Immature Development of Cotesia vestalis (Hymenoptera: Braconidae), an Endoparasitoid of Plutella xylostella (Lepidoptera: Plutellidae)

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1 MORPHOLOGY, HISTOLOGY, AND FINE STRUCTURE Immature Development of Cotesia vestalis (Hymenoptera: Braconidae), an Endoparasitoid of Plutella xylostella (Lepidoptera: Plutellidae) RUI-XIAN YU, MIN SHI, FANG HUANG, AND XUE-XIN CHEN 1 Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou , China Ann. Entomol. Soc. Am. 101(1): 189Ð196 (2008) ABSTRACT Cotesia vestalis (Haliday) (Hymenoptera: Braconidae) is one of the most important biological control agents of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Immature development of C. vestalis was studied by dissecting parasitized hosts in the laboratory at 25 1 C and 60Ð65% RH. Results showed that the egg matured in 2 d. Larvae seemed to have three instars; the Þrst two larval instars molted inside the host, and the third instar emerged from the host to spin a cocoon. The Þrst, second, and third instars lasted 2, 5, and 1 d, respectively. The Þrst instar is transparent and caudate-mandibulate with a large head, distinct mandibles, and a caudal projection. The second instar is semitransparent with an anal vesicle. The third instar larva is yellowish green and the body is slightly curved. The pupa develops for 5 d and is bright yellow with transparent antennae, wings, and legs. All stages were fully documented with microscopic photos. KEY WORDS Braconidae, larval development, immature stages, biological control, parasitoid Parasitoids are widespread and diverse with two different kinds of life histories. Idiobionts develop in a nongrowing host, whereas koinobionts develop in hosts that continue to feed and grow during the initial stages of parasitism (Askew and Shaw 1986, Harvey et al. 1999). In studies of parasitoid development, most attention has focused on the physiology of parasitoidð host interactions, whereas the biology and morphology of larval stages are little studied, especially in endoparasitic, koinobiont species. In many cases, endoparasitic species developing inside their hosts undergo hypermetamorphosis. Specialized morphologies are often seen in Þrst (L1) and second (L2) instars, such as the anal vesicle and sickle-like mandibles (Odebiyi and Oatman 1972, Mackauer 1990, Shaw and Huddleston 1991, Carignan et al. 1995, Quicke 1997, Mayhew and Van Alphen 1999, Kaeslin et al. 2006, Xu et al. 2007). Cotesia vestalis (Haliday) (Hymenoptera: Braconidae) is an endoparasitoid of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), one of most destructive pests of crucifer crops worldwide (Talekar and Shelton 1993). Although the development of C. vestalis has been preliminarily studied (Chiu and Chien 1972), more detailed observations on the developmental biology and morphology of C. vestalis are presented in this article. Note that C. vestalis has commonly been called Cotesia (or formerly Apanteles) plutellae (Kurdjumov) in the literature, but now should be referred to as C. vestalis (Haliday) (Shaw 2003). 1 Corresponding author, xxchen@zju.edu.cn. Materials and Methods Insect Collection and Rearing. Pupae and parasitized larvae of P. xylostella were initially collected from cabbage (Brassica spp.) Þelds in the suburbs of Hangzhou, Zhejiang Province, China. Both P. xylostella and C. vestalis colonies were raised on cabbage grown at 25 1 C, 60Ð65% RH, and a photoperiod of 14:10 (L:D) h. Adult wasps were fed with 20% (vol:vol) honey solution and propagated using P. xylostella larvae. Development of C. vestalis. Parasitized P. xylostella larvae were dissected in phosphate-buffered saline, ph 7.4 (0.138 M NaCl, M KCl, M Na 2 HPO 4, and M KH 2 PO 4 ) with the aid of a dissecting microscope (MZ APO, Leica) at 2Ð4 h intervals after parasitism until all stages of parasitoid development were recorded. Developmental time was counted from the moment the parasitoid egg was oviposited into the host. All stages of the wasp were photographed using a stereomicroscope (MZ 16A Leica) and an inverted phase contrast microscope (DM IRB, Leica) equipped with an image manager (IM1000, Leica). Length and width of eggs and larvae also were also measured (Table 1). Ovarian eggs were observed by teasing the ovaries out of the abdomens of C. vestalis. To obtain eggs or Þrst instars, a small cut was made in the caudal end of the host larva. Then, the eggs and Þrst instars ßowed out with the body ßuid of the host larvae. To obtain second instars, the Þrst or second segment of the host abdomen was gently pressed to force the parasitoid larva out of the host. Third instars (L3) were observed /08/0189Ð0196$04.00/ Entomological Society of America

2 190 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 1 Table 1. Time after oviposition Size of immature stages of C. vestalis Stage Body length (mm) SE Body width (mm) SE Sample size Ovarian egg 0 h Egg d Egg d Egg d L d L d L d L d L d L d L d L as they left the host body to pupate externally. The development of prepupae and pupae was observed by dissecting cocoons. Results Egg. Ovarian and newly laid eggs were spindle shaped and transparent, with a narrow pedicel at the end (Fig. 1A). The newly laid eggs were always adhered Þrmly to host tissues, especially to the gut (Fig. 1B). Three-layered egg membranes were visible in the developing embryo (Fig. 1C). Approximately 15 h after oviposition, a group of cells grew out from both the anterior and posterior poles of the egg to spread all over the surface of the embryo (Fig. 1D), forming a complete outer membrane, the serosal membrane (Fig. 1E). The membrane comprised a syncytium in the abdominal region and discrete polar cells, which gave rise to teratocytes (Fig. 1H). Approximately 20Ð24 h after oviposition, the gut became visible (Fig. 1F). Thirty-six hours after oviposition, the embryo was already a distinguishable larva within the egg (Fig. 1G). Forty-eight hours after oviposition, the Þrst instar was visible through the transparent chorion, with a distinct cephalic capsule and segmentation (Fig. 1H). Most of the internal organs were visible as well. At this time, the egg was about to hatch, 2 d after oviposition at 25 1 C. C. vestalis has three instars. the Þrst two instars molted inside the host, whereas the third instar exited the host to spin a cocoon and pupate. First Instar. At hatching, the chorion ruptured and the discrete polar teratocytes seemed to have dissociated into the hostõs hemolymph. The Þrst instar was transparent and caudate-mandibulate, with a large head. Segmentation was not yet clearly visible because Fig. 1. Eggs of C. vestalis. (A) Newly laid egg, with arrow indicating the pedicel. (B) Egg adhered to the host. (C) Developing embryo, showing three-layered egg membrane. (D) Egg after 15 h; the serosal membrane is forming. (E) Serosal membrane surrounding the embryo (arrow). (F) Gut (arrow). (G) Egg after 36 h. (H) Egg after 48 h, with arrow indicating discrete teratocytes.

3 January 2008 YU ET AL.: IMMATURE DEVELOPMENT OF C. vestalis 191 Fig. 2. First instar of C. vestalis. (A) Newly hatched larva, with arrow indicating the falcate mandibles. (B) Dissociating serosal membrane. (C) Teratocyte formation. (D) Larva 1 d after hatching, with obvious segments. (E) Dissociating amnion. (F) Larva 40 h after hatching, with arrow indicating the anal vesicle. of the serosal membrane, which was still attached to the larva (Fig. 2A). Mandibles were falcate and sclerotized. They measured mm in length. The serosal membrane began to dissociate to form teratocytes several hours after hatching (Fig. 2B and C). One day after hatching, another membrane, i.e., the amnion, attached to the embryo had dissociated (Fig. 2E). At this time, 13 segments of the larva were visible (Fig. 2D). A transparent anal vesicle was observed 40 h after hatching (Fig. 2F). Forty-eight hours after hatching, the Þrst instar was close to molting and had taken 2 d to develop at 25 1 C. Second Instar. The second instar was present from the Þfth day after oviposition. Development lasted 5 dat25 1 C. The early second instar was transparent, with obvious segments and a visible gut. The head capsule was similar to that of the Þrst instar. At this stage, the tail disappeared, and it was replaced by the anal vesicle (Fig. 3A). Six days after oviposition, the head capsule and mandibles also disappeared (Fig. 3B). Close observation showed that the mouthparts were three lobed and included lateral maxillae and labrum (Fig. 3C). The anal vesicle was connected to the digestive duct with an obvious constriction (Fig. 3D). The anal vesicle, therefore, seemed to be an evagination of the hindgut. As the larva aged, it became more vermiform. Seven days after oviposition, the larva was opaque with a cream color and light green gut (Fig. 3E). At this time, the tracheal system was visible through the integument (Fig. 3F). One day later, the larva was a white-greenish, and the gut was dark green (Fig. 3G). The tracheal system was also clearly visible (Fig. 3H). At 9 d, the larva was light yellow, and the Þrst several segments of the abdomen were dark yellow. The anal vesicle began to shrink back as the second instar was about to molt. Third Instar. During molting, the larva emerged from side of the hostõs abdomen. The exuvium of the second instar must have been left behind in the host body, because there was no visible cast larval skin attached to the emerged larva (Fig. 4A). Exit of the larvae required only 4 or 5 min. The third instar was vermiform and yellowish green, with a slightly curved body. The mature larva was white with a light yellow tint. It was recurved anteriorly and lacked an anal vesicle (Fig. 4C). The mature larva emerged from the fourth instar of P. xylostella 9 d after oviposition at 25 1 C. Cocoon. The parasitoid larva began to spin a cocoon before emergence and continued to spin after emergence (Fig. 4B). The cocoon was spun within 4 h. The host lived one or 2 d after the parasitoid exited. The cocoon was oval and white, and it measured 3.49 mm in length and 1.30 mm in width (Fig. 5A). Prepupa. Newly pupated wasps were visible in their cocoons 10 d after oviposition at 25 1 C. The head and the thorax of the prepupa was primrose, and the abdomen was ivory-white (Fig. 5B). The prepupa had

4 192 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 1 Fig. 3. Second instar of C. vestalis. (A) Second instar larva 5 d after oviposition. (B) Second instar after 6 d. (C) Head. (D) Anal vesicle. (E) Larva after 7 d, with arrow indicating light green gut. (F and H) Tracheal system. (G) Larva after 8 d, with arrow indicating gut. (I) Larva after 9 d, with arrow indicating exuvium of the second instar. 13 segments and orange red eyes. It measured 3.08 mm in length, shorter than the mature larva. Through the cuticle, the gut seemed to be Þlled with dark meconia. Structures of the mouthpart were visible, i.e., the mandibles, labium, labial palpi, and maxillary palpi (Fig. 5F). Three to 5 h after pupation, the body was yellow, and the meconia were clearly visible (Fig. 5C). Five to 10 h after pupation, the meconium could be observed at the end of the cocoon (Fig. 5D). By dissecting the cocoon, it was clear that the black meconium had been expelled from the prepupa and was attached to the posterior end of the abdomen (Fig. 5E). At this time, three yellow dorsal ocelli could be observed (Fig. 5G). Pupa. Two days after pupation, the pupa was bright yellow and slightly transparent, with two red-brown eyes and three orange-red dorsal ocelli (Fig. 6A). Legs, wings, and antennae were visible with the same color as the body (Fig. 6B). The exuvium of the third instar and the meconium could be observed attaching to the end of the abdomen (Fig. 6E). The exuvium was yellow and thick, unlike the exuvium of the Þrst and second instars (Fig. 6F). Three days after pupation, the head and thorax began to blacken until the whole body was black (Fig. 6C). The exuvium and meconium were left in the cocoon after emergence. Five days after pupation, the adult made a circular cut at the thin end of the cocoon using its mouthpart, and then

5 January 2008 YU ET AL.: IMMATURE DEVELOPMENT OF C. vestalis 193 Fig. 4. Third instar of C. vestalis. (A) Larva emerging from the host. (B) Larva spinning cocoon. (C) Mature larva. emerged (Fig. 6D). The pupa completed development in5d. Discussion Flanders (1942) described two types of parasitoid eggs. Anhydropic eggs have enough yolk for subsequent embryonic development, whereas hydropic eggs do not and absorb nutrient from the host through the chorion (Ferkovich and Dillard 1986). Once laid inside the host, hydropic eggs usually swell greatly over a period of hours to days (Quicke 1997). The eggs of euphorine braconids may swell to 1,000 times the volume of the ovarian eggs (Jackson 1928, Smith 1952). We found that after 48 h, C. vestalis eggs had doubled in length and increased almost six-fold in width. Our observations clearly indicated that eggs of C. vestalis are hydropic. Fig. 5. Prepupae of C. vestalis. (A) Cocoon. (B) newly pupated wasp, with arrow indicating meconium. (C) Prepupa 3Ð5 h after pupation, with arrow indicating meconium. (D) Cocoon after 5Ð10 h, with arrow indicating meconium. (E) Prepupa 5Ð10 h after pupation, with arrow indicating meconium. (F) Ventral observation of the head of the newly formed prepupa. (G) Dorsal observation of the head of the prepupa 5Ð10 h after pupation, with arrow indicating dorsal ocelli.

6 194 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 1 Fig. 6. Pupae of C. vestalis. (A) Dorsal observation of the pupa 2 d after pupation. (B) Ventral observation of the pupa 2 d after pupation. (C) Pupa 3 d after pupation. (D) Cocoon after emergence. (E) Close observation of the posterior end of the abdomen. (F) Exuvium of the third instar. (G) Meconium. Beckage and DeBuron (1994) showed that, in the microgastrine braconid Cotesia congregate (Say), the single-cell thick serosal membrane actually comprised two membranes: the true serosa immediately below the chorion, and an amnion, which occurs inside the chorion. When C. congregata eggs hatched, the amnion was found to remain attached to the embryo for a while after the serosa had dissociated to form teratocytes (Beckage and DeBuron, 1994). In our study, the serosal membrane of C. vestalis also began to dissociate to form teratocytes several hours after hatching. One day after hatching, another membrane was visibly attached to the embryo, the amnion, and then it dissociated from the embryo. Data on the number of instars in parasitic Hymenoptera is difþcult to obtain (Hagen 1964), and previous records may be unreliable, especially for endoparasitoids. In our study, we found that the exuviae of the Þrst two instars of C. vestalis larvae were thin and transparent, making them difþcult to observe. Based on the research of Chiu and Chien (1972) and our own observations, we conclude that C. vestalis has three instars. The Þrst two instars molted inside the host, whereas the third instar exited from the host to spin a cocoon and pupate. In many endoparasitoids, the larva undergoes hypermetamorphosis wherein specialized early instar forms give rise to more or less uniformly hymenopteriform later instars. In some species, the larva passes through three distinct morphological forms. For example, scelionids of the genus Trissolcus have a teleaform Þrst instar, a sacciform intermediate stage, and hymenopteriform Þnal stage (Volkoff and Colazza 1992). In most braconids, the Þrst and last larval instars are conserved morphologically (Odebiyi and Oatman 1972, Shaw and Huddleston 1991, Carignan et al. 1995, Xu et al. 2007). For example, the Þrst instar of Peristenus digoneutis Loan is caudate-mandibulate, with a distinct sclerotized head capsule armed with sickleshaped mandibles. The third instar is vermiform with sclerotized mouthparts. The second instar is vermiform, with a round and ßeshy head (Carignan et al. 1995). As in most braconid larvae, the Þrst instar of C. vestalis is caudate-mandibulate, with a distinct sclerotized head capsule and sickle-like mandibles. The third instar is vermiform. However, the second instar of C. vestalis is vesiculate, with an anal vesicle at the posterior end. The mandibles of Þrst instars of parasitoid larvae seem to serve in the destruction of supernumerary

7 January 2008 YU ET AL.: IMMATURE DEVELOPMENT OF C. vestalis 195 Fig. 7. Larval competition between C. vestalis. (A) Development of eggs in the same host. (B) First instar biting the abdomen of another Þrst instar. (C) Dead Þrst instar from superparasitized host. larvae in superparasitism and multiparasitism (Mackauer 1990). When P. xylostella was superparasitized by C. vestalis, all supernumerary eggs developed to the Þrst instar (Fig. 7A). Usually, the Þrst laid egg hatched earliest and ultimately survived alone in the host. Other larvae were killed by the mandibles of the Þrst hatched larva (Fig. 7B). Wounds on the abdomen of dead larvae were clearly observed (Fig. 7C). Our results indicate the existence of larval siblicide, as found in some but not all braconids (Salt 1961). For example, Þrst instars of Aphaereta genevensis (Fischer) use their large mandibles to kill other parasitoid eggs or larvae in the same host (Mayhew and Van Alphen 1999). However, the congener A. pallipes often lays more than one egg in each host, and several offspring may complete development, although A. pallipes also have large mandibles (Mayhew and Van Alphen 1999). A number of endoparasitic braconids have been shown to possess anal vesicles in the Þrst and second instars (Quicke 1997). In our study, the anal vesicle of second instars of C. vestalis could be used to separate the second from the third instar. Wigglesworth (1953) suggested that anal vesicles of the early instars of some braconid larvae may function in water balance, ion balance, or both. Lewis (1970) noted a pair of welldeveloped tubules associated with the anal vesicle of the braconid Microplitis croceipes (Cresson) and suggested that these tubules may have a secretory function. Late second instar larvae of Chelonus near curvimaculatus Cameron have been shown to produce a 27-kDa protein in the anal vesicle (Reed and Brown 1998), also suggesting a secretory role for the anal vesicle. Edson and Vinson (1977) conþrmed that the anal vesicle of M. croceipes was important in the uptake of simple food molecules. An electron microscopic study of the anal vesicle of Chelonus inanitus (L.) revealed large epithelial cells with microvilli on their apical side, which also suggests uptake of nutrients from the hostõs hemolymph (Kaeslin et al. 2006) or perhaps gas exchange. It seems likely that the anal vesicle has both secretory and uptake functions. The development of the gut is variable in the Hymenoptera, including in parasitoid wasps. The guts of most Apocrita remain incomplete until pupation (Quicke 1997). In these species, fusion of the midgut and hindgut usually occurs at the end of the Þnal instar, allowing expulsion of undigested food and nitrogenous waste in the form of meconia. Opiine and alysiine braconids are exceptions; parasitoids of these subfamilies do not void the meconium until adult eclosion. In C. vestalis, the meconium pellet was expulsed at the prepupal stage (Fig. 5E), which means that the digestive system was complete before the prepupal stage. In conclusion, the development of C. vestalis is in many respects similar to other braconids. However, the development of few species has been studied in as much detail as we have done here. By documenting the development of the immature stages of C. vestalis, we have produced a solid foundation for further studies on physiological interactions between P. xylostella and C. vestalis. Some of these studies have been already carried out in our laboratory (Bai et al. 2005a,b; Wang et al. 2006; Yu et al. 2007). Ongoing research in our laboratory is focusing on the microstructure of the developing C. vestalis larvae. Acknowledgments We thank C. van Achterberg (Leiden, The Netherlands) for critical review of the Þrst draft of this article, F. Zaidi for improvement of the text, and T. R. Collier (University of Wyoming, Laramie, WY) for editing the manuscript for content and grammar. We also thank two anonymous reviewers for valuable comments. The project was supported by funds from 973 Program (2006CB102005), National Science Fund for Distinguished Young Scholars ( ), National Science Foundation of China ( ), Program for New Century Excellent Talents in University, China (NCET ), and the Innovation Research Team Program of the Ministry of Education of China (IRT0355). References Cited Askew, R. R., and M. R. Shaw Parasitoid communities: their size, structure and development, pp. 225Ð264. In J. Waage and D. Greathead [eds.], Insect parasitoids. Academic, London, United Kingdom.

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