Insect Immunity

Large scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts. The S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences--567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were s...

The research was aimed to study the efficacy of botanicals and physical methods to manage storage insect pests of maize. The efficacy of two botanicals abeyi seed (Maesa lanceolata) and bekenisa leaf (Croton macrostachyus), both at 5% (w/w), teff (Eragrostis teff) admixture 50% (w/w), exposing maize grain to sun heat by black polyethylene sheet were evaluated to manage maize storage insect pests. The exposure of grain to sun heat using black polyethylene sheet caused the highest mortality of both maize weevil and Angoumois moth. Teff admixture constantly caused mortality of weevils and Angoumois moth through the experimental period. During the early stage, the botanicals were also equally performed mortality. All treatments reduced the grain damage and weight loss compared to the untreated check thereby, improved the seed germination compared the untreated check. However, the highest seed germination was recorded under the grain treated by teff admixture and the black polyethylene sheet. Finally, we concluded that solar heat, teff admixture as well as botanical plants are the potential and safe management options to control of maize storage insect pests, suggesting further investigation to extract the potential compound from these botanicals and test their efficacy for the future formulation of safe pesticides.

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-B and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide.

Microbial consortia accompanied to all eukaryotes can be inherited from ancestors, environment, and/or from various food source. Gut microbiota study is an emerging discipline of biological sciences that expands our understanding of the ecological and functional dynamics of gut environments. Microorganisms associated with honey bees play an important role in food digestion, colony performance, immunity, pollination, antagonistic effect against different pathogens, amelioration of food and many more. Although, many repots about honey bee gut microbiota are well documented, microbiome with other key components of honey bees such as larvae, adults, their food (pollen, beebread, and honey), honey combs, and floral nectar are poorly understood. Mutual interactions and extent of the roles of microbial communities associated with honey bees are still unclear and demand for more research on the nutritional physiology and health benefits of this ecologically and economically important group. Here in this study, we highlighted all the honey bee microbiome that harbored from different life stages and other relevant components. The anatomical parts of honey bee (larvae, adults), food source (pollen, beebread, and honey), honey combs, and floral nectar were highly flourished by numerous microorganisms like bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Actinomycetes, Bacilli, Bacteroidetes, Cocci, Clostridia, Coliforms, Firmicutes, Flavobacteriia, Mollicutes) and fungi (Dothideomycetes, Eurotiomycetes, Mucormycotina, Saccharomycetes, Zygomycetes, Yeasts, Molds). Some distinctive microbial communities of a taxonomically constrained species have coevolved with social bees. This contribution is to enhance the understanding of honey bee gut microbiota, to accelerate bee microbiota and microbiome research in general and to aid design of future experiments in this growing field.

Background: Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models. Results: Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes. Conclusions: The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.

In order to survive microbe encounters, insects rely on both physical barriers as well as local and systemic immune responses. Most research focusses on adult or larval defenses however, whereas insect eggs are also in need of protection. Lately, the defense of eggs against microbes has received an increasing amount of attention, be it through endogenous egg defenses, trans-generational immune priming (TGIP) or parental investment. Here we studied the endogenous immune response in eggs and adults of Tenebrio molitor. We show that many immune genes are induced in both adults and eggs. Furthermore, we show that eggs reach comparable levels of immune gene expression as adults. These findings show that the eggs of Tenebrio are capable of an impressive endogenous immune response, and indicate that such inducible egg defenses are likely common in insects.

Parasitoid virulence and host resistance are complex interactions depending on metabolic rate and cellular activity, which in aphids additionally implicate heritable secondary symbionts among the Enterobacteriaceae. As performance of the parasitoid, the aphid host and its symbionts may differentially respond to temperature, the success or failure of aphid parasitism is difficult to predict when temperature varies. We tested the hypothesis that resistance of the pea aphid Acyrthosiphon pisum to the parasitoid Aphidius ervi, which is linked to aphid secondary symbionts, may depend on temperature in several resistant and non-resistant aphid clonal lineages of different geographic origin and of known bacterial symbiosis, using experiments in controlled environments. Complete immunity to A. ervi at 20 1C in three different aphid clones whose symbiosis is characterized by the possession of Hamiltonella defensa reversed to high susceptibility at 25 1C and especially 30 1C, suggesting that the aphid's immune responses to the establishment and early development of the parasitoid is strongly reduced at moderately high temperatures. There was no evidence that a pea aphid control genotype that was susceptible to A. ervi at 20 1C could become more resistant as temperature increases, as has been suggested for insect fungal pathogens. By contrast, our results suggest that aphid clonal resistance to A. ervi and related parasitoids is characteristic of cool temperature conditions, similar to various other fitness attributes of aphids. Based on evidence that H. defensa symbionts characterized all three A. ervi resistant pea aphid clones studied, but was absent in control aphids that remained susceptible at all temperatures, we suggest that secondary symbiosis plays a key role in the heat sensitivity of aphid clonal resistance. Our study may also indicate that aphid natural control of variably susceptible host populations by aphid parasitoids is more likely at moderate to high temperatures. r

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes ($50%), granulocytes ($35%), prohemocytes ($8%), oenocytes ($4%) and spherulocytes ($3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply ($12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.

The red flour beetle, Tribolium castaneum, is an established genetically tractable model insect for evolutionary and developmental studies. Therefore, it may also represent a valuable model for comparative analysis of insect immunity. Here, we used the suppression subtractive hybridization method to identify Tribolium genes that are transcriptionally induced in response to injection of crude lipopolysaccharide (LPS). Determined genes encode proteins that share sequence similarities with counterparts from other insects known to mediate sensing of infection (e.g. Toll and PGRP) or to represent potential antimicrobial effectors (e.g. ferritin, c-type lysozyme, serine proteinase inhibitors, and defensins). Especially significant is the identification of thaumatin-like peptides, representing ancient antifungal peptides originally reported from plants, that are absent from the genomes of many other insects such as Drosophila, Anopheles, and Apis. We produced recombinant thaumatin-1 in bacteria and we found that it represents an antimicrobial peptide against filamentous fungi in Tribolium. Additionally, septic injury induces expression of genes involved in stress adaptation (e.g. heat-shock proteins) or insecticide resistance (e.g. cytochrome P450s) in Tribolium, suggesting that there may be crosstalk between the immune and stress responses.

Background: The 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.

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

other lepidopteran insects as well as to hen egg lysozyme. Further results presented in this paper give indications for the existence of soluble molecules which are released by the cells and which enhance the LPS-triggered activation. 0 1997 Elsevier Science Ltd. q

The egg parasitoid Avetianella longoi Siscaro attacks two species of eucalyptus longhorned borers (Phoracantha semipunctata F. and P. recurva Newman) in southern California. During the past decade, P. recurva has replaced P. semipunctata as the dominant borer species, apparently due to diVerential rates of parasitism. The present study reveals that this replacement is due in part to a physiological defensive response mounted by one species (P. recurva) against parasitoid eggs and larvae, similar to the encapsulation/melanization immune response observed in larval host/parasitoid systems. Fluorescence microscopy and vital dyes conWrmed that the defensive response was cellular in nature. Both Phoracantha species exhibited a cellular wound-healing response around the wasp egg pedicel, but the encapsulation of the wasp eggs/larvae was elicited only in eggs of P. recurva. This is the Wrst conclusive evidence that hosts in the egg stage can mount a cellular immune response against a metazoan parasitoid, and may provide insight into the mechanisms underlying the host speciWcity of many egg parasitoids.

In the recent years a strong resemblance has been observed between the insect immune system and the mammalian innate immune mechanisms suggesting their common origin. Among the insects, only the dipterans (Drosophila and various mosquito species) have been widely investigated for their immune responses towards diverse pathogens. In the present study we constructed and analysed the immune transcriptome of the lepidopteran Antheraea mylitta, an economically important Indian tasar silkmoth with a view to unravel the potential immune-related genes and pathways. An expressed sequence tag (EST) library was constructed from mRNA obtained from fat bodies of A. mylitta larvae that had been challenged by infection with Escherichia coli cells. We identified 719 unique ESTs from a total of 1412 sequences so generated. A third of the transcriptome showed similarity with previously characterized immune-related genes that included both the known and putative immune genes. Of the four putative nove...

Social insects are able to mount both group-level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome-wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one-third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens.

Much work has elucidated the pathways and mechanisms involved in the production of insect immune effector systems. However, the temporal nature of these responses with respect to different immune insults is less well understood. This study investigated the magnitude and temporal variation in phenoloxidase and antimicrobial activity in the mealworm beetle Tenebrio molitor in response to a number of different synthetic and real immune elicitors. We found that antimicrobial activity in haemolymph increased rapidly during the first 48h after a challenge and was maintained at high levels for at least 14 days. There was no difference in the magnitude of responses to live or dead Escherichia coli or Bacillus subtilis. While peptidoglylcan also elicited a long-lasting antimicrobial response, the response to LPS was short lived. There was no long-lasting upregulation of phenoloxidase activity, suggesting that this immune effector system is not involved in the management of microbial infections over a long time scale.

In this study, we report the analysis of the immune-related transcriptome from an apterygote insect, the firebrat Thermobia domestica (Zygentoma, Lepismatidae), which currently emerges as a suitable model insect for evolutionary and developmental studies. The suppression subtractive hybridization method was used for targeted screening of genes that are up-regulated in response to injected bacterial lipopolysaccharide (LPS). A subtracted cDNA library enriched in immune-inducible genes was constructed and analysis of 288 cDNAs resulted in identification of 26 novel genes in T. domestica. Among these immune-related transcripts we found homologues of genes from other insects which are involved in the regulation of signaling cascades and six novel putative antimicrobial peptides. The identified genes implicate the presence of sophisticated regulatory mechanisms in insect immune signaling and give insight into evolutionarily conserved features of insect innate immunity. r

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes ($50%), granulocytes ($35%), prohemocytes ($8%), oenocytes ($4%) and spherulocytes ($3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply ($12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.

Background Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models. Results Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes. Conclusions The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies.

During population outbreaks, top-down and bottom-up factors are unable to control defoliator numbers. To our knowledge, details of biotic interactions leading to increased population density have not been studied during real population outbreaks. We experimentally assessed the strength of plant defenses and of insect immunocompetence, assumed to contribute to active insect resistance against parasitoids and pathogens, in the geometrid Epirrita autumnata during a steep increase in population density. We demonstrated rapid (same-season) induced resistance in the foliage of its host, mountain birch. The response was systemic, spreading throughout the tree, and retarded larval growth rate by ϳ10%. On the other hand, no direct delayed carry-over effects were found in the next season in larval growth rate, mortality, or pupal mass. Larval damage to a tree during the previous year, however, significantly (by ϳ13%) accelerated the advance of the immune response (measured as melanization of an implant inserted into the pupal hemocoel). The encapsulation rate correlated positively with larval mortality in trees in which larvae had been introduced the previous year, but not in control trees. Both of these observations suggest that induced plant defense was associated with an increased insect immunocompetence during the population increase.

Three inducible bacteriolytic proteins, designated P7, P9A and P9B, from the hemolymph of immunized pupae of the giant silk moth Hyalophora cecropia have been purified using a two-step procedure with cation-exchange chromatography. Purified protein P7 has a molecular weight of 15000 and its amino acid composition shows a great similarity to that of the lysozyme from the wax moth Galleria mellonella. Moreover, heat stability, pH-rate profile and bacteriolytic specificity also indicate that protein P7 is a lysozyme. The other purified proteins, P9A and P9B, are highly potent against Escherichia coli and some other gram-negative bacteria. The amino acid compositions of proteins P9A and P9B are very similar, although the contents of glutamic acid and methionine were different. The molecular weights of these very basic proteins are around 7000. The P9 proteins are heat stable; their activities were retained after 30 min incubation at 100 degrees C. Both forms of protein P9 clearly differ from the lysozyme class of enzymes and they may represent a new type of bacteriolytic protein.

Background Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models. Results Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize imm...

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this “symbiosis of pathogens” remain largely obscure. In order to identify genes encoding potential virulence factors, we performed ∼2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to theEscherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), ...

Several dipteran insects are vectors of parasites causing major human infectious diseases. Among these, the tsetse fly, Glossina spp., is responsible for the transmission of trypanosomes, the pathogens responsible for sleeping sickness in Africa. A better understanding of insect-parasite interactions will help establish new strategies to fight this important often fatal disease. Antimicrobial peptides (AMPs) are part of the humoral immune response in insects during bacterial, fungal and parasitic infections. Here, we studied the immune response of Glossina morsitans to bacteria and to Trypanosoma brucei brucei by analyzing the synthesis of AMPs as markers of the humoral immune response. By reversed-phase chromatography, mass spectrometry analysis, Edman degradation and in vitro antimicrobial assays of the hemolymph of immune-challenged adults of G. morsitans, we identified three AMPs: a cecropin, an attacin and a defensin. These three AMPs were found to be induced upon systemic bacterial infection and also after per os infections by bacteria and parasites.

Recent in vitro studies have revealed several important aspects of the biochemical and cellular processes involved in insect blood clotting. However, in vivo empirical studies of the functional consequences of clotting are lacking, despite the role of coagulation in wound-healing, preventing infection, and its homology with vertebrate wound repair. Here we present results of the in vivo effects of haemolymph coagulation and its consequences on the spatial disposition of immune activity, in the American cockroach Periplaneta americana. Our results demonstrate that clotting: (1) localises immune effectors in the vicinity of a breach of the cuticle; (2) restricts the spread of invasive particles across the haemocoel, and is greater when wounding is associated with non-self. Our results demonstrate that haemolymph coagulation has major functional consequences, the most important of which is the compartmentalisation of the open haemocoel. r Please cite this article as: Eleanor R. Haine et al., Functional consequences of blood clotting in insects, Developmental and Comparative Immunology (2006), Please cite this article as: Eleanor R. Haine et al., Functional consequences of blood clotting in insects, Developmental and Comparative Immunology (2006),

Dopachrome Conversion Factor (DCF) was found in the plasma of the locust Locusta migratoria. It has an apparent molecular mass of 85,000. Its K m was 0.2 mM at 22°C and pH 7 with l-dopachrome as substrate. It had a high substrate specificity for ldopachrome, methyl-L-dopachrome and l-dopachrome methyl ester but no activity on the corresponding D-isomers or on dopaminechrome. DCF was devoid of any phenoloxidase activity. Under action of DCF, l-dopachrome was converted into dihydroxyindole, which showed that a decarboxylation occured in the course of reaction. Locust DCF was inhibited by indole-3-propionic acid but not by indole-3-or indole-2-carboxylic acid. It was also inhibited by l-tryptophan in a competitive manner. Inhibition and substrate specificity suggest that a carboxyl group, either free or as a methyl ester, is necessary but not sufficient for enzyme recognition. When purified prophenoloxidase was activated and then added to dihydroxyindole either prepared by chemical synthesis or obtained by action of purified DCF on dopachrome, black pigments with a maximum absorption at 540 nm were generated. Therefore in the eumelanin pathway of locust plasma, phenoloxidase can catalyze the reaction that converts the product generated by DCF.

Cecropins are insect antibacterial peptides that are part of the insect humoral immune response and could, therefore, be potential targets of natural selection. In Drosophila, the Cec genes constitute a multigene family whose members are arranged in tandem. The complete Cec family was isolated in two obscura group species: D. subobscura and D. pseudoobscura. The chromosomal regions encompassing the Cec genes were subsequently sequenced and mapped by in situ hybridization. In D. pseudoobscura, as in species of the D. melanogaster complex and in D. virilis, the Cec genes constitute a single cluster with five genes. In D. subobscura, unlike in the rest of the species, the eight members of the family are split into two clusters located in different parts of the same chromosome. Remarkable differences in levels of divergence were observed between copies in both species. The genomic organization and the phylogenetic relationships among members of this family in the genus Drosophila indicate (i) that the presence of two clusters is the derived state of the family, (ii) repeated gene duplication within species, (iii) nonfunctionalization and loss of some Cec copies, and (iv) the presence of both highly divergent and highly similar copies within species. These features are better explained by the birth-and-death model of molecular evolution, which posits that the high instability of the Cec multigene family in Drosophila is simply determined by the duplication rate and the subsequent loss of duplicated loci.

In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.

Much work has elucidated the pathways and mechanisms involved in the production of insect immune effector systems. However, the temporal nature of these responses with respect to different immune insults is less well understood. This study investigated the magnitude and temporal variation in phenoloxidase and antimicrobial activity in the mealworm beetle Tenebrio molitor in response to a number of different synthetic and real immune elicitors. We found that antimicrobial activity in haemolymph increased rapidly during the first 48 h after a challenge and was maintained at high levels for at least 14 days. There was no difference in the magnitude of responses to live or dead Escherichia coli or Bacillus subtilis. While peptidoglylcan also elicited a long-lasting antimicrobial response, the response to LPS was short lived. There was no long-lasting upregulation of phenoloxidase activity, suggesting that this immune effector system is not involved in the management of microbial infections over a long time scale.

Hemocytes and the (prophenol-) phenoloxidase system constitute the immediate innate immune system in insects. These components of insect immunity are present at any post-embryonic life stage without previous infection. Differences between individuals and species in these immune parameters can reflect differences in infection risk, life expectancy, and biological function. In honeybees which show an age-related division of labor within the worker caste, previous studies demonstrated that foragers show a strongly reduced number of hemoctyes compared to the younger nurse bees. This loss of immune competence has been regarded advantageous with respect to an already high mortality rate due to foraging and to redistribution of energy costs at the colony level. Based on the idea that abandoning hemocytes in all adults would be a reasonably direct regulatory mechanism, we posed the hypothesis that abandoning hemocytic immunity is not restricted to worker honeybees. We tested our hypotheses by performing a comprehensive analysis of hemocyte number and phenoloxidase (PO)-activity levels in immunologically naive workers, queens, and drones. We found that in all three adult phenotypes hemocyte number is dramatically reduced in early adult life. In contrast, we found that the dynamics of PO-activity levels have sex and caste-specific characteristics. In workers, PO activity reached a plateau within the first week of adult life, and in queens enzyme levels continuously increased with age and reached levels twice as high as those found in workers. PO-activity levels slightly declined with age in drones. These data support our hypothesis, from which we infer that the previously reported reduction of hemocyte in foragers is not worker specific but represents a general phenomenon occurring in all honeybee adult phenotypes. r

Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus-HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24 h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.

The tobacco hornworm Manduca sexta is widely used as a model organism to investigate the biochemical basis of insect physiological processes but little transcriptome information is available. To get a broad view of the larval hemolymph proteins, particularly those related to immunity, we synthesized and sequenced cDNA fragments from a mixture of eight total RNA samples: fat body and hemocytes from larvae injected with killed bacteria, fat body, hemocytes, integument and trachea from naïve larvae, and fat body and hemocytes from wandering larvae. Using massively parallel pyrosequencing, we obtained 95,458 M. sexta expressed sequence tags (ESTs) at an average size of 185 bp per read. A majority of the sequences (69,429 reads) could be assembled into 7,231 contigs with an average size of 300 bp, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only ~8% (606) of the contigs matched known M. sexta cDNA sequences, representing 186 of the 375 unique NCBI entries. The remaining 6,625 contigs represented newly discovered cDNA segments from this well studied biochemical model insect. A search of the 7,231 contigs using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity (E-value < 1×10 −5 ). These included 218 previously unknown M. sexta sequences coding for putative defense molecules such as pattern recognition receptors, serine proteinases, serpins, Spätzle, Toll-like receptors, intracellular signaling molecules, and antimicrobial peptides.

Background: In the recent years a strong resemblance has been observed between the insect immune system and the mammalian innate immune mechanisms suggesting their common origin. Among the insects, only the dipterans (Drosophila and various mosquito species) have been widely investigated for their immune responses towards diverse pathogens. In the present study we constructed and analysed the immune transcriptome of the lepidopteran Antheraea mylitta, an economically important Indian tasar silkmoth with a view to unravel the potential immune-related genes and pathways. Results: An expressed sequence tag (EST) library was constructed from mRNA obtained from fat bodies of A. mylitta larvae that had been challenged by infection with Escherichia coli cells. We identified 719 unique ESTs from a total of 1412 sequences so generated. A third of the transcriptome showed similarity with previously characterized immune-related genes that included both the known and putative immune genes. Of the four putative novel defence proteins (DFPs) annotated by PSI-BLAST three showed similarity to extracellular matrix proteins from vertebrates implicated in innate immunity, while the fourth was similar to, yet distinct from, the anti-microbial protein cecropin. Finally, we analysed the expression profiles of 15 potential immune-related genes, and the majority of them were induced more prominently with E. coli compared to Micrococcus luteus. We also identified several unknown proteins, some of which could have probable immunerelated functions based on the results of the ProDom analysis. Conclusion: The present study has identified many potential immune-related genes in A. mylitta some of which are vertebrate homologues and others are hitherto unreported putative defence proteins. Several genes were present as members of gene families, as has also been observed in other insect species.

The simultaneous presence of predators and a limited time for development imposes a conflict: accelerating growth under time constraints comes at the cost of higher predation risk mediated by increased foraging. The few studies that have addressed this tradeoff have dealt only with life history traits such as age and size at maturity. Physiological traits have largely been ignored in studies assessing the impact of environmental stressors, and it is largely unknown whether they respond independently of life history traits. Here, we studied the simultaneous effects of time constraints, i.e., as imposed by seasonality, and predation risk on immune defense, energy storage, and life history in lestid damselflies. As predicted by theory, larvae accelerated growth and development under time constraints while the opposite occurred under predation risk. The activity of phenoloxidase, an important component of insect immunity, and investment in fat storage were reduced both under time constraints and in the presence of predators. These reductions were smaller when time constraints and predation risk were combined. This indicates that predators can induce sublethal costs linked to both life history and physiology in their prey, and that time constraints can independently reduce the impact of predator-induced changes in life history and physiology.

Phenoloxidase (PO), a melanin-synthesizing enzyme known to play an important role in insect defense, is found as a zymogen (ProPO) in hemolymph and cuticle, where it is activated by proteolysis. We characterized the first proPO cDNA in an eusocial insect, the Apis mellifera honey bee. The AmproPO cDNA contains an ORF of 2079 bp encoding 693 amino acids, and is composed of 9 exons and 8 introns. Southern blot of digested genomic DNA suggested that only one copy of the proPO gene is present in A. mellifera. The molecular mass of the deduced ProPO and the active enzyme was predicted to be 80.1 and 74.4 kDa, respectively. The calculated pI was 6.28. BLASTp search of the deduced amino acid sequence, and neighbor-joining analysis, showed similarity with ProPOs from other insects, ranging from 47% to 63%. Protein signature analyses revealed four conserved regions, including the two copper binding sites characteristic of arthropod ProPOs. RT-PCR and Southern blot showed the highest amount of AmproPO transcripts in workers whole body, followed by queens and drones. Expression was also detected in hemocytes and integument. Real time RT-PCR showed higher amounts of AmproPO transcripts in adults and older pupae than in younger pupae and larvae, suggesting a function of AmproPO in adult exoskeleton melanization and differentiation.

Updated information and services can be found at: These include: SUPPLEMENTAL MATERIAL Supplemental material REFERENCES http://aem.asm.org/content/72/2/1653#ref-list-1 at: This article cites 34 articles, 14 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:

Ceratotoxins are antibacterial peptides produced in the female reproductive accessory glands of the medfly Ceratitis capitata. Their expression is not affected by bacterial infection, but is enhanced after mating and is modulated by juvenile hormone. Three different peptides, named ceratotoxins A, B and C, have been previously purified from the female accessory gland secretion and their amino acid and cDNA sequences have been determined. We report here the complete nucleotide sequences of four genes encoding closely related ceratotoxin peptides. One of them encodes a novel peptide, which we named ceratotoxin D. Restriction and nucleotide sequence analysis indicate that these ceratotoxin genes are organized in a large cluster spanning more than 26 kilobases of DNA. All ceratotoxin genes are coordinately expressed. Ceratotoxin transcripts appear in 2-3 day old adult females, and they reach a maximum in 6-7 day old females. The presence of highly conserved motifs in the upstream regions of all the sequenced ceratotoxin genes suggests the presence of common regulatory elements for all ceratotoxins.

Although the study of thermoregulation in insects has shown that infected animals tend to prefer higher temperatures than healthy individuals, the immune response and energetic consequences of this preference remain unknown. We examined the effect of environmental temperature and the energetic costs associated to the activation of the immune response of Tenebrio molitor larvae following a lipopolysaccharide (LPS) challenge. We measured the effect of temperature on immune parameters including phenoloxidase (PO) activity and antibacterial responses. Further as proximal and distal costs of the immune response we determined the standard metabolic rate (SMR) and the loss of body mass (m b ), respectively. Immune response was stronger at 30°C than was at 10 or 20°C. While SMR at 10 and 20°C did not differ between immune treatments, at 30°C SMR of LPS-treated larvae was almost 25-60% higher than SMR of PBS-treated and naïve larvae. In addition, the loss in m b was 1.9 and 4.2 times higher in LPS-treated larvae than in PBStreated and naïve controls. The immune responses exhibited a positive correlation with temperature and both, SMR and m b change, were sensitive to environmental temperature. These data suggest a significant effect of environmental temperature on the immune response and on the energetic costs of immunity.

Insects respond to microbial infection by the rapid and transient expression of several genes encoding antibacterial peptides. In this paper we describe a powerful technique, two-dimensional difference gel electrophoresis, that, when combined with mass spectrometry, can be used to study the immune response of Drosophila melanogaster at the protein level. By comparatively analyzing the hemolymph proteome of 2,000 third-instar Drosophila larvae, we identified 10 differential proteins that appear in the fruit fly hemolymph very early after an immune-challenge with lipopolysaccharides. These proteins can be assigned to the immune response, because they are not induced after sterile injury. Reduction of intergel variability or quantification problems related to conventional two-dimensional electrophoresis and improvement of image analysis were achieved by the use of two fluorescent dyes to label the two different protein samples. Some of the immune-induced proteins, such as thioestercontaining protein 2, can be assigned to specific aspects of the immune response; others were already reported as being involved in stress response. An immune-induced protein (CG18594) is homologous to a mammalian serine protease inhibitor that mediates the mitogen-activated protein kinase and the NF-B signaling pathways. In addition, a number of proteins that had not been associated with the immune response before were isolated and identified, and some of these were still present in the hemolymph 4 h after injury. Determining the function of all of these immune-induced proteins represents an exciting challenge for increasing our knowledge of insect immunity.

The introduction of novel biochemical, genetic, molecular and cell biology tools to the study of insect immunity has generated an information explosion in recent years. Due to the biodiversity of insects, complementary model systems have been developed. The conceptual framework built based on these systems is used to discuss our current understanding of mosquito immune responses and their implications for malaria transmission. The areas of insect and vertebrate innate immunity are merging as new information confirms the remarkable extent of the evolutionary conservation, at a molecular level, in the signaling pathways mediating these responses in such distant species. Our current understanding of the molecular language that allows the vertebrate innate immune system to identify parasites, such as malaria, and direct the acquired immune system to mount a protective immune response is very limited. Insect vectors of parasitic diseases, such as mosquitoes, could represent excellent models to understand the molecular responses of epithelial cells to parasite invasion. This information could broaden our understanding of vertebrate responses to parasitic infection and could have extensive implications for anti-malarial vaccine development.