Banya Kar

First evidence of comparative responses of Toll-like receptor 22 (TLR22) to relatively resistant and susceptible Indian farmed carps to Argulus siamensis infection.

Toll-like receptor 22 (TLR22) is present in teleost but not in mammals. Among Indian farmed carps, Catla catla is relatively more resistant than Labeo rohita to Argulus siamensis lice infection. TLR22 is believed to be associated with innate immunity against ectoparasite infection. To investigate the TLR22 mediated immunity against argulosis, we have cloned and characterized TLR22 genes of L. rohita (rTLR22) and C. catla (cTLR22). The full-length cDNAs of rTLR22 and cTLR22 contained an open reading frame of 2838 and 2841 nucleotides, respectively; bearing the typical structural features. Phylogenetically rTLR22/cTLR22 was most closely related to Cyprinus carpio (common carp) counterpart, having highest sequence identity of 86.0%. The TIR domain remained highly conserved with 90% identity within freshwater fishes. The sequence information of cDNA and genomic DNA together revealed that the rTLR22/cTLR22 genes are encoded by uninterrupted exons. The co-habitation challenge study with A. siamensis infection confirmed that C. catla is comparatively more resistant than L. rohita. Further, comparative mRNA expression profile in immune relevant tissues also suggested about the participatory role of TLR22 during lice infection. However, TLR22 might not solely be involved in conferring relative resistance among carp species against argulosis.

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.

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.

Mixed infection of Argulus japonicus and Argulus siamensis (Branchiura, Argulidae) in carps (Pisces, Cyprinidae): loss estimation and a comparative invasive pattern study

The genus Argulus is economically one of the most important taxa of crustacean ectoparasites in freshwater aquaculture systems throughout the world. This study investigates the occurrence and effect of a mixed infestation of two species of Argulus, Argulus japonicus Thiele, 1900 and Argulus siamensis Wilson, 1926, in a carp culture farm in Mandi, Himachal Pradesh, India. In this study the economic loss due to argulosis was estimated to be 67 102.00 INR (US

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

1428)/ha per year, taking into account factors like mortality, reduced growth rate, and costs associated with drug application. The parasite population of the farm under study was dominated by A. japonicus, which constituted 93.5% of the population, the rest being A. siamensis. The dominance of A. japonicus can be explained by a comparison of the invasiveness of both species through determination of their off-host survival period, fecundity, and infestation pattern. A. japonicus was found to be better adapted to resist starvation, as the maximum off-host survival period for A. japonicus was 6 days, compared to 4 days in A. siamensis at 28°C. The average clutch size of A. japonicus was experimentally found to be 187 eggs, versus 120 for A. siamensis. An experimental mixed infestation of both species was established on Labeo rohita (Hamilton, 1822) to assess their infestation patterns, which proved A. japonicus to be more virulent than A. siamensis. The wide distribution of A. japonicus in comparison to A. siamensis may thus be attributed to its relatively prolonged off-host survival period, higher fecundity, and stronger virulence.

Molecular characterization of interleukin 15 mRNA from rohu, Labeo rohita (Hamilton): Its prominent role during parasitic infection as indicated from infection studies.

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.

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

Interleukin 15 (IL-15) is an important cytokine of fish immune system. Sequence characterization of IL-15 from rohu, Labeo rohita revealed a mRNA sequence of 1064 bp with coding sequence of 567 bp and signal peptide of 16 amino acids. There are four characteristic sequence features viz., presence of four out-of-frame AUG initiation codons, four highly conserved cysteine residues, constitutive expression in all tissues and evolutionary similarity. The ontogeny study revealed maternal transfer of this molecule and higher expression up to 3 h post-fertilization in fertilized embryos. Its expression was down-regulated in anterior and posterior kidneys, intestine and liver tissues of rohu infected with Aeromonas hydrophila. Mild up-regulation in liver and higher expression in spleen was noticed in rohu stimulated with poly I:C (poly ionosinic:cytidylic), whereas down-regulation was observed in intestine and kidney tissues. However, a consistent higher expression was noticed in kidney and skin tissues during Argulus siamensis infection. Therefore, rohu IL-15 might possess more defensive role during early development and parasitic infection.

Evaluation of ribosomal P0 peptide as a vaccine candidate against Argulus siamensis in Labeo rohita

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 1β) 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, β2 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.

The immune response in rohu, Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Abstract Argulus spp. are important ectoparasites of fish, and the current approach of their control using chemical pesticides has numerous drawbacks. Vaccination is a promising alternative but identification of protective antigens is a limiting step. The ribosomal protein P0, essential for protein synthesis, has been studied as a vaccine candidate. We generated sequence information of the P0 protein of the ectoparasite Argulus siamensis and the host Labeo rohita. The region of the parasite P0 protein with less sequence similarity with that of the host P0 protein and high predicted antigenicity was used for peptide synthesis. The peptide was conjugated with keyhole limpet hemocyanin (KLH) for immunization of rohu at a dose of 1.5 μg/g body weight. Dot blot assays confirmed production of antibodies against pP0-KLH in immunized fish. We evaluated the efficiency of pP0-KLH as a vaccine antigen by challenge of the immunized fish with A. siamensis. Although there was no significant difference in parasite load between both groups, a reduced and delayed mortality of 59% (15 days post-infection) in immunized group was noticed as compared to 75% mortality (within 7–15 days post-infection) in control group. The partial protection observed indicated the need for further optimization of this molecule to develop it into a vaccine candidate.