Jyotirmaya Mohanty

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.

Efficacy of two avermectins, doramectin and ivermectin against Argulus siamensis infestation in Indian major carp, Labeo rohita

The efficacy of two avermectins, doramectin and ivermectin via oral and parenteral routes and their prophylactic effects against Argulus siamensis infestation in Indian major carp, Labeo rohita was studied. Doramectin and ivermectin were fed once to L. rohita sub-adults naturally infested with A. siamensis parasites at graded levels of 1000, 750 and 500 μg/kg body weight and 500, 300 and 200 μg/kg body weight, respectively. The mean time of clearance of parasites was 24 ± 0.149 h for 1000 and 24 ± 0.210 h for 750 μg of doramectin/kg body weight fish, however, the dose of 1,000 μg resulted in adverse reactions and mortality in fish. A single dose of 500μg/kg body weight of ivermectin also cleared the infestation in 24 ± 0.258 h. In second experiment, L. rohita naturally infested with A. siamensis were administered with doramectin and ivermectin intramuscularly as single dose of 200, 150 and 100 μg/kg body weight. Both doramectin and ivermectin at 200 μg/kg body weight cleared the infestation in 4 ± 0.149 and 4 ± 0.258 h respectively, however, those resulted in adverse reactions viz., black pigmentation, lethargy and poor appetite in fish. The mean time of clearance of parasites from fish was 6 ± 0.210 and 12 ± 0.314 h for doramectin and ivermectin, respectively, when given at a lower dose of 150 μg/kg body weight. The prophylactic effects of both drugs were also evaluated by challenging naïve rohu with metanaupliar larvae of A. siamensis after feeding the fish single dose of the doramectin and ivermectin at 750 and 500 μg/kg body weight, respectively. The effects of both the drugs were found to last for 17-18 days. The effects of bath treatment with doramectin and ivermectin at different concentrations on adult A. siamensis and metanaupliar larvae of A. siamensis were also evaluated. The results suggested that ivermectin could act more rapidly compared to doramectin. However, detailed studies on the toxicity and pharmacokinetics of doramectin and ivermectin upon their administration to aquatic organisms and the environmental impacts of their usage are required to be undertaken before finally recommending the safe use of these drugs for control of parasitic infections.

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

CHARACTERIZATION OF SERUM IMMUNOGLOBULIN IN CHANNA STRIATA (BLOCH) AND KINETICS OF ITS RESPONSE TO AEROMONAS HYDROPHILA ANTIGEN

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.

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

The immunoglobulin (Ig) from the serum of Channa striata was isolated by gel electroelution and characterized further to understand its nature and subsequent applications in studying the immune response. The purity of the sample was confirmed with the presence of a single band on native gradient PAGE and the molecular weight of ∼897 kDa was determined from the gel. In SDS-PAGE, C. striata Ig was reduced to produce two bands corresponding to H (heavy) (∼72 kDa) and L (light) (∼27 kDa) chain subunits. Polyclonal antiserum against the purified Ig was raised in a rabbit and adsorbed with 10% liver tissue homogenate of C. striata to enhance its specificity. By an indirect ELISA standardized using the adsorbed rabbit antiserum, the normal serum Ig concentration in C. striata was estimated to be 3.48 mg/mL. Further, a kinetic study of specific immunoglobulin response to formalin-killed Aeromonas hydrophila antigen was undertaken using another indirect ELISA, which showed a significant increase in serum immunoglobulin titer from day 2 onwards and reached its peak at day 14. Subsequently, the Ig titer was dropped from day 21 onwards till the completion of the experiment at day 42, although it was at a significantly higher level than the control.

Purification and Molecular Characterization of IgM in Olive Barb, Puntius Sarana

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.

Development of a western blot method for detection of fish ectoparasite Argulus siamensis antigens

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.

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

ABSTRACT A study was undertaken to develop a western blot method for detection of immunogenic proteins of fish ectoparasite, Argulus siamensis for its further use as potential vaccine candidates. Argulus antigens were prepared by homogenization and injected to rohu (Labeo rohita) juveniles for development of immune serum. The serum was used to immunostain the antigens in western blot. The other reagents added in sequence were rabbit anti-rohu serum, goat anti-rabbit ALP conjugate and substrate (BCIP-NBT). However, similar banding patterns were observed with both control and immunized rohu serum. Hence, a possible cross-reaction was suspected and verified in number of western blot experiments. A typical cross-reaction observed was of rabbit serum reacting directly with Argulus antigens. Hence, the rabbit anti-rohu serum was replaced with guinea pig anti-rohu serum. Another cross-reaction of goat anti-guinea pig ALP conjugate with rohu serum was eliminated by using goat anti-rabbit ALP conjugate with guinea pig serum. Thus, the final western blot method consisting of Argulus antigens → rohu serum → guinea pig anti-rohu serum → goat anti-rabbit ALP conjugate → substrate, yielded distinguishing results between control and Argulus-immunized rohu serum samples. The developed test has tremendous downstream applications, particularly in immunoproteomic studies of Argulus antigens.

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.