Immune System Of Invertebrates Research Articles

Immunotoxicity in green mussels under perfluoroalkyl substance (PFAS) exposure: Reversible response and response model development.

The immunotoxicity of 4 commonly detected perfluoroalkyl substances (PFASs), namely, perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) was investigated by measuring biomarkers of the immune profile of green mussels, Perna viridis. The biomarkers included neutral red retention, phagocytosis, and spontaneous cytotoxicity, all of which were tested on mussel hemocytes. Hemocytes are an important component of the invertebrate immune system. We found that exposure to PFASs could lead to reduced hemocyte cell viability and suppress immune function by up to 50% of normal performance within the experimental exposure range. The results indicate that PFASs have an immunotoxic potential and thus could pose severe health risks to aquatic organisms. The reported immunotoxicity is likely to result from the compounds' direct and indirect interactions with the hemocyte membrane, and therefore likely to affect the functionality of these cells. The immunotoxic response was found to be related to the organism's burden of PFASs, and was reversible when the compounds were removed from the test organisms. Based on this relationship, models using an organism's PFAS concentration and bioaccumulation factor (BAF) as the independent variables were established to quantify PFAS-induced immunotoxicity. The models help us to gain a better understanding of the toxic mechanism of PFASs, and provide a tool to evaluate adverse effects for the whole group of compounds with one mathematical equation. Environ Toxicol Chem 2018;37:1138-1145. © 2018 SETAC.

Role of Antheraea pernyi serpin 12 in prophenoloxidase activation and immune responses.

Serine protease inhibitors play a key role in the immune system of invertebrates by controlling proteolytic cascades. Besides its importance, the knowledge on immune functions of serpins in most of insects is fragmentary. In the present study, we identified serpin-12 from Antheraea pernyi encoding a predicted 402 amino acid residue protein (Apserpin-12). We expressed the recombinant protein in Escherichia coli and the purified protein was used for the synthesis of rabbit anti-Apserpin-12 polyclonal antibodies and functional studies. Quantitative real-time ploymerase chain reaction (qRT-PCR) analysis revealed that the knock-down of Apserpin-12 enhanced the prophenoloxidase (PPO) cascade stimulated by Micrococcus luteus in hemolymph, whereas addition of recombinant Apserpin-12 protein along with same elicitor led to down-regulate PPO activation. Following different microbial challenge (E. coli, Beauveria bassiana, M. Luteus, and nuclear polyhedrosis virus), the expression of Apserpin-12 mRNA was induced significantly. Furthermore, the Apserpin-12 double-stranded RNA administration elicited the expression of antimicrobial peptides, while the treatment with recombinant protein suppressed their expression. Tissue profile of Apserpin-12 indicated that it is expressed in all examined tissues, that is, hemolymph, malpighian tubules, midgut, silk gland, integument, and fat body with variation in their transcript levels. We concluded that Apserpin-12 may regulate PPO activation and inhibit the production of antimicrobial peptides in A. pernyi, suggesting important role in its immune system.

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach, Periplaneta americana.

Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar-binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, Periplaneta americana with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion-exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of P. americana. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from P. americana. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.

Immune-related genes in gastropods and bivalves: a comparative overview

The biological diversity of molluscs and their adaptation to highly diverse environments offer a unique opportunity for studying the evolution of the innate immune system in invertebrates. This review provides an updated account about the progresses made over the past few years in the study of the molecular players involved in the recognition of pathogen associated molecular patters (PAMPs), in the transduction of immune signaling and in the elimination of potentially pathogenic microbes in gastropod and bivalve molluscs. A major focus will be put on the differences and peculiarities of the molecular immune system of the two major molluscan classes, which have developed specific adaptations to cope with diverse living environments, pathogenic and non- pathogenic microbes over the course of several hundred million years of independent evolution. Intriguing but still poorly understood aspects, such as antiviral response and immune priming, will be also explored, highlighting the present challenges and opportunities connected to the application of modern genomics techniques to the study of the immune system in these fascinating metazoans.

Analysis of the shapes of coelomocytes of Aphelasterias japonica in vitro (Echinodermata: Asteroidea).

A description and formal classification of in vitro spreading coelomocytes from the Aphelasterias japonica sea star was performed using 39 parameters of linear and nonlinear morphometry based on the correlation, factor, and cluster analysis. The comparison of a variety of clustering models revealed the optimum classification parameters and algorithms. As a result, four morphological types of spreading cells significantly differing in a number of structural parameters were identified. This approach may be an important alternative or addition to classical methods of classification of polymorphic, irregularly shaped cells, in particular, cell elements of the invertebrate immune system. It provides the optimum methodology for structural analysis and classification of cells as a part of their further investigation in terms of structure, function, ontogeny, and diversity.

Invertebrate Models for Investigating the Impact of Nanomaterials on Innate Immunity: The Example of the Marine Mussel Mytilus spp.

Evaluating the interactions of nanomaterials (NMs) with the immune system is becoming an essential part of assessing nanosafety, not only for human health, but also for organisms living in different environments. The interactions between NMs and the components of the immune system in wildlife have been recently intensively investigated. Invertebrates represent more than 90% of animal species and are widespread in all environments, where they are subjected to a wide range of stressors. Despite invertebrates lack an adaptive immunity, they have developed a potent and complex innate immune system showing many commonalities to that of vertebrates. Conservation of the main mechanisms of innate immunity may greatly help understanding the possible interactions of NMs with the immune system across different taxa. However, the utilization of invertebrate models for immunosafety studies requires a thorough basic knowledge on the physiological regulation of the immune response of the tested species, together with information on particle behavior in the receiving environment, as well as routes of exposure in different cells and organisms. In this work, available data on the effects of NMs on the immune system of invertebrates are summarized. In particular, the results obtained in the marine bivalve, the mussel Mytilus, are summarized, demonstrating that mussel immune cells, the haemocytes, represent a suitable model for investigating the impact of NMs on innate immunity. These results underline how the utilization of invertebrate models represents a promising field for designing environmentally safer, "green" nanomaterials. Keywords: Nanosafety, innate immunity, invertebrates, marine bivalves, Mytilus, haemocytes, immunomodulation, nanosafety.

Hsp70 gene polymorphisms in farmed marine shrimp Litopenaeus vannamei populations exposed to white spot disease and infectious myonecrosis.

Single nucleotide polymorphisms (SNPs) are the best genetic markers for associative studies of the immune system in invertebrates. In the marine shrimp Litopenaeus vannamei, SNPs linked to disease resistance have been reported for some genes, such as hemocyanin, anti-lipopolysaccharide factor, and heat-shock protein 70 (Hsp70). In the present study, polymorphisms in the Hsp70 gene were investigated among three commercial L. vannamei populations bred in Northeast and South Brazil. The first population withstood a strong white spot disease outbreak; the second population suffered extended exposure to infectious myonecrosis; the third population was a high health population, which was experimentally infected with white spot syndrome virus (WSSV) in the present study. All five previously known SNPs (C661A, T712C, C782T, C892T, and C1090T) were detected in the coding region of Hsp70, by Sanger sequencing of 119 shrimp. Significant differences in genetic and genotype frequencies among populations were observed for C661A, C892T, and C1090T. In the population submitted to WSSV challenge, no frequency differences were found between dead and surviving shrimp groups. These results indicate that the Hsp70 polymorphisms described here cannot be associated with WSSV tolerance. However, significant frequency differences were observed for the population exposed to infectious myonecrosis virus. This is the first time that L. vannamei Hsp70 gene polymorphisms were studied in correlation with these important shrimp viruses.

The functions of serpin-3, a negative-regulator involved in prophenoloxidase activation and antimicrobial peptides expression of Chinese oak silkworm, Antheraea pernyi

Serpins are a superfamily of proteins engaged in various physiological processes in all kingdoms of life. To date, many striking results have demonstrated serpins are involved in the invertebrate immune system by regulating the proteolytic cascades. However, in most insect species, the immune functions of serpins in response against pathogen invasion remain obscure. In this study, we identified a full-length cDNA sequence of serpin, named serpin-3, from the Chinese oak silkworm Antheraea pernyi. Sequence alignments have indicated that Apserpin-3 might regulate the melanization reaction via inhibiting prophenoloxidases-activating protease(s) in plasma. Furthermore, it was detected to be primarily transcribed within the fat body, epidermis and hemocytes with significant induction following immune-challenge. Further studies have shown that the knockdown of serpin-3 up-regulated the prophenoloxidases cascade stimulated by pathogen in hemolymph, while the addition of recombinant serpin-3 along with the same elicitor led to the suppressed activation of prophenoloxidase. Besides, the injection of dsRNA of serpin-3 caused the elevated expression of antimicrobial peptides. Altogether, we arrived at a conclusion that serpin-3 might act as a negative-regulator in prophenoloxidases activation and inhibit the production of antimicrobial peptides in Antheraea pernyi larvae.

A Cumulative Strategy to Predict and Characterize Antimicrobial Peptides (AMPs) from Protein Database

Antimicrobial peptides (AMPs) are indivisible part of the innate immune system in invertebrates. AMPs have been proven to have crucial role with a wide range of biological activities, mainly with immunomodulatory and broad spectrum of antimicrobial activity against various pathogens. The unique and salient features of the AMPs show its exceptional nature of therapeutic activity and serves as an alternative agent for conventional antibiotics. The search for potential AMPs persist, as the emergence of multiple drug resistant bacterial strains has been spreading in higher number. Here, the putative antimicrobial peptide sequences were identified from 19,915 sequences of prawn transcriptome and analyzed with various in silico tools such as EXPASY, AMPA, and helical wheel projection and so on. The characteristic antimicrobial properties have been determined for 660 putative AMPs with above mentioned tools. We have demonstrated an efficient bioinformatics approach to derive and analyze the AMPs from the transcriptome data of Macrobrachium rosenbergii. Even though, 660 peptide regions were identified among those five peptide sequences were demonstrated comprehensively with each characteristic property contributes the antimicrobial activity. In this study, we have proposed a rapid and successful protocol that would help to predict AMP in sequential procedure using various in silico methods. Also, we have shown a distinctive method to shortlist the AMPs based on their various physico-chemical properties. Until now, no sequential protocol has been developed to identify and characterize the AMPs from protein database.

A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon

Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.

Wright–Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright–Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph.

Contrasting invertebrate immune defense behaviors caused by a single gene, the Caenorhabditis elegans neuropeptide receptor gene npr-1.

BackgroundThe invertebrate immune system comprises physiological mechanisms, physical barriers and also behavioral responses. It is generally related to the vertebrate innate immune system and widely believed to provide nonspecific defense against pathogens, whereby the response to different pathogen types is usually mediated by distinct signalling cascades. Recent work suggests that invertebrate immune defense can be more specific at least at the phenotypic level. The underlying genetic mechanisms are as yet poorly understood.ResultsWe demonstrate in the model invertebrate Caenorhabditis elegans that a single gene, a homolog of the mammalian neuropeptide Y receptor gene, npr-1, mediates contrasting defense phenotypes towards two distinct pathogens, the Gram-positive Bacillus thuringiensis and the Gram-negative Pseudomonas aeruginosa. Our findings are based on combining quantitative trait loci (QTLs) analysis with functional genetic analysis and RNAseq-based transcriptomics. The QTL analysis focused on behavioral immune defense against B. thuringiensis, using recombinant inbred lines (RILs) and introgression lines (ILs). It revealed several defense QTLs, including one on chromosome X comprising the npr-1 gene. The wildtype N2 allele for the latter QTL was associated with reduced defense against B. thuringiensis and thus produced an opposite phenotype to that previously reported for the N2 npr-1 allele against P. aeruginosa. Analysis of npr-1 mutants confirmed these contrasting immune phenotypes for both avoidance behavior and nematode survival. Subsequent transcriptional profiling of C. elegans wildtype and npr-1 mutant suggested that npr-1 mediates defense against both pathogens through p38 MAPK signaling, insulin-like signaling, and C-type lectins. Importantly, increased defense towards P. aeruginosa seems to be additionally influenced through the induction of oxidative stress genes and activation of GATA transcription factors, while the repression of oxidative stress genes combined with activation of Ebox transcription factors appears to enhance susceptibility to B. thuringiensis.ConclusionsOur findings highlight the role of a single gene, npr-1, in fine-tuning nematode immune defense, showing the ability of the invertebrate immune system to produce highly specialized and potentially opposing immune responses via single regulatory genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2603-8) contains supplementary material, which is available to authorized users.

Whole transcriptome analysis using next-generation sequencing of sterile-cultured Eisenia andrei for immune system research.

Recently, earthworms have become a useful model for research into the immune system, and it is expected that results obtained using this model will shed light on the sophisticated vertebrate immune system and the evolution of the immune response, and additionally help identify new biomolecules with therapeutic applications. However, for earthworms to be used as a genetic model of the invertebrate immune system, basic molecular and genetic resources, such as an expressed sequence tag (EST) database, must be developed for this organism. Next-generation sequencing technologies have generated EST libraries by RNA-seq in many model species. In this study, we used Illumina RNA-sequence technology to perform a comprehensive transcriptome analysis using an RNA sample pooled from sterile-cultured Eisenia andrei. All clean reads were assembled de novo into 41,423 unigenes using the Trinity program. Using this transcriptome data, we performed BLAST analysis against the GenBank non-redundant (NR) database and obtained a total of 12,285 significant BLAST hits. Furthermore, gene ontology (GO) analysis assigned 78 unigenes to 24 immune class GO terms. In addition, we detected a unigene with high similarity to beta-1,3-glucuronyltransferase 1 (GlcAT-P), which mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1 (human natural killer-1, also known as CD57), a marker of NK cells. The identified transcripts will be used to facilitate future research into the immune system using E. andrei.

Upregulated transcription of phenoloxidase genes in the pharynx and endostyle of Ciona intestinalis in response to LPS

We investigated the role of phenoloxidases (POs) in ascidians inflammatory reaction, a components of a copper-containing protein family involved in invertebrate immune system. In Ciona intestinalis two phenoloxidases (CinPO-1, CinPO-2) have been sequenced. In the present study, real time PCR analysis showed that both CinPO-1 and CinPO-2 genes were modulated by LPS inoculation suggesting that they are inducible and highly expressed in the inflamed pharynx. In situ hybridization disclosed CinPO-1 and CinPO-2 transcripts in pharynx hemocytes (granulocytes) and, mainly, in unilocular refractile granulocytes (URG) which mainly populated the inflamed tunic matrix. Interestingly, the genes are also upregulated by LPS in the endostyle (zones 7, 8 and 9) that is considered homolog to the vertebrate thyroid.

Function of two novel single-CRD containing C-type lectins in innate immunity from Eriocheir sinensis

C-type lectin is one of the pattern-recognition proteins of the non-self-innate immune system in invertebrates. In this study, two novel C-type lectin cDNAs (EsCTL1 and EsCTL2) of Eriocheir sinensis were cloned and characterized. EsCTL1 has 169 amino acids, whereas EsCTL2 has 164 amino acids. These two lectins contain one carbohydrate-recognition domain. Phylogenetic analysis showed that EsCTL1 and EsCTL2 were not clustered with other reported lectins from crabs. EsCTL1 and EsCTL2 were expressed only in the hepatopancreas, as detected by real-time PCR. When healthy crabs were challenged with lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, or Aeromonas hydrophila, the expression levels of EsCTL1 and EsCTL2 were significantly regulated. The recombinant EsCTL1 and EsCTL2 can agglutinate both Gram-positive (S. aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and A. hydrophila) in a Ca2+-dependent manner. The recombinant EsCTL1 and EsCTL2 can directly bind to LPS and PGN and to all tested microorganisms (S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila). Furthermore, rEsCTL1 and rEsCTL2 may facilitate the clearance of V. parahaemolyticus in vivo. These results suggest that EsCTL1 and EsCTL2 may have important roles in the anti-bacterial immunity of Chinese mitten crab.

Shrimp Immune System-Special Focus on Penaeidin

In this review paper, we summarize the immune system of penaeid shrimp. This review includes the clotting system comprising clot protein and clotting enzyme (transglutaminase II), as well as the prophenoloxidase (proPO) activation system including proPO-activating enzyme (PPAE), proPO and clip domain serine protease homolog (c-SPH). In addition, several shrimp cytokines or cytokine-like molecules found in tiger shrimp Penaeus monodon participating in the defense responses are also discussed. C-SPH could enhance hemocyte adhesion. Shrimp astakine promoted hemocyte proliferation in the hematopoietic tissues and could be down-regulated at the post-transcriptional level by the binding of intracellular molecules such as transglutaminase I and crustin Pm4 to its 3'-untranslatd region. Penaeidin is found to be a dual function molecule as an antimicrobial peptide and an autocrine-acting cytokine. Yet, other antimicrobial substances such as quinone and melanine which are generated during the proPO activation pathway and the reactive oxygen and nitrogen intermediates (ROI and RNI) which are generated during the phagocytic process can also kill and clear the invading microorganisms directly. In addition, Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF) which plays an essential role in the alternative adaptive immune system of invertebrates, has also been characterized in shrimp.Penaeid shrimp is one of the most important commercially available aquaculture resources. Although, the culture techniques and field management skills have significantly improved in the recent years, infectious diseases are still a threat to the industry. The study of shrimp immune system could be important in designing the strategies against pathogen infection. For shrimp, the innate immune responses play a major role in combatting invading pathogens and prevent them against diseases. The immune responses involve different factors such as physical barrier, bacteria clearance, encapsulation, clotting reactions, prophenoloxidase system, reactive oxygen intermediates, and antimicrobial activity. The innate immune system uses a variety of pattern recognition receptors (PRR) on the hemocyte membrane to detect the pathogenassociated molecule pattern (PAMP) and transduce signals during pathogen invasion. Hemocytes are then activated and the defense molecules are degranulated against the pathogens.

Corals and their potential applications to integrative medicine.

Over the last few years, we have pursued the use and exploitation of invertebrate immune systems, most notably their humoral products, to determine what effects their complex molecules might exert on humans, specifically their potential for therapeutic applications. This endeavor, called “bioprospecting,” is an emerging necessity for biomedical research. In order to treat the currently “untreatable,” or to discover more efficient treatment modalities, all options and potential sources must be exhausted so that we can provide the best care to patients, that is, proceed from forest and ocean ecosystems through the laboratory to the bedside. Here, we review current research findings that have yielded therapeutic benefits, particularly as derived from soft and hard corals. Several applications have already been demonstrated, including anti-inflammatory properties, anticancer properties, bone repair, and neurological benefits.

New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes.

Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.

<i>In vitro</i> Studies on Cellular Mediated Immune Response in Haemocytes of Crab— <i>Episesarma tetragonum</i>

Studying the biological interaction with the invertebrate immune system is attractive for the advancement of a basic knowledge on invertebrate and vertebrate general immunity, because it offers various possible alternatives for disease management. The invertebrate immune system involves encapsulation, nodule formation and phagocytosis process by haemocytes (cellular mediated). In the present study, we investigated the morphology of the haemocytes of Episesarma tetragonum , their role in cellular mediated immune responses such as encapsulation, phagocytosis and viability assays. Encapsulation response of haemocytes is tested with the CM cellulose, Sepharose CL-6B and DEAE Sepharose 6CLB beads. DEAE Sepharose 6CLB beads showed (80%) good cellular response. Phagocytic activity of haemocytes showed pathogen engulfing activity against the microbial patterns (N-acteyl muramic acid (NAM), N-acetyl glutamic acid (NAG), lipopolysaccharide, β-1,3-glucans, chitins, laminarins, zymosan and peptidoglycans) present on the surface of the pathogens. Phagocytic activities of the haemocytes were decreased when increasing the incubation time. Viability of haemocytes was decreased when the time intervals of absorption of tryphan blue were increased. The present investigation enlightens the role of hemocytes in the cellular mediated immune responses of crustacean crab including all invertebrates.

Molecular mechanisms of the shrimp clotting system

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.