Structure of the ovipositor, associated sensilla and spermathecal system of entomoparasitic pipunculid flies (Diptera Pipunculidae)

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Structure of the ovipositor, associated sensilla and spermathecal system of entomoparasitic pipunculid flies (Diptera Pipunculidae) Article in Journal of Natural History · July 1273 DOI: 10.1080/00222939700770701

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JOURNALOF NATURALHISTORY,1997,31, 1273-1288

Structure of the ovipositor, associated sensilla and spermathecal system of entomoparasitic pipunculid flies (Diptera Pipunculidae) M. KOZÀNEKt

and A. BELCARlt

t

!nstitute o/ Zoology, Slovak Academy o/ Sciences, Dubravska cesta 9, 84206 Bratislava, Slovak. Republic tIstituto di Patologia e Zoologia Forestale e Agraria, Università degli Studi, Piazzale delle Cascine, 28, 50144 Firenze, !taly (Accepted

1 February

1997)

The extemai parts of the female reproductive system in entomoparasitic tlies of the famiIy PipuncuIidae form a compact, heaviIy sclerotized ovipositor for inserting the egg into the host. Two types of pipunculid ovipositor have been recognized: The Nephrocerus type is bilaterally asymmetricai and consists of modified abdominai segments 8 and 9. There is no spiracle. The genitai opening is situate ventrally, approximately in the middie ofthe ovipositor. The Pipunculus type ovipositor Ìs biIateralIy symmetricai and originates from abdominai segments 7, 8 and 9. A pair of spiracles are Iocated venterolaterally near the basaI margin; the genital opening is situated close to the apex. Two types of campaniform and two types of haired sensilla have been found (tergum 9 only). Campaniform sensilla have been observed in apical third to apical half of the ovipositor of all studied species. Ten long haired sensilla arranged in two lateral rows on apex have been recognized only in species of Nephrocerus. Short haired sensilla have been found in all Chalarinae and Pipunculinae, situated in basaI half of the ovipositor piercer. The spermathecal system consists of three spermathecae; w~ll sclerotized (Nephrocerinae, Chaiarinae) or soft and membraneous (Pipunculinae); and three spermiducts which can be undifferentiated and tubular (Nephrocerus) or differentiated to muscular, glandular and ductal parts (Chaiarinae, PipuncuIinae). In the Nephrocerus type ovipositor, spermathecae are situated in the distal half of abdominal segment 6; all other Pipunculidae have spermathecae located in the basaI part of the ovipositor. Ovipositor, campaniform sensilla, haired sensilla, spermathecae, spermiducts, Pipunculidae, Diptera.

KEYWORDs:

Introduction The farni1y Pipuncu1idae are situated in the Cyclorrhapha, Atriata, infrapha1anx Syrphidea (Griffiths, 1972). Representatives of this group are usuallysmall, dark, inconspicuous flies which can be easi1y recognized by the 1arge compound eyes occupying most of the subhemispherica1 or hemispherica1 head, and by the wing venation (De Meyer, 1989a). Pipuncu1idae are parasitoids of Auchenorrhyncha (Homoptera) during their 1arva1 stage. Rearing experiments have shown that many species have a distinct host specificity (Sander, 1985; Wa10ff & Jervis, 1987). The pipuncu1id fema1e has a piercing, heavi1y sclerotized ovipositor enab1ing egg injection 0022-2933/97 $12·00 © 1997 Taylor & Francis Ltd.

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M. Kozanek and A. Belcari

into the host body. The shape and structure of ovipositor provide some species specific characters for determination (Hardy, 1987). The functional significance of interspecific differences in ovipositor architecture is unclear, and there is no obvious relationship between the structure of ovipositor and the kind of host attacked. On the contrary, there is very IittIe intraspecific variabiIity in ovipositor structure (Jervis, 1992). Despite the taxonomic, physioIogicaI and evolutionary importance of the pipunculid ovipositor, we have still poor knowIedge about its fine structure. The pipunculid ovipositor consists of modified and fused abdominal segments 7, 8 and 9 (Hardy, 1987) but the homoIogy of components still requires clarification. According to Jervis (1992), the piercer of Chalarus ovipositor bears numerous sensilla, including campaniform and two other types of sensilla. Nothing is known about the morphoIogy and distribution of ovipositor sensilla in other pipunculid genera. The internaI space of the ovipositor base contains a spermathecal system consisting of three spermathecae connected with three tubuIous spermiducts (Harris, 1966). The aim of this work was to study the ovipositor structure of representatives of various pipunculid genera, including shape and distribution of associated sensilla as well as construction of the spermathecal system. Material and methods

External and internaI structures of pipunculid ovipositors were studied using Iight and scanning electron microscopy (SEM). To identify tergal and sternal components of the ovipositor, cuticle was partially depigmented in KOH solution. Specimens stored in 70% ethanol were washed in distilled water and placed in 4% KOH for 24 to 48 h (depending on specimen size) and subsequent1y rewashed twice in water. Following this procedure, ovipositors were separated from the body in gIyceroI and photographed or drawn using WILD M5A and LEITZ DIALUX 20 microscopes with WILD MPS 45 photoequipment. The spermathecal system was dissected in gIyceroI from ovipositors of intact specimens stored in 70% ethanol. For SEM anaIysis, ovipositors of specimens stored in 70% ethanol were dissected, dehydrated in ascending ethanol concentrations, dried on criticaI point drying (CPD) equipment and goId sputtered. Specimens were examined with PhiIips 505 scanning eIectron microscope. The resu1ts of this work are based on investigation of following species: Verrallia aucta (Fallén), Jassidopahaga pillosa (Zetterstedt), J. se tosa (Verrall), J. villosa (vonRoser), Chalarus basalis (Loew), Ch. pughi Coe, Ch. spurius (Fallén), Chalarus sp., Nephrocerusjlavicornis Zetterstedt, N. scutellatus (Macquart), Pipunculus campestris Latreille, P. spinipes Meigen, P. -tenuirostris Kozanek, P. thomsoni Becker, Parapipunculus sp., Cephalops aeneus Fallén, C. semifumosus (Kowarz), C. vittipes (Zetterstedt), Beckerias pannonicus AczéI, Microcephalops vestitus (Becker), Cephalosphaera panamensis (Hardy), Eudorylas elephas (Becker), E. horridus (Becker), E. ruralis (Meigen), E. slovacus Kozanek, E. subterminalis Collin, E. zonellus Collin, Elmohardyia sp., Moriparia gigas Kertész, Dorylomorpha confusa (Verrall), D. xanthocera (Kowarz), Tomosvaryella geniculata (Meigen) and T. sylvatica (Meigen). The investigated specimens originated from the Malaise traps collections from numerous Iocalities of SIovakia (all Europen species), Costa Rica (Parapipunculus sp., Cephalosphaera panamensis, Elmohardyia sp.) and South Korea (Mori paria gigas). Single (non-European) to three (European) specimens of each species were used for SEM anaIysis. At Ieast, six representative of seIected species were used for the study of ovipositor internaI structures. The following keys were

Structure of entomoparasitic pipunculid flies used for species identification: Coe, 1966; De Meyer, 1989b; Albrecht, Jervis, 1992.

1275

1990;

Results

Gross structure of pipunculid ovipositor. The external parts of the female reproductive system in Pipunculidae form a compact heavily sc1erotized ovipositor with a cylindrical to semiglobular basaI part and thin tubular piercer gradually tapering to an apex. When not in use, the ovipositor is he1d under the abdomen. The proximal margin of ovipositor articulates to abdominal segment 6 (Chalarinae and Pipunculinae) or abdominal segment 7 (Nephrocerus); the character of articulation allows the female rather extensive vertical and less horizontal movement of ovipositor during the injection of the egg into the host. The dorsal surface of the basaI part is covered with more or less dense, short pilosity and few, sparce, long setae. No sensilla were found in the basaI part of the ovipositor. On the contrary, the thinner apical part-piercer is free of pilosity (at most with a few short hairs near the connection with the basaI part) and bears numerous campaniform and haired sensilla. Anal opening is situated dorsally, possessing paired cerci covered with numerous long setae. The genital opening is placed ventrally, usually c10se to the apex. The study of both external and internaI characters reveals that two types of ovipositor can be distinguished within the family Pipunculidae. Nephrocerus type ovipositor. External structure. The Nephrocerus type ovipositor is a robust, bilaterally asymmetrical, compact structure (Figs 1, 2, 5). It consists of fused and modified abdominal segments 8 and 9 articulating with segment 7, the last free abdominal segment. Tergum 8 forms a rather narrow ring extending from the proximal margin of ovipositor to the hind margin of anal opening and is covered with dense short hairs and sparse long setae. The intertergal space between terga 8 and 9 lacks pilosity. The border between fused terga 8 and 9 can be c1ear1yseen after partial depigmentation of the ovipositor cutic1e. Tergum 9 is large, its right side protrudes to apically pointed, triangular piercing lamella. The pilosity on tergum 9 is restricted to a narrow area near the connection with tergum 8 and few long setae dispersed in basaI half. Broad median longitudinal groove is situated ventrally. Sterna 8 and 9 are shortened and broad, weakly sc1erotized. The genital opening is located ventrally approximately in the middle of tergum 9. The anal opening is rather large, elipsoid, placed dorsally between terga 8 and 9. No spirac1es are present in Nephrocerus type ovipositor. This type of ovipositor occurs only in females of genus Nephrocerus. Sensilla. The apical half of tergum 9 bears two types, haired and campaniform sensi1la. In Nephrocerus jlavicornis and N. scutellatus two rows of five long haired sensilla are arranged laterally at the tip (Fig. Il). This type of long haired sensilla have been found only in the Nephrocerus type ovipositor. About 120 campaniform sensilla subtype A are dispersed irregular1y on the surface of apical third of the ovipositor. No short haired sensilla have been found in Nephrocerus type ovipositor. Spermathecal system. The spermathecal system in species of Nephrocerus consists of three well sc1erotized spherical spermathecae and tubular undifferentiated spermiducts (Figs 33, 35). Spermathecae are situated in abdominal segment 6 in its distaI half. Each is covered with a soft membraneous sheath. Spermiducts enter the oviduct in the cavity of segment 8 together with well deve10ped accessory glands. Pipunculus type ovipositor. External structure. This type of ovipositor is characteristic for species of subfamilies Chalarinae and Pipunculinae; it is bilaterally

1276

M. Kozanek and A. Belcari St9

St8

TeB Ce,An

StB Te8 FIGS

1-2. Schematic drawings of Nephrocerus type ovipositor: (1) lateral view; (2) ventral view. An = ana! opening, Ce = cerci, Go = genital opening, St = stemite, Te = tergite.

symmetrical compact structure (Figs 3, 4). It articulates with abdominal segment 6 and consists of modified and fused segments 7, 8 and 9. Segment 7 is cylindrical to semiglobular, rather robust and stout part of ovipositor. In most Chalarinae (Verrallia, Jassidophaga and many Chalarus sp.) the tergum 7 is separated from entirely sclerotized pleura by tergal suture (Figs 6, 7); in all species of Pipunculinae this suture is absent. Tergum 7 is usually covered with dense short pilosity in many species with admixture of long setae. The pilosity of tergum 7 in some species of genus Pipunculus is more or less restricted. Pleural and stemal parts of segment 7 are usually covered with short hairs, the density of these hairs varies. Long setae are usually absent in pleural part; in stemum 7 setae absent in all studied species. In Verrallio, Jassidophaga and many Chalarus sp. segment 7 is separated from remaining part of ovipositor with distinct transversal suture (Figs 6, 7). In Pipunculinae and few Chalarus sp. this suture is absent and the border between segments 7 and 8 forms more or less distinct impression always free of pilosity (Figs 8, 9, lO). Segment 8 is an entirely sclerotized, short ring forming the connection

Structure of entomoparasitic pipunculid flies St 7

1277 So

Stél

Te7

Go Te9

St9 TeB

Stél

Sì7

So Te7

FIGs~. Schematic drawings of Pipunculus type ovipositor: (3) lateral view; (4) ventral view. An = anal opening, Ce =cerci, Go = genital opening, So = spiracular opening, St = sternite, Te = tergite.

between segment 7 and piercing part of ovipositor, segment 9. It is usually pilosity free or at most covered with short sparse haÌrs in tergal parto Stemal part of segment 8 is swollen in some species (i.e. Pipunculus kozlovi, Eudorylas zonellus) and forms distinct protuberance. Segment 9 in all species forms pier-cing apparatus by which female introduces the egg into the host body. Segment 9 is awl-like, more or less continually tapering to apex, stright, or slightly to strongly curved. Tergum 9 forms the body of the piercer, ventrally overlapped for most of its length with narrow, long stemum 9. In some Dorylomorpha species tergum 9 is dorsoventrally flattened with distinct lateral flanges. Pleura are elastic, unsclerotized, allowing expansion of inner space during oviposition. Tergum 9 bears numerous sensilla in most of its length; no sensilla were found on stemum 9 of all studied species. SEM analyses revealed the occurrence of two types of ovipositor cuticle arrangement within the family: sparsely pitted (Nephrocerus jlavicornis, N. lapponicus, Chalarus sp., Pipunculus spinipes, Microcephalops vestitus, Tomosvaryella geniculata,

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M. Kozanek and A. Belcari

FrGs 5-10. Ovipositors of some Pipunculidae representatives: (5) Nephrocerus scutellatus; lateral view; (6) Verrallia aucta, lateral view; (7) Chalarus spurius, lateral view; (8) Microcephalops vestitus, lateral view; (9) Eudorylas horridus, ventral view; (lO) Dorylomorpha confusa, lateral view. Scale (5,7,8,9) 0·1 mm; in (6,10) 1·0 mm. T. sylvatica) (Figs 11, 13, 14, 16) and dense1y pitted (most species of Verrallia, Jassidophaga, Dorylomorpha, Cephalops, Cephalosphaera and Eudorylas) (Figs

12, 15, 17). In Pipunculus type ovipositor the ana1 opening is situated dorsally in the intersegmenta1 space between tergum 8 and 9. Two cerci covered with numerous long setae occupy its cavity. No sensi11a were found on cerci surface. Genita1 opening is arranged ventrally near the apex of segment 9 (Fig. 31). Segment 7 possesses a pair of spirac1es 10cated ventra1aterally in p1eura1 area near the basaI margin of ovipositor (Figs 27, 28). The spiracu1ar opening is simp1e, roundish or ovate usually with enhanced margin (Fig. 29). The spiracu1ar atrium contains trabecu1ar filtering apparatus (Fig. 30). Sensi11aof Pipunculus type ovipositor. Studies with SEM revea1ed that a1most the entire tergum 9 bears sensilla. Two basic types of sensi11a,campaniform and

Structure

FIGS

11-18.

of entomoparasitic

pipunculid

flies

1279

The arrangement of sensilla in apica1 part of ovipositor: (11) Nephrocerus scutel!atus, ventra1 view; (12) Verrallia aucta, 1atera1view; (13) Chalarus spurius, 1atera1 view; (14) Pipunculus spinipes, ventra1 view; (15) Cephalops semifumosus, ventra1 view; (16) Microcephalops vestitus, dorso1atera1 view; (17) Eudorylas horridus, ventro1atera1 view; (18) Dorylomorpha confusa, dorso1atera1 view. Scale lO J.lm; (11) 0·1 mm.

M. Kozanek and A. Belcari

1280

FIGS

19-26.

Types of pipuncu1id ovipositor sensilla. Campaniform sensilla: (19) Nephrocerus scutellatus; (20) Chalarus spurius; (21) Pipunculus spinipes; (22) Eudorylas horridus; (23) Microcephalops vestitus. Haired sensilla: (24) Microcephalops vestitus; (25) Cephalops semifumosus; (26) Tomosvaryella geniculata. Scale 1 jlm, (26) lO jlm.

Structure of entomoparasitic pipunculid flies

FIGS 27-30.

The ovipositor spiracle and its location: (27) Chalarus spurius; vestitus; (30) Eudorylas horridus. Scale lO 11m; (28) 0·1 mm.

1281

(28, 29)

Microcephalops

FIG.31.

The location of genital opening in apical part of Chalarus spurius ovipositor. Scale lO 11m.

FIG. 32. Small homopteran wax particles (brocosomes) present on Dorylomorpha ovipositor surface. Scale 111m.

confusa

short haired, have been recognized; both were found in all studied species with this type of ovipositor. Campaniform sensilla occurred in apicai third to apicai half of tergum 9; number of campaniform sensilla varied in studied species from 12 (Cephalops semifumosus) to 60 (Pipunculus spinipes) (Tabie. 1). We have observed two subtypes of campaniform sensilla. Subtype A, the body of sensillum is semiglobuIar to gIobuIar, emerged from the sensillum cavity (Figs 19, 20). It has been found in all species of subfamiIy Chaiarinae and two species of genus Cephalops. Subtype B, the body of sensillum is flattened, frequently with more or Iess deep scar in the

M. Kozanek and A. Belcari

1282

FIG. 33.

Schematic drawing of spermathecal systems in Nephrocerus type ovipositor. Ag= accessory gland; Ov = oviduct; Spd = spermiduct; Spt = spermatheca; Te = tergite.

Va

Ag Spdrn

Spdt Spdg

Spt

FIG. 34.

Schematic drawing of spermathecal system in Pipunculus type ovipositor. Ag= accessory gland; Ov = oviduct; Spdg = glandular part of spermiduct; Spdm = muscular part of spermiduct; Spdt = tubular part of spermiduct; Spt = spermatheca; Va = vagina.

middle, usually submerged (Figs 21, 22, 23). This subtype has been observed in most species of Pipunculinae. Short haired sensilla are situated in the basaI third to basaI half of tergum 9 with the exception of Microcephalops vestitus where short haired sensilla are also Iocated in the ovipositor apex (Figs 16, 24, 25, 26). The number of short haired

1283

Structure of entomoparasitic pipunculid flies

35

FIGS

35-37.

Types of pipunculid spermatheca: (35) Nephrocerus spurius; (37) Pipunculus thomsoni.

FIG.

38. The differentiated spermiduct of Pipunculus thomsoni. Spdg=glandular Spdm = muscular part, Spdt = tubular parto

FIGS

39-40.

scutellatus;

(36) Chalarus part,

The position of spermathecae. (39) Jassidophaga pillosa, (40) Chalarus spurius.

sensilla varied from lO (Cephalops semifumosus) to 100 (Pipunculus spinipes). No long haired sensilla were found in species possessing Pipunculus type ovipositor. Spermathecal system. In all Chalarinae species, there are three, well sclerotized bean-like spermathecae. They are situated in the basaI part of ovipositor (abdominal segment 7) although in some specimens of Verrallia aucta, Jassidophaga pillosa, J. se tosa and Chalarus basalis only two spermathecae descended into the cavity of the ovipositor base and a third spermatheca was located in abdominal segment 6 near its connection with ovipositor (Figs 39, 40). Spermiducts in Chalarinae are

1284 Table I.

M. Kozanek and A. Belcari The number of ovipositor sensilla counted in a single Pipunculidae species with SEM.

Species Chalarus spurius Veralia aucta Jassidophaga setos Nephrocerus scutellatus Parapipunculus sp. Pipunculus campestris Pipunculus spinipes Cephalosphaerapanamensis Cephalops semifumosus Cephalops vittipes Microcephalops vestitus Eudorylas horridus Eudorylas imperfectus Eudorylas ruralis Eudorylas slovacus Moriparia gigas Elmohardyia sp. Dorylomorpha confusa Dorylomorpha xanthocera

Campaniform sensilla 46 40 40 120 24 20 60 30 12 15 16 34 28 20 32 28 26

50 52

Haired sensilla 20 40 50 lO

54 70 100 48 lO

l3 50 12 56 46 40 44 56 50 70

rather short, differentiated to basaI muscular part (sometimes distinctly shortened), welI developed centraI glandular part and short tubular apical part entering the spermathecae. In alI studied Pipunculinae we have found three membraneous, unsclerotized irregularly globular spermathecae (Figs 34, 37). Spermiducts are welI differentiated to basaI muscular, centraI glandular and apical tubular parts (Fig. 38). Spermiducts and accessory glands enter the common oviduct in centraI part of ovipositor (segment 8). A small narrow sclerotized structure separating spermiducts and oviduct shortly before their fusion have been found in alI species with Pipunculus type ovipositor. This structure is situated in the internaI space close to the proximal margin of the anal opening. During the SEM investigations of pipunculid ovipositors we have occasionalIy found smalI, regularly shaped wax particles on the ovipositor surface (Fig. 32). According to Vidano and Arzone (1984) these particles, called brocosomes, frequently cover the body of CicadelIidae. The presence of these particles on the ovipositor surface indicates that the female has already been in touch with a leafhopper for oviposition. Discussion In higher Brachycera the segments beyond segment 5 and 6 form a more or less tapered tube with various modifications for egg laying. Species inserting the egg into plant or animaI tissue produce female terminalia which differentiate into a more or less sclerotized ovipositor, alIowing penetration into the host body. Also in Pipunculidae, the entomoparasitic kind of life in immature stages and the need to insert the egg into the host body resulted in extensive modification of terminaI abdominal segments and the evolution of a highly specialized compact piercing organ, the ovipositor. Comparing the pipunculid ovipositor with ovipositors of brachycerous flies laying eggs in plant hosts (i.e. Tephritidae) revealed some principal

Structure or entomoparasitic pipunculid flies

1285

differences. In Pipunculidae, the ovipositor is a compact structure and intersegmental connections are entirely sclerotized. This functional adaptation corresponds with specific ovipositing behaviour. No special organs (see poison gland in parasitic Hymenoptera) have been developed for immobilization of the host. Host protective and escape behaviour is eliminated by the velocity of the attacking female as well as rapidity of oviposition. The female penetrates with ovipositor through the intersegmental membrane and lays the egg in the abdominal cavity of the host (Sander, 1985; Waloff and Jervis, 1987; Morakote and Yano, 1988). The intersegmental membrane in the ovipositor of non-entomoparasitic brachycerous flies is unsclerotized and, on the contrary, tendency to reduce the sclerotized parts is obvious. It has been well documented by Berube and Zacharuk (1983) that the tephritid ovipositor consists of a heavily sclerotized distaI portion formed from abdominal segment 9; a flexible, tubular median portion, formed from abdominal segment 8; and a conical oviscape formed from abdominal segment 7. Oviposition takes longer and the female repeatedly tests the surface prior to laying (Stoffolano and Yin, 1987). Although the ovipositor of entomoparasitic Pipunculidae shows some differences from the ovipositor of Tephritidae laying their eggs within plant tissue, which is caused by the different oviposition strategy, some common features in ovipositor gross structure can be recognized. The piercing part of ovipositor in both families represents modified abdominal segment 9, the only segment from the ovipositor bearing sensilla. The genital opening leads from the apex of segment 9. In contrast, segment 7 is the less modified segment of the ovipositor and can be free (Tephritidae, Nephrocerus) or fused with segment 8 (Chalarinae, Pipunculinae). The last pair of abdominal spiracles in both families are located in segment 7. The anal and genital openings are separated in Pipunculidae as in most brachycerous flies but not in Tephritidae, in which these openings are fused (Berube and Zacharuk, 1983; Stoffolano and Yin, 1987). In both Nephrocerus and Pipunculus ovipositor types, the anal opening is situated dorsally between terga 8 and 9. On the other hand, there is a remarkable difference in the genital opening position. In Pipunculus type the genital opening is situated ventroapically, close to the apex; in Nephrocerus it is located ventromedially, in the basaI third of abdominal segment 9. The host and ovipositing behaviour of Nephrocerus species are unknown. The unusual position of genital opening as well as some other ovipositor characters (stout apical part, asymmetrical architecture, well developed accessory glands, the presence of long haired sensilla at apex) indicate that the oviposition strategy of Nephrocerus species could be different to other Pipunculidae. Due to the wide interspecific differences in structure of ovipositor but very small intraspecific variation, the shape, size and other ovipositor characters are frequently used in the taxonomy of Pipunculidae. For taxonomic purposes the ovipositor used to be divided into a conical or semiglobular base and an apical, tubular, more or less continually tapering piercer. Hardy (1943) was probably the first to investigate the origin of the pipunculid ovipositor and he stated 'The well adapted ovipositing structure of the female is composed of modified seventh, eighth and ninth abdominal segments; the seventh segment sometimes is extruded to give the ovipositor greater length as in Jassidophagajasciata (Hardy). The seventh segment makes up the basaI portion and the eighth and ninth are combined to form the actual piercing structure'. Up to now, no work dealing with comparative morphology of pipunculid ovipositors and analysing the homology of ovipositor parts has been published, although some authors revising various genera (De Meyer, 1989b; Albrecht, 1990; Jervis, 1992)

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M. Koz{mek and A. Belcari

evaluated some ovipositor characters and reported many new data concerning interspecific differences. Two types of ovipositor occur within the family Pipunculidae, Nephrocerus type consisting of segments 8 and 9 and Pipunculus type developed by modification and fusion of segments 7, 8 and 9. In Nephrocerus type terga of both segments are large and form the body of ovipositor. Sterna are reduced, fused and form the operculum overlapping the genital opening. Compared with terga, sterna are very slight1y sclerotized. In contrast to Pipunculus type ovipositor, the pleura are still distinguishable and narrow, unsclerotized strip connecting sternal genital operculum with tergal body of ovipositor. In Pipunculus type ovipositor consists of modified and fused abdominal segments 7,8 and 9. The abdominal segment 7 forms rather large cylindrical to semiglobular base of ovipositor. Pleural part is entirely sclerotized and provides continuaI fusion of tergal and sternal parts resulting in syntergosternum. In Verrallia, Jassidophaga and some Chalarus species the tergum is surrounded by a tergal suture which is absent in all Pipunculinae. In species lacking the tergal suture, the pleural area of segment 7 and sclerotized intersegmental connections can be traced after partial depigmentation of ovipositor in 4% KOH, as light yellowish parts contrasting with dark brown tergal and sternal cuticle. Abdominal segment 8 represents shortened syntergosternum in all species studied and forms a narrow ring connecting segment 7 (base) and segment 9 (piercer). The tergum and sternum of abdominal segment 9 are heavily elongated and form a tubular, thin, continually tapering piercer. The pleura of segment 9 are flexible and unsclerotized, allowing expansion of inner space and extrusion of egg into the host. The number of studies on the structure and distribution of sensilla of ovipositor of flies is stilllimited. The ovipositor of Phormia regina (Wallis, 1962) and Musca autumnalis (Hooper et al., 1972) bears bilateral sensory fields with several types of sensilla. The ovipositor of Lucilia cuprina possesses 14 tactile hairs in three size groups, one campaniform, two olfactory pegs and five contact chemosensitive hairs. The terminalia of both male and female Aedes aegypti have several types of sensilla, all of which are mechanoreceptory (Rossignol & McIver, 1977). Stoffolano & Yin (1987) found the ovipositor of Rhagoletis pomonella possesses chemosensilla as well as hair-like and campaniform mechanosensilla. Jervis (1992) has recognized campaniform and other two types of sensilla on the ovipositor piercer in some Chalarus speCles. Two subtypes of campaniform sensilla and long and short haired sensilla have been recognized on the ovipositor of Pipunculidae. Long haired sensilla are present in two lateral rows only on Nephrocerus ovipositor, the position and shape is similar to that of some non-entomoparasitic flies. In all studied species with Pipunculus type ovipositor the presence of two subtypes of campaniform sensilla located in the apical third to apical half and short haired sensilla distributed in basaI half of tergum 9 have been found. Campaniform sensilla are generally accepted as mechanoreceptors (Zacharuk, 1985). The distribution of campaniform sensilla at the apex of tergum 9 and short haired sensilla in its basaI third to half indicates that the former are responsible for monitoring the ovipositor position in the host body and the latter control the depth of ovipositor penetration. Both campaniform and short haired sensilla probably also play an important role during copulation. Harris (1966) studied the spermathecal system of eight British genera and recognized three types of spermathecae within the family Pipunculidae. Type 1 are represented by spherical or reniform, well sclerotized spermathecae occurring in

Structure of entomoparasitic pipunculid flies

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Nephrocerinae and Chalarinae. Type 2, filiform, has been found in some Cephalops, Eudorylas, Dorylomorpha and Tomosvaryella species and type 3 tubular occur only within the genus Pipunculus. The spermathecae observed were invariably three in number. As Harris (1966) pointed out, the observations have been made mostly on dried specimens, only very little live material was used. Jervis (1992) analysed differences in spermathecal structure between Chalarus species but he found no interspecific differences with respect to these structures. We have found three spermathecae in alI studied species. The well sclerotized spermathecae have been confirmed in Nephrocerinae (spherical) and Chalarinae (reniform). Species of subfamily Pipunculinae possess weak, membraneous, more or less spherical spermathecae of hyaline appearance, covered with membraneous sheath. These very soft structures are undetectable in dry specimens and even in live or alcohol preserved specimens can be easily destroyed during the preparation of the spermathecal system. The filiform or tubular types of pipunculid spermathecae of Harris (1966) are in fact the distaI portions of spermiducts or remains of a membraneous sheath. The spermiducts of Pipunculidae can be simple, tubular and undifferentiated (Nephrocerus) or differentiated (alI other Pipunculidae) to basaI muscular, centraI glandular and apical tubular sections. The length ratio of sections distinct1y varies with species. The present study on the morphology of Pipunculidae ovipositors opens some questions which should be investigated in order to understand the oviposition physiology of this group. Despite the fact that the hosts of many pipunculid species are known, we still have very poor information about the process of oviposition itself. The unusual architecture of the ovipositor, well developed accessory glands and exceptional location of long haired sensilla at the apex of Nephrocerus type ovipositor indicates that the oviposition strategy in these species could be different from other Pipunculidae. More information conceming the role of some sensilla (mainly haired sensilla) may be obtained by using various chemical treatments of sensilla surfaces providing observation of their porous and aporous structure and hence to identify their physiological function. The presence of various sensilla types at the ovipositor of some species indicates the multifunctional role of this sensorial apparatus. The study of sensilla innervation could help to elucidate wheather one type of sensilla is included in one or more behavioural responses. Acknowledgements

We would like to thank the Consiglio Nazionale delle Ricerche (CNR, Italy) , MURST (Italy) and Slovak Academy of Sciences (SAS, Slovakia) for the financial support of our work. Thanks are also due to Mrs Elisabetta Bruno (Firenze) for her helpful assistance at SEM, Dr Marc De Meyer (Belgium) and Dr. Alan E. Mill (Great Britain) for their criticaI reading of manuscript. References ALBRECHT,A., 1990, Revision, phylogeny and classification of the genus Dorylomorpha (Diptera, Pipunculidae), Acta Zoologica Fennica, 188, 1-240. BERUBE, D.E. and ZACHARUK, R.Y., 1983, The abdominal musculature associated with oviposition in two gall-forming tephritid fruit flies in the genus Urophora, Canadian Journal o/ Zoology, 61, 1805-1814. COE,R.L., 1966, Diptera, Pipunculidae. Handbooks for the Identification of British Insects, X 2(c), 83 pp.

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DE MEYER,M., 1989a, A synthesis of the present of the present knowledge of Pipunculidae (Diptera) in Belgium, Bulletin de L'Institut Royalles Sciences Naturelles de Belgique (supplement), pp. 373-377. DE MEYER,M., 1989b, The West-Palaearctic species of the pipunculid genera Cephalops and Beckerias (Diptera): c1assification, phylogeny and geographical distribution, JournaloJ Natural History, 23, 725-765. GRIFFITHS, G.C.D., 1972, The phylogenetic c1assification of Diptera Cyc1orrhapha. Junk, The Hague, 341 pp. HARDY,D.E., 1943, Revision of the Nearctic Dorilaidae (Pipunculidae), Kansas University Science Bulletin, 29, 1-231. HARDY,D.E., 1987, Pipunculidae. In: J.F. McAlpine (Ed.), Manual of Nearctic Diptera. Ottawa: Research Branch Agriculture Canada. Monograph No. 28, voI. 2, pp. 745-748. HARRIs,K.M., 1966: Pre1iminary notes on spermatheca1 structure in British Pipunculidae (Diptera), Proceedings oJ the Royal Entomological Society oJ London (A), 41, 130-132. HOOPER,R.L., PITTS,C.W. and WESTFALL, J.A., 1972: Sense organs on the ovipositor of the face fly, Musca autumnalis, Annals oJ Entomological Society oJ America, 65,577-586. JERVIS,M.A., 1992, A taxonomic revision of the pipunculid fly genus Cha1arus Walker, with particular reference to the European fauna, Zoological Journal oJ the Linnean Society, 105,243-352. MORAKOTE, R. and YANO,K., 1988, Adult behavior of some Japanese Pipunculidae (Diptera) parasitizing Nephotettix cincticeps (Hemiptera, De1tocephalidae), Kontyu, Tokyo, 56, 653-658. ROSSIGNOL, P.A and McIVER, S.B., 1977, Fine structure and role in behavior of sensilla on the terminalia of Aedes aegypti (L.)(Diptera: Culicidae), Journal oJ Morphology, 151, 419-438. SANDER,F.W., 1985, Zikadenfeinde in Rasengesellschaften der DDR: Augenfliegen (Diptera, Pipunculidae)-Bemerkungen zu Entwick1ung, Verha1ten und Wirtsbeziehungen, WissenschaJtliche Zeitschrift Fridrich-Schiller-Universitiit Jena, NaturwissenschaJtiche R., 34, 605-624. STOFFOLANO, J.G. JR. and YIN, L.R.S., 1987, Structure and function of the ovipositor and associated sensilla of the apple maggot, Rhago1etis pomonella (Walsh) (Diptera: Tephritidae), International Journal oJ Insect Morphology and Embryology, 16,41-69. VIDANO,C. and ARZONE,A., 1984, 'Wax area' in Cicadelids and its connetions with brochosomes from Malpighian tubules, Mitteilungen der entomologische GesselschaJt, 57, 444-445. WALLIS,D.I., 1962, Olfactory stimuli and oviposition in the b1owfly, Phormia regina Meigen, Journal oJ Experimental Biology, 39,603-615. WALOFF,N. and JERVIS,M.A., 1987, Communities ofparasitoids associated with leafhoppers and p1anthoppers in Europe, Advances in Ecological Research, 17,281-402. ZACHARUK,R.Y., 1985: Antennae and Sensilla. In: G.A. Kerkut and L.I. Gilbert (Eds), Comprehensive insect physiology, biochemistry and pharmacology. Pergamon Press, New York, voI. 6, pp. 1-71.