Host Fat Body Research Articles

First Record, Distribution and Occurrence of A Protistan Entomopathogen, Adelina mesnili Perez (Coccidia: Adeleidae) in the Indian Meal Moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) Populations in Türkiye.

Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) originating from South America is one of the important insect pests that damages storage products and is found on every continent. There is a new interest in using enthomopathogens for microbial control of P. interpunctella as well as other stored product pests. Coccidia as a group of protistan entomopathogens are host specific and their pathogenic effects on the hosts are more pronounced. Although this pathogenic effect results in increased host mortality or higher susceptibility to insecticides, the suppressive potential of coccidia in natural populations has not been adequately studied. In this study, characterization, distribution and occurrence of a coccidian entomopathogen was aim to show its natural suppressing potential in P. interpunctella populations. During the three years (from 2019 to 2021), a total of 3.432 P. interpunctella samples (2.047 dead and 413 living larvae, 932 adults and 40 pupae) were collected from fourteen populations. After macroscopic examination, suspected samples were dissected in Ringer's solution and then prepared wet smears including host fat body were examined for presence of coccidian pathogens under a light microscope at a magnification of 400-1000X. The oocysts of the coccidian were measured and photographed using a microscope with a digital camera and soft imaging system. The pathogen was observed in the fat bodies of the larvae, pupae and adults. Oocysts measured as 29.52±3.32 (25.27- 35.08) μm in diameter and they include 8 sporocysts. Sporocysts measured as 9.11±0.61 (8.90-9.85) μm. Forty-five of 3.432 P. interpunctella larvae, pupae and adults were found to be infected. Coccidian infections have also reached to the levels that can be considered high in some populations, as significant as 29.2%. The infection was observed in the three (21.4%) of the examined fourteen populations. The coccidian entomopathogen presented in this study is the first Adeleid coccidian record from P. interpunctella populations in Türkiye. The detection of Adelina mesnili Perez (Coccidia: Adeleidae) in at least three populations and the infection rate reaching 29.2 percent, confirms that this pathogen has a considerable effect P. interpunctella populations that cannot be underestimated. Our results confirm that the coccidian pathogen is very effective in the larval stage.

Cloak Scavenges the Reactive Oxygen Species around the Larvae of Drino inconspicuoides (Diptera: Tachinidae).

Drino inconspicuoides (Diptera: Tachinidae) is an endoparasitoid that develops inside the lepidopteran host. When the larva of D. inconspicuoides penetrates into the host, Mythimna separata (Lepidoptera: Noctuidae), the larva creates a cap-like structure, called the funnel, by using host hemocytes, forming a respiratory attachment to permit efficient respiration. A newly described cloudy and cottony structure, called the "cloak", is formed outside the funnel within 24 h of parasitism. The cloak contains the host fat body and hemocytes. In this study, we aimed to clarify the function of the cloak, which has to date remained unknown. We hypothesized that the funnel generates reactive oxygen species (ROS) through melanization, and that the cloak detoxifies them. We confirmed that the black pigments of the funnel were caused by melanization, which inevitably generates ROS that are potentially harmful to the D. inconspicuoides larva inside the funnel. The cloak showed high activities of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase. These results suggest that the cloak scavenged the ROS from the melanized funnel through the diversion of antioxidant enzymes in the fat body, thereby protecting the D. inconspicuoides larva from oxidative damage.

Parasite reliance on its host gut microbiota for nutrition and survival

Studying the microbial symbionts of eukaryotic hosts has revealed a range of interactions that benefit host biology. Most eukaryotes are also infected by parasites that adversely affect host biology for their own benefit. However, it is largely unclear whether the ability of parasites to develop in hosts also depends on host-associated symbionts, e.g., the gut microbiota. Here, we studied the parasitic wasp Leptopilina boulardi (Lb) and its host Drosophila melanogaster. Results showed that Lb successfully develops in conventional hosts (CN) with a gut microbiota but fails to develop in axenic hosts (AX) without a gut microbiota. We determined that developing Lb larvae consume fat body cells that store lipids. We also determined that much larger amounts of lipid accumulate in fat body cells of parasitized CN hosts than parasitized AX hosts. CN hosts parasitized by Lb exhibited large increases in the abundance of the bacterium Acetobacter pomorum in the gut, but did not affect the abundance of Lactobacillus fructivorans which is another common member of the host gut microbiota. However, AX hosts inoculated with A. pomorum and/or L. fructivorans did not rescue development of Lb. In contrast, AX larvae inoculated with A. pomorum plus other identified gut community members including a Bacillus sp. substantially rescued Lb development. Rescue was further associated with increased lipid accumulation in host fat body cells. Insulin-like peptides increased in brain neurosecretory cells of parasitized CN larvae. Lipid accumulation in the fat body of CN hosts was further associated with reduced Bmm lipase activity mediated by insulin/insulin-like growth factor signaling (IIS). Altogether, our results identify a previously unknown role for the gut microbiota in defining host permissiveness for a parasite. Our findings also identify a new paradigm for parasite manipulation of host metabolism that depends on insulin signaling and the gut microbiota.

Varroa destructor mites vector and transmit pathogenic honey bee viruses acquired from an artificial diet.

The ectoparasitic mite Varroa destructor is one of the most destructive pests of the honey bee (Apis mellifera) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) (i.e., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa-mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in ingested host tissues or the mites themselves. Here we describe a system that includes an artificial diet free of insect tissue-derived components for maintaining Varroa mites for in vitro experimentation. Using this system, together with the novel engineered cDNA clone-derived genetically tagged VDV-1 and wild-type DWV, we demonstrated for the first time that Varroa mites provided an artificial diet supplemented with engineered viruses for 36 hours could acquire and transmit sufficient numbers of virus particles to establish an infection in virus-naïve hosts. While the in vitro system described herein provides for only up to five days of mite survival, precluding study of the long-term impacts of viruses on mite health, the system allows for extensive insights into the dynamics of Varroa-mediated vectoring and transmission of honey bee viruses.

Lipidomics reveals how the endoparasitoid wasp Pteromalus puparum manipulates host energy stores for its young

Endoparasitoid wasps inject venom along with their eggs to adjust the physiological and nutritional environment inside their hosts to benefit the development of their offspring. In particular, wasp venoms are known to modify host lipid metabolism, lipid storage in the fat body, and release of lipids into the hemolymph, but how venoms accomplish these functions remains unclear. Here, we use an UPLC-MS-based lipidomics approach to analyze the identities and concentrations of lipids in both fat body and hemolymph of host cabbage butterfly (Pieris rapae) infected by the pupal endoparasitoid Pteromalus puparum. During infection, host fat body levels of highly unsaturated, soluble triacylglycerides (TAGs) increased while less unsaturated, less soluble forms decreased. Furthermore, in infected host hemolymph, overall levels of TAG and phospholipids (the major component of cell membranes) increased, suggesting that fat body cells are destroyed and their contents are dispersed. Altogether, these data suggest that wasp venom induces host fat body TAGs to be transformed into lower melting point (more liquid) forms and released into the host hemolymph following infection, allowing simple absorption and nutritional acquisition by wasp larvae. Finally, cholesteryl esters (CEs, a dietary lipid derived from cholesterol) increased in host hemolymph following infection with no concomitant decrease in host cholesterol, implying that the wasp may provide this necessary food resource to its offspring via its venom. This study provides novel insight into how parasitoid infection alters lipid metabolism in insect hosts, and begins to uncover the wasp venom proteins responsible for host physiological changes and offspring development.

Effects of plant-mediated differences in host quality on the development of two related endoparasitoids with different host-utilization strategies

Among parasitoids that develop inside the bodies of feeding, growing hosts (so-called ‘koinobiont’ endoparasitoids), two strategies have evolved to dispose of host resources. The larvae of one group consumes most host tissues before pupation, whereas in the other the parasitoid larvae consume only host hemolymph and fat body and at maturity emerge through the host cuticle to pupate externally. Here we compared development and survival (to adult emergence) of two related larval endoparasitoids (Braconidae: Microgastrinae) of the diamondback moth, Plutella xylostella. Larvae of Dolichogenidea sicaria are tissue feeders whereas larvae of Cotesia vestalis are hemolymph feeders. Here, development of P. xylostella and the two parasitoids was compared on three populations (one cultivar [Cyrus], two wild, [Winspit and Kimmeridge]) of cabbage that have been shown to vary in direct defense and hence quality. Survival of P. xylostella and C. vestalis (to adult eclosion) did not vary with cabbage population, but did so in D. sicaria, where survival was lower when reared on the wild populations than on the cultivar. Furthermore, adult herbivore mass was significantly higher and development was significantly shorter in moths reared on the cultivar. The tissue-feeing D. sicaria was larger but took longer to develop than the hemolymph-feeder C. vestalis. The performance of both parasitoids was better on the cabbage cultivar than on the wild populations, although the effects were less apparent than in the host. Our results show that (1) differences in plant quality are diffused up the food chain, and (2) the effects of host quality are reflected on the development of both parasitoids.

Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H2 O2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv.

Development of two related endoparasitoids in larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

We compare the growth and development of two related solitary endoparasitoids (Braconidae, Microgastinae) in different instars (second and third) of the diamondback moth Plutella xylostella. Cotesia vestalis is a well-studied parasitoid whose larvae feed primarily on host hemolymph and fat body whereas Dolichogenidea sicaria is a parasitoid whose larvae consume the entire host caterpillar before pupation. Little is known about the biology of D. sicaria including its association with P. xylostella. When developing in L2 and L3 host instars, survival of both parasitoids to adult was similar. However, development time was longer in D. sicaria than in C. vestalis but the adult wasps were also larger. Both species were protandrous and exhibit sexual size dimorphism, where females were the larger sex. Our results suggest that D. sicaria is a promising new biological control agent of P. xylostella, augmenting better studied parasitoids such as C. vestalis.

Morphological and molecular characterization of a microsporidian parasite, Takaokaspora nipponicus n. gen., n. sp. from the invasive rock pool mosquito, Ochlerotatus japonicus japonicus

A new genus and species of Microsporidia, Takaokaspora nipponicus n. gen., n. sp. is described from Ochlerotatus japonicus japonicus (Theobald) and Ochlerotatus hatorii (Yamada) based on light microscope and ultrastructural morphology, developmental features, transmission cycles and comparative sequence analyses of the small subunit ribosomal DNA (SSU rDNA). The microsporidium is both vertically and horizontally transmitted, exhibits dimorphic development alternating between diplokaryotic and monokaryotic stages and produces two morphologically distinct spores, one in larvae and another in adult females. Horizontal transmission of infection to larval mosquitoes occurs via direct oral ingestion of uninucleate spores that are produced in vertically-infected larval hosts. Development in horizontally-infected hosts is diplokaryotic following karyokinesis of uninucleate schizonts and binary fission to produce small (4.3μm×2.0μm) membrane free, ovoid, binucleate spores that are confined to adult female reproductive tissues (ovariole sheath and oviducts). Vertical transmission of the microsporidium from adult females to larval progeny takes place via surface contamination of the egg (transovum). Microsporidian development in vertically-infected larvae is haplophasic with unpaired nuclei throughout, producing rosette-shaped sporogonial plasmodia contained within a thin non-persistent sporophorous vesicle and culminating in the formation of membrane free, uninucleate, conical spores (7.0μm×2.8μm). Development is confined to host fat body tissue which appears as swollen white masses in the thorax and selected segments of the abdomen causing larvae to appear abnormally distorted and results in death during the third and fourth instar stages. The SSU rDNA sequences obtained from the two morphologically identical microsporidia isolated from Oc. j. japonicus and Oc. hatorii were nearly identical and unique when compared with GenBank entries of all other mosquito-parasitic species. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and bootstrap analyses using the Neighbor Joining search parameter yielded similar typologies. In each case, the novel microsporidium was the sister group to the clade containing Parathelohania species from Anopheles mosquitoes and the monotypic Novothelohania ovalae from Ochlerotatus caspius showing approximately 10–13% sequence divergence to those two genera providing strong support for establishment as a separate genus.

Expression of immune-response genes in lepidopteran host is suppressed by venom from an endoparasitoid, Pteromalus puparum.

BackgroundThe relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae) inject venom along with eggs into their hosts. P. puparum does not inject polydnaviruses during oviposition. For this reason, P. puparum and its pupal host, the small white butterfly Pieris rapae (Lepidoptera: Pieridae), comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. P. puparum venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we tested our hypothesis that P. puparum venom influences host gene expression in the two main immunity-conferring tissues, hemocytes and fat body.ResultsAt 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified in silico, including 62 unknown contigs) derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified in silico, including 123 unknown contigs) from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH) data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection.ConclusionsOur findings support our hypothesis that P. puparum venom influences gene expression in host hemocytes and fat body. Specifically, the venom treatments led to reductions in expression of a large number of genes. Many of the down-regulated genes act in immunity, although others act in non-immune areas of host biology. We conclude that the actions of venom on host gene expression influence immunity as well as other aspects of host biology in ways that benefit the development and emergence of the next generation of parasitoids.

Inorganic elements in the fat bodies of Diatraea saccharalis (Lepidoptera: Crambidae) larvae parasitized by Cotesia flavipes (Hymenoptera: Braconidae)

Koinobiont parasitoids use several strategies to regulate the host's physiological processes during parasitism. Although many aspects of host-parasitoid interactions have been explored, studies that attempted to assess the effects of parasitism on the availability of inorganic elements in the host are virtually nonexistent. Therefore, we aimed to evaluate the effects of parasitism on the concentrations of inorganic elements in the fat bodies of larvae of Diatraea saccharalis (Lepidoptera: Crambidae) during the development of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae), by using total reflection X-ray fluorescence (TXRF). TXRF analysis allowed comparisons of the changes in the availability of the elements P, S, K, Ca, Cr, Fe, Ni, Cu, and Zn in the fat body tissues of D. saccharalis larvae parasitized by C. flavipes. Overall, the concentration of inorganic elements was higher early in parasitoid development (1 and 3days after parasitism) compared to non-parasitized larvae, but much lower towards the end of parasitoid development (7 and 9days after parasitism). Ca, K, and S were reduced after the fifth day of parasitism, which affected the total abundance of inorganic elements observed in the fat bodies of the parasitized hosts. The regulatory mechanisms or pathological effects related to the observed variation of the host inorganic elements induced by the parasitoid remain unknown, but there might be a strategy to make these elements available to the parasitoid larvae at the end of their development, when higher metabolic activity of the host fat body is required to sustain parasitoid growth. The observed variation of the host's inorganic elements could also be related to the known effects of parasitism on the host's immune response.

Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata

The solitary endoparasitoid Meteorus pulchricornis can parasitize many lepidopteran host species successfully. In the case of parasitization of Pseudaletia separata, developmental duration of M. pulchricornis was 8–9 days from egg to larval emergence and 6 days from prepupa to adult emergence. Successful parasitism by M. pulchricornis decreased with host age. Following parasitization of day-0 4th host instar, the parasitoid embryo, whilst still enclosed in serosal cell membrane, hatched out of the egg chorion 2 days after oviposition. Subsequently, the 1st instar parasitoid emerged from the surrounding serosal cell membrane. Serosal cells dissociated and developed as teratocytes 3.5 days after oviposition. One embryo of M. pulchricornis gave rise to approximately 1200 teratocytes, a number that remained constant until 6 days after parasitization, but decreased drastically to 200 at 7 days post-oviposition. The teratocytes of M. pulchricornis were round- or oval-shaped and grew from 65 μm at 4 days to 200 μm in the long axis at 6 days post-parasitization. At 4 days post-parasitization, many cells or cell clusters with lipid particles were observed in the hemocoels of parasitized hosts. In addition, paraffin sections of parasitized hosts revealed that many teratocytes were attached to the host's fat body and contributed to disrupting the fat body tissue. Further, examination of the total hemocyte count (THC) during parasitization revealed that THC was maintained at low levels. Surprisingly, a temporal decrease followed by restoration of THC was observed in hosts injected with virus-like particles of M. pulchricornis (MpVLPs) plus venom, which contrasts with the constant THC suppression seen in parasitized hosts. This indicates that MpVLP function is temporal and is involved in regulation of the host during early parasitism. Therefore, teratocytes, a host regulation factor in late parasitism, could be involved in keeping THC at a low level.

Characterization of the IκB-like gene family in polydnaviruses associated with wasps belonging to different Braconid subfamilies

Polydnaviruses (PDVs) are obligate symbionts of hymenopteran parasitoids of lepidopteran larvae that induce host immunosuppression and physiological redirection. PDVs include bracoviruses (BVs) and ichnoviruses (IVs), which are associated with braconid and ichneumonid wasps, respectively. In this study, the gene family encoding IkappaB-like proteins in the BVs associated with Cotesia congregata (CcBV) and Toxoneuron nigriceps (TnBV) was analysed. PDV-encoded IkappaB-like proteins (ANK) are similar to insect and mammalian IkappaB, an inhibitor of the transcription factor nuclear factor kappaB (NF-kappaB), but display shorter ankyrin domains and lack the regulatory domains for signal-mediated degradation and turnover. Phylogenetic analysis of ANK proteins indicates that those of IVs and BVs are closely related, even though these two taxa are believed to lack a common ancestor. Starting from a few hours after parasitization, the transcripts of BV ank genes were detected, at different levels, in several host tissues. The structure of the predicted proteins suggests that they may stably bind NF-kappaB/Rel transcription factors of the tumour necrosis factor (TNF)/Toll immune pathway. Accordingly, after bacterial challenge of Heliothis virescens host larvae parasitized by T. nigriceps, NF-kappaB immunoreactive material failed to enter the nucleus of host haemocytes and fat body cells. Moreover, transfection experiments in human HeLa cells demonstrated that a TnBV ank1 gene product reduced the efficiency of the TNF-alpha-induced expression of a reporter gene under NF-kappaB transcriptional control. Altogether, these results suggest strongly that TnBV ANK proteins cause retention of NF-kappaB/Rel factors in the cytoplasm and may thus contribute to suppression of the immune response in parasitized host larvae.

Divergences in Protein Activity and Cellular Localization within the Campoletis sonorensis Ichnovirus Vankyrin Family

Ichnoviruses (IVs) occur in obligate symbiotic associations with endoparasitic ichneumonid wasps. IVs are injected with eggs during parasitization, where viral infection and gene expression alter host physiology to ensure endoparasitoid survival. The seven Campoletis sonorensis IV (CsIV) vankyrin genes encode proteins that possess ankyrin repeat domains resembling the inhibitory domains of NF-kappaB transcription factor inhibitors (IkappaBs). The CsIV vankyrins are divided into two subclasses: those expressed primarily in the host fat body (three genes) and those expressed in host hemocytes (four genes). CsIV vankyrin proteins showed limited antigenic similarity when analyzed by Western blotting. Cellular localization and expression patterns of recombinant vankyrin proteins in High Five and Sf9 insect cells differed within and between the subclasses and in cells exposed to lipopolysaccharide, laminarin, or viral immune challenge. In unstimulated Sf9 cells, five vankyrins were detected in cell nuclei. The remaining two proteins localized predominantly to cytoplasmic granules. Immune stimulation of cells resulted in a nuclear-to-cytoplasmic shift of three vankyrins but did not affect localization of other variants. When expressed from recombinant Autographa californica multiple nucleopolyhedroviruses (AcMNPVs), all vankyrins showed a nuclear localization during early stages of infection with patterns resembling those of immune-challenged cells as the infection progressed. Two fat body vankyrins also produced unique biological effects when expressed from recombinant AcMNPV. Insect cells infected with these viruses exhibited enhanced longevity compared to those infected with viruses expressing other vankyrins. Together, these data suggest that vankyrin proteins in CsIV have divergent physiological functions.

Ultrastructural analysis supports transferring Nosema whitei Weiser 1953 to the genus Paranosema and creation a new combination, Paranosema whitei

The current ultrastructural description of Nosema whitei is in agreement with the genus definition of Paranosema [Sokolova, Y.Y., Dolgikh, V.V., Morzhina, E.V., Nassonova, E.S., Issi, I.V., Terry, R.S., Ironside, J.E., Smith, J.E., Vossbrinck, C.R., 2003. Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg. Journal of Invertebrate Pathology 84, 159–172]. In addition to exhibiting similar spore morphology and sporogony type, N. whitei, like P. grylli and Paranosema locustae, possesses a distinct meront–sporont transitional stage in the life cycle; develops in the host fat body, and produces secretory material arranged in “tubular structures” during sporogony. Ultrastructural analysis supports the similarity of N. whitei to P. grylli and to P. locustae as predicted on the basis SSrDNA sequence data (GenBank Accession Nos. AY305323, AY305325, and AY305324). Comparative studies of these three related species provide a good example of the consistency of morphological and sequence data, and support both the placement of N. whitei inside the genus Paranosema and the validity of the new combination Paranosema whitei (Weiser) . Supported by Russian Foundation for Basic research: Grants 04-04-49314 and 03-04-49629.

Iκβ-Related vankyrin Genes in the Campoletis sonorensis Ichnovirus: Temporal and Tissue-Specific Patterns of Expression in Parasitized Heliothis virescens Lepidopteran Hosts

Polydnaviruses (PDVs) are unusual insect viruses that occur in obligate symbiotic associations with parasitic ichneumonid (ichnoviruses, or IVs) and braconid (bracoviruses, or BVs) wasps. PDVs are injected with eggs, ovarian proteins, and venom during parasitization. Following infection of cells in host tissues, viral genes are expressed and their products function to alter lepidopteran host physiology, enabling endoparasitoid development. Here we describe the Campoletis sonorensis IV viral ankyrin (vankyrin) gene family and its transcription. The seven members of this gene family possess ankyrin repeat domains that resemble the inhibitory domains of the Drosophila melanogaster NF-kappabeta transcription factor inhibitor (Ikappabeta) cactus. vankyrin gene expression is detected within 2 to 4 h postparasitization (p.p.) in Heliothis virescens hosts and reaches peak levels by 3 days p.p. Our data indicate that vankyrin genes from the C. sonorensis IV genome are differentially expressed in the tissues of parasitized hosts and can be divided into two subclasses: those that target the host fat body and those that target host hemocytes. Polyclonal antibodies raised against a fat-body targeting vankyrin detected a 19-kDa protein in crude extracts prepared from the 3 days p.p. fat body. Vankyrin-specific Abs localized to 3-day p.p. fat-body and hemocyte nuclei, suggesting a role for vankyrin proteins in the nuclei of C. sonorensis IV-infected cells. These data are evidence for divergent tissue specificities and targeting of multigene families in IVs. We hypothesize that PDV vankyrin genes may suppress NF-kappabeta activity during immune responses and developmental cascades in parasitized lepidopteran hosts of C. sonorensis.

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8 h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24 h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.

Host regulation and the embryonic development of the endoparasitoid Toxoneuron nigriceps (Hymenoptera: Braconidae)

Insect endoparasitoids modulate the host physiology through the injection of maternal-derived substances into the host, inducing physiological and hormonal changes in the host's internal environment to benefit parasitoid development. These changes are direct to control host development and regulate nutrient availability to the developing parasitoid, and they are synchronized with parasitoid development. Eggs of some of these parasitoids have low yolk content and require nutrients from the host hemolymph to initiate and complete embryogenesis. We report changes in the amino acid composition and protein profile of the host hemolymph of the endoparasitoid Toxoneuron nigriceps, and improved the in vitro culture of pre-germ band stage eggs. The protein profile of parasitized larvae was similar to controls throughout the embryonic development, but total amino acid concentration decreased in the first 2 h after parasitization, significantly increasing in the following hours up to 8 h. Amino acid levels were higher in parasitized larvae from 16 to 28 h after parasitization. Comparison of single amino acids indicated amino acids involved in energy metabolism (Krebs cycle) followed a trend during parasitoid embryogenesis, and their changes were correlated with embryonic development. Improvement in the in vitro development of 6 h-old eggs of T. nigriceps was obtained by adding factors released by the host fat body to the artificial medium, while a cell lysate stimulated embryogenesis and allowed the full development of newly laid eggs in vitro.

Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host’s fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A 2, B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.

Fate of polydnavirus DNA of the egg–larval parasitoid Chelonus inanitus in the host Spodoptera littoralis

In situ hybridizations show that 5 min after parasitization, polydnavirus DNA is in close vicinity of the parasitoid egg, but 5 h later also in the yolk and partially in the host embryo. Fifteen hours after parasitization, the viral DNA is seen all over the host embryo and hardly in the yolk. The tissue distribution of the viral DNA was analysed and quantified by dot blots in the fifth instar parasitized larvae. On a per host basis, haemocytes and fat body contained the highest amount of viral DNA, while nervous tissue, intestinal tract and carcass contained less. Of the three viral segments tested, all were found in all tissues. Relative to the quantity of host DNA, viral DNA was most abundant in haemocytes, about five times less abundant in fat body and nervous tissue and about 25 times less abundant in intestinal tract. The total quantity of viral DNA per host was 444±145 pg which is similar to the quantity injected by the wasp; thus, the viral DNA persists throughout parasitization. The parasitoid larva contains 820±80 pg viral DNA integrated in the genome. This illustrates that the dose of viral DNA injected in virions represents approximately one third of the total viral genomic information present in a host at a late stage of parasitism.