Melanotic Encapsulation Response Research Articles

Effect of rearing density on female investment in reproduction and melanotic encapsulation response in the sand cricket (Gryllus firmus) (Orthoptera: Gryllidae)

Abstract The density-dependent prophylaxis hypothesis posits that because pathogens are expected to be transmitted among conspecifics in crowded conditions, individuals living in such conditions should minimize their chance of infection and maximize survival by elevating their baseline immunity. Further, if individuals developing in crowded conditions indeed allocate more resources to immunity then resource allocation to other life-history traits, such as reproduction, should decline. We tested these hypotheses by rearing sand crickets (Gryllus firmus) at either low or high density in the laboratory and quantified immune function (melanotic encapsulation response) and reproductive investment (ovary mass, egg size) at adulthood. The results did not support the density-dependent prophylaxis hypothesis, but we did find that rearing density significantly affected investment in two reproductive traits, with average ovary mass and egg size both being larger in low-density crickets. We discuss possible explanations for the lack of a trade-off between immunity and reproduction in our study.

A fat body transcriptome analysis of the immune responses of Rhodnius prolixus to artificial infections with bacteria

BackgroundRhodnius prolixus is an important vector of Trypanosoma cruzi, the causal agent of Chagas disease in humans. Despite the medical importance of this and other triatomine vectors, the study of their immune responses has been limited to a few molecular pathways and processes. Insect immunity studies were first described for holometabolous insects such as Drosophila melanogaster, and it was assumed that their immune responses were conserved in all insects. However, study of the immune responses of triatomines and other hemimetabolous insects has revealed discrepancies between these and the Drosophila model.MethodsTo expand our understanding of innate immune responses of triatomines to pathogens, we injected fifth instar nymphs of R. prolixus with the Gram-negative (Gr−) bacterium Enterobacter cloacae, the Gram-positive (Gr+) bacterium Staphylococcus aureus, or phosphate-buffered saline (PBS), and evaluated transcript expression in the fat body 8 and 24 h post-injection (hpi). We analyzed the differential expression of transcripts at each time point, and across time, for each treatment.ResultsAt 8 hpi, the Gr− bacteria-injected group had a large number of differentially expressed (DE) transcripts, and most of the changes in transcript expression were maintained at 24 hpi. In the Gr+ bacteria treatment, few DE transcripts were detected at 8 hpi, but a large number of transcripts were DE at 24 hpi. Unexpectedly, the PBS control also had a large number of DE transcripts at 24 hpi. Very few DE transcripts were common to the different treatments and time points, indicating a high specificity of the immune responses of R. prolixus to different pathogens. Antimicrobial peptides known to be induced by the immune deficiency pathway were induced upon Gr− bacterial infection. Many transcripts of genes from the Toll pathway that are thought to participate in responses to Gr+ bacteria and fungi were induced by both bacteria and PBS treatment. Pathogen recognition receptors and serine protease cascade transcripts were also overexpressed after Gr− bacteria and PBS injections. Gr- injection also upregulated transcripts involved in the metabolism of tyrosine, a major substrate involved in the melanotic encapsulation response to pathogens.ConclusionsThese results reveal time-dependent pathogen-specific regulation of immune responses in triatomines, and hint at strong interactions between the immune deficiency and Toll pathways.Graphical abstract

Combination of label-free quantitative proteomics and transcriptomics reveals intraspecific venom variation between the two strains of Tetrastichus brontispae, a parasitoid of two invasive beetles

The venom apparatus is a conserved organ in parasitoids that shows adaptations correlated with life-style diversification. Combining transcriptomics and label-free quantitative proteomics, here we explored the venom apparatus components of the endoparasitoid Tetrastichus brontispae (Eulophidae), and provide a comparison of the venom apparatus proteomes between its two closely related strains, T. brontispae-Octodonta nipae (Tb-On) and T. brontispae-Brontispa longissima (Tb-Bl). Tb-Bl targets the B. longissima pupa as its habitual host. However, Tb-On is an experimental derivative of Tb-Bl, which has been exposed to the O. nipae pupa as host consecutively for over 40 generation. Results showed that approximately 1505 venom proteins were identified in the T. brontispae venom apparatus. The extracts contained novel venom proteins, such as 4-coumarate-CoA ligase 4. A comparative venom proteome analysis revealed that significant quantitative and qualitative differences in venom composition exist between the two strains; although the most abundant venom proteins were shared between them. The differentially produced proteins were mainly enriched in fatty acid biosynthesis and melanotic encapsulation response. Six of these enriched proteins presented increased levels in Tb-On, and this result was validated by parallel reaction monitoring (PRM) analysis. Overall, our data reveal that venom composition can evolve quickly and respond to host selection.

Sexually dimorphic effect of mating on the melanotic encapsulation response in the harem‐defending Wellington tree weta, Hemideina crassidens (Orthoptera: Tettigonioidea: Anostostomatidae)

AbstractReproductive activities are generally costly to immune responsiveness because limited resources required by reproduction are diverted away from immunity (and vice versa). Reproduction, however, is not expected to affect the immune response in males and females similarly as mating is expected to negatively affect male immunity more so than female immunity. Here, I test the phenotypic plasticity hypothesis in the Wellington tree weta (Hemideina crassidens), a sexually dimorphic orthopteran insect that is endemic to New Zealand. My laboratory experiment showed that although males had higher rates of melanotic encapsulation than females, contrary to prediction, females were the only sex significantly affected by mating and the effect was positive. In addition to immunity differing between the sexes, immune function can differ intrasexually, particularly when males are polymorphic and different investment strategies are used to maximize fitness. Male H. crassidens exhibit alternative mating strategies that are represented by three different morphotypes. I therefore explored whether the morphs differed in their melanotic encapsulation response and whether mating affected the morphs differently. I found no difference among morphs or an effect of mating on male immune response.

Blue light-induced immunosuppression in Bactrocera dorsalis adults, as a carryover effect of larval exposure.

Detrimental effects of ultraviolet (UV) light on living organisms are well understood, little is known about the effects of blue light irradiation. Although a recent study revealed that blue light caused more harmful effects on insects than UV light and blue light irradiation killed insect pests of various orders including Diptera, the effects of blue light on physiology of insects are still largely unknown. Here we studied the effects of blue light irradiation on cuticular melanin in larval and the immune response in adult stage of Bactrocera dorsalis. We also evaluated the effects of blue light exposure in larval stage on various age and mass at metamorphosis and the mediatory role of cuticular melanin in carryover effects of larval stressors across metamorphosis. We found that larvae exposed to blue light decreased melanin contents in their exoskeleton with smaller mass and delayed metamorphosis than insects reared without blue light exposure. Across metamorphosis, lower melanotic encapsulation response and higher susceptibility to Beauveria bassiana was detected in adults that had been exposed to blue light at their larval stage, thereby constituting the first evidence that blue light impaired adult immune function in B. dorsalis as a carryover effect of larval exposure.

Starvation and Imidacloprid Exposure Influence Immune Response by Anoplophora glabripennis (Coleoptera: Cerambycidae) to a Fungal Pathogen.

In several insect systems, fungal entomopathogens synergize with neonicotinoid insecticides which results in accelerated host death. Using the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), an invasive woodborer inadvertently introduced into North America and Europe, we investigated potential mechanisms in the synergy between the entomopathogenic fungus Metarhizium brunneum Petch and the insecticide imidacloprid. A potential mechanism underlying this synergy could be imidacloprid's ability to prevent feeding shortly after administration. We investigated whether starvation would have an impact similar to imidacloprid exposure on the mortality of fungal-inoculated beetles. Using real-time PCR to quantify fungal load in inoculated beetles, we determined how starvation and pesticide exposure impacted beetles' ability to tolerate or resist a fungal infection. The effect of starvation and pesticide exposure on the encapsulation and melanization immune responses of the beetles was also quantified. Starvation had a similar impact on the survival of M. brunneum-inoculated beetles compared to imidacloprid exposure. The synergy, however, was not completely due to starvation, as imidacloprid reduced the beetles' melanotic encapsulation response and capsule area, while starvation did not significantly reduce these immune responses. Our results suggest that there are multiple interacting mechanisms involved in the synergy between M. brunneum and imidacloprid.

Larval UV exposure impairs adult immune function through a trade‐off with larval investment in cuticular melanin

Summary Despite the strong impact of ultraviolet (UV) radiation on invertebrates, it is unknown whether it affects immune function across metamorphosis. More generally, the mechanisms on how larval stressors bridge metamorphosis and shape adult fitness in animals with a complex life cycle remain poorly understood. We studied whether cuticular melanin content is upregulated under UV exposure in the larval stage of the damselfly Coenagrion puella and whether this is traded off across metamorphosis against a key component of the invertebrate immune response, the melanotic encapsulation response, in the adult stage. Larvae exposed to UV increased the melanin content in their exoskeleton and metamorphosed later and at a smaller mass than animals reared without UV. Across metamorphosis, this was associated with a reduced melanotic encapsulation response, thereby constituting the first proof for a UV‐driven impaired immune response in an invertebrate. The demonstrated costs of UV exposure in terms of age and mass at metamorphosis and reduced adult immune response likely translate into reduced adult fitness. Path analysis indicated that the immunosuppressive property of larval UV exposure was not mediated by age and mass at metamorphosis, but instead that the adult immune response was traded off against larval cuticular melanin investment. Melanin‐based trade‐off across metamorphosis provides a new pathway by which effects of larval stressors are carried over to the adult stage and thereby advances our understanding of the still largely enigmatic mechanisms of carryover effects of larval stressors across metamorphosis. Given the mechanistic basis, this carryover effect of larval UV exposure on adult immune function is expected to be general and may constitute a widespread and important cost of UV exposure in invertebrates.

A role for nematocytes in the cellular immune response of the DrosophilidZaprionus indianus

SUMMARYThe melanotic encapsulation response mounted by Drosophila melanogaster against macroparasites, which is based on haemocyte binding to foreign objects, is poorly characterized relative to its humoral immune response against microbes, and appears to be variable across insect lineages. The genus Zaprionus is a diverse clade of flies embedded within the genus Drosophila. Here we characterize the immune response of Zaprionus indianus against endoparasitoid wasp eggs, which elicit the melanotic encapsulation response in D. melanogaster. We find that Z. indianus is highly resistant to diverse wasp species. Although Z. indianus mounts the canonical melanotic encapsulation response against some wasps, it can also potentially fight off wasp infection using two other mechanisms: encapsulation without melanization and a non-cellular form of wasp killing. Zaprionus indianus produces a large number of haemocytes including nematocytes, which are large fusiform haemocytes absent in D. melanogaster, but which we found in several other species in the subgenus Drosophila. Several lines of evidence suggest these nematocytes are involved in anti-wasp immunity in Z. indianus and in particular in the encapsulation of wasp eggs. Altogether, our data show that the canonical anti-wasp immune response and haemocyte make-up of the model organism D. melanogaster vary across the genus Drosophila.

Phenylalanine Metabolism Regulates Reproduction and Parasite Melanization in the Malaria Mosquito

The blood meal of the female malaria mosquito is a pre-requisite to egg production and also represents the transmission route for the malaria parasite. The proper and rapid assimilation of proteins and nutrients in the blood meal creates a significant metabolic challenge for the mosquito. To better understand this process we generated a global profile of metabolite changes in response to blood meal of Anopheles gambiae, using Gas Chromatography-Mass Spectrometry (GC-MS). To disrupt a key pathway of amino acid metabolism we silenced the gene phenylalanine hydroxylase (PAH) involved in the conversion of the amino acid phenylalanine into tyrosine. We observed increased levels of phenylalanine and the potentially toxic metabolites phenylpyruvate and phenyllactate as well as a reduction in the amount of tyrosine available for melanin synthesis. This in turn resulted in a significant impairment of the melanotic encapsulation response against the rodent malaria parasite Plasmodium berghei. Furthermore silencing of PAH resulted in a significant impairment of mosquito fertility associated with reduction of laid eggs, retarded vitellogenesis and impaired melanisation of the chorion. Carbidopa, an inhibitor of the downstream enzyme DOPA decarboxylase that coverts DOPA into dopamine, produced similar effects on egg melanization and hatching rate suggesting that egg chorion maturation is mainly regulated via dopamine. This study sheds new light on the role of amino acid metabolism in regulating reproduction and immunity.

Reproductive Costs of the Immune Response of the Autogenous Mosquito Culex pipiens molestus Against Inoculated Dirofilaria immitis

Culex pipiens molestus is an autogenous mosquito, a vector of Dirofilaria immitis in Madeira Island, and it mounts a melanotic encapsulation response when inoculated intrathoracically with D. immitis microfilariae (mf). Because Cx. p. molestus is autogenous, this mosquito is a good model from which to gain a better understand the relationship between oviposition and melanization, independent of the signaling pathways related to blood feeding. The present work assessed the impact follicle growth might have on melanization of intrathoracically inoculated mf. The ovaries from mosquitoes undergoing melanotic encapsulation developed more eggs than those that could not melanize the mf. Possible explanations are discussed in this article.

Penetration of the mosquito midgut is not required for Brugia pahangi microfilariae to avoid the melanotic encapsulation response of Armigeres subalbatus

Insect vectors of disease have the capacity to respond to, and prevent further development of, parasites and pathogens using a response known as melanotic encapsulation. The naturally-occurring Armigeres subalbatus-Brugia spp. system provides an excellent way to investigate melanotic encapsulation and immune recognition in a mosquito host, because Brugia malayi microfilariae (mf) acquired via a blood meal are rapidly melanized in the body cavity of Ar. subalbatus, but Brugia pahangi mf evade or suppress the immune response and develop normally into infective stage larvae. Previous studies have suggested that B. pahangi mf are changed in some manner in the process of exiting the mosquito gut, thereby facilitating escape from, or suppression of, the melanotic encapsulation response. By inoculating mosquitoes with parasites, thus circumventing the midgut, we show that ∼88% of B. pahangi mf escape the melanotic encapsulation response while approximately 90% of inoculated B. malayi mf are melanized. Methods to isolate parasites for this procedure are described. These results mimic those observed in Ar. subalbatus against Brugia spp. mf that are ingested following blood feeding, and demonstrate that midgut penetration is not required for B. pahangi mf to avoid the melanotic encapsulation response of Ar. subalbatus.

Drosophila serpin 27A is a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response

Avirulent strains of the endoparasitoid Leptopilina boulardi succumb to a blood cell-mediated melanotic encapsulation response in host larvae of Drosophila melanogaster. Virulent wasp strains effectively abrogate the cellular response with substances introduced into the host that specifically target and effectively suppress one or more immune signaling pathways, including elements that control phenoloxidase-mediated melanotic encapsulation. The present study implicates involvement of the Drosophila Toll pathway in cellular innate immunity by regulating the serine protease inhibitor Serpin 27A ( Spn27A), which normally functions as a negative regulator of phenoloxidase. The introduction of Spn27A into normally highly immune competent D. melanogaster larvae significantly reduced their ability to form melanotic capsules around eggs of L. boulardi. This study confirms the role of Spn27A in the melanization cascade and establishes that this pathway and associated blood cell responses can be activated by parasitization. The activation of phenoloxidase and the site-specific localization of the ensuing melanotic response are such critical components of the blood cell response that Spn27A and the signaling elements mediating its activity are likely to represent prime targets for immune suppression by L. boulardi.

The role of phenylalanine hydroxylase in melanotic encapsulation of filarial worms in two species of mosquitoes

Melanin formation has a significant influence on mosquito vector competence by limiting the development of metazoan parasites. Tyrosine, the rate-limiting substrate of melanin production, can be obtained exogenously or derived from phenylalanine by phenylalanine hydroxylase (PAH). The characteristics of this defense mechanism, such as temporal expression of constituent enzymes involved in the biosynthetic pathway, can vary considerably between mosquito species. We investigated the functional role of PAH in the melanotic encapsulation response in Aedes aegypti and Armigeres subalbatus, two mosquito species with markedly different melanization responses. We used double-stranded RNA (dsRNA) to knock down PAH and observed the phenotypic effects on melanin formation. PAH transcripts were dramatically reduced in both mosquito species after gene knock down. The abundance of PAH proteins was decreased in gene knockdown mosquitoes that were inoculated with Dirofilaria immitis microfilariae (mf) as compared to inoculation controls. A significant reduction of mf melanization also was observed in these knockdown mosquitoes as compared to inoculation controls. Our data suggest that PAH is required for a fully functional melanotic encapsulation response in both mosquito vectors.

Density‐dependent prophylaxis and condition‐dependent immune function in Lepidopteran larvae: a multivariate approach

Summary The risk of parasitism and infectious disease is expected to increase with population density as a consequence of positive density‐dependent transmission rates. Therefore, species that encounter large fluctuations in population density are predicted to exhibit plasticity in their immune system, such that investment in costly immune defences is adjusted to match the probability of exposure to parasites and pathogens (i.e. density‐dependent prophylaxis). Despite growing evidence that insects in high‐density populations show the predicted increase in resistance to certain pathogens, few studies have examined the underlying alteration in immune function. As many of these species show increased cuticular melanism at high densities, the aim of this study was to use a multivariate approach to quantify relative variation in the allocation of resources to immunity associated with both rearing density (solitary vs. crowded) and cuticular colour (pale vs. dark) in a phase‐polyphenic Lepidopteran species (Spodoptera littoralis Boisduval). Relative to pale individuals, dark larvae (the high‐density phenotype) exhibited higher haemolymph and cuticular phenoloxidase (PO) activity and a stronger melanotic encapsulation response to an artificial parasite inserted into the haemocoel. However, they also exhibited lower antibacterial (lysozyme‐like) activity than pale larvae. Larval density per se had little effect on most of the immune parameters measured, though capsule melanization and antibacterial activity were significantly higher in solitary‐reared than crowded larvae. Correcting for variation in larval body condition, as estimated by weight and haemolymph protein levels, had little effect on these results, suggesting that variation in immune function across treatment groups cannot be explained by condition‐dependence. These results are examined in relation to pathogen resistance, and the possibility of a trade‐off within the immune system is discussed.

Quantitative trait loci in Anopheles gambiae controlling the encapsulation response against Plasmodium cynomolgi Ceylon

BackgroundAnopheles gambiae females are the world's most successful vectors of human malaria. However, a fraction of these mosquitoes is refractory to Plasmodium development. L3-5, a laboratory selected refractory strain, encapsulates transforming ookinetes/early oocysts of a wide variety of Plasmodium species. Previous studies on these mosquitoes showed that one major (Pen1) and two minor (Pen2, Pen3) autosomal dominant quantitative trait loci (QTLs) control the melanotic encapsulation response against P. cynomolgi B, a simian malaria originating in Malaysia.ResultsWe have investigated the response of L3-5 to infection with P. cynomolgi Ceylon, a different but related parasite species, in crosses with the susceptible strain 4Arr. Refractoriness to this parasite is incompletely recessive. Infection and genotyping of F2 intercross females at genome-spanning microsatellite loci revealed that 3 autosomal QTLs control encapsulation of this species. Two loci map to the regions containing Pen2 and Pen3. The novel QTL maps to chromosome 3R, probably to polytene division 32 or 33. Thus the relative contribution of any QTL to oocyst encapsulation varies with the species of parasite. Further, different QTLs were most readily identified in different F2 families. This, like the F1 data, suggests that L3-5 is not genetically homogeneous and that somewhat different pathways may be used to achieve an encapsulation response.ConclusionWe have shown here that different QTLs are involved in responses against different Plasmodium parasites.

Reproductive Costs Associated with Resistance in a Mosquito-Filarial Worm System

The mosquito Armigeres subalbatus can encapsulate and kill > 80% of Brugia malayi microfilariae (mf) within 36 hr following ingestion. The cascade of biochemical events constituting this melanotic encapsulation response is also important in other mosquito biological events, including egg-chorion tanning. Certain biochemical entities, including a tyrosine precursor, are thought to be shared among these biological activities. Because of this purported tyrosine link, and because the blood meal both initiates egg development and is the source of mf, we evaluated the possibility that reproductive cost is incurred by the resistant host when undergoing a response to mf acquired in an infected blood meal. Mean time to oviposition was significantly longer for mosquitoes responding to parasites than for controls (77.7 versus 66.5 hr). Tyrosine levels in ovaries from infected mosquitoes were less than half those of controls at 24 and 48 hr, and were still significantly reduced at 72 hr following blood feeding. Ovary development, assessed via measurements and total protein content, also was delayed significantly in the experimental group, with ovary width and protein content never attaining levels found in control mosquitoes. Sections from 24-hr post-blood meal ovaries demonstrated that the normal processes of egg development, including vitelline accumulation, was drastically altered as well. The biological implications of these results are considered.

Reduced cellular immune competence of a temperature-sensitive dopa decarboxylase mutant strain of Drosophila melanogaster against the parasite Leptopilina boulardi

1. 1. The melanotic encapsulation response made by larvae of a temperature-sensitive dopa decarboxylase (DDC) mutant stain of Drosophila against the parasitic wasp Leptopilina was severely compromised in hosts with reduced levels of DDC. 2. 2. Dopa and 5,6-dihydroxyindole (DHI) were two hemolymph components identified in hosts exhibiting a melanotic encapsulation response. 3. 3. This is the first study to implicate DDC in insect cellular immune responses, and to provide chemical evidence that the pigment formed during such responses is eumelanin derived from tyrosine.

Aedes aegypti: Characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80–90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.

Effect of γ irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

The effect of γ irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15–20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15–20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External γ irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in γ-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.