Immunoproteomic analysis of fish ectoparasite, Argulus siamensis antigens.

AIM: An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. MATERIALS AND RESULTS: The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4-7 and SDS-PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti-A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver-stained gel and subjected to mass spectrometry in MALDI-TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against A siamensis is discussed in the paper. CONCLUSION: Three of the identified proteins namely, bromodomain-containing protein, anaphase-promoting complex subunit 5 and elongation factor-2 could possibly serve as vaccine candidates against argulosis in carps.

Transcriptional changes in three immunoglobulin isotypes of rohu, Labeo rohita in response to Argulus siamensis infection.

Immunoglobulin heavy chains of three isotypes viz., IgM, IgD and IgT/IgZ are described in teleosts. In this study, a challenge experiment with an ectoparasite Argulus siamensis was conducted to evaluate the changes in adaptive immune response by quantitation of expression of Ig heavy chains in skin, head kidney and mucus of infected rohu, Labeo rohita. Rohu were challenged with 100 metanauplii of A. siamensis/fish. Head kidney, skin and mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d and 30 d by sacrificing four fish each from infected and control groups at each time point. The expression of IgM, IgD and IgZ in these tissues were measured by reverse transcription real time quantitative PCR. IgM level was found to reach its peak significantly 30 d post-infection in head kidney tissue, while IgM transcripts were below detectable range in skin and mucus at all time points. IgZ and IgD levels were significantly up-regulated post-infection in all the three tissue samples. Early up-regulation of IgD was observed in skin and mucus, compared to head kidney. This study showed that parasitic invasion can trigger varied expressions of immunoglobulin types to provide systemic as well as local protection in the host. In particular, the appearance of high level of expression of IgZ and IgD in skin and mucus will pave the way for vaccine development against A. siamensis which feeds on those tissues.

Purification and molecular characterization of IgM in olive barb, Puntius Sarana.

In the present article, immunoglobulin (Ig) of Puntius sarana (a vulnerable medium carp species) was purified by affinity chromatography, characterized, and identified as only IgM type with a native molecular weight of 879 kDa having one heavy (88 kDa) and one light (26 kDa) chain. Further, the developed rabbit antisera against IgM was found to be quite specific to P. sarana IgM and used in ELISA to measure the antibody titer in P. sarana at different time periods, against an antigen (hemocyanin) injection with and without adjuvant. The antibody titer was significantly higher in most of the time periods in both groups, however, the adjuvant-treated group showed higher antibody titer at days 43 and 90, compared to non adjuvant-treated group. Further, the partial IgM sequence was amplified and its expression level was checked during ontogenesis. The IgM transcript was detected from unfertilized egg stage to 4 days post fertilization (dpf) and again reappeared at 21 dpf whereas during infection with Aeromonas hydrophila, significantly marked up-regulation of the gene was observed at 12 hr, 24 hr, and 7 days post-infection time periods indicating the role of IgM during early embryonic time period as well as during bacterial pathogenesis.

Physiological Responses to Acute Heat Stress in Rohu, Labeo rohita: Insights from Liver Proteomics.

Heat stress is a major problem in aquaculture species, causing changes in physiology such as decreased feed intake, growth rate, reproduction, and internal cellular damage, thereby affecting fish's health. The effects of an acute heat stress simulating a daily rise and fall in temperature on summer days were evaluated in the liver proteome of rohu (Labeo rohita) fingerlings in the present study. The fish maintained at 30 °C were gradually exposed to a higher temperature of 36 °C at an increment rate of 1 °C per 1.5 h, and after 3 h at that temperature, it was gradually reduced to 30 °C. The liver tissue samples were collected at 5 am, 5 pm, and 5 am the next day from the exposed and control fish. Protein samples were prepared from the liver tissues, and the extracted proteins were compared using 2-dimensional (2D) gel electrophoresis (2DGE) and mass spectrometry (MS) using a MALDI-TOF/TOF mass spectrometer. A total of 44 differentially expressed protein spots were visualized in 2D gel analysis from heat stress exposed fish at three time points, out of which 21 proteins including one hypothetical protein could be identified by MS. The abundance of five selected differentially expressed proteins (DEPs) was validated using qPCR. The majority of DEPs were found to be involved primarily in lipid, protein and energy metabolism, immune system regulation, cytoskeletal stability, and ROS management. The findings of this study would help in the development of strategies to mitigate heat stress in L. rohita.

De novo whole transcriptome analysis of the fish louse, Argulus siamensis: first molecular insights into characterization of Toll downstream signalling molecules of crustaceans.

Argulus siamensis is a major ectoparasitic pathogen of freshwater fish capable of causing substantial economic loss. None of the available control measures have been able to address the problem of argulosis resourcefully. To combat this pathogen effectively, it is necessary to have a comprehensive understanding of its life processes with information on various genes involved. The transcriptome studies can generate introductory information about genes participating in physiological processes of the parasite which could be targeted for their control. In this study, the transcriptome sequencing of A. siamensis was performed on Illumina HiSeq 2000 platform which generated 75,126,957 high quality reads. A total of 46,352 transcript contigs were assembled with average length of 1211bp and N50 length of 2302bp. In total, 19,290 CDS including 184 novel CDS and 59,019 open reading frames (ORFs) were identified from the assembled contigs. Gene ontology and Kyoto Encylopedia of Genes and Genomes pathway analysis were performed to classify contigs into their functional categories and regulation pathways. Additionally, 1171 simple sequence repeats were identified from the assembled contigs. Further, twelve contigs with high similarity with downstream molecules of the mammalian toll like receptor (TLR) pathway were validated by their inductive expressions in response to lipopolysaccharide (LPS) of Gram negative bacteria, Escherichia coli and Gram positive bacteria, Staphylococcus aureus. The transcriptome of an ectoparasite A. siamensis was sequenced, assembled, annotated, and the downstream signalling molecules of Toll pathway characterized. The transcriptome data generated will facilitate studies on functional genomics that will subsequently be applied for vaccine development and other control strategies against the parasite.

Characterization of a Lipopolysaccharide- and Beta-1,3-Glucan Binding Protein (LGBP) from the Hepatopancreas of Freshwater Prawn, Macrobrachium rosenbergii, Possessing Lectin-Like Activity.

The study focuses on the isolation, characterization, and expression analysis of a lectin from the hepatopancreas of Macrobrachium rosenbergii. The protein was isolated by affinity chromatography on a melibiose-agarose column. The molecular weight of the native protein was found to be ~120 kDa which consists of a single polypeptide of ~39.5 kDa. On mass spectrometric analysis, the protein was identified as lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP). LGBP showed hemagglutination with rabbit RBC like a lectin and its carbohydrate-binding specificity was determined by the hemagglutination inhibition test. The protein also showed antibacterial activity against two Gram-negative bacteria Vibrio harveyi and Aeromonas sobria, and one Gram positive bacteria Bacillus cereus in the disc diffusion test. Rabbit antiserum was raised against the purified LGBP and used to develop a sandwich ELISA system for quantitation of the protein in hepatopancreas and serum samples of M. rosenbergii. The expression of the LGBP transcripts in muscle, hepatopancreas, and gill tissues from M. rosenbergii juveniles at 72 h post-challenge of V. harveyi was not modulated as noticed in qPCR analysis. However, significant increases in the concentrations of LGBP protein in hepatopancreas (5.23 ± 0.45 against 3.43 ± 0.43 mg/g tissue in control) and serum (1.08 ± 0.14 against 0.61 ± 0.08 µg/ml in control) were observed in the challenged group of prawns in ELISA suggesting its putative role against bacterial infections. The study for the first time characterized the native LGBP of M. rosenbergii showing a multifunctional role in immunity.

Isolation and characterization of a lectin-like chitinase from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii.

A lectin was isolated from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii by affinity chromatography using mucin-sepharose matrix. The purity of the isolated lectin was confirmed in native gradient PAGE that showed a single protein band of ∼37.9 kDa. In SDS-PAGE also one band of ∼43.3 kDa molecular weight was observed that indicated the protein to be a monomer. The band from the SDS-PAGE gel was identified through mass spectrometry as chitinase 1. The purified chitinase (50 µg/ml) hemagglutinated rabbit RBCs and, mucin and glucose inhibited hemagglutination with minimum concentrations of 0.1 mg/ml and 100 mM, respectively. Bacterial agglutination with Gram -ve Vibrio harveyi, Aeromonas sobria and Escherichia coli was also observed by this protein. Thus, chitinase 1 showed lectin-like properties besides its chitin hydrolytic activity. In western blot with hepatopancreas sample, rabbit antiserum against chitinase 1 cross-reacted to two additional proteins namely, chitinase 1C and obstructor E (a chitin-binding protein, CBP), besides its specific reactivity. An indirect ELISA was developed with the antiserum to quantify chitinases/CBP in hepatopancreas and serum samples of M. rosenbergii. The assay was used in samples from juvenile prawns following V. harveyi challenge. At 72 h post-challenge, significantly higher levels of chitinases/CBP were quantified in the hepatopancreas of the challenged group (1.8 ± 0.2 mg/g tissue) compared to the control (1.2 ± 0.1 mg/g tissue). This study suggests that the chitinase 1 protein with lectin-like properties is possibly induced at the protein level and can be putatively involved in the innate immune response of M. rosenbergii.

Molecular cloning, sequence characterization, and expression analysis of C-type lectin (CTL) and ER-Golgi intermediate compartment 53-kDa protein (ERGIC-53) homologs from the freshwater prawn, Macrobrachium rosenbergii.

Lectin protein families are diverse and multi-functional in crustaceans. The carbohydrate-binding domains (CRDs) of lectins recognize the molecular patterns associated with pathogens and orchestrate important roles in crustacean defense. In this study, two lectin homologs, a single CRD containing C-type lectin (CTL) and an L-type lectin (LTL) domain containing endoplasmic reticulum Golgi intermediate compartment 53 kDa protein (ERGIC-53) were identified from the freshwater prawn, Macrobrachium rosenbergii. The open reading frames of MrCTL and MrERGIC-53 were 654 and 1,515 bp, encoding polypeptides of 217 and 504 amino acids, respectively. Further, MrCTL showed a 20-amino acid transmembrane helix region and 10 carbohydrate-binding residues within the CRD. MrERGIC-53 showed a signal peptide region, a type-I transmembrane region, and a coiled-coil region at the C-terminus. Phylogenetic analysis revealed a close relationship between MrCTL and MrLectin and M. nipponense CTL (MnCTL), whereas MrERGIC-53 shared high sequence identity with Eriocheir sinensis ERGIC-53 and Penaeus vannamei MBL-1. A homology-based model predicted small carbohydrate-combining sites with a metal-binding site for ligand binding (Ca2+ binding site) in MrCTL and beta-sheets connected by short loops and beta-bends forming a dome-shaped beta-barrel structure representing the LTL domain of MrERGIC-53. Quantitative real-time polymerase chain reaction detected MrCTL and MrERGIC-53 transcripts in all examined tissues, with particularly high levels observed in hemocytes, hepatopancreas, and mucosal-associated tissues, such as the stomach and intestine. Further, the expression levels of MrCTL and MrERGIC-53 transcripts were remarkably altered after V. harveyi challenge, suggesting putative function in host innate immunity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10499-022-00845-3.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Identification and functional characterization of a g-type lysozyme gene of Labeo rohita, an Indian major carp species.

Lysozyme, an important secretory innate immune component, possesses antimicrobial activity against broad spectrum of bacteria and viruses. In the present study, complete CDs (558 bps) of g-type lysozyme of rohu (Labeo rohita) was amplified and translated for a putative protein of 185 amino acids. The domain architecture and tertiary structure was also predicted for the protein. Its expression profile was studied in three infection models (bacteria: Aeromonas hydrophila, poly I:C, a dsRNA viral analogue and an ectoparasite: Argulus siamensis) in liver and kidney tissues of rohu. An up-regulation of 630-fold and 420-fold of the gene was observed at 48 h in liver and anterior kidney tissues respectively, after A. hydrophila infection. Significant increase in transcript level was noticed in both liver (0.8-fold) and kidney (480-fold) after 1 h and 12 h of poly I:C induction, respectively. Similarly, expression of lysozyme g transcripts was increased 6000-fold after 7 d of A. siamensis infection in liver tissue. The recombinant protein of g-type lysozyme of rohu (rLr-lysG) of 20.19 kDa was produced in Escherichia coli system and the lysozyme activity of rLr-lysG was found to be most active at pH 6.0 and temperature 35 °C. The potential lytic activity was found to be against A. hydrophila (UL = 0.53) followed by for E. tarda (UL = 0.45) whereas the lytic activity was the least against S. aureus (UL = 0.35) and M. lysodeikticus (UL = 0.34), at pH 6.0 and temperature 35 °C. The normal serum level of protein was estimated using indirect ELISA and was found to be very low (0.12-0.15 µg/ml). These results suggested that g-type lysozyme of rohu might be a potent immunostimulant against microbial infections, with a major role in innate immunity.

Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets.

Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.

Transcriptional analysis of immune-relevant genes in the mucus of Labeo rohita, experimentally infected with Argulus siamensis.

The knowledge of mucosa-associated molecular events that occur during infections is scarce despite the well-established importance of mucus in fish immunity. Using qRT-PCR, we analyzed the immune gene expression patterns in mucus of Labeo rohita experimentally infected with an ectoparasite Argulus siamensis. Mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d, and 30 d post challenge of L. rohita with metanauplii of A. siamensis. All interleukins studied herein (IL 6, IL 15, and IL 1ß) showed significant upregulation of expression levels in mucus of A. siamensis-infected fish compared to control samples. Further, the expression levels of molecules involved in pathogen recognition, toll like receptor 22, and pathogen presentation, ß2 microglobulin, were found to be significantly upregulated in experimental samples until 7 d post challenge compared to control samples. The upregulated expression of lysozyme G at all time points post infection indicated the early activation of acute phase responses in mucus of infected L. rohita. Moreover, the expression levels of natural killer cell enhancing factor B were found to be higher in infected fish than they were in the control fish. The early upregulation of the immune genes observed herein reinforces the role of mucus as the first line of defense against pathogenic assault; furthermore, it expands our understanding of mucosal-immune responses to A. siamensis infection, which can aid development of immunological interventions.

Differential affinity of natural haemagglutinin of Macrobrachium rosenbergii towards vertebrate erythrocytes: effect of sex, size and moult stage on haemagglutination titre.

Lectins play important role in innate immunity of animals. The affinity of the natural haemagglutinin of the giant freshwater prawn Macrobrachium rosenbergii towards vertebrate erythrocytes and its level with relation to sex, size and moult stages were studied. The strongest agglutinating titres in haemolymph of prawns were marked against guinea pig, chicken, Clarias batrachus, and rabbit erythrocytes, and the weakest towards cattle, dog, horse and goat erythrocytes. A moderately agglutinating titre was evident in duck and human erythrocytes. The haemolymph of adult, male or intermoult stage prawns weighing more than 100 g had the highest haemagglutinating activity as compared to their respective counterparts with varied responses observed towards various erythrocytes.

Variation in susceptibility pattern of fish to Argulus siamensis: Do immune responses of host play a role?

Branchiuran ectoparasites of the genus Argulus can have extensive damaging effects on cultured fish. There exist no systematic studies that evaluate susceptibility or resistance of various carp species to Argulus sp. and the underlying mechanisms. The present study aimed at identifying the most susceptible and resistant cultured species, studying settlement and survival of parasite on these species, and finally unravelling the variations of immune response in both resistant and susceptible species. Fish from eight species (Labeo rohita, Cirrhinus mrigala, Catla catla, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idella, Carassius auratus, Labeo fimbriatus) were individually challenged with metanauplii of A. siamensis (100 metanauplii/fish) before rearing them in single tank in triplicate for 45 days. Based on the observed parasite load on each species, L. rohita was found to be the most susceptible and C. idella the resistant species. The settlement and survival of the parasite on L. rohita and C. idella was compared at 24, 48, 72 and 96h post experimental infection. Survival was significantly low at 72h onwards in C. idella indicating it is an unsuitable/poorly preferred host for A. siamensis. The inflammatory responses which are known to be related to susceptibility were analysed. Individuals of both the species were exposed to A. siamensis (100 parasites/fish), and after 24h and 3 d, skin samples directly from the attachment site and non-attachment sites were assessed for transcriptomic profiles of selected innate defence genes. Artificial skin abrasion permitted comparisons between abrasion associated injury and louse-associated injury. The inflammatory responses varied significantly between both species indicating their role in determining susceptibility of a host to A. siamensis. The expression of major histocompatibility class II and matrix metalloproteinase 2 was significantly higher in C. idella compared to L. rohita and therefore appeared to be involved in the early protective response against A. siamensis. It is essential to study the expression pattern of more participatory genes of the inflammation related pathways to understand species specific susceptible patterns.

Proteogenomics of Candida tropicalis--An Opportunistic Pathogen with Importance for Global Health.

The frequency of Candida infections is currently rising, and thus adversely impacting global health. The situation is exacerbated by azole resistance developed by fungal pathogens. Candida tropicalis is an opportunistic pathogen that causes candidiasis, for example, in immune-compromised individuals, cancer patients, and those who undergo organ transplantation. It is a member of the non-albicans group of Candida that are known to be azole-resistant, and is frequently seen in individuals being treated for cancers, HIV-infection, and those who underwent bone marrow transplantation. Although the genome of C. tropicalis was sequenced in 2009, the genome annotation has not been supported by experimental validation. In the present study, we have carried out proteomics profiling of C. tropicalis using high-resolution Fourier transform mass spectrometry. We identified 2743 proteins, thus mapping nearly 44% of the computationally predicted protein-coding genes with peptide level evidence. In addition to identifying 2591 proteins in the cell lysate of this yeast, we also analyzed the proteome of the conditioned media of C. tropicalis culture and identified several unique secreted proteins among a total of 780 proteins. By subjecting the mass spectrometry data derived from cell lysate and conditioned media to proteogenomic analysis, we identified 86 novel genes, 12 novel exons, and corrected 49 computationally-predicted gene models. To our knowledge, this is the first high-throughput proteomics study of C. tropicalis validating predicted protein coding genes and refining the current genome annotation. The findings may prove useful in future global health efforts to fight against Candida infections.

Egg laying strategies and effect of temperature on egg development of Argulus siamensis.

Argulus siamensis is the most damaging fish parasite prevalent in the freshwater aquaculture systems of India. In an attempt to further understand the behavior of this economically important parasite, the means of biological transmission, egg laying strategies and effect of temperature on development of eggs was studied. A. siamensis showed opportunistic egg laying behavior where in it used both living and non-living substrata for egg laying. It was marked that the parasites used the shells of freshwater snails of the family Viviparidae, the runners of the water weeds of genus Nymphoides and dead fish in the culture ponds for laying of eggs. This study confirmed that the maximum eggs were laid by the parasite in the habitat usage zone of the host fish. The optimum temperature for development of the eggs of A. siamensis into the infective naupliar stage and hatching was found to be 28 °C. These new insights into the behavior of A. siamensis would be helpful to devise biological control methods against the parasite.

Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies.

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.