Distribution of growth-blocking peptide in the insect central nervous tissue

Parasitization of the armyworm Pseudaletia separata by the endoparasitic wasp Cotesia kariyai inhibits larval growth and delays pupation, conditions necessary for proper maturation of the parasite larvae. Parasitization is correlated with an elevated level of a 25-amino-acid hormone-like peptide, growth-blocking peptide (GBP, ENFSGGCVAGYMRTPDGRCKPTFYQ). Injection of synthetic GBP into nonparasitized larvae dose dependently mimics the effects of parasitization by delaying the larval development. Here we studied the relationship between parasitization and both the production and distribution of GBP in central nervous tissues. We found that parasitization is correlated with an elevated expression of GBP mRNA, and increased concentrations of both proGBP and GBP in the host insect brain and subesophageal ganglion. The increase in proGBP precedes that of the mature GBP by about 12 h. In situ hybridization analysis using sections of parasitized and nonparasitized larval brains showed strong expression of GBP mRNA in perineural cells and/or class I neuroglia in the rind of both larval brains. The expression in parasitized larval brain-subesophageal ganglion is approximately two- to threefold higher than that in nonparasitized larvae. The presence of GBP in insect neural tissue, and its role in inhibiting growth, suggest an involvement in the regulation of neurosecretory cells.     

A putative new juvenile peptide hormone in lepidopteran insects.

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.     

Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae

A proteinaceous factor that represses plasma juvenile hormone esterase activity in parasitized insect larvae has been isolated and partially characterized from last instar larvae of the armyworm Pseudaletia separata parasitized with the wasp Apantales kariyai. Purification procedures consisted of extraction with 25% ethanol, gel filtration and reversed phase high performance liquid chromatography. Plasma juvenile hormone esterase activity in Day 3 last instar larvae was repressed by 50% when larvae were injected on Days 1 and 2 with 6.5 pmol of the purified peptide, which has a molecular weight of about 4,500 Da. The application of the factor also causes more than a 2-day delay in the onset of pupation. The sequence of 23 amino acid residues at the amino terminus of the factor was determined as follows: H-Glu-Asn-Phe-Ser-Gly-Gly-Xaa-Val-Ala-Gly-Tyr-Met- Arg-Thr-Pro-Asp-Gly-Arg-Xaa-Lys-Pro-Thr-Phe-Tyr-Gln-.     

C-terminal Elongation of Growth-blocking Peptide Enhances Its Biological Activity and Micelle Binding Affinity

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1–23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1–28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1–28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1–28 GBP undergoes a conformational transition from a random coiled state to an α-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.Growth-blocking peptide (GBP)² was initially identified from the hemolymph of armyworm Pseudaletia separata as a 25-amino acid peptide (1–25 GBP) that prevents the onset of pupation of the host by parasitization of wasp Cotesia kariyai (1–4). Injection of GBP into nonparasitized armyworm larvae early in the last instar delays larval growth and retards pupation for more than a few days. Our previous studies showed that GBP is a hormone-like biogenic peptide of the host armyworm (5, 6). In nonparasitized larvae, the concentrations of GBP were much higher in the early larval stages than in the latter ones. However, parasitization by wasp induces an elevation of GBP in the last larval stages. This elevation was shown to lead to growth retardation via repression of juvenile hormone esterase activity (7–9). Interestingly, a cDNA analysis indicated that the cDNA encodes a 23-amino acid GBP (1–23 GBP), although GBP purified from parasitized armyworm plasma consists of 25 amino acid residues. GBP was expressed as a 23-residue peptide (1–23 GBP) in nonparasitized armyworm larvae, whereas 1–25 GBP, containing Tyr²⁴ and Gln²⁵, was purified from the parasitized larvae. Moreover, the preliminary peptide sequencing of GBP prepared from parasitized larval hemolymph showed the 26^th and 27^th residues on rare occasions (Leu and Ile, respectively) (6). On the basis of these results, we concluded that the TAG stop codon for the 24^th amino acid was unusually decoded as Tyr upon parasitization by parasitoid wasps (10) and predicted that an intact and mature GBP synthesized in the parasitized armyworm larvae would consist of 28 amino acid residues (1–28 GBP).GBP has multiple functions: adhesion and spreading of a specific class of immune cells (plasmatocytes), proliferation of various cultured cells, and induction of larval paralysis (11–13). More than 10 GBP homologous peptides have been identified in Lepidopteran insects, and based on their N-terminal consensus sequences (Glu¹-Asn²-Phe³), they have been categorized as the ENF peptide family (14). The tertiary structure of 1–25 GBP consists of a disordered N-terminal region (residues Glu¹–Gly⁶), a well ordered core region (residues Cys⁷–Thr²²) stabilized by a disulfide bond and a short antiparallel β-sheet, and a short unstructured C-terminal region (Phe²³–Glu²⁵) (15). Because no GBP receptor or its gene has been isolated yet, the nature of either of them at the cellular and molecular levels is poorly understood at present. In contrast, the relationship between the structure and activity of GBP has been well studied by analyzing the biological activities of several variants of GBP and plasmatocyte-spreading peptide (one of the ENF family peptides). Especially, extensive studies on the N termini (residues 1–6) of GBP and plasmatocyte-spreading peptide demonstrated the importance of Phe³ for exerting their hemocyte stimulating activity, thereby suggesting a possible mechanism for receptor activation that requires binding of the aromatic ring of Phe³ and a closely spaced primary amine with receptor activating properties (16–19).In contrast, the C termini of GBP and other ENF peptides have received less attention, because of the weak secondary structure predictions. Therefore, in this study we focused on the C terminus region of GBP and analyzed its contribution to the expression of some biological activities and to the tertiary structure of this peptide. Especially, we prepared GBP with 28 amino acids and characterized the C-terminal region of 1–28 GBP (residues Phe²³–Thr²⁸), because we knew that GBP is present as a 23-amino acid peptide in nonparasitized healthy larvae and that GBP with 28 amino acids has been found only in parasitized host larvae. Our results suggest that the elongation of the C-terminal region of Phe²³–Thr²⁸ greatly reinforced GBP binding with the membrane. Further, the elongation increased GBP inhibition of larval growth.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Host regulation effects on Heliothis virescens (F.) larvae inducd by teratocytes of Cardiochiles nigriceps Viereck (Lepidoptera,Noctuidae-Hymenoptera,Braconidae)

Teratocytes deriving from the serosal membrane of Cardiochiles nigriceps Viereck, obtained “in vitro” from embryos hatched on a semidefined medium, were injected at different numbers and in different developmental stages of nonparasitized Heliothis virescens (F.) last instar larvae. Host development was affected by teratocyte injections and the responses registered ranged from normal to complete inhibition of pupation, according to the number of teratocytes injected and the developmental stage of the larva at time of injection. Complete pupation failure was observed when teratocytes derived from 4C nigriceps embryos were injected into 1st day 5th instar (new-slender stage) host larvae. Complete pupation occurred when teratocytes from 2 embryos were injected into 3rd or 4th day 5th instars (burrow-digging or day 1 cell formation stage). Intermediate responses, such as the formation of pupal cuticle without ecdysis or with only partial ecdysis, were obtained with intermediate teratocyte numbers, or host developmental stages. All pupae derived from teratocyte injected larvae failed to develop into adults normally obtained from control injected larvae. The larval weight just before pupation was negatively affected only when teratocyte injections were performed on 1st day 5th instar H. virescens larvae. Teratocyte injections altered the hemolymph protein titer to a level similar to that occurring in parasitized larvae. At the same time the ecdysteroid titer was characterized by a late significant increase, which reached values almost 3 times greater than found in normally parasitized larvae, and also surpassed the highest values registered for nonparasitized larvae. Ligation of parasitized larvae between the meso- and metathorax demonstrated that when the prothoracic glands were excluded, there was almost no ecdysteroid production posterior to the ligation. Ligations performed on parasitized larvae to isolate parasitoid eggs before hatching in the last abdominal segments, demonstrated that only virus and venom determined a reduction of the ecdysteroid titer. On the basis of these results the possible role of teratocytes in affecting the biological activity of ecdysteroids is postulated and discussed in a wider context of host-parasitoid physiological interactions.     

Structure of a growth-blocking peptide present in parasitized insect hemolymph

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.     

Endocrine alteration and precocious premetamorphic behaviors in the greater wax moth larvae,Galleria mellonella,parasitized by an endoparasitoid,Apanteles galleriae

Parasitization of Galleria mellonella (Lepidoptera: Pyralididae) larvae by a larval endoparasitoid Apanteles galleriae (Hymenoptera: Braconidae) leads to the precocious expression of premetamorphic behavior in the sixth (normally penultimate) instar host larvae prior to the parasitoid's emergence. We investigated the role of parasitization with A. galleriae on the alteration of development and/or behavior of its host. The ecdysteroid titer in the hemolymph of parasitized sixth instar larvae (the last instar of parasitized larvae) was higher than that of unparasitized ones, and the high ecdysteroid concentrations induced premetamorphic behaviors such as wandering and cocoon spinning. However, the epidermis of the parasitized larvae was not pupally committed through this stage. The activity of JH esterase in the parasitized larvae remained low, and application of a JH analogue to these larvae caused the production of a larval-type cocoon. These facts suggest that the parasitization by A. galleriae induces precocious premetamorphic behaviors of G. mellonella larvae by changing host endocrine conditions without causing the typical larval-pupal metamorphosis. Arch. Insect Biochem. Physiol. 34:257–273, 1997. © 1997 Wiley-Liss, Inc.     

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda (J. E. Smith), associated with parasitism by the braconid parasitoid Cotesia marginiventris (Cresson)

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda, associated with parasitism by the parasitoid Cotesia (= Apanteles) marginiventris were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis. As early as hour 4 after parasitization treatment, several electrophoretically slow-migrating, high-molecular-weight proteins were detected in the host's haemolymph. These proteins were detected earlier in haemolymph from parasitized larvae than in haemolymph from control larvae, and their concentrations were higher in heavily parasitized host larvae (≥ 3 eggs/host) than in lightly parasitized larvae (1 egg/host). Additionally, unique proteins that migrated electrophoretically with bovine serum albumin appeared in the haemolymph of parasitized larvae at hour 8 after parasitization treatment and were evident in haemolymph collected through to hour 64.     

Inhibition of testicular growth and development in Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata

Tobacco hornworm larvae parasitized by the gregarious larval endoparasitoid Cotesia congregata exhibited an inhibition in testicular growth and development, the extent of which was determined by the age and developmental stage of the host at the time of parasitization. The degree of parasitic castration, as assessed by measurements of testicular volume, was correlated with the stadium in which parasitization occurred. A mathematical formula requiring the measurement of testicular length, width and depth was used to calculate testicular volume. The use of the depth parameter revealed a negative correlation between host weight and testicular volume in parasitized larvae. Testicular volumes of fifth instar hosts, which had been parasitized in the first stadium, were significantly smaller than those originally parasitized as fourth or fifth instar larvae and were not correlated with parasitoid load. Effects of natural parasitism were not duplicated by injections of C. congregata polydnavirus and venom, topical treatment with the juvenile hormone analog methoprene, or starvation of nonparasitized larvae. Larvae receiving virus plus venom or methoprene grew larger due to delayed wandering and had larger testes than controls. Deleterious effects on host testes may be due to the effects of nutrient competition between the developing parasitoid progeny and the gonads, combined with the juvenilizing effects believed to be caused by the polydnavirus.     

Developmental and behavioural responses of larval Trichoplusia ni to parasitization by an imported braconid parasite Chelonus sp

ABSTRACT. Larval Trichoplusia ni (Hübner) (Noctuidae) parasitized by Chelonus sp. (near curvimaculatus ) (Braconidae) precociously initiated pupation during the penultimate fourth instar. The temporal sequence of developmental markers exhibited by parasitized T. ni closely matched the temporal sequence in normal, pupating larvae. The parasitized larvae did not complete pupation, but consistently stopped development at a stage recognizable by a certain set of markers. This halt was observed in hosts from which parasites emerged and from hosts which had been stung but from which no parasites emerged. Weight gain and food consumption by parasitized hosts were significantly lower than normal, although most reached the fourth instar at the same time as normal larvae. Measurement of head capsule widths indicated that the width in precociously pupating larvae was less than the critical width associated with attainment of the pupation instar of normal larvae.     

Insect cytokine growth-blocking peptide may regulate density-dependent phase trait of cuticular melanization in the larval armyworm,Mythimna separata

Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found in lepidopteran insects that has diverse biological activities such as larval growth regulation, paralysis induction, cell proliferation and stimulation of immune cells. Density-dependent phase polyphenism is a phenomenon of phenotypic plasticity in which the expression of a variety of traits can be affected by local population density. In the present study, the armyworm Mythimna separata larvae with four rearing densities (1 larva/vial, 2 larvae/vial, 4 larvae/vial and 6 larvae/vial) were tested for cuticular melanization and body weight throughout the third-fifth instar, and the functional role of GBP in regulating the changes was investigated. The results indicated that when reared at high densities, the larvae exhibited less body weight and more degree of cuticular melanization than larvae reared at low densities. The gene expression of GBP in armyworm larvae showed an initial rise and then decline trend with increased rearing densities in the third to fifth instar. Compared with control, more degree of cuticular melanization was observed in GBP-injected larvae (500 ng/larva in volume 50 μL) than that in Ringer’s solution-injected counterparts. Furthermore, the gene expression level of dopa decarboxylase and prophenoloxidase increased significantly in GBP-injected fifth instar larvae from 6 h to 12 h after injection, suggesting the role of GBP in modulating density-dependent phase trait of armyworm cuticular melanization.     

Juvenile hormone synthesis, metabolism, and resulting haemolymph titre in Heliothis virescens larvae parasitized by Toxoneuron nigriceps

Last instar larvae of the tobacco budworm, Heliothis virescens F., fail to pupate and have little 20-hydroxyecdysone when parasitized by Toxoneuron nigriceps (Viereck). In this paper, we extend these observations to juvenile hormone (JH) to determine if parasitism by this wasp affects other endocrine systems. To this end, we compared the production of JH by corpora cardiaca-corpora allata complexes (CC-CA), the metabolism of JH by haemolymph enzymes, and the haemolymph titre of JH in parasitized and non-parasitized control larvae of H. virescens during the last larval instar. CC-CA from parasitized and control larvae had similar peaks of JH synthesis on day 1 of the fifth instar, with JH II accounting for more than 90% of total JH in both groups. On subsequent days, JH synthesis dropped to undetectable levels more quickly in non-parasitized controls than in parasitized larvae. JH metabolism by haemolymph of parasitized and control animals increased from low levels on day 1 of the fifth instar to high levels on days 2 and 3 of the instar. JH metabolism was significantly higher in control larvae than in parasitized larvae. After day 3, JH metabolism decreased in both groups, but was significantly higher in parasitized larvae. The major metabolite of JH in both groups was JH acid, though traces of JH diol and JH acid diol were also detected. The haemolymph titre of JH in both groups peaked on day 1 of the fifth instar and, similar to the synthesis of JH by CC-CA, decreased more rapidly in control larvae. As a result, non-parasitized animals had significantly lower JH titres on day 2. The higher JH titres observed in parasitized larvae during the early fifth instar may contribute to their developmental arrest. The possible role of these JH alterations in the host developmental and metabolic redirection is discussed and a more comprehensive physiological model accounting for host-parasitoid interactions is proposed.     

Venom of ectoparasitoid,Euplectrus sp near plathypenae (Hymenoptera: Eulophidae) regulates the physiological state of Pseudaletia separata (Lepidoptera: Noctuidae) host as a food resource

Euplectrus sp. near plathypenae is an ectoparasitoid that can parasitize from 3rd to day 0-6th instar Pseudaletia separata. The developmental period of the parasitoid from the egg to the pupal stage is about 13 days. Parasitized hosts are developmentally arrested and never molt to the next stadium. The injection of venom fluid results in similar effects on P. separata larvae as does parasitization. The inhibitory effect of the venom on molting was dose dependent. Injection of 0.3 female equivalents of venom into day 0-5th host instar resulted in a similar developmental arrest as seen in parasitized hosts. The amount of total lipid in the hemolymph of the host increased as a function of the amount of venom injected, while the lipid content of the fat body was similar to lipid levels in the fat body of parasitized larvae. The amount of total protein in the hemolymph also increased when venom was injected, whereas the protein level of the fat body did not increase. The lipid concentration within the parasitoid larva was maintained at the same level throughout larval development, but increased before pupation. We conclude that the injected venom increased the hemolymph content of lipid and protein to support the growth and development of the ectoparasitoid larva.     

Juvenile hormone inhibits the synthesis of major larval haemolymph proteins in rice moth, Corcyra cephalonica

Larval haemolymph proteins (LHP), LHP49 and LHP46 are produced in the penultimate and last larval instars. Starvation during the early and mid stage of last instar development prevents the production of both LHPs. Decapitation in early and mid last instar stimulates LHP synthesis and their concentration in haemolymph increases, while ligation in last instar larvae blocks the production of LHPs. Application of exogenous JH lowers the synthesis of LHP49 and LHP46 in Corcyra. These observations suggests that LHP49 and LHP46 synthesis is activated during the periods when JH titres are either low or undetectable.     

Does superparasitism improve host suitability for parasitoid development? A case study in the Microplitis rufiventris-Spodoptera littoralis system

Superparasitism refers to the oviposition behavior of parasitoid females who lay their eggs in an already parasitized host. Recent studies have shown that allocation of additional eggs to an already parasitized host may be beneficial under certain conditions. In the present work, mortality of Microplitis rufiventris wasps was significantly influenced by both host instar of Spodoptera littoralis larvae at parasitism and level of parasitism. In single parasitization, all host instars (first through sixth) were not equally suitable. Percentage of emergence success of wasp larvae was very high in parasitized first through third (highly suitable hosts), fell to 60% in the fourth instar (moderate suitable) and sharply decreased in the penultimate (5th) instars (marginally suitable). Singly parasitized sixth (last) instar hosts produced no wasp larvae (entirely unsuitable), pupated and eclosed to apparently normal adult moths. The scenario was different under superparasitism, whereas supernumerary individuals in the highly suitable hosts were almost always killed as first instars, superparasitization in unsuitable hosts (4th through 6th) had significant increase in number of emergence success of wasp larvae. Also, significantly greater number of parasitoid larvae successfully developed in unsuitable hosts containing three wasp eggs than counterparts containing two wasp eggs. Moreover, the development of surplus wasp larvae was siblicidal in earlier instars and nonsiblicidal gregarious one in the penultimate and last “sixth” instars. It is suggested that the optimal way for M. rufiventris to deal with high quality hosts (early instars) is to lay a single egg, while the optimal way to deal with low quality hosts (late instars) might be to superparasitize these hosts.     

Parasitism-induced effects on host growth and metabolic efficiency in Plutella xylostella larvae parasitized by Cotesia vestalis or Diadegma semiclausum

The nutritional physiology of the diamondback moth, Plutella xylostella, larvae was examined after parasitization by the solitary endoparasitoids Cotesia vestalis or Diadegma semiclausum. Examinations were performed in two phases, one was examined at the time point of 24 h post‐parasitization, and the other was done at the end of the 4th instar larvae of host. Rates of growth, food consumption, assimilation, excretion, and respiration were calculated as well as approximate digestibility and the rate ratios ECI (percent efficiency of conversion of ingested food to body substance), and ECD (percent efficiency of conversion of digested food to body substance). Parasitization by C. vestalis resulted in significant decrease in the rates of growth, feeding, excretion, assimilation, and respiration, but the final dry rate of respiration at the end of last larval stadium was elevated. The ECI and ECD were also reduced as the result of parasitization, but digestibility was increased. All these parameters in the larvae parasitized by D. semiclausum at 24 h post‐parasitization were also significantly changed compared to the control; however, these differences were quantitatively, but not qualitatively before pupation, similar to those resulted from parasitization by C. vestalis. In spite of the similarities of the parasitism‐induced effects caused by these endoparasitoids, the final metabolic rate, that is, the rate of intake of nutrients required to compensate for metabolism, was much lower in the larvae parasitized by C. vestalis than that of the larvae parasitized by D. semiclausum. All of the results discussed here will contribute toward explaining the different ways these two wasps regulate the parasitoid‐host relationship.     

Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis

The cDNA corresponding to an inhibitor of apoptosis (IAP) from the Egyptian armyworm, Spodoptera littoralis, was cloned by RT-PCR. Sequence analysis showed that the IAP of S. littoralis (SlIAP) contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger, an organization which is very similar to that of other lepidopteran IAPs. SlIAP mRNA was detected in ovary, testis, salivary gland, fat body, epidermis, brain and midgut of S. littoralis. During the last larval instar, prepupal and pupal stages, brain mRNA levels remained approximately constant, whereas those of midgut showed a large peak centred in the prepupal stage. Midgut morphology changed during metamorphosis from a semi-transparent, cylindrical structure in last instar larvae to a brownish globular mass in pupae. TUNEL assays, LysoTracker staining and caspase-3 immunohistochemistry, indicated that programmed cell death in midgut starts actively at the onset of pupation process, coinciding with the dramatic decrease of SlIAP mRNA levels observed at the same time.     

Inhibitory effects of parasitism by the gregarious endoparasitoid Cotesia congregata on host testicular development

Cotesia congregata is a gregarious larval endoparasitoid of the tobacco hornworm, Manduca sexta. Parasitized larvae exhibit a variety of physiological and developmental aberrations, the most obvious of which is the induction of developmental arrest characterized by the absence of wandering behavior and suppression of pupation. This arrest appears attributable to continued maintenance of an elevated titer of juvenile hormone and reduced levels of hemolymph juvenile hormone esterase activity. Injection of the wasp's polydnavirus into nonparasitized larvae also causes arrest and the larvae eventually form larval-pupal intermediates instead of normal pupae, indicating the virus may be partially responsible. Aside from causing arrested host development, parasitism also inhibits the normal development and differentiation of testes in male host larvae, so that the testes atrophy instead of growing synchronously with other larval tissues. Here we report that parasitism has pronounced disruptive cytological effects on the developing reproductive organs of male hosts, in addition to causing them to atrophy. Parasitism results in a reduction in testicular volume attributable to a reduction in the number of developing germ cells. Microscopy revealed that the structural integrity of the sheaths surrounding the testicular follicles also is disrupted, so that the tissues appear grossly abnormal compared to those of nonparasitized larvae. Intrahemocoelic injection of purified C. congregata polydnavirus in combination with venom into nonparasitized fourth instar larvae, or topical application of 100 μg of methoprene to fourth instar larvae, also alters sheath integrity and reduces the numbers of developing germ cells, but not to the same degree as the pattern observed in truly parasitized hosts. The occurrence of cell death in the male gonad was documented using the vital dyes acridine orange and ethidium bromide. Arch. Insect Biochem. Physiol. 36:95–114, 1997. © 1997 Wiley-Liss, Inc.