The probable significance of tracheal tufts in the 8th abdominal segment of Heliothis virescens (F.) on the development of its parasitoid, Toxoneuron nigriceps (Viereck)

The 8th abdominal segment of Heliothis virescens (Fabricius) larvae contains aerating trachea and tracheole tufts that end in the hemocoel of the 8th segment, unlike the tracheae that invade tissues in other segments. These tracheal tufts from the 8th abdominal segment extend to the tokus region, which along with the telson cavity is known to act as a “lung” for hemocytes in Calpodes ethlius and a few other lepidopteran larvae. The goal of this research was to study the effects of these tracheal tufts in the 8th abdominal segment on parasitoid development inside the host larvae, H. virescens. The first objective was to determine if the eggs of the parasitoid, Toxoneuron nigriceps, are predominantly located among the tracheal tufts of the 8th abdominal segment compared to other body cavity regions irrespective of their oviposition site or the position of the host larvae. The results showed that several hours after oviposition most of the eggs are found in the 8th abdominal segment irrespective of the oviposition site or the position of the host larvae. The second objective was to study the effect of varying oxygen concentrations in vitro on various developmental stages of the egg. The results showed that decreasing oxygen concentrations adversely affects the parasitoid egg development in vitro. A third objective was to determine the oxygen concentration in 8th abdominal segment of the host larvae and compare it to other regions of the body using an oxygen sensor placed in vivo. The results suggested relatively high concentration of oxygen in the 8th abdominal segment compared to other regions of the host, thus supporting our hypothesis that the increased oxygen level in the 8th abdominal segment is important to the development of the parasitoid eggs.     

Larval morphology and development of Coptera occidentalis

The morphology and development of immature stages of the solitary internal parasitoid Coptera occidentalis were studied within the pupae of their factitious host, Ceratitis capitata. Parasitoid eggs are of the hymenopteriform type. Three larval instars are described. The first instar is of the mandibulate type bearing prominent submandibular appendages and a pair of terminal lobes on the last abdominal segment. The terminal lobes are covered with cuticular spines. The integument also bears cuticular spines arranged in paired dorsal welts on each of body segments V--IX. The second and third instar larvae are hymenopteriform with simple mandibles, reduced submandibular appendages and smooth integument. The third instar has an open tracheal system with three pairs of spiracles on segments II--IV. Considerable variation in development rates of parasitoid immature stages and high percentage of superparasitism were observed. Parasitoid eggs nearly doubled in size during the 96-h incubation. Development from egg to pupa took minimum 25 days and emergence of imagines started on day 42 after parasitization. Superparasitism was recorded in 56% of the examined hosts. The average number of eggs/host was 5.04 (range 1--22). Supernumerary occurrence of successive larval instars per one host was also observed.     

Immune response of the hibiscus mealybug, Maconellicoccus hirsutus Green (Homoptera: Pseudococcidae), to oviposition of the parasitoid Anagyrus kamali Moursi (Hymenoptera: Encyrtidae)

Anagyrus kamali Moursi has been recently introduced into the Caribbean as a biological agent against the hibiscus mealybug, Maconellicoccus hirsutus Green. This host has a cellular defense reaction that involves encapsulation and melanization of the endoparasitoid egg. The impact of this immune response on the parasitoid progeny was assessed, as well as the response of the parasitoid countermeasures to overcome it. Under laboratory conditions, significant differences in the immune response were found for different developmental stages of M. hirsutus. The intensity of the immune response varied between second instar, third instar and adult mealybugs. After 30 h, the level of encapsulation was the highest for eggs oviposited in adults: 58% of eggs were encapsulated, followed by third (32%) and second (4%) instars. Three days after oviposition 23, 44 and 86% of the parasitoid eggs oviposited, respectively, in adult, third and second instars were not encapsulated. The unencapsulated parasitoid eggs could hatch and continue their development. Adult mealybugs required 30 h to encapsulate 50% of the eggs, whereas in second and third instars, 50% level encapsulation was never reached. Superparasitism had a saturating effect on the immune system; reduced levels of encapsulation occurred when more than 10 eggs were oviposited in a single mealybug. Wasp larvae were never encapsulated by M. hirsutus.     

Ultrastructural studies on the fungus,Nomuraea rileyi,infecting the velvetbean caterpillar,Anticarsia gemmatalis

Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Histopathology of Nomuraea rileyi in Plathypena scabra larvae

Green cloverworm larvae. Plathypena scabra, were inoculated with Nomuraea rileyi by “tumbling” larvae in a vial of conidia. The ontogeny of the pathogen was followed by using standard histological techniques. N. rileyi conidia germinated on green cloverworm integument within 12 hr after inoculation. Germ tubes penetrated larval cuticle 36 hr after inoculation, then grew parallel to endocuticular laminae. After hyphal penetration of the epidermis ca. 4.5 days after inoculation, hyphal bodies were produced and were transported throughout the hemocoel. Hyphal bodies and hemocytes cohabited the hemocoel, but gut epithelial and muscle tissues were not invaded by Day 5. Hemocytes lysed and mycelia completely ramified throughout all larval tissues by 7 days after inoculation. Death of larvae was followed by conidiogenesis ca. 7.5 days after inoculation.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Effects of the Diadromus pulchellus ascovirus,DpAV-4, on the hemocytic encapsulation response and capsule melanization of the leek-moth pupa,Acrolepiopsis assectella

DpAV-4 is a symbiotic ascovirus found in natural populations of the solitary endoparasitoid wasp Diadromus pulchellus. The female wasp injects this virus into the pupae of the leek-moth Acrolepiopsis assectella during oviposition. The ascovirus replicates in the pupal tissues and the consequent lysis of the cells occurs synchronously with egg hatching and the development of the wasp larva. We show here that encapsulation and capsule melanization were activated when minute nylon monofilaments were implanted into the hemocoel of non parasitized leek-moth pupae and that encapsulation and melanization were inhibited in pupae parasitized by D. pulchellus. When the pupae were infected by DpAV-4, melanization of the nylon monofilaments was abolished, but a capsule was still always formed. These results indicate that DpAV-4 is a free virus able to alter the defence system of the parasitized host so as to improve the development of the parasitoid wasp, D. pulchellus.     

Oviposition and offspring survival within eggs of European corn borer: discrimination of the host embryo by female Trichogramma nubilale

Oviposition preference and offspring survival relative to the location of the host embryo was tested in the egg parasitoid Trichogramma nubilale on European corn borer host eggs. Females preferentially oviposited near to the embryo on hosts about 24 h old. Survival of Trichogramma was lower when females oviposited far from the embryo, and a higher proportion of host embryos hatched. Females did not show preferences for drilling or oviposition within very young hosts relative to where the embryo would develop, and these eggs did not produce wasps or host larvae. Female behavior was apparently adaptive in day-old hosts, not to very young hosts.     

Cotesia kariyai larvae need an anchor to emerge from the host Pseudaletia separata

Mature larvae of the gregarious endoparasitoid Cotesia kariyai construct cocoons for pupation approximately 10 days after parasitization and emerge from their host Pseudaletia separata under a long day photo-regime (16L8D) at 25 +/- 1 degrees C. The parasitoid larvae make capsules in the host hemocoel just prior to their emergence. These capsules function as "anchors," which enable them to press against the host integument from inside the host. It was predicted that this anchor might be composed of silk proteins secreted from the parasitoid larvae, because a previous study showed that the anchor was made up of a glycoprotein and that the silk gland of parasitoid larvae developed from 2nd larval stage. Fibroin-like proteins in C. kariyai larva mainly consist of two proteins with molecular masses of the 300.6 and 46.7 kDa estimated by SDS-PAGE. The fibroin-like proteins with the same molecular mass were detected from the anchor proteins just prior to parasitoid emergence. These results indicate that the anchor was assembled with fibroin-like proteins and was formed just before parasitoid emergence while in the host body cavity. Injection of bovine pancreatic trypsin inhibited the emergence of parasitoid larvae from the host because the anchor was decomposed by trypsin. Trypsin activity in the parasitized host hemolymph increased only after parasitoid emergence.     

The structure and modification of integumental tissues in Pagurus bernhardus (L.) (Decapoda: Anomura)

The histological structure of cephalothoracic and abdominal integuments has been studied in the hermit crab Pagurus bernhardus (L.). In the branchial region of the carapace, the integument shows a similar structure as described hitherto in a number of other decapod species; there are a thin epicuticle, an exocuticle, and a relatively thick endocuticle, followed by a layer of columnar epithelium and underlying connective tissue. This pattern is repeated on the inner surface of the carapace fold but with generally thinner cuticular layers. Within the connective tissue there are tegumental glands, haemocytes, and some reserve inclusions. The abdominal integument shows a modified cuticle structure which is probably related to its specific function as an adhesive organ attaching the hermit crab to the inner surface of the gastropod shell. The cuticle is uncalcified and it shows deep wrinkles and grooves. Endocuticle and exocuticle are thick and layered whereas the epicuticle is very thin. Large funnel-shaped ducts with secretions occur frequently in the abdominal integument. The cells that are responsible for these secretions are described. The chemical nature of integumental structures has been studied with histochemical tests.     

The dynamics of egg production, oviposition and resorption in a parasitoid wasp

1. The extent to which parasitoid wasps are limited by their egg supply is very important in understanding their reproductive strategies. Egg reserves are dynamic, with most wasps maturing new eggs throughout their life (synovigeny) and many species resorbing eggs that are not used in oviposition. We investigated the extent to which a parasitoid modulates its egg reserves in the light of its experience in finding hosts.
2. The egg dynamics of the Encyrtid Wasp Leptomastix dactylopii , a solitary parasitoid of mealybugs, were studied in the laboratory. This species is synovigenic and practises egg resorption.
3. We allowed newly emerged wasps to experience one of four environments of increasing value in terms of reproductive opportunities. We proposed that wasps that experienced good quality environments would maintain more mature eggs ready for oviposition. Dissection of wasps subject to different periods of host deprivation after the experimental treatment failed to confirm the hypothesis: egg load was independent of experience.
4. We also proposed that any adjustment of egg supply to make up for eggs oviposited would be effected through a reduction in egg resorption. Instead, we found that the wasp quickly made up for eggs oviposited by increased egg production.     

Stage-dependent strategies of host invasion in the egg-larval parasitoid Chelonus inanitus

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid which lays its eggs into eggs of Spodoptera littoralis (Noctuidae); the parasitoid larva then develops in the haemocoel of the host larva. Host embryonic development lasts approx. 3.5 days while parasitoid embryonic development lasts approx. 16 h. All stages of host eggs can be successfully parasitized, and we show here that either the parasitoid larva or the wasp assures that the larva eventually is located in the host's haemocoel. (1) When freshly laid eggs, up to almost 1-day-old, are parasitized, the parasitoid hatches while still in the yolk and enters the host either after waiting or immediately through the dorsal opening. (2) When 1-2-day-old eggs are parasitized, the host embryo has accomplished final dorsal closure and is covered by an embryonic cuticle when the parasitoid hatches; in this case the parasitoid larva bores with its moving abdominal tip into the host. (3) When 2.5-3.5-day-old eggs are parasitized, the wasp oviposits directly into the haemocoel of the host embryo; from day 2 to 2.5 the embryo is still very small and the wasps, after probing, often restrain from oviposition for a few hours.     

Trichogramma egg parasitism of Helicoverpa armigera on pigeonpea and sorghum in southern India

Trichogramma spp. (Hymenoptera: Trichogrammatidae) only rarely parasitize eggs of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on pigeonpea (Cajanus cajan (L.) Millspaugh), while on other plants including sorghum (Sorghum bicolor (L.)) high parasitism levels of this host are found. In this study two strategies designed to increase the parasitoid activity on pigeonpea were tested in the field: intercropping pigeonpea with sorghum and mass-releasing T. chilonis Ishii. Neither strategy led to an increase in parasitism. On pigeonpea, H. armigera oviposited >74.8% of its eggs on calyxes and pods. Parasitism levels in host eggs collected from different plant structures varied significantly with 3.6, 0.3, and 40.7% of eggs on calyxes, pods, and leaves parasitized. Earlier studies have shown that calyxes and pods possess long glandular and non-glandular trichomes, and are covered by sticky trichome exudates which inhibit parasitoid searching behaviour. Parasitism levels between 27.9 and 100% were recorded from host eggs on the intercropped sorghum. Trichogramma chilonis was the dominant parasitoid species. The mean clutch size was 2.03, but up to six parasitoids emerged per egg. Progeny sex ratio (% females) decreased with clutch size, from 63.1% at a clutch size of one to 46.0% at a clutch size of five. Sticky trap catches showed that while the parasitoid population in sorghum increased when H. armigera started ovipositing, the population within pigeonpea did not benefit from either a high parasitoid population in sorghum or a high host egg density on pigeonpea. During one of five seasons studied, however, high parasitism levels (up to 73%) were recorded on pigeonpea. During this season, H. armigera oviposited on pigeonpea plants in the vegetative growth stage and a high proportion of eggs were collected from leaves. Parasitism levels were positively correlated with the percentage of eggs collected from leaves. This study shows that the parasitization efficiency of Trichogramma spp. on pigeonpea depends mainly on the location of the host eggs. This explains why parasitism levels of H. armigera eggs on pigeonpea did not increase when intercropped with sorghum or after mass-releasing T. chilonis.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

The synthesis of hemolymph proteins by the larval epidermis of an insect Calpodes ethlius (Lepidoptera: Hesperiidae)

Sheets of the dorsal abdominal integument from fifth instar larvae of Calpodes ethlius (Lepidoptera: Hesperiidae) were incubated in artificial hemolymph in the presence of [³⁵S]methionine to investigate protein synthesis and vectorial secretion. The epidermis synthesizes and secretes at least 13 polypeptides basally and 15 apically. Two dimensional analysis of proteins labeled in vitro and in vivo showed that (a) most of the polypeptides secreted on apical and basal surfaces are different, (b) in vitro apical secretions are the same as in vivo cuticular proteins, (c) at least four of the basal secretions can be demonstrated in hemolymph labeled in vivo.Antibodies made against whole hemolymph recognized five basally secreted polypeptides and one apically secreted polypeptide both on fluorograms of immunoprecipitates and immunoblots. Arylphorin is secreted from both surfaces. Arylphorin synthesized in vitro has been identified through its precipitation by antibodies to hemolymph arylphorin in epidermis, cuticle and medium. We conclude that insect epidermis has bi-directional secretion. Cuticular proteins are carried to the apical face. A different set of proteins are carried basally to the hemolymph.     

The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5''-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.     

Genetic control of epidermis differentiation in Drosophila

In arthropods, the animal body is isolated from the external environment by a protective exoskeleton called the cuticle. The cuticle of young larvae has certainly been the most scrutinized structure in Drosophila and genetic studies of the pattern of cuticular extensions has provided the main source of our comprehension of the control of embryonic development. However, the complex structure of the cuticle remains poorly understood and analysis of the underlying epidermis has started only recently. Here I review different aspects of epidermis differentiation with the aim of presenting an integrated view of the organisation of the Drosophila integument. Although profound differences in epidermis organisation are observed across species, accumulated results suggest that epidermis formation and differentiation might share an unsuspected number of homologies between Drosophila and vertebrates.     

Comparative assessments of Gonatocerus ashmeadi and the ‘new association’ parasitoid Gonatocerus tuberculifemur (Hymenoptera: Mymaridae) as biological control agents of Homalodisca vitripennis (Hemiptera: Cicadellidae)

Egg age preference, competitive ability, and behavior of Gonatocerus tuberculifemur (‘new association’ parasitoid) and Gonatocerus ashmeadi (‘old association’ parasitoid) were investigated in the laboratory to determine if one species exhibited competitive superiority. When searching concurrently for Homalodisca vitripennis egg masses, G. ashmeadi consistently outperformed G. tuberculifemur by parasitizing 25–53% more eggs under three different experimental systems in the laboratory with varying host densities, egg ages, and exposure times. G. ashmeadi parasitism in control vials containing one parasitoid ranged from 81–97% across all egg ages. G. tuberculifemur in control vials parasitized 60–66% of eggs 1 and 3 days old, and just 18% of eggs 5 days old. G. ashmeadi produced 5–16% more female offspring than G. tuberculifemur for all experimental conditions. In comparison to G. ashmeadi, G. tuberculifemur was observed off leaves with host eggs 20% more frequently and it oviposited 15% less frequently. G. ashmeadi and G. tuberculifemur when confined together allocated ∼1% of behaviors to antennating or aggressively chasing competitors off egg masses, and up to 2% of behaviors to antennating host egg masses and/or ovipositing into eggs from the opposite side of the leaf. These latter behaviors did not occur when parasitoids were confined alone with host eggs.     

In-vitro Assays of Meloidogyne incognita and Heterodera glycines for Detection of Nematode-antagonistic Fungal Compounds

In-vitro methods were developed to test fungi for production of metabolites affecting nematode egg hatch and mobility of second-stage juveniles. Separate assays were developed for two nematodes: root-knot nematode (Meloidogyne incognita) and soybean cyst nematode (Heterodera glycines). For egg hatch to be successfully assayed, eggs must first be surface-disinfested to avoid the confounding effects of incidental microbial growth facilitated by the fungal culture medium. Sodium hypochlorite was more effective than chlorhexidine diacetate or formaldehyde solutions at surface-disinfesting soybean cyst nematode eggs from greenhouse cultures. Subsequent rinsing with sodium thiosulfate to remove residual chlorine from disinfested eggs did not improve either soybean cyst nematode hatch or juvenile mobility. Soybean cyst nematode hatch in all culture media was lower than in water. Sodium hypochlorite was also used to surface-disinfest root-knot nematode eggs. In contrast to soybean cyst nematode hatch, root-knot nematode hatch was higher in potato dextrose broth medium than in water. Broth of the fungus Fusarium equiseti inhibited root-knot nematode egg hatch and was investigated in more detail. Broth extract and its chemical fractions not only inhibited egg hatch but also immobilized second-stage juveniles that did hatch, confirming that the fungus secretes nematode-antagonistic metabolites.