Immune System Of Crustaceans Research Articles

A Hybrid Design for the Functional Assay of LvLRRm (Protein Containing LRR Domain) of the White Leg Shrimp, Litopenaeus vannamei

In the crustacean immune system, leucine-rich repeat (LRR) is one of the major structures for recognizing pathogen-associated molecular patterns (PAMPs). LRR domain-containing proteins belong to the LRR family, which is a large group of proteins with more than 6000 genes in the database. They are involved in very diverse physiological functions, mainly by interacting with other proteins. In a previous study, the LvLRRm, a transmembrane protein containing only LRR domain, was identified in the white leg shrimp, Litopenaeus vannamei. Its versatile role in performing multiple immunomodulation activities has been reported. However, there is still a lack of research on its efficient function at the protein level. To investigate its interactions with other proteins, we applied a convenient method called the ‘Hybrid LRR technique’ to produce a recombinant LvLRRm. The LvLRRm and hagfish’s variable lymphocyte receptors (VLRs) fragments were fused to the conserved LxxLxLxxN motif while retaining the β-strand. In addition, we established interactions between hybrid proteins and the flagellin of Salmonella typhimurium by performing surface plasmon resonance (SPR) analysis. The results of the SPR analysis demonstrated notable affinity for both LvLRRm and hybrid proteins towards Salmonella flagellin. The designed LvLRRm hybrid proteins bring insight for universal applications without losing protein functions.

The effect of reactive oxygen species (ROS) in immunity and WSSV infection of Scylla paramamosain

Reactive oxygen species (ROS) are typically regarded as being generated by the cellular respiratory chain or by cells under pathological damage, which play a crucial role as signaling molecules in promoting hemocytes circulation and normal cellular physiological functions. In this study, the antioxidant N-acetylcysteine (NAC) was used to reduce ROS in vivo and in vitro, which to analyze the effect of ROS on innate immunity and viral infection of mud crab. The total hemocyte count (THC), phenoloxidase (PO), superoxide dismutase (SOD) activity, immune-relative genes were analyzed, respectively. Moreover, the effect of ROS on WSSV infection was analyzed by THC and hemocytes apoptosis. The data showed that NAC could effectively remove and inhibit intracellular ROS. The THC of NAC group was reduced at 12 h and 24 h compared with that of control. And the inhibition of ROS by NAC could increase the SOD activity with control group, while increased the PO activity caused by early WSSV infection. And NAC could up-regulate the expression of MCM7, JAK, TLR and proPO significantly, while down-regulate the expression of Astakine, proPO, caspase and p53. Similarly, NAC could inhibit WSSV-induced apoptosis of S. paramamosain hemocytes. The data illustrated that ROS participates in the interaction between hemocytes and virus infection by regulating innate immunity. Especially, after NAC inhibited ROS, the expression of hemocytes proliferation gene Astakine was also inhibited, which may indicate that ROS is related to the process of hemocytes proliferation. The data will show a preliminary exploration on the regulatory role of ROS in crustacean immune system.

Characterization of peroxiredoxin from Neocaridina denticulata sinensis and its antioxidant and DNA protection activity analysis

Peroxiredoxin (Prx) is an antioxidant protein that widely exists in various organisms. To further investigate the role of Prx in the antioxidant and immune responses of Neocaridina denticulata sinensis, the full-length cDNA sequence of a Prx gene (Nd-Prx) from N. denticulata sinensis was obtained. The open reading frame (ORF) of Nd-Prx is 597 bp and encodes 198 amino acids. Amino acid similarity alignment showed that Nd-Prx contained a conserved sequence region “FYPLDFTFVCPTEI”. qRT-PCR assay showed that Nd-Prx was expressed in all tested tissues and its expression was highest in the ovary. Nd-Prx was most highly expressed at 36 h after copper stimulation. Nd-Prx expression levels in hepatopancreas were significantly upregulated after Vibrio parahaemolyticus challenge (P < 0.05). In addition, the recombinant Nd-Prx was prepared and its enzyme activity was most stable at 70 °C with pH of 6.0. The antioxidant activity and DNA protection of recombinant Nd-Prx was also demonstrated. In summary, this study investigated the role of Prx in antioxidant and immune responses of N. denticulata sinensis, which might provide a foundation for further exploring Prx in immune system of crustaceans and for the application in disease control.

Shrimp immune response: A transcriptomic perspective

AbstractDuring the last 15 years, a significant amount of data related to the crustacean immune system has been generated, mainly led by the advances made in the field of transcriptomic analysis through massive mRNA sequencing technology. In order to shed new light on the genes and metabolic pathways associated with the immune system in shrimp species, we have carried out a meta‐analysis using the current literature and the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI), which has 1195 published transcriptomic data sets until 31 December 2020, from different tissues and 14 Penaeus species. The transcriptomic analyses published so far have explored the effects of the challenge from viral and bacterial pathogens, as well as abiotic stressors, such as ammonia, temperature, salinity, oxygen deprivation and pH. They have also explored the effect of dietary factors and beneficial microbes (pre‐ and probiotics) in the immune response. In order to harness the vast amount of information available, multidisciplinary efforts have to generate better genomic tools to overcome the complexity of the genome and to implement the knowledge gained into commercial applications for the improvement of the culture conditions, adaptation of more sustainable feeds and producing strains showing more resilience/tolerance to diseases.

Impacts of pH on the Fitness and Immune System of Pacific White Shrimp

The atmospheric partial pressure of CO2 (pCO2) has been increasing dramatically since the beginning of the industrial revolution and about 30% of the CO2 produced by anthropogenic activities was absorbed by the ocean. This led to a perturbation of the seawater carbonate chemistry resulting in a decrease of the average surface ocean pH by 0.1 and termed ocean acidification (OA). Projections suggest that pCO2 may reach 900 μatm by the end of the twenty-first century lowering the average pH of the surface ocean by 0.4 units. The negative impacts of OA on many species of marine invertebrates such as mollusks, echinoderms, and crustaceans are well documented. However, less attention has been paid to the impacts of low pH on fitness and immune system in crustaceans. Here, we exposed Pacific white shrimps to 3 different pHs (nominal pH 8.0, 7.9, and 7.6) over a 100-days experiment. We found that, even though there were no significant effects on fitness parameters (survival, growth and allometries between length and weight), some immune markers were modified under low pH. A significant decrease in total hemocyte count and phenoloxidase activity was observed in shrimps exposed to pH 7.6 as compared to pH 8.0; and phagocytosis rate significantly decreased with decreasing pH. A significant increase in superoxide production was also observed at pH 7.6 as compared to pH 8.0. All these results suggest that a 100-days exposure to pH 7.6 did not have a direct effect on fitness but lead to a modulation of the immune response.

Neuroendocrine‐immune regulation mechanism in crustaceans: A review

AbstractThe development of aquaculture is closely related to human life. Crustaceans play essential roles in aquaculture. However, the cultivation of crustaceans has been plagued by various diseases, causing substantial economic losses. At present, the occurrence of shrimp diseases is directly related to environmental stress and physiological dysfunction, and especially environmental stress has become an important factor inducing diseases. Therefore, how to regulate the immune defence of crustaceans under environmental stress has become the primary problem in the prevention and control of shrimp diseases. The neuroendocrine‐immune system plays a vital role in maintaining homeostasis and enhancing environmental adaptability. Besides, in the evolutionary sense, with the theory that the nervous system and the immune system have a common origin put forward, the research on the neuroendocrine‐immune system of invertebrates has gradually become a hot spot. This review describes the evolution and communication mechanism of the neuroendocrine system and immune system in vertebrates and invertebrates, summarizes the characteristics of the neuroendocrine system and immune system in crustaceans, and focuses on how environmental stress affects the regulation of the neuroendocrine system on the immune system in crustaceans, as well as the influence of these regulations on host immune defence. This review also introduced the immune regulation of crustacean intestines under environmental stress and the effect of pathogen stress on crustacean immunity, hoping to lay a theoretical foundation for the follow‐up study of crustacean stress resistance and provide theoretical guidance for the healthy aquaculture of crustaceans.

Bursicon homodimers regulate the expression of anti-microbial peptide genes via relish in Macrobrachium nipponense

Bursicon (burs) is a neuropeptide hormone composed of two cystine knot-like proteins (burs α and burs β) that regulate the tanning of the cuticle of insect epidermis and the extension of new wings. In insects, burs regulate the secretion of antimicrobial peptides (AMPs). In this study, we characterized burs homodimers (named Mnbursα and Mnbursβ) and explored their roles in the synthesis of AMPs in Macrobrachium nipponense for the first time. The full cDNA lengths of Mnbursα and Mnbursβ are 946 and 1357 bp containing 444 and 414 nucleotides of an open reading frame that encodes 147 and 137 amino acids of protein, respectively. Tissue distribution analysis showed that Mnbursα and Mnbursβ were widely expressed in various tested immune tissues, and they were most prominently expressed in the hepatopancreas. At multiple time points after being challenged by bacteria (Vibrio parahaemolyticus and Staphylococcus aureus) and virus (white spot syndrome virus), the expression levels of Mnbursα and Mnbursβ in the gills and stomach were significantly upregulated, suggesting that pathogen invasion was involved in regulating the transcription of Mnbursα and Mnbursβ. We further explored the relationship between the burs homodimers and the synthesis of AMPs [crustin (Cru), anti-lipopolysaccharide factor (ALF), and carcinin (Car)] through RNA interference. Knockdown of Mnbursα and Mnbursβ evidently downregulated the expression of six tested AMPs (Cru1, Cru2, Cru3, ALF1, ALF3, and Car1). Recombinant r-Mnbursα-α and r-Mnbursβ-β proteins could obviously increase the expression levels of the six AMPs, indicating that Mnbursα and Mnbursβ play positive regulatory roles in the synthesis of AMPs. NF-κB transcription factors are essential in the production of AMPs in humoral immunity. Further research showed that the upregulation roles of r-bursicon homodimers on three tested AMPs (Cru1, ALF1, and ALF3) disappeared after silencing the MnRelish transcription factor. Our findings suggest that the synthesis of AMPs is induced by the bursicon homodimers via Relish in the innate immunity of M. nipponense. This study provides new insights in the function of bursicon in the immune system of crustaceans.

Transcriptome analysis of immune-related genes in Sesarmops sinensis hepatopancreas in reaction to peptidoglycan challenge

Sesarmops sinensis is a dominant omnivorous crab species, which plays an important ecological function in salt marsh ecosystems. To better understand its immune system and immune related genes under pathogen infection, the transcriptome was analyzed by comparing the data of S. sinensis hepatopancreas stimulated by PBS and PGN. A set of assembly and annotation identified 39,039 unigenes with an average length of 1105 bp, obtaining 1300 differentially expressed genes (DEGs) in all, which included 466 remarkably up-regulated unigenes and 834 remarkably down-regulated unigenes. In addition, based on mensurable real time-polymerase chain reaction and high-throughput sequencing, several immune responsive genes were found to be markedly up-regulated under PGN stimulation. In conclusion, in addition to enriching the existing transcriptome data of S. sinensis, this study also clarified the immune response of S. sinensis to PGN stimulation, which will help us to further understand the crustacean's immune system.

Characterization and antimicrobial evaluation of a new Spgly-AMP, glycine-rich antimicrobial peptide from the mud crab Scylla paramamosain

Antimicrobial peptide (AMP) is a crucial component of the innate immune system in crustaceans. In mud crab, Scylla paramamosain, a commercially important species, a glycine-rich antimicrobial peptide (Spgly-AMP) gene was newly identified and putatively encoded a 26aa signal peptide and 37aa mature peptide. To understand the function of Spgly-AMP, the expression profile of Spgly-amp gene was characterized, which showed Spgly-amp was expressed widely in most tissues of adult crabs with the highest expression level in hemocytes. After Vibrio parahaemolyticus, PGN, or Poly I:C stimulations, the expression level of Spgly-amp was significantly up-regulated in the hemocytes. In antimicrobial assays, chemically synthesized Spgly-AMP peptides exhibited strong antibacterial activities against both Gram-positive and Gram-negative bacteria and high thermal stability after high-temperature heating. These findings in the present study verified the importance of the Spgly-AMP in defense of pathogenic bacteria infection in the mud crab and provided a promising candidate of antimicrobial agents in the crab aquaculture.

Acute BPA exposure-induced oxidative stress, depressed immune genes expression and damage of hepatopancreas in red swamp crayfish Procambarus clarkii

Bisphenol A is a typical endocrine disrupting chemicals (EDCs) and produce various toxic effects on animals due to its potential endocrine disruption, oxidative damage effect, mutagenic effect and hypomethylation. To study its effect on the immune system of crustaceans, the Procambarus clarkii were utilized to detect the immune related indicators after 225 μg/L BPA exposure for 1 week. Hepatopancreatic histology and ultrastructure analysis showed that the brush border disappeared, the lumen increased, and the connection between the hepatic tubules fade away in BPA treated group. BPA could significantly increase the level of ROS, inhibit the activities of antioxidant-related enzymes (SOD, POD, and CAT), and thereby cause the oxidative stress. The enzyme activities of AKP, ACP and lysozyme in hepatopancreas after BPA exposure were also depressed even after Aeromonas hydrophila infections. The relative expression profiles of immune-related genes after BPA exposure and bacterial infection showed suppressed trends of most selected genes. Under A. hydrophila infections, the cumulative mortality of 225 μg/L BPA-treated crayfish was significantly higher than other groups. All these results indicated that BPA exposure had adverse effects on the immune ability of P. clarkii. The present study will provide an important foundation for further understanding the effects of EDCs on crustacean immune functions.

The immune function of prophenoloxidase from red swamp crayfish (Procambarus clarkii) in response to bacterial infection

Prophenoloxidase (proPO) is the zymogen form of phenoloxidase (PO), a key enzyme in melanization cascade that has been co-opted in invertebrate immune reactions. There have been reported that proPO plays many essential roles in the crustacean immune system. However, little is known about the function of proPO from red swamp crayfish (Procambarus clarkii) which is an important cultured species worldwide. Here, we cloned and expressed proPO gene from red swamp crayfish (PcproPO). Subsequently, specific antibody against PcproPO was generated. The immune function of PcproPO was further characterized in vitro and in vivo. The results showed that the expression of PcproPO mRNA could be significantly up-regulated during the challenge of Gram-positive-negative (Vibrio parahaemolyticus) and Gram-positive-positive bacterial (Staphylococcus aureus). Furthermore, the purified recombinant PcproPO protein had a strong affinity binding to both bacteria and polysaccharides. In vivo knockdown of PcproPO could significantly reduce the crayfish bacterial clearance ability, resulting in the higher mortality of the crayfish during V. parahaemolyticus infection. In addition, in vitro knockdown of PcproPO in the hemocytes significantly reduced the phenoloxidase (PO) activity and the bacterial clearance ability, indicating that PcproPO might involve in hemocyte-mediated melanization. Our results will shed a new light on the immune function of PcproPO in the crayfish.

Transcriptional Identification of Related Proteins in the Immune System of the Crayfish Procambarus clarkii.

The freshwater crayfish Procambarus clarkii is an animal model employed for physiological and immunological studies and is also of great economic importance in aquaculture. Although it is a species of easy husbandry, a high percentage of its production is lost annually as a result of infectious diseases. Currently, genetic information about the immune system of crustaceans is limited. Therefore, we used the abdominal nerve cord from P. clarkii to obtain its transcriptome using Next Generation Sequencing (NGS) to identify proteins that participate in the immune system. The reads were assembled de novo and consensus sequences with more than 3000 nucleotides were selected for analysis. The transcripts of the sequences of RNA were edited for annotation and sent to the GenBank database of the National Center for Biotechnology Information (NCBI). We made a list of accession numbers of the sequences which were organized by the putative role of the immune system pathway in which they participate. In this work, we report on 80 proteins identified from the transcriptome of crayfish related to the immune system, 74 of them being the first reported for P. clarkii. We hope that the knowledge of these sequences will contribute significantly to the development of future studies of the immune system in crustaceans.

C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca2+-dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain.

SpCrus3 and SpCrus4 share high similarity in mud crab (Scylla paramamosain) exhibiting different antibacterial activities

Type I crustins are crucial effectors of crustacean immune system. Various type I crustins with high sequence diversity possess different antimicrobial activities. To date, the mechanism on how the sequence diversity of type I crustins affects their antimicrobial activities is largely unclear, and how different crustins function together against bacterial invasion still remains unknown. In this study, we identified two novel type I crustins, namely, SpCrus3 and SpCrus4, from an economically important crab, Scylla paramamosain. Either SpCrus3 or SpCrus4 was highly expressed in gill. After challenges with Vibrio parahemolyticus or Staphylococcus aureus, SpCrus4 was up-regulated, whereas SpCrus3 was down-regulated. No significant expression change of SpCrus3 and SpCrus4 was observed after white spot syndrome virus injection, suggesting that these two genes may not participate in the antiviral immune responses. SpCrus3 and SpCrus4 had the common 5′ terminus and high similarity of 66.06%, but SpCrus4 exhibited stronger antimicrobial activity than that of SpCrus3. Microorganism-binding assay results revealed that both SpCrus3 and SpCrus4 exhibited binding ability to all tested microorganisms. Furthermore, the polysaccharide-binding assay showed that these two proteins exhibited strong binding activity to bacterial polysaccharides, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN). SpCrus3 and SpCrus4 exhibited stronger binding activity to LPS or LTA than to PGN. Moreover, SpCrus4 showed stronger binding activity to LTA than that of SpCrus3, which may be responsible for the significantly distinct antimicrobial activity between these two proteins. In addition, SpCrus4 displayed stronger agglutination activity against several kinds of microorganisms than that of SpCrus3. This increased agglutination activity may also contribute to the strong antibacterial activity of SpCrus4. On the basis of all these results, a possible antibacterial mode exerted by SpCrus3 and SpCrus4 was proposed as follows. SpCrus3 was highly expressed in normal crabs to maintain low-level antibacterial activity without bacterial challenges. When crabs were challenged with bacteria, large amount of SpCrus4 was generated to exhibit strong antibacterial activity against bacterial invasion. This study provides new insights to understand the antibacterial functions and mechanisms of type I crustins.

Molecular isolation and characterization of a haemocyanin ofMacrobrachium rosenbergiireveal its antibacterial activities

Haemocyanin is a multi-subunit protein complex found in the haemolymph and is involved in the immune system of crustaceans. In this study, a haemocyanin gene of Macrobrachium rosenbergii, designated MrHc, was successfully isolated. The MrHc gene contained an open reading frame (ORF) of 1,992 nucleotides, encoding a protein of 663 amino acid residues with a molecular mass of 76.5 kDa. The deduced amino acid sequence contained distinct structural motifs of the haemocyanin superfamily, including an all-alpha domain, a copper-containing domain and an immunoglobulin-like domain. Based on the phylogenetic analysis, the MrHC protein demonstrated a close relationship with the haemocyanins of Palaemon carinicauda and Macrobrachium nipponense. The MrHc gene was expressed in various shrimp tissues, including the hepatopancreas, gill, haemocytes, stomach and muscle. After Macrobrachium rosenbergii nodavirus (MrNV) challenge tests, the MrHc gene was up-regulated 237-fold at day 2. A recombinant protein of the MrHc immunoglobulin-like domain exhibited antibacterial activity against Vibrio vulnificus, V. parahaemolyticus, Aeromonas caviae, A. veronii, A. hydrophila and Bacillus cereus. This study suggested that MrHc may play important roles in the shrimp innate immune response to MrNV infection and bacterial infection.

Participation of lectins in crustacean immune system

Several lectins have been found in crustaceans, and it has been suggested that they play roles in cell signalling, cell–cell interaction, protein synthesis and pathogen recognition. These functions are performed through their specificity for carbohydrates. This review analyses the carbohydrate specificity, tissue distribution and participation in the immune responses of lectins in crustaceans. Furthermore, we explore some data showing that lectin expression seems to be stimulated by pathogens, favouring crustacean survival.

Modeling of macromolecular proteins in prophenoloxidase cascade through experimental and computational approaches.

Prophenoloxidase (ProPO) cascade is a principal defense system in crustaceans, which consists of a variety of pattern recognition proteins (lipopolysaccharide and β-glucan-binding protein [β-GBP], β-GBP, and β-glucan recognition protein), proteases (serine protease), and protease inhibitors (α2-macroglobulin and pacifastin) to regulate the protection mechanism in crustaceans. In the prophneoloxidase pathway, the protein-protein interactions (PPIs) and other immune-related analyses still have not been reported. Moreover, the structural features of ProPO cascade proteins have not yet been reported, hence we constructed the three-dimensional structural features for all ProPO pathway proteins. Their PPIs were studied through an in silico approach. Laminarin has been identified as a triggering activator and it showed energetic binding with homology modeled β-GBP and activated the β-GBP, followed by the protein-protein complex formation leading to phenoloxidase synthesis. These findings provided a novel view of the ProPO mechanism and enhanced our knowledge of the innate immune system in crustaceans via computation. In conclusion, we propose a combined experimental and computational approach to analyze the mechanism of ProPO cascade proteins.

Phenoloxidase an Important Constituent in Crustacean Immune System - A Review

Phenoloxidase an Important Constituent in Crustacean Immune System - A Review

Next-Generation Sequencing and the Crustacean Immune System: The Need for Alternatives in Immune Gene Annotation.

Next-generation sequencing has been a huge benefit to investigators studying non-model species. High-throughput gene expression studies, which were once restricted to animals with extensive genomic resources, can now be applied to any species. Transcriptomic studies using RNA-Seq can discover hundreds of thousands of transcripts from any species of interest. The power and limitation of these techniques is the sheer size of the dataset that is acquired. Parsing these large datasets is becoming easier as more bioinformatic tools are available for biologists without extensive computer programming expertise. Gene annotation and physiological pathway tools such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology enable the application of the vast amount of information acquired from model organisms to non-model species. While noble in nature, utilization of these tools can inadvertently misrepresent transcriptomic data from non-model species via annotation omission. Annotation followed by molecular pathway analysis highlights pathways that are disproportionately affected by disease, stress, or the physiological condition being examined. Problems occur when gene annotation procedures only recognizes a subset, often 50% or less, of the genes differently expressed from a non-model organisms. Annotated transcripts normally belong to highly conserved metabolic or regulatory genes that likely have a secondary or tertiary role, if any at all, in immunity. They appear to be disproportionately affected simply because conserved genes are most easily annotated. Evolutionarily induced specialization of physiological pathways is a driving force of adaptive evolution, but it results in genes that have diverged sufficiently to prevent their identification and annotation through conventional gene or protein databases. The purpose of this manuscript is to highlight some of the challenges faced when annotating crustacean immune genes by using an American lobster (Homarus americanus) transcriptome as an example. Immune genes have evolved rapidly over time, facilitating speciation and adaption to highly divergent ecological niches. Complete and proper annotation of immune genes from invertebrates has been challenging. Modulation of the crustacean immune system occurs in a variety of physiological responses including biotic and abiotic stressors, molting and reproduction. A simple method for the identification of a greater number of potential immune genes is proposed, along with a short introductory primer on crustacean immune response. The intended audience is not the advanced bioinformatic user, but those investigating physiological responses who require rudimentary understanding of crustacean immunological principles, but where immune gene regulation is not their primary interest.

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity.

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system.