Sagar Nayak

Microsporidians are highly specialised intracellular parasites adapted to an obligate parasitic lifestyle. Although more than 1,500 species have been described to date, their true diversity is likely much higher, particularly in poorly-studied aquatic environments. Here, we applied an optimised tissue-based metabarcoding approach to investigate the diversity and distribution of microsporidians in aquatic invertebrates from three nearby fish ponds in South Bohemia in the Czech Republic. We analysed more than two thousand individuals of chironomids and oligochaetes, which represent important aquatic invertebrate hosts of microsporidians, and compared microsporidian communities across ponds and host morphogroups. In total, 66 microsporidian OTUs were detected across both host groups. Metabarcoding and phylogenetic analyses revealed broad sequence diversity within the studied localities, with parasite sequences clustering into four main clades. The high proportion of OTUs without close matches to described taxa (more than 95%) highlights substantial undocumented diversity of microsporidians in freshwater ecosystems. The occurrence of shared OTUs amongst different invertebrate taxa is consistent with multi-host strategies and/or complex life cycles. Overall, our findings emphasise the ecological complexity of microsporidians in aquatic habitats. Further experimental work, including parasite localisation and transmission studies, is needed to determine whether the observed host associations represent active infections or incidental detection.

The effect of dietary omega-6 long-chain polyunsaturated fatty acid (LC-PUFA) on host microbiome and gut associated immune function in fish is unexplored. The effect of dietary supplementation with the omega-6 LC-PUFA-rich microalga Lobosphaera incisa wild type (WT) and its delta-5 desaturase mutant (MUT), rich in arachidonic-acid and dihomo-gamma-linolenic acid (DGLA), respectively, on intestinal gene expression and microbial diversity was analyzed in zebrafish. For 1 month, fish were fed diets supplemented with broken biomass at 7.5% and 15% (w/w) of the two L. incisa strains and a control nonsupplemented commercial diet. Dietary supplementation resulted in elevated expression of genes related to arachidonic acid metabolism-cyclooxygenase 2 (cox-2), lipoxygenase 1(lox-1), anti-inflammatory cytokine-interleukin 10 (il-10), immune defense-lysozyme (lys), intestinal alkaline phosphatase (iap), complement (c3b), and antioxidants-catalase (cat), glutathione peroxidase (gpx). Microbiome analysis of the gut showed higher diversity indices for microbial communities in fish that were fed the supplemented diets compared to controls. Different treatment groups shared 237 operational taxonomic units (OTUs) that corresponded to the core microbiome, and unique OTUs were evident in different dietary groups. Overall, the zebrafish gut microbiome was dominated by the phylum Fusobacteria and Proteobacteria (averaging 38.4% and 34.6%, respectively), followed by Bacteroidetes (12.9%), Tenericutes, Planctomycetes, and Actinobacteria (at 3.1–1.3%). Significant interaction between some of the immune-related genes and microbial community was demonstrated.

Arachidonic acid (ARA, 20:4n-6) and dihomo-γ-linolenic acid (DGLA, 20:3n-6) are omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), which are key precursors for lipid mediators of the immune system and inflammatory response. The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages (78 and 68%, respectively, as compared to only 46% in the control group). The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.

Elucidation, through molecular analyses, of bacterial afflictions in commercially important aquaculture‐reared shrimps is pivotal for the prevention and/or control of disease outbreaks. In this study, we examined the phylogenetic relatedness and compared the possible immune‐related functional roles of both translationally controlled tumour protein (TCTP) and ferritin genes with previous studies. Both TCTP and ferritin genes were substantially upregulated in the Indian white prawn, Fenneropenaeus indicus (H. Milne Edwards), post‐larvae following bath challenge with the virulent strain of bacteria, Vibrio harveyi D3. Full‐length cloning of these genes by rapid amplification of complementary DNA ends ‐polymerase chain reaction (RACE‐PCR) yielded 727‐base pair (bp)‐long TCTP and 1212‐bp‐long ferritin gene sequences. Their open reading frames (ORFs) were 507 and 510 bp, respectively. The TCTP‐ORF coded for 168 amino acids with three substitutions at positions 37, 141, 155, and the ferritin ORF coded for 170 amino acids with no species‐specific substitutions. Phylogenetic analysis suggested the closest relatedness of both TCTP and ferritin from F. indicus to Chinese white prawn, Fenneropenaeus chinensis (Osbeck). In addition to reporting the full‐length sequences of these immune‐relevant genes, this study highlighted their conserved natures, which perhaps make them important defence‐related proteins in the innate immune system of F. indicus.

As many as 71 marine bacterial DNA extracts were PCR screened for L‐asparaginase (ansA), a key gene in anti‐cancer molecular‐searches. Over 62% (44) of them were positive for ansA gene. The positive cultures were from genera Bacillus and Staphylococcus. The ansA gene cloned from isolate NIOS4 belonging to recently described Bacillus tequilensis is 1099 bp in length with a 990 bp ORF coding for 329 amino acids. BLASTx analysis revealed this sequence to be 98% similar to earlier reported ansA sequence from B. subtilis (Accession no. NP390239.1). By comparing its deduced amino acid sequence with other bacterial asparaginase sequences six substitutions at positions 305(Thr), 313(Lys), 314(Leu), 315(Asp), 318(Arg), and 320(Gln) are observed. Key residues like Thr(12), Thr(85), Asp(86), Lys(156), and Phe(165) taking part in active‐site formation and imparting catalytic properties are conserved. The phylogenetic tree based of the ansA amino acid sequences revealed close relatedness of the NIOS4 ansA sequence with B. subtilis (Accession no. NP 390239.1). It's very close genetic resemblance to B. subtilis and conservation of certain key amino acid residues suggest it as a prospective candidate for evaluation and, production of L‐asparaginases.