iTRAQ-based quantitative proteomic profiling of the immune response of the South African abalone, Haliotis midae.

The South African abalone Haliotis midae is a commercially important species farmed at high densities in land-based aquaculture systems. Disease outbreaks have had a severe financial impact on the abalone industry yet the molecular mechanisms underlying the immune response of H. midae remain obscure. In this study, a comparative shotgun proteomics approach using iTRAQ coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A total of 118 non-redundant, unique haemocyte proteins were identified and quantified, with 16 proteins significantly regulated. Hierarchical clustering and pathway analysis uncovered a coordinated response dominated by calcium and cAMP signalling via activation of MAPK cascades. Early up-regulated biological processes involve phagocytosis, nitric oxide production and ATP-synthesis, whilst down-regulated responses were predominantly involved in the regulation of apoptosis. The late up-regulated response involved protein kinase activity and detoxification processes. Expression of selected proteins was validated by Western blot. A putative allograft inflammatory factor-1 protein was further selected to establish its functional molecular role in haemocytes. Confocal imaging revealed that allograft inflammatory factor-1 regulates phagocytosis via a functional interaction with filamentous actin. This is the first time a high-throughput proteomics approach has been used to investigate the immune response of H. midae.

Bombyx mori hemocyte extract has anti-inflammatory effects on human phorbol myristate acetate-differentiated THP­1 cells via TLR4-mediated suppression of the NF-κB signaling pathway.

Hemolymph is the circulating fluid of insects and is a key component of their immune system. However, little is known concerning hemocyte identification, development, differentiation and related cellular immune responses. The present study aimed to determine whether a hemocyte extract prepared from Bombyx mori larvae had anti­inflammatory effects; THP­1 (a human monocytic leukemia cell line) cells that had been differentiated into macrophage­like cells by treatment with phorbol myristate acetate (PMA) were used. THP­1 cells were cultured with different concentrations of a B. mori hemocyte extract prior to exposure to lipopolysaccharide (LPS) to induce an inflammatory response. The effects of the B. mori hemocyte extract on anti­inflammatory pathways were determined using reverse transcription­quantitative polymerase chain reaction and western blotting to assess the expression of pro­inflammatory molecules. The B. mori hemocyte extract inhibited the LPS­induced mRNA expression of Toll­like receptor 4 in addition to LPS­induced interleukin (IL)­1ß, IL­6, IL­8 and tumor necrosis factor­α. Treatment of PMA­differentiated THP­1 cells with B. mori hemocyte extract also inhibited inducible nitric oxide synthase and cyclooxygenase­2 transcription and translation. Nuclear factor­κB activation and phosphorylation also decreased. Further in­depth functional studies are required to understand the mechanism underlying the anti­inflammatory effects of silkworm hemocyte extract.

Validation of Comet assay in Oregon-R and Wild type strains of Drosophila melanogaster exposed to a natural radioactive environment in Brazilian semiarid region.

Natural radiation of geological origin is a common phenomenon in Brazil, a country where radioactive agents such as uranium may be often found. As an unstable atom, uranium undergoes radioactive decay with the generation of a series of decay by-products, including radon, which may be highly genotoxic and trigger several pathological processes, among which cancer. Because it is a gas, radon may move freely between cracks and gaps in the ground, seeping upwards into the buildings and in the environment. In this study, two Drosophila melanogaster Meigen (Diptera, Drosophilidae) strains called Oregon-R and Wild (collected in a non-radioactive environment) were exposed to atmospheric radiation in the Lajes Pintadas city, in the semiarid zone of northeastern Brazil. After six days of environmental exposure, the organisms presented genetic damage significantly higher than that of the negative control group. The genotoxic effects observed reinforce the findings of other studies carried out in the same region, which warn about the environmental risks related to natural radioactivity occurrence. The results also validate the use of the Comet assay in hemocytes of D. melanogaster as a sensitive test to detect genotoxicity caused by natural radiation, and the use of a recently collected D. melanogaster strain in the environmental of radon.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill.

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea.

Purification and Characterization of a Cysteine-Rich 14-kDa Antibacterial Peptide from the Granular Hemocytes of Mangrove Crab Episesarma tetragonum and Its Antibiofilm Activity.

Antimicrobial peptide (AMP) crustin is a type of immune molecule present in the immune system of crustaceans and response against microbial invasion. In the present study, we have identified and characterized the cationic, amphipathic structure consisting of AMP crustin from a mangrove crab Episesarma tetragonum using CM Sepharose-based cation exchange column chromatography. E. tetragonum crustin showed a single band of 14 kDa on SDS-PAGE and the homogeneity showed retention time of 8.4 min in RP-HPLC. Functional studies of E. tetragonum crustin exhibits the antibacterial activity (2-4 µg/ml) and biofilm inhibition (20 µg/ml) against the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis. Hydrophobicity and extrapolysaccharide production of Gram-positive bacteria were inhibited through the bactericidal inhibitory concentration. In situ visualization analysis of biofilm inhibition was observed through light and confocal laser scanning microscopy. Surface morphology and the bacterial biofilm inhibition were viewed by scanning electron and atomic force microscopy. This study emphasizes the potential activity of E. tetragonum crustin, an interesting candidate for the development of novel broad-spectrum antimicrobial agent against bacterial pathogens. Graphical Abstract Antimicrobial peptide synthesis and host-pathogen interaction lead to production of immune molecules directed to destruction of pathogens.

Comparison of intermittent and continuous exposures to inorganic mercury in the mussel, Mytilus edulis: accumulation and sub-lethal physiological effects.

Aquatic organisms are often subject to intermittent exposure to pollutants in real ecosystems. This study aimed to compare mercury accumulation and the physiological responses of mussels, Mytilus edulis during continuous and intermittent exposure to the metal. Mussels were treated in a semi-static, triplicated design to either a control (no added Hg) or 50 µg l(-1) Hg as HgCl2 in continuous (daily) or intermittent (2 day exposure, 2 days in clean seawater alternately) exposure for 14 days. A time-dependent increase in Hg accumulation was observed in the continuous exposure, while the intermittent treatment showed step-wise changes in Hg concentrations with the exposure profile, especially in the gills. At the end of the experiment, tissue Hg concentrations were significantly increased in the continuous compared to the intermittent exposure for digestive gland (4 fold), gonad and remaining soft tissue (>2 fold), but not for the gill and adductor muscle. There was no observed oxidative damage at the end of the experiment as measured by the thiobarbituric acid reactive substances (TBARS) concentrations in tissues from all treatments. However, total glutathione was significantly decreased in the gill and digestive gland of both the continuous and intermittent exposure by the end of the experiment. The neutral red retention ability of the haemocytes was not affected, but total haemocyte counts were significantly decreased (<2 fold) in the intermittent compared to the continuous exposure. Histopathological examinations showed less pathology in the gill, but more inflammation in the digestive gland of mussels for the intermittent compared to the continuous exposure. Overall, the results showed that Hg accumulation from intermittent exposure was less than that of the continuous exposure regime, but the sub-lethal responses are sometimes more severe than expected in the former.

Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the expression regulation of immunity-related genes.

The tobacco hornworm Manduca sexta has served as a model for insect biochemical and physiological research for decades. However, knowledge of the posttranscriptional regulation of gene expression by microRNAs is still rudimentary in this species. Our previous study (Zhang et al., 2012) identified 163 conserved and 13 novel microRNAs in M. sexta, most of which were present at low levels in pupae. To identify additional M. sexta microRNAs and more importantly to examine their possible roles in the expression regulation of immunity-related genes, we constructed four small RNA libraries using fat body and hemocytes from naïve or bacteria-injected larvae and obtained 32.9 million reads of 18-31 nucleotides by Illumina sequencing. Mse-miR-929 and mse-miR-1b (antisense microRNA of mse-miR-1) were predicted in the previous study and now found to be conserved microRNAs in the tissue samples. We also found four novel microRNAs, two of which result from a gene cluster. Mse-miR-281-star, mse-miR-965-star, mse-miR-31-star, and mse-miR-9b-star were present at higher levels than their respective mature strands. Abundance changes of microRNAs were observed after the immune challenge. Based on the quantitative data of mRNA levels in control and induced fat body and hemocytes as well as the results of microRNA target site prediction, we suggest that certain microRNAs and microRNA*s regulate gene expression for pattern recognition, prophenoloxidase activation, cellular responses, antimicrobial peptide synthesis, and conserved intracellular signal transduction (Toll, IMD, JAK-STAT, MAPK-JNK-p38, and small interfering RNA pathways). In summary, this study has enriched our knowledge on M. sexta microRNAs and how some of them may participate in the expression regulation of immunity-related genes.

The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.

Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity.

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded ß-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.

Altered membrane lipid composition and functional parameters of circulating cells in cockles (Cerastoderma edule) affected by disseminated neoplasia.

Membrane lipid composition and morpho-functional parameters were investigated in circulating cells of the edible cockle (Cerastoderma edule) affected by disseminated neoplasia (neoplastic cells) and compared to those from healthy cockles (hemocytes). Membrane sterol levels, phospholipid (PL) class and subclass proportions and their respective fatty acid (FA) compositions were determined. Morpho-functional parameters were evaluated through total hemocyte count (THC), mortality rate, phagocytosis ability and reactive oxygen species (ROS) production. Both morpho-functional parameters and lipid composition were profoundly affected in neoplastic cells. These dedifferentiated cells displayed higher THC (5×), mortality rate (3×) and ROS production with addition of carbonyl cyanide m-chloro phenylhydrazone (1.7×) but lower phagocytosis ability (½×), than unaffected hemocytes. Total PL amounts were higher in neoplastic cells than in hemocytes (12.3 and 5.1 nmol×10(-6) cells, respectively). However, sterols and a particular subclass of PL (plasmalogens; 1-alkenyl-2-acyl PL) were present in similar amounts in both cell type membranes. This led to a two times lower proportion of these membrane lipid constituents in neoplastic cells when compared to hemocytes (20.5% vs. 42.1% of sterols in total membrane lipids and 21.7% vs. 44.2% of plasmalogens among total PL, respectively). Proportions of non-methylene interrupted FA- and 20:1n-11-plasmalogen molecular species were the most impacted in neoplastic cells when compared to hemocytes (â…“× and »×, respectively). These changes in response to this leukemia-like disease in bivalves highlight the specific imbalance of plasmalogens and sterols in neoplastic cells, in comparison to the greater stability of other membrane lipid components.

A simple protocol for extracting hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster, the mosquitoes Aedes aegypti and Anopheles gambiae and the tobacco hornworm, Manduca sexta. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild. Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms.

Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri.

Phenoloxidases (POs) and haemocyanins constitute a family of copper-containing proteins widely distributed among invertebrates. Both of them are able, under appropriate conditions, to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of two haemocyanin-like proteins from two colonial ascidians: Botryllus schlosseri from the Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between the above sequences and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Botryllus HLP grouped in the same cluster with Ciona POs, whereas Polyandrocarpa HLP clustered with arthropod haemocyanins; all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the genes are transcribed inside morula cells, a characteristic haemocyte type in ascidians where PO activity is located, at the beginning of their differentiation. These results represent a first attempt to identify candidate molecules responsible of the PO activity in compound ascidians.

A protocol for collecting and staining hemocytes from the yellow fever mosquito Aedes aegypti.

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens(1, 2). The innate immune system of mosquitoes consists of several lines of defense (3). Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut (4) enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens (5, 6). Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages (7). The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis (8), and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen (9, 10). These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively (11). More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues (11). While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

Pyrosequencing-based expression profiling and identification of differentially regulated genes from Manduca sexta, a lepidopteran model insect.

Although Manduca sexta has significantly contributed to our knowledge on a variety of insect physiological processes, the lack of its genome sequence hampers the large-scale gene discovery, transcript profiling, and proteomic analysis in this biochemical model species. Here we report our implementation of the RNA-Seq cDNA sequencing approach based on massively parallel pyrosequencing, which allows us to categorize transcripts based on their relative abundances and to discover process- or tissue-specifically regulated genes simultaneously. We obtained 1,821,652 reads with an average length of 289 bp per read from fat body and hemocytes of naïve and microbe-injected M. sexta larvae. After almost all (92.1%) of these reads were assembled into 19,020 contigs, we identified 528 contigs whose relative abundances increased at least 5- and 8-fold in fat body and hemocytes, respectively, after the microbial challenge. Polypeptides encoded by these contigs include pathogen recognition receptors, extracellular and intracellular signal mediators and regulators, antimicrobial peptides, and proteins with no known sequence but likely participating in defense in novel ways. We also found 250 and 161 contigs that were preferentially expressed in fat body and hemocytes, respectively. Furthermore, we integrated data from our previous study and generated a sequence database to support future gene annotation and proteomic analysis in M. sexta. In summary, we have successfully established a combined approach for gene discovery and expression profiling in organisms lacking known genome sequences.

Leureptin: a soluble, extracellular leucine-rich repeat protein from Manduca sexta that binds lipopolysaccharide.

Leucine-rich repeat containing proteins are involved in immune response in many capacities. In insects, these include Toll-like receptors and the Anopheles gambiae proteins APL1 and LRIM1. Here we describe the identification and characterization of leureptin, a novel extracellular protein with 13 leucine-rich repeats from hemolymph of the insect Manduca sexta. After injection of bacteria, leureptin mRNA level increased in fat body, but protein levels in plasma decreased, an indication that leureptin is consumed during the immune response. Leureptin bound to bacterial lipopolysaccharide (LPS). Microscopy using leureptin antiserum showed that leureptin associates with hemocytes after injection of bacteria, an indication that leureptin is involved in hemocyte responses to bacterial infection. Sequence database searches suggest similar proteins are present in other Lepidopteran species.

Is activated hemocyanin instead of phenoloxidase involved in immune response in woodlice?

In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod hemocyanin are reflected by the quaternary structure of the hemocyanin dodecamers that differs from that of other crustacean hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60 degrees against each other. Accordingly, the sequence of Porcellio hemocyanin can be distinguished clearly from other crustacean hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod hemocyanins. We propose a peracarid-type hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.

Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate, Halocynthia papillosa.

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram-positive and Gram-negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP.

Hemocyte-lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus.

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses.

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 185bp per read. A majority of the sequences (69,429 reads) could be assembled into 7231 contigs with an average size of 300bp, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only approximately 8% (606) of the contigs matched known M. sexta cDNA sequences, representing 186 of the 375 unique NCBI entries. The remaining 6625 contigs represented newly discovered cDNA segments from this well studied biochemical model insect. A search of the 7231 contigs using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity (E-value <1 x 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.

The role of hemocytes, serine protease inhibitors and pathogen-associated patterns in prophenoloxidase activation in the desert locust, Schistocerca gregaria.

The prophenoloxidase-activating system is an important component of the innate immune response of insects, involved in wound healing and melanotic encapsulation. In this paper we show that in the desert locust, Schistocerca gregaria, hemocytes, challenged with microbial elicitors, are indispensable for the limited proteolytic activation of prophenoloxidase (proPO) in plasma. In addition, we assessed the influence of serine protease inhibitors on the induction of PO-activity in plasma. While soybean Bowman-Birk inhibitor (SBBI) inhibited the PO activation by laminarin-treated hemocytes, the endogenous pacifastin-related inhibitors, SGPI-1 (S. gregaria pacifastin-related inhibitor-1) and SGPI-2 did not affect the PO-activity under similar conditions. On the other hand, real-time PCR analysis revealed that the transcripts, encoding SGPI-1-3, were more abundant in the fat body of immune challenged animals, as compared to control animals.