Development of Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) in Helicoverpa (Lepidoptera: Noctuidae) host eggs

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1 Australian Journal of Entomology (2002) 41, Development of Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) in Helicoverpa (Lepidoptera: Noctuidae) host eggs Ekhlass A Jarjees* and David J Merritt Department of Zoology and Entomology, University of Queensland, Qld 4072, Australia. Abstract Key words Morphological studies of development of the egg parasitoid Trichogramma australicum Girault in the cotton bollworm, Helicoverpa armigera (Hübner), were conducted to provide benchmarks for assessing developmental rates in both natural hosts and artificial diets. Observations of living embryos and histological sections show that embryos proceed rapidly through cleavage and blastoderm formation and show a characteristic pinching or rotation 8 h after deposition. Eggs progressively increase in volume, primarily by increasing in diameter at the widest point. At 29 ± 1 C the duration of the egg stage is h, the larval stage 27 h, the prepupal stage h, and pupa 85 h. Larvae undergo dramatic shape changes as they ingest food but do not show signs of larval moults, reinforcing observations that there is only one larval instar. Criteria for staging the embryonic and postembryonic development in natural hosts will be used for future studies aimed at developing and refining artificial diets for Trichogramma. embryonic development, Helicoverpa armigera, postembryonic development, Trichogramma australicum. INTRODUCTION Trichogramma australicum (Girault) is a potential biological control agent for Helicoverpa armigera (Hübner) in cotton in Australia (Dahlan & Gordh 1996, 1998; Nurindah et al. 1997). The recent successful rearing of T. australicum in an artificial diet comprising cells from an insect cell line along with conventional components (Notarte & Merritt 2001) has led to more detailed investigations of the rate of development of T. australicum. The immature stages of Trichogramma wasps are very small and are not well known because development occurs within the confines of a host egg. Embryonic and larval development of Trichogramma is quite rapid, seen as an adaptation allowing the exploitation of the egg stage of their hosts (Strand 1986), generally lepidopteran eggs. Embryonic development has been described in detail in two species, T. evanescens (Westwood) and T. chilonis (Ishii) (Gatenby 1917; Tanaka 1985a,b). The eggs of Trichogramma contain very little yolk and hatch about 24 h after deposition (Pak & Oatman 1982). Upon eclosion, larvae undergo rapid growth within the host egg (Dahlan & Gordh 1996). The larvae are soft-bodied, about 0.4 mm long and 0.1 mm wide, expanding as they ingest food and increasing in volume 40-fold (Wu et al. 2000). *Author to whom correspondence should be addressed ( ekhlass@hawaii.edu). Present address: Department of Plant and Environmental Protection Sciences, University of Hawai i at Manoa, 3050 Maile Way, 310 Gilmore Hall, Honolulu, Hawai i 96822, USA. The number of larval instars remains uncertain. One to five instars have been described based upon features such as mandible size, larval size and shape, and presence of exuviae. For example, studies using larval shape as a primary criterion have decided upon two (Flanders 1937; Clausen 1962), three (Kochetova 1969; Manweiler 1986), four (Pak & Oatman 1982) or five (Hagen 1964) instars. More recent studies indicate that there is one larval instar, based upon an absence of larval exuviae and the lack of growth of larval mandibles (Volkoff et al. 1995; Dahlan & Gordh 1996; Jarjees et al. 1998; Wu et al. 2000). The presence of urate cells around the gut is conventionally used to indicate the onset of the prepupal stage (Dahlan & Gordh 1996; Jarjees et al. 1998). Pupation occurs on day 5 and the pupal period lasts approximately 3 days at between 25 and 28 C (Volkoff et al. 1995; Dahlan & Gordh 1996; Nordlund et al. 1997). Significant embryonic and larval mortality can occur in unsuitable artificial diets (Grenier et al. 1995; Nordlund et al. 1997) and rates of development can be significantly altered in unsuitable diets or natural host eggs of different ages (Ruberson & Kring 1993; Cônsoli & Parra 1996; Nordlund et al. 1997). Life-cycle staging criteria such as those established for T. cacoeciae Marchel (Volkoff et al. 1995) are necessary to determine the rate of progression of T. australicum through embryonic and postembryonic development. Our study aimed to develop criteria to differentiate between stages of T. australicum development and establish the duration of the larval, prepupal and pupal stages in the laboratory. Embryonic development was assessed by examination of living embryos dissected from host eggs and from histological sections of embryos in situ within eggs of

2 Development of Trichogramma australicum 311 H. armigera. In addition, we have used the staging criteria to investigate the specific stages at which embryonic and larval development is delayed in less suitable hosts. MATERIALS AND METHODS Insect maintenance A T. australicum culture was established from individuals collected from H. armigera eggs from sorghum on the Darling Downs, south-eastern Queensland (Dahlan & Gordh 1996). The isofemale culture was kept in shell vials in an incubator (29 ± 1 C, 75% RH and 12L:12D photoperiod). Histology Helicoverpa armigera eggs were attached to strips of paper towel and placed in glass shell vials (4.5 by 1.5 cm) for parasitoid oviposition. The eggs were collected within 1 h of deposition and exposed to mated T. australicum females for parasitisation. After 1 h, the females were removed. Parasitised host eggs were incubated at 29 ± 1 C and fixed at 4 h intervals postparasitisation, that is, at 4 h, 8 h, 12 h, etc. The fixation method was similar to the phase-partition procedure originally used for Drosophila embryos (Zalokar & Erk 1977). Eggs were placed in 50% bleach and agitated for 5 10 min or until the chorion sloughed. Eggs were then fixed for 20 min in 3.7% formaldehyde in phosphate-buffered saline mixed with an equal amount of heptane. After the fixative was removed, the eggs were devitellinized by adding an equal volume of 100% methanol to the heptane. After dehydration through an ethanol series, eggs were infiltrated overnight in LR White medium-grade resin, placed in gelatine capsules in fresh resin and cured overnight at 60 C. Sections (2 µm) were cut on glass Ralph knives, floated onto an acetone water bath, collected onto slides, dried on a hotplate and stained with 1% toluidine blue in borax. A coverslip was placed over the preparation and the egg sections were examined under a Zeiss compound microscope (Carl Zeiss, Jena, Germany). Images were recorded using a Dage DC-330E CCD camera (Dage-MTI, Michigan City, IN, USA), a Scion frame-grabber (Scion Corporation, Frederick, MD, USA) and Scion-Image software package. Adobe Photoshop software was used for image layout. Measurements of live Trichogramma eggs and larvae Eggs were parasitised and incubated at 29 ± 1 C for dissection in saline on a microscope slide at 4-h intervals. Embryos, larvae or pupae were removed from the host tissues, placed in a drop of saline and coverslipped. Eggs and early larvae were observed with a Zeiss Axioskop equipped with DIC optics and photographed. Late larvae, prepupae and pupae were observed with Ziess Stemi SV 6 stereoscope. Measurements of the length and width of embryos, larvae and pupae were made by comparing photographs of specimens to photographs of a stage micrometer taken using the same lens combination. RESULTS Embryonic development In freshly deposited host eggs, Trichogramma larvae maintained at 29 ± 1 C eclose from the egg h after oviposition. The newly laid egg is ovoid, about 0.14 mm long and 0.04 mm wide. The egg is broader at the anterior end (Fig. 1a). The embryo and egg gradually increase in volume as development proceeds. Measurements made at 4-h intervals show a progressive increase in egg volume, primarily because of an increase in the diameter of the egg at its widest point. At 1 h after oviposition, the egg measures 0.20 mm long by 0.05 mm wide; at 12 h, 0.21 mm by 0.09 mm, and at 24 h, 0.22 mm by 0.08 mm. Sections show that the egg is initially yolkless with a small number of large nuclei in a syncitium (Fig. 1a). Two hours postoviposition, the embryo is cellularised and cleavage is complete (Figs 1b,c). Discrete cells with large, spherical nuclei approximately 0.35 µm in diameter occupy the full volume of the egg (Fig. 1b). Some cells show signs of differentiation, for example, cells at the posterior pole are larger and more elongate than others (Fig. 1b). Four hours after oviposition, signs of invagination at both the stomodaeum and proctodaeum appear (Fig. 1d). At 8 h after oviposition, a haemocoel cavity is present, indicating that gastrulation has occurred. At this stage, the embryo shows a constriction along its length, perhaps associated with gastrulation (Figs 1e,f). By 12 h after oviposition, midgut formation is well underway (Figs 1g,h). Sixteen hours after oviposition, the stomodaeaum is well developed (Figs 1i,j). An epithelium of cuboid cells surrounds a narrow midgut lumen, continuous with the stomodaeal lumen (Fig. 1j). By 20 h, the cells forming the midgut epithelium enlarge (Figs 1k,l). The oral cavity, a component of the foregut, and the anal vesicle, the most posterior part of the hindgut, are well-developed. Postembryonic development At eclosion (22 24 h after oviposition), the embryonic exuvium slides backward along the body (Fig. 2a) as described by Jarjees et al. (1998). The epidermal cells become squamate and the pharynx is fully developed (Figs 2b,c). Larvae are opaque, unsegmented and lack obvious setae or other external morphological characters. The newly hatched larva is about 0.42 mm long and 0.11 mm wide. As the larva feeds it increases in size and changes in shape from vermiform to pyriform (0.35 mm long and 0.15 mm wide) (Fig. 2d), to sacciform by h after oviposition, 5 h after eclosion. The fully fed, sacciform larva is about 1.55 mm long and 0.80 mm wide (Fig. 2e) when the host-egg contents have been totally ingested and shows no further shape changes for h. From extensive observations of both living and

3 312 EA Jarjees and DJ Merritt Fig. 1. Embryonic development of Trichogramma australicum. (a) Cleavage stage showing ( ) nuclei (energids). (b,c) Cellular blastoderm stage, 2 h postparasitisation showing ( in b) larger posterior cells in section. The outline of a single cell is indicated by ( in c). (d) Section of an embryo 4 h after oviposition showing ( ) the proctodaeal invagination. (e,f) Whole mount and section of embryos 8 h after oviposition showing ( ) rotation and pinching of the embryo. (*) Midgut primordia. (g,h) 12 h after oviposition ( ) proctodaeal invagination is underway, ( ) stomodaeal invagination and (*) midgut epithelium is formed. (i,j) 16 h after oviposition ( ) the body cavity is well developed. ( ) The mouth opening and pharynx are visible. (*) The anal vesicle. (k,l) 20 h postoviposition ( ) rosette cells are visible, ( ) the oral cavity is well developed and (*) the anal vesicle further enlarged. Scale bar = 10 µm for a, b, d, f, h, j and l; 20 µm for c, e, g, i and k.

4 Development of Trichogramma australicum 313 Fig. 2. Postembryonic development of Trichogramma australicum. (a) Eclosion occurs h postoviposition. ( ) The embryonic exuvium slides along the body wall. (b) Histological section and (c) whole mount of the newly hatched (vermiform) larva with ( ) exuvium attached to the posterior end of the larvae. ( ) The anal vesicle is fully developed and feeding has begun as (*) host-egg contents are visible in the gut in (c). The muscular portion of the pharynx is indicated by arrowheads. (d) Pyriform larva showing ( ) the exuvium attached to the posterior end of the feeding larva. The gut is filled with (*) host egg contents. (e) Sacciform larva, h postoviposition has enlarged further. (*) Anal vesicle. (f) Prepupa, h after oviposition showing ( ) urate cells. (g) Pupa, h postoviposition showing ( ) ocellus. Scale bars = 20 µm for a; 10 µm for b; 50 µm for c and d; 100 µm for e g. fixed larvae under the compound microscope, no evidence of larval moults has been detected. The embryonic exuvium remains attached to the proctodeum throughout larval development, although it is reduced to a remnant by the sacciform stage. The onset of the prepupal stage is indicated by the appearance of urate bodies about h after oviposition scattered around the midgut (Fig. 2f). The immobile prepupa is about 1.24 mm in length and 0.77 mm in width. The prepupal stage lasts h. The compound eyes and ocelli become red in the pupal stage about h postoviposition. The early pupa is about 1.25 mm long and 0.67 mm wide. The pupal stage (Fig. 2g) lasts h. The adult emerges h after oviposition. DISCUSSION The eggs of Trichogrammatidae show adaptations to their habit of egg parasitism by being small in size and lacking yolk (Flanders 1937; Klomp & Teerink 1967; Pak & Oatman 1982). They increase in volume after being deposited, apparently absorbing nutrient directly from the host through the egg membranes (Fisher 1971). Trichogramma australicum eggs are typical of Trichogrammatidae: they are thin-walled, contain little yolk when laid and expand in volume as the embryo matures. Within H. armigera host eggs, embryonic cleavage is complete 2 h postoviposition. A pinching or rotation of the embryo is a transient marker occurring at 8 h of development, perhaps corresponding with the formation

5 314 EA Jarjees and DJ Merritt of the longitudinal groove and initiation of gastrulation described in T. evanescens (Gatenby 1917). The formation of the stomodaeum, proctodaeum and midgut closely follows the process described in detail in T. chilonis (Tanaka 1985b). In T. australicum, shallow invaginations representing the first signs of the stomodaeum and proctodaeum formation and a well-defined body cavity are visible after 8 h. The duration of embryonic development in T. australicum (22 24 h at 29 C) is comparable to other Trichogramma species, reported to take from 20 to 29 h at temperatures ranging from 25 to 29 C (Flanders 1937; Pak & Oatman 1982; Strand 1986; Volkoff et al. 1995; Dahlan & Gordh 1996). Because the immature stages of Trichogramma are small and development occurs within the confines of a host egg, the immature stages are not well known and the number of larval instars remains controversial. We measured and described postembryonic immature stages directly after removal from the host egg. In many observations of both living and sectioned larvae, we saw no signs of pharate stages, no evidence of exuvia and no larvae undergoing ecdysis. Further, the embryonic exuvium remains attached to the proctodaeum of the feeding larva up to the prepupal stage. These observations support the presence of a single larval instar. The designations of larval instars by earlier workers usually relied on larval morphology, but we consider that larval shape and size changes can be attributed to swelling from the ingestion of food. Trichgramma australicum larvae ingest the host-egg contents within 4 h of eclosion and become sacciform in shape (Jarjees et al. 1998). Similarly, Volkoff et al. (1995) reported that the larva of T. cacoeciae (Marchal) reared on eggs of Ephestia kuehniella (Zeller) at 25 C consume the host-egg contents and reach maximum size within only 8 h of eclosion. The absorption of host-egg components during embryonic development of the parasitoid has implications for the use of artificial diets. Solutes and ionic contents of the artificial diets must be appropriate for Trichogramma embryonic development to continue. Similarly, during larval development, the diet must be nutritionally adequate as well as being of a consistency that allows the larva to ingest the food (Jarjees et al. 1998; Wu et al. 2000). Our study has provided a number of morphological landmarks that allow assessment of the rate of embryonic and larval development of T. australicum in artificial diets and in host eggs of different species and different ages. ACKNOWLEDGEMENTS Financial support came from the Queensland State Government s Supporting Green Industries Initiative. The project was initiated by Professor Gordon Gordh. REFERENCES Clausen CP Entomophagous Insects. Hafner Publishing Company, New York, USA. 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6 Development of Trichogramma australicum 315 Volkoff AN, Daumal J, Barry P, Francois MC, Hawlitzky N & Rossi MM Development of Trichogramma cacoeciae Marchal (Hymenoptera: Trichogrammatidae): Timetable and evidence for a single larval instar. International Journal of Insect Morphology and Embryology 24, Wu ZX, Cohen AC & Nordlund DA The feeding behaviour of Trichogramma brassicae: new evidence for selective ingestion of solid food. Entomologia Experimentalis et Applicata 96, 1 8. Zalokar M & Erk I Phase-partition fixation and staining of Drosophila eggs. Stain Technology 52, Accepted for publication 8 July 2002.