovary of Parasitoids to aid control of navel orangeworm. California Proterops Wesmael (Hymenoptera, Braconidae) described Agricultural, 18: 10-12. by IWATA(1959) but differs from the ovary decribed by Ensor- (K.M.) & VINSON(S.B.), 1979.- A comparative morphology IWATA(1959) in that it has an enlarged calyx. Similar to of the venom apparatus of female braconids (Hymenoptera: Braconidae). Canadian Entomologists, Ill: 1Ol3- 1024. the reproductive system of Cardiochiles nigriceps Viereck EOSON (K.M.), VINSON (S.B.), STOLTZ(D. B.) & SUMMERS(M.D.), (Hymenoptera, Braconidae) (VINSON, 1969), it has a 1981.- Virus and parasitoid wasp: supression of the cellular im- clearly differentiated well developed germanium and each mune response in the parasitoid's host. Science, 211: 528-583. ovariole has no contact with the other one of the pair all FERRAN (A.) & DAUMAL (1.), 1973.- Consequences sur les larves along its length. d'Anagasta kuehniella Zeller (Lep., Phycitidae) de I'elirnination There is one accesory structure associated with the precoce par le froid de son endoparasite Phanerotomaflavitestacea Fischer (Hyrn., Braconidae). Comptes Rendus de l'Academie de ovary; this structure is partially responsible for the Sciences, Paris, ser, 0, 277: 869-872. changes observed in the parasitized host (SHAW, 1981; FERRAN(A.) & LAFORGE(J.P.), 1974.- Mise en evidence d'une ac- VINSON,1977 b). The venom apparatus of P. (P.) ocularis tion toxique des secretions ovariennes des femelles d'un insecte opens into the common oviduct (Fig. lA). The venom ap- entomophage [Phanerotoma flavitestacea Fischer (Hyrn., paratus of this species is Type 11(EDSON& VINSON,1979) Braconidae)] sur les larves de son hote, Anagasta kuehniella Zeller consisting of a thin-walled reservoir surrounded by (Lep., Phycitidae). Comptes Rendus de l'Academie de Sciences, relatively few muscles. The Type 11venom apparatus also Paris, ser. 0, 279: 1341-1344. HAWLITZKY (N.), 1978.- Biologie, regimes alimentaires et typically presents two gland filaments of one cell in caracterisation des reserves chez un insecte entomophage, thickness surrounding a central lumen (EDSON& VINSON, Phanerotoma flavitestacea Fisch. (Hymenoptere, Braconidae). 1979) and the venom duct extends from the base of the Action de la larve parasite sur son hote de laboratoire, Anagasta reservoir to the ovipositor. kuehniella Zell.(Lepidoptere, Pyralidae). These, Doctorat es- In P. (P.) ocularis a variation of the Type 11gland oc- Sciences, Universite de Paris-Sud, Paris, 225 pp. curs: the reservoir is placed at the distal extreme of the I\VATA(K.), 1959.- The comparative anatomy of the ovary in Hymenoptera. Part Ill. Braconidae (including Aphidiidae) with only gland filament. Moreover the venom gland duct ex- descriptions of ovarian eggs. Kontyu, 27: 231-240. tends from the base of the gland filament to the common LE MASURIER (A.D.), 1987.- A comparative study of the relation- oviduct. This modified Type 11venom apparatus has been ship between host size and brood size in Apanteles spp. 56 J. MORENO & R. JIMENEZ

(Hymenoptera: Braconidae). Ecological Entomology, 12: 383-393. in insects. Advances in Virus Research, 24: 125-171. MORENO(L), 1991.- Phanerotomini: biologia y efectos del fenoxy- STOLTZ (D.B.), VINSON (S.B.) & MACKINNON (E.A.), 1976.- carb sobre el complejo Ephestia kuehniella Zeller (Lepidoptera, Baculovirus-like particles in the reproductive tracts of female Pyralidae) - Phanerotoma (Phanerotomaj ocularis Kohl parasitoid wasps. Canadian Journal of Microbiology, 22: (Hymenoptera, Braconidae). Tesis Doctoral, Facultad de Cien- 1013-1023. cias Biologicas, Universitat de Valencia,Valencia, 574 pp. VI so (S.B.), 1969.- General morphology of the digestive and in- MORE 0 (1.), HAWLlTZKY(N.) & JIMENEZ (R.), 1992.- Relaciones ternal reproductive systems of adult Cardiochiles nigriceps hospedador-parasitoide: el complejo Phanerotoma (Phanerotoma) (Hymenoptera: Braconidae). Annals of the Entomological Society ocularis Kohl (Hyrn., Braconidae) - Ephestia kuehniella Zeller of America, 62: 1414-1419. (Lep., Pyralidae). Boletin de la Asociaci6n Espaiiola de En- VINSON (S.B.), 1970.-Development and possible functions of tomologia, 15: 325-332. teratocytes in the host-parasite association. Journal of Invertebrate NORTON(W.N.), VINSON (S.B.) & STOLTZ (D.B.), 1975.- Nuclear Pathology, 16: 93-101. secretory particles associated with the calyx cells of the icneumonid VINSON (S.B.), 1972.- Factors involved in successful attack on parasitoid Campoletis sonorensis (Cameron). Cellular Tissues Heliothis virescens by the parasitoid Campoletis sonorensis. Jour- Research, 169: 195-208. nal of Invertebrate Pathology, 20: 118-123. SALT(G.), 1973.- Experimental studies in insect parasitism. XVI. VINSON (S.B.), 1974.- The role of the foreing surface and female The mechanism of the resistance of Nemeritis to defence reac- parasitoid reactions on the inmune response of an insect. tions. Proceedings of the Royal Society of London, 183: 337-350. Parasitology, 68: 27-33. SHAW (M.R.), 1981.- Delayed inhibition of host development by VINSON (S.B.), 1977 a.- Microplitis croceipes inhibitions of the the non paralyzing venoms of parasitic wasps. Journal of In- Heliothis zea defense reaction to Cardiochiles nigriceps. Ex- vertebrate Pathology, 37: 215-221. perimental Parasitology, 41: 112-117. STOLTZ(D. B.) & VINSON(S.B.), 1977.- Baculovirus-Iike particles in VINSON (S.B.), 1977 b.- Insect host responses against parasitoids the reproductive tracts of female parasitoid wasps. H. The genus and the parasitoid's resistance: with emphasis on the Lepidoptera- Apanteles. Canadian Journal of Microbiology, 23: 28-37. Hymenoptera association. In: Comparative Pathobiology (L.A.Jr. STOLTZ (D.B.) & VINSON (S.B.), 1979.- Viruses and parasitism Bulla & T'C, Chen edit.), Plenum Press, New York, 3: 103-125.