Insect Immunity

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.

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.

Drosophila melanogaster, like other invertebrates, relies solely on its innate immune response to fight invading microbes; by definition, innate immunity lacks adaptive characteristics. However, we show here that priming Drosophila with a sublethal dose of Streptococcus pneumoniae protects against an otherwise-lethal second challenge of S. pneumoniae. This protective effect exhibits coarse specificity for S. pneumoniae and persists for the life of the fly. Although not all microbial challenges induced this specific primed response, we find that a similar specific protection can be elicited by Beauveria bassiana, a natural fly pathogen. To characterize this primed response, we focused on S. pneumoniae-induced protection. The mechanism underlying this protective effect requires phagocytes and the Toll pathway. However, activation of the Toll pathway is not sufficient for priming-induced protection. This work contradicts the paradigm that insect immune responses cannot adapt and will promote the search for similar responses overlooked in organisms with an adaptive immune response. Citation: Pham LN, Dionne MS, Shirasu-Hiza M, Schneider DS (2007) A specific primed immune response in Drosophila is dependent on phagocytes. PLoS Pathog 3(3): e26.

The ubiquitous fungal pathogen Metarhizium anisopliae kills a wide range of insects. Host hemocytes can recognize and ingest its conidia, but this capacity is lost on production of hyphal bodies. We show that the unusual ability of hyphal bodies to avoid detection depends on a gene (Mcl1) that is expressed within 20 min of the pathogen contacting hemolymph. A mutant disrupted in Mcl1 is rapidly attacked by hemocytes and shows a corresponding reduction of virulence to Manduca sexta. Mcl1 encodes a three domain protein comprising a hydrophilic, negatively charged N-terminal region with 14 cysteine residues, a central region comprising tandem repeats (GXY) characteristic of collagenous domains, and a C-terminal region that includes a glycosylphosphatidylinositoldependent cell wall attachment site. Immunofluorescence assay showed that hyphal bodies are covered by the N-terminal domains of MCL1. The collagen domain became antibody accessible after treatment with DTT, suggesting that the N termini are linked by interchain disulfide bonds and are presented on the cell surface by extended collagenous fibers. Studies with staining reagents and hemocyte monolayers showed that MCL1 functions as an antiadhesive protective coat because it masks antigenic structural components of the cell wall such as ␤-glucans, and because its hydrophilic negatively charged nature makes it unattractive to hemocytes. A survey of 54 fungal genomes revealed that seven other species have proteins with collagenous domains suggesting that MCL1 is a member of a patchily distributed gene family.

In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes GAPDH, RPS18, actin and RPL13a as the most stable ones, only differing in their ranking order. BestKeeper identified RPS18 as being the reference gene with the least overall variation, but also actin and GAPDH were found to be the second and third most stable expressed gene. By a combination of three software programs the genes actin, RPS18 and GAPDH were found suitable reference genes in the honeybee head in the context of bacterial infection.

The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the G...

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.

Inhibition of eicosanoid formation in larvae of the tobacco hornworm Manduca sexta, using specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase, severely weakened the ability of larvae to clear the bacterium Serratia marcescens from their hemolymph. The reduced capability to remove bacteria is associated with increased mortality due to these bacteria. There is a dose-dependent relationship between the phospholipase A2 inhibitor dexamethasone and both the reduced bacterial clearance and increased larval mortality. The dexamethasone effects on larval survival were reversed by treatment with arachidonic acid. Maleic acid, a nonspecific antioxidant, did not interfere with the insects' ability to remove bacterial cells from hemolymph. The larvae were shown to contain all of the C20 polyunsaturated fatty acids necessary for eicosanoid biosynthesis and to be capable of converting radioactive arachidonic acid into several primary prostaglandins. These results strongly suggest that eicosanoids mediate transduction of bacterial infection signals into the complex of cellular and humoral responses that comprise

Since 1991, when a baculovirus was first shown to inhibit apoptosis of its host insect cells, considerable contributions to our knowledge of apoptosis have arisen from the study of these viruses and the anti-apoptotic genes they encode. Baculovirus anti-apoptotic genes include p35, which encodes the most broadly acting caspase inhibitor protein known, and iap (inhibitor of apoptosis) genes, which were the first members of an evolutionarily conserved gene family involved in regulation of apoptosis and cytokinesis in organisms ranging from yeast to humans. Baculoviruses also provide an ideal system to study the effects of an apoptotic response on viral pathogenesis in an animal host. In this review, I discuss a number of interesting recent developments in the areas of apoptotic regulation by baculoviruses and the effects of apoptosis on baculovirus replication and pathogenesis. Cell Death and Differentiation (2001) 8, 137 ± 143.

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.

Apolipophorin 111 (apoLp-111) was isolated from the haemolymph of last instar larvae of Galferia melfonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-111 from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-111 was determined to be 18077 Da using mass spectrometry. The heat treatment (90"C, 30 rein) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-111 without change in the molecular mass, indicating that a confirmational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-111 into last instar G. meUoneZla larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-111 enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-111 in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein.

The effects of Drosophila and Hyalophora cecropins were tested against different fungi, both insect pathogens and fungi from the normal environment of Drosophila. The fungi were generally found to be as susceptible to the cecropins as most bacteria, the only exception being the insect pathogen Beauveria bassiana which is completely resistant. This is also the only fungus tested which is virulent to Drosophila, giving 100% lethality within 5 days after injection. Lethal concentrations of cecropins against other fungi tested ranged between 0.4 and 4 μM. Andropin is less fungicidal than the cecropins, and Drosophila cecropin A is somewhat more potent than cecropin B. Even dense cultures of Saccharomyces cerevisiae can be cleared by micromolar concentrations of cecropin, whereas Geotrichum candidum is unaffected by cecropin when tested in a dense culture.

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.

Insect pests such as termites cause damages to crops and man-made structures estimated at over $30 billion per year, imposing a global challenge for the human economy. Here, we report a strategy for compromising insect immunity that might lead to the development of nontoxic, sustainable pest control methods. Gram-negative bacteria binding proteins (GNBPs) are critical for sensing pathogenic infection and triggering effector responses. We report that termite GNBP-2 (tGNBP-2) shows β( 1 , 3 )-glucanase effector activity previously unknown in animal immunity and is a pleiotropic pattern recognition receptor and an antimicrobial effector protein. Termites incorporate this protein into the nest building material, where it functions as a nest-embedded sensor that cleaves and releases pathogenic components, priming termites for improved antimicrobial defense. By means of rational design, we present an inexpensive, nontoxic small molecule glycomimetic that blocks tGNBP-2, thus exposing term...

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

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.

Hoffmann, J. A., and Christensen, B. M. 1999. Mosquito-Plasmodium immunity; defensin, malaria. interactions in response to immune activation of the vector. Experimental Parasitology 91, 59-69. During the development of Plasmodium sp. within the mosquito midgut, the parasite undergoes a series of developmental changes. The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina.

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

Polydnaviruses are symbiotic proviruses of some ichneumonid and braconid wasps that modify the physiology, growth and development of host lepidopteran larvae. Polydnavirus infection targets neuroendocrine and immune systems, altering behavior, stunting growth, and immobilizing immune responses to wasp eggs and larvae. Polydnavirus-mediated disruption of cellular and humoral immunity renders parasitized lepidopteran larvae suitable for development of wasp larvae as well as more susceptible to opportunistic infections. Evidence from the Campoletis sonorensis polydnavirus system indicates that the unique genomic organization of polydnaviruses may have evolved to amplify the synthesis of immunosuppressive viral proteins. Immunosuppressive viruses have been essential to elucidating vertebrate immunity. Polydnaviruses have similar potential to clarify insect immune responses and may also provide novel insights into the role of insect immunity in shaping polydnavirus genomes.

Abbreviations : PAP, prophenoloxidase-activating protease; PO, phenoloxidase; proPO, prophenoloxidase; RCL, reactive center loop 2 A serpin from the parasitoid wasp Leptopilina boulardi targets the Drosophila phenoloxidase cascade Abstract The insect phenoloxidase (PO) cascade is known to be tightly regulated by serine proteases and serine protease inhibitors of the serpin family. As a key component of the insect immune system, it is also suspected to be inhibited by several endoparasitoid wasps, insects that develop inside other arthropods as hosts. However, the underlying mechanisms of this inhibition are largely undescribed. Here, we report the characterization of a gene encoding a serpin, LbSPNy, highly expressed in the venom of the wasp Leptopilina boulardi (ISy type), and we show that either the venom or the recombinant LbSPNy inhibit the PO cascade in the hemolymph of Drosophila yakuba host larva. Altogether, our results identify the first serpin used as a virulence factor by a parasitoid wasp and show that it disrupts the activation pathway of the PO in the Drosophila host.

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25 th generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses.

Background: Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV.

including phagocytosis, microaggregation, nodulation, encapsulation, cell spreading and hemocyte migration toward a source of a bacterial peptide. We also describe our most recent work on the influence of one group of eicosanoids, prostaglandins, on gene expression in an established insect cell line.

Hemolin is a bacteria-inducible protein of the immunoglobulin superfamily identified in the silk mothHyalophora cecropia.The role of this protein, in hemocyte aggregation and phagocytosis, was studiedin vitro.Hemocyte aggregation, stimulated by phorbol myristate acetate or lipopolysaccharide (LPS), was prevented by hemolin in a dose-dependent fashion, but hemolin did not disrupt aggregates once they had been formed. Furthermore, hemolin was able to stimulate phagocytic activity in both hemocytes and hemocytic mbn-2 cells and this activity was enhanced by LPS. The enhanced phagocytosis produced by a combination of hemolin and LPS was prevented by the protein kinase C (PKC) inhibitors staurosporine and H-7, and PKC activity in hemocyte crude extracts was enhanced by hemolin and LPS, with the highest activity observed in the presence of both. Hemolin affected tyrosine phosphorylation of hemocyte proteins, enhancing the phosphorylation of two proteins of 20 and 30 kDa and preventing tyrosine phosphorylation of two proteins of 35 and 40 kDa. These results suggest that hemolin is involved in the regulation of the cellular immune responses via a pathway that includes PKC activation and protein tyrosine phosphorylation.

The endoplasmic reticulum type I signal peptidase complex (ER SPC) is a conserved enzyme that cleaves the signal peptides of secretory or membrane preproteins. The deletion of this enzyme leads to the accumulation of uncleaved proteins in biomembranes and cell death. However, the physiological functions of ER SPC in insects are not fully understood. Here, a catalytic subunit gene of ER SPC, LmSPC1, was cloned from Locusta migratoria manilensis and its physiological functions were analysed by RNA interference (RNAi). The LmSPC1 open reading frame encoded a protein of 178 amino acids with all five conserved regions of signal peptidases. RNAimediated knockdown of LmSPC1 resulted in high mortality. Sixty-nine per cent of dead nymphs died of abnormal moulting, corresponding to decreased activity of moulting fluid protease. Moreover, insects in the RNAi group experienced a decline in food intake, and a decrease in the secretion of total protein and digestive enzymes from midgut tissues to the midgut lumen. Furthermore, the females produced fewer eggs and eggs with disrupted embryogenesis. These results indicate that LmSPC1 is required for the secretion of secretory proteins, affects physiological functions, including moulting, feeding, reproduction and embryonic development, and is essential for survival. Therefore, LmSPC1 may be a potential target for locust control.

Parasitic wasps and flies (parasitoids) exert high mortality on caterpillars, and previous studies have demonstrated that most primary and secondary defenses do not protect caterpillars against parasitoids. We investigated the efficacy of tertiary defenses (i.e., immune responses) against parasitoids. Using a bead injection technique to measure the immune response and a 15-year database to measure parasitism, we compared the immune response for 16 species of caterpillars in nine different families. We found that caterpillar species with a strong immune response had the lowest incidence of parasitism, and when statistically compared to other defensive traits, the immune response was the best predictor of parasitism. Parasitoids either avoid attacking caterpillar species with a capacity for high levels of melanization or are killed once they have parasitized. In either case, the immune response is clearly one of the most effective defenses that caterpillars have against parasitism, and elucidating consistent predictors of variation in encapsulation could improve understanding of parasitism patterns in time and space and could enhance biological control efforts.

House ßies are carriers of Ͼ100 devastating diseases that have severe consequences for human and animal health. Despite the fact that it is a passive vector, a key bottleneck to progress in controlling the human diseases transmitted by house ßies is lack of knowledge of the basic molecular biology of this species. Sequencing of the house ßy genome will provide important inroads to the discovery of novel target sites for house ßy control, understanding of the house ßy immune response, rapid elucidation of insecticide resistance genes, and understanding of numerous aspects of the basic biology of this insect pest. The ability of the house ßy to prosper in a remarkably septic environment motivates analysis of its innate immune system. Its polymorphic sex determination system, with male-determining factors on either the autosomes or the Y chromosome, is ripe for a genomic analysis. Sequencing of the house ßy genome would allow the Þrst opportunity to study the interactions between a pest insect and its parasitoid (Nasonia vitripennis) at the whole genome level. In addition, the house ßy is well placed phylogenetically to leverage analysis of the multiple Dipteran genomes that have been sequenced (including several mosquito and Drosophila species). The community of researchers investigating Musca domestica are well prepared and highly motivated to apply genomic analyses to their widely varied research programs.

Approximately 4 kb of the Cecropin cluster region have been sequenced in nine lines of Drosophila melanogaster and one line of the sibling species D. simulans, D. mauritiana, and D. sechellia. This region includes three functional genes (CecA1, CecA2, and CecB), which are involved in the insect immune response, and two pseudogenes (Cec⌿1 and Cec⌿2). The level of silent polymorphism in the three Cec genes is rather high (0.028), and there is no excess of nonsynonymous polymorphism. There is no evidence of gene conversion in the history of these genes. The interspecific comparison has revealed that in the three species of the simulans cluster the CecA2 gene is partially deleted and has therefore lost its function and become a pseudogene; in each of the species, subsequent deletions have accumulated. Divergence estimates indicate that the Cec⌿1 and Cec⌿2 pseudogenes are highly diverged, both between themselves and relative to the other three Cec genes. However, both Cec⌿1 and Cec⌿2 have conserved transcriptional signals and splice sites, and they present an open reading frame; also, correctly spliced transcripts have been detected for both Cec⌿1 and Cec⌿2. The data support that these genes are either active genes with some null alleles or young pseudogenes. orientation (Engströ m 1997). In particular, the two Genetics 150: 157-171 (September 1998) 158 S. Ramos-Onsins and M. Aguadé or cycle sequenced (Perkin Elmer cycle sequencing kit; Nor-CecA genes of Drosophila present B-like, GATA and walk, CT) and separated on an ABI 377 automated DNA se-R1 motifs; the CecB gene presents both the B-like and quencer. The sequences reported in this article have been R1 motifs, and the CecC gene presents the B-like, deposited in the EMBL sequence database library under ac-GATA, and GAAANN motifs. cession numbers Y16852-Y16863.

Cells of the moth immune system are derived from organs that loosely envelop the four wing imaginal discs. The immune response in these insects is believed to depend on the activities of two main classes of hemocytes: plasmatocytes and granular cells. The fates of cells that arise from these hematopoietic organs have been followed by immunolabeling with plasmatocyte-specific and granular-cell-specific antibodies. Cells within each hematopoietic organ differ in their coherence and in their expression of two plasmatocyte-specific surface proteins, integrin and neuroglian. Within an organ there is no overlap in the expression of these two surface proteins; neuroglian is found on the surfaces of the coherent cells while integrin is expressed on cells that are losing coherence, rounding up, and dispersing. A granular-cellspecific marker for the protein lacunin labels the basal lamina that delimits each organ but only a small number of granular cells that lie on or near the periphery of the hematopoietic organ. When organs are cultured in the absence of hemolymph, all cells derived from hematopoietic organs turn out to immunolabel with the plasmatocyte-specific antibody MS13. The circulating plasmatocytes derived from hematopoietic organs have higher ploidy levels than the granular cells and represent a separate lineage of hemocytes.

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.

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.

A family of hemolymph peptides was previously identified in several lepidopteran insects, which exhibited multiple biological activities including rapid paralysis, blockage of growth and development, or stimulation of plasmatocyte spreading and aggregation. We synthesized Manduca sexta paralytic peptide 1 (PP1) and found that after it was injected into larvae, bleeding from wounds was dramatically reduced. PP1 also stimulated spreading and aggregation behavior of M. sexta plasmatocytes in vitro. Stimulation of plasmatocyte aggregation and adherence to the body wall may explain a decrease observed in the number of circulating plasmatocytes after injection of PP1. Such aggregates might rapidly form plugs in wounds to prevent bleeding. We cloned a cDNA for a Manduca paralytic peptide precursor, using polymerase chain reactions and cDNA library screening. The active 23-residue PP2 peptide encoded by this clone is at the carboxyl-terminal end of a precursor protein predicted to be 107 amino acid residues long after cleavage of a secretion signal peptide. Active PP2 was produced by processing of recombinant proPP2 by bovine factor X a . A single proPP2 mRNA was present in fat body but not in hemocytes. The level of this mRNA was not affected by injection of bacteria into larvae. We produced recombinant proPP2 in Escherichia coli and used this protein to produce an antiserum. The antiserum detected proPP2 in plasma and was used to observe rapid proteolytic processing of proPP2 after hemolymph collection.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3P-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pETvector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae. ß 0167-4838 / 99 / $^see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 3 8 ( 9 9 ) 0 0 1 4 8 -X

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.

Bacterial challenge induced the appearance of a strong antibacterial activity in the cell-free hemolymph of adult Pdomena prusina (Hemiptera). We isolated several peptides that were capable of causing this activity and determined their primary sequence by Edman degradation and mass spectrometry.