A survey to gather perspectives of DBT/Wellcome Trust India Alliance-funded researchers on public engagement with science.

Lately, the Indian research ecosystem has seen an upward trend in scientists showing interest in communicating their science and engaging with non-scientific audiences; however, the number and variety of science communication or public engagement activities undertaken formally by scientists remains low in the country. There could be many contributing factors for this trend. To explore this further, the science funding public charity in India, DBT/Wellcome Trust India Alliance (India Alliance), in a first of its kind of study by a funding agency in India, surveyed its 243 research grantees in November 2020 requesting their views on public engagement with science in India through an online survey. The survey included both quantitative as well as open-ended questions to assess the understanding of, participation in, and attitude of India Alliance Fellows/Grantees towards public engagement with research, identify the enablers, challenges, and barriers to public engagement for India Alliance Fellows/Grantees, understand the specific needs (training/capacity-building, funding, etc.) and develop recommendations for India Alliance as well as for the larger scientific ecosystem in the country. The survey showed that India Alliance grantees are largely motivated to engage with the public about science or their research but lack professional recognition and incentives, training and structural support to undertake public engagement activities.

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.

Apolipoprotein A-I in Labeo rohita: Cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection.

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 µg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila.

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.

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.

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

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.

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.

Effect of Dactylogyrus catlaius (Jain 1961) infection in Labeo rohita (Hamilton 1822): innate immune responses and expression profile of some immune related genes.

The monogenean ectoparasite, Dactylogyrus sp. is a major pathogen in freshwater aquaculture. The immune responses in parasitized fish were analyzed by quantitation of innate immune factors (natural agglutinin level, haemolysin titre, antiprotease, lysozyme and myeloperoxidase activities) in serum and immune-relevant gene expression in gill and anterior kidney. The antiprotease activity and natural agglutinin level were found to be significantly higher and lysozyme activity was significantly lower in parasitized fish. Most of the genes viz., beta2-microglobulin (beta2M), major histocompatibility complex I (MHCI), MHCII, tumor necrosis factor alpha (TNFalpha) and toll-like receptor 22 (TLR22) in gill samples were significantly down-regulated in the experimental group. In the anterior kidney, the expression of superoxide dismutase and interleukin 1beta (IL1beta) were significantly up-regulated whereas a significant down regulation of MHCII and TNFalpha was also observed. The down-regulation of most of the genes viz, MHCI, beta2M, MHCII, TLR22 and TNFalpha in infected gills indicated a well evolved mechanism in this parasite to escape the host immune response. The modulation of innate and adaptive immunity by this parasite can be further explored to understand host susceptibility.

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.

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.

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.