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Abstract Tetracapsuloides bryosalmonae is a malacosporean endoparasite that infects a wide range of salmonids and causes proliferative kidney disease (PKD). Brown trout serves as a carrier host whereas rainbow trout represents a dead-end host. We thus asked if the parasite adapts to the different hosts by changing molecular mechanisms. We used fluorescent activated cell sorting (FACS) to isolate parasites from the kidney of brown trout and rainbow trout following experimental infection with T. bryosalmonae. The sorted parasite cells were then subjected to RNA sequencing. By this approach, we identified 1120 parasite transcripts that were expressed differentially in parasites derived from brown trout and rainbow trout. We found elevated levels of transcripts related to cytoskeleton organisation, cell polarity, peptidyl-serine phosphorylation in parasites sorted from brown trout. In contrast, transcripts related to translation, ribonucleoprotein complex biogenesis and subunit organisation, non-membrane bounded organelle assembly, regulation of protein catabolic process and protein refolding were upregulated in rainbow trout-derived parasites. These findings show distinct molecular adaptations of parasites, which may underlie their distinct outcomes in the two hosts. Moreover, the identification of these differentially expressed transcripts may enable the identification of novel drug targets that may be exploited as treatment against T. bryosalmonae. We here also describe for the first time how FACS based isolation of T. bryosalmonae cells from infected kidney of fish fosters research and allows to define differentially expressed parasite transcripts in carrier and dead-end fish hosts.

Abstract Background The genus Huffmanela Moravec, 1987 (Nematoda, Trichosomoididae, Huffmanelinae), represents a group of nematodes that infect both marine and freshwater fish, and the main gross feature of infection with different species of the genus is the presence of noticeable dark spots or tracks within the parasitized tissues. The purpose of this study was to describe morphologically and morphometrically the eggs of a new marine species of Huffmanela (Huffmanela persica sp. nov.), which was found in the form of black spots in the ovary and the tunica serosa of the stomach of the daggertooth pike conger (Muraenesox cinereus). The new species differs from Huffmanela hamo, another species reported from musculature of this host in Japan, in egg metrics, eggshell features and targeted organ. Molecular identification and pathological examination of the lesions caused by the new species are also reported. Methods Nematode eggs with varying degrees of development were separated from the infected tissues (ovary and tunica serosa of stomach) and investigated using light and scanning electron microscopy. Different species-specific markers (small subunit ribosomal DNA, 18S; large subunit ribosomal DNA, 28S; internal transcribed spacer, ITS) were used for molecular identification and phylogenetic study of the new species. Infected tissues were fixed in buffered formalin for pathological investigations. Results The fully developed eggs of H. persica sp. nov. are distinguished from those previously described from this host on the basis of their measurements (size, 54–68 × 31–43 µm; polar plugs, 6.4–9.7 × 8.4–12 µm; shell thickness, 3.5–6.1 µm) and a delicate but ornate uterine layer (UL) covering the entire eggshell including the polar plugs. Histopathological examination revealed a fibro-granulomatous inflammation in the ovary and the serosal layer of the stomach of infected fish. Maximum-likelihood (ML) phylogenetic analysis recovered a sister relationship between the new species of marine origin and Huffmanela species previously collected from freshwater hosts. Conclusions The present study is the first to report the molecular characterization and phylogenetic position of a teleost-associated marine species of the genus Huffmanela. A comprehensive list of nominal and innominate populations of Huffmanela is also provided. Graphical abstract

Lignin nanoparticles (LNPs) have a wide range of potential uses in the biomedical and environmental fields. They are used to prepare antioxidants, food packaging material, energy storage, cosmetics, thermal/light stabilizers, reinforced materials, and drug delivery. In this study, LNPs were prepared from soda lignin obtained from Oxytenanthera abyssinica using dioxane, acetone, ethanol, and acid-base solvents through the nanoprecipitation method, followed by an ultrasonic process. These methods produced four different LNPs with yields in the range of 28.5% to 88.9%. Scanning electron microscope (SEM) showed that the spherical-shaped LNPS (100–400 nm) were obtained using the acetone solvent, while irregularly shaped LNPs were formed by dioxane (150–400 nm), ethanol (40–200 nm) and acid-base (100–800 nm) solvents. The maximum zeta potential of the LNPAS samples was |-35.1 mV|, determined by the dynamic light scattering (DLS), with a particle size distribution between 91.28 and 458.7 nm. According to the energy-dispersive X-ray spectrometer (EDX) results, the sample contains the elements C, O, Si, Na, and Mo. X-ray powder diffraction (XRD) analysis showed very small crystallinity sizes ranging from 0.31 to 0.35 nm. Proton nuclear magnetic resonance (H-NMR) and Fourier transformation infrared Spectroscopy (FTIR) were used to find the functional groups in the synthesized LNPs. The Thermogravimetric analysis (TGA) peak showed that LNPS was thermally stable, and rapid mass loss occurred between 300 and 415 °C. Differential Scanning Calorimetry (DSC) data shows that the highest exothermic peaks are obtained at 345.2 °C. Synthesis of LNPs by dissolving isolated soda lignin in acetone, ethanol, dioxane, and acid-base solvents was quickly recovered, cheap, and eco-friendly. The synthesized are free of sulfur; they do not use toxic solvents such as Tetrahydrofuran (THF), Dimethyl sulfoxide (DMSO), and Dimethylformamide (DMF). Thus, they provide benefits for high-value-added Nanocomposite applications such as bio-based nonmaterial and food packaging materials. Acid-base nanoprecipitation procedure is the best approach regarding stability and size, but the acetone nanoprecipitation method is preferable regarding shape.

The study aimed to understand the molecular evolution of the pathogen recognition peptidoglycan (PGN) proteins and their role in regulating the immune response against infectious diseases in Drosophila melanogaster. D. melanogasterWe obtained the PGRP proteins from 11 different species of Drosophila and analyzed the different evolutionary trends that might be associated with them. We were able to identify the evidence of strong positive selection taking place for these proteins. We investigated the diversity and function of the PGN proteins in D. melanogaster and related species through a combination of bioinformatics approaches. They found that the PGN proteins have undergone rapid and diverse evolution, with some undergoing positive selection and others experiencing gene duplication and loss. The study also revealed that different PGN proteins play distinct roles in regulating the immune response to bacterial infections, with some responding specifically to certain types of bacteria. The research provides valuable insights into the evolution and function of the PGN proteins in the immune response of D. melanogaster. It highlights their potential relevance to pathogen recognition and immune defence in other organisms.

Abstract Background The cnidarian myxozoan parasite Tetracapsuloides bryosalmonae causes chronic proliferative kidney disease (PKD) in salmonids. This parasite is a serious threat to wild and cultured salmonids. T. bryosalmonae undergoes intra-luminal sporogonic development in the kidney of brown trout (Salmo trutta) and the viable spores are released via urine. We investigated the alternative splicing pattern in the posterior kidney of brown trout during PKD. Results RNA-seq data were generated from the posterior kidney of brown trout collected at 12 weeks post-exposure to T. bryosalmonae. Subsequently, this data was mapped to the brown trout genome. About 153 significant differently expressed alternatively spliced (DEAS) genes, (delta PSI = 5%, FDR P-value

Freshwater turtles are among the least studied faunas in the Andaman and Nicobar Islands, India. Here, we report the presence of freshwater turtles (Cuora amboinensis) from Car Nicobar, a small remote island in the Nicobar archipelago comprising the Andaman and Nicobar Islands. Semi-structured interviews were conducted with the people belonging to Nicobarese tribes (n = 233) to gather their local ecological knowledge as supportive information to obtain a better understanding of the status of turtles in Car Nicobar. Most interviewees (90%) opined that freshwater turtles are found to be rare or very rare on the Island. All the respondents (100%) expressed willingness to contribute to future conservation projects. Community-level awareness emerged as a prominent issue for future conservation using the Garrett ranking method. A stream network map was prepared using the digital elevation model to visualize streams and potential habitats of turtles. This report on the freshwater turtles is essential from an ecological perspective since information is scarce on the freshwater turtles in Car Nicobar. This study emphasizes the importance of the engagement of stakeholders in conservation projects and recommends providing adequate attention to the conservation and protection of freshwater turtle diversity in Car Nicobar.

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that causes proliferative kidney disease (PKD) in wild and farmed salmonids in Europe and North America. The life cycle of T. bryosalmonae completes between invertebrate bryozoan and vertebrate fish hosts. Inside the fish, virulence factors of T. bryosalmonae are induced during infection or interactions with host cells. T. bryosalmonae genes expressed in vivo are likely to be important in fish pathogenesis. Herein, we identify in vivo induced antigens of T. bryosalmonae during infection in brown trout (Salmo trutta) using in vivo induced antigen technology (IVIAT). Brown trout were exposed to the spores of T. bryosalmonae and were sampled at different time points. The pooled sera were first pre-adsorbed with antigens to remove false positive results. Subsequently, adsorbed sera were used to screen a T. bryosalmonae cDNA phage expression library. Immunoscreening analysis revealed 136 immunogenic T. bryosalmonae proteins induced in brown trout during parasite development. They are involved in signal transduction, transport, metabolism, ion-protein binding, protein folding, and also include hypothetical proteins, of so far unknown functions. The identified in vivo induced antigens will be useful in the understanding of T. bryosalmonae pathogenesis during infection in susceptible hosts. Some of the antigens found may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against T. bryosalmonae in salmonids.

The Indian coal fly ash (CFA) is composed of 5-15% ferrous fractions. The variation in percentage depends on the source of coal and the operating conditions of the thermal power plants. The present research work reports the recovery of ferrous particles from CFA by the wet magnetic separation method. The morphological, elemental, and chemical properties of the extracted ferrous fractions were analyzed. In order to achieve surface-modified ferrospheres, the extracted ferrospheres were treated with concentrated HCl followed by sonication. The instrumental analysis reported the ferrous composition is around 16% by weight and belongs to class F. The toxicity of CFA was determined on normal human lung (BEAS-2B) cells using MTS assay. The results showed that CFA’s induced cell toxicity in a dose-dependent manner. The ferrous particles were spherically shaped with various sizes ranging from 200 nm to 7000 nm. It was crystalline in nature and is a mixture of hematite and magnetite. The particles were found to be associated with alumina, silica, oxygen, and traces of Ca, Mg, Ti, and C. The surface-modified ferrospheres were used for the remediation of Azure A dye by batch adsorption study. The removal percentage of dye was 25.03%, within 30 minutes at neutral pH, i.e., 7.2. The surface-modified ferrospheres show potential as an alternate, more economical, and reusable adsorbent for the remediation of Azure A dye in the industries or in common effluent treatment plants. Moreover, the recovery of surface-modified ferrospheres using an external magnet and the reuse of the particles make the material much economical for dye removal at an industrial scale.

Incense stick ash (ISA) has traces of toxic heavy metals which have an adverse effect on the environment. Every year, tonnes of ISA are disposed of in rivers and other water bodies which leads to water pollution and affects the natural water resources. ISA has several value-added minerals which could be modified or functionalized for environmental cleanup. Here, in the current research work, ISA was transformed into a flower-like noble porous material by mixing ISA and NaOH in a 1 : 1 ratio followed by calcination at 600°C for six hours in a muffle furnace. The developed material was analyzed by sophisticated instruments for the identification of the properties. The microscopic techniques revealed the micron-sized flower-like structure, while the XRD showed peaks at 30–33° which indicates the transformation of the calcite and silicate phases into new-phase mineral. FTIR also revealed bands in regions of 500–1200 cm−1 and new bands near 450 cm−1. EDS confirmed the presence of Na in the sintered product and the transformation of the ISA. Finally, the sintered product potential was assayed for the removal of methylene blue dye from wastewater using an adsorption mechanism. The removal efficiency of dye reached up to 70% within one hour only. It was found that the ISA sintered product has the potential to remove MB dye efficiently from wastewater and also reduce solid waste pollution. Microculture tetrazolium assay (MTT) and lactate dehydrogenase (LDH) assays were performed to evaluate the cytotoxicity of the sintered incense stick ash product on RTG-2 cells. The sintered incense stick ash product induced cytotoxicity on RTG-2 cells in a dose-dependent manner. Sintered ISA products have the potential to remove methylene blue dye efficiently from wastewater and reduce solid waste pollution.

Abstract Background Tetracapsuloides bryosalmonae is a myxozoan parasite which causes economically important and emerging proliferative kidney disease (PKD) in salmonids. Brown trout, Salmo trutta is a native fish species of Europe, which acts as asymptomatic carriers for T. bryosalmonae. There is only limited information on the molecular mechanism involved in the kidney of brown trout during T. bryosalmonae development. We employed RNA sequencing (RNA-seq) to investigate the global transcriptome changes in the posterior kidney of brown trout during T. bryosalmonae development. Methods Brown trout were exposed to the spores of T. bryosalmonae and posterior kidneys were collected from both exposed and unexposed control fish. cDNA libraries were prepared from the posterior kidney and sequenced. Bioinformatics analysis was performed using standard pipeline of quality control, reference mapping, differential expression analysis, gene ontology, and pathway analysis. Quantitative real time PCR was performed to validate the transcriptional regulation of differentially expressed genes, and their correlation with RNA-seq data was statistically analyzed. Results Transcriptome analysis identified 1169 differentially expressed genes in the posterior kidney of brown trout, out of which 864 genes (74%) were upregulated and 305 genes (26%) were downregulated. The upregulated genes were associated with the regulation of immune system process, vesicle-mediated transport, leucocyte activation, and transport, whereas the downregulated genes were associated with endopeptidase regulatory activity, phosphatidylcholine biosynthetic process, connective tissue development, and collagen catabolic process. Conclusion To our knowledge, this is the first RNA-seq based transcriptome study performed in the posterior kidney of brown trout during active T. bryosalmonae development. Most of the upregulated genes were associated with the immune system process, whereas the downregulated genes were associated with other metabolic functions. The findings of this study provide insights on the immune responses mounted by the brown trout on the developing parasite, and the host molecular machineries modulated by the parasite for its successful multiplication and release.

Abstract Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. In fish, the intestine represents an important site of nutrient uptake, host–pathogen interactions, and defense. The posterior intestine can be inflamed, reddened, and filled with an opaque, yellowish fluid during Y. ruckeri infection. Herein, we report an investigation on the proteome alteration in the posterior intestinal mucosa of rainbow trout (Oncorhynchus mykiss) after exposure to Y. ruckeri. The intestinal mucosal proteins were identified and quantified by a shotgun proteomic approach by applying data-independent quantification with sequential windowed acquisition of all theoretical mass spectra (SWATH). A total of 437 proteins were found to be differentially up- or downregulated in the posterior intestine. Gene ontology of upregulated proteins pointed to their involvement into exopeptidase, endopeptidase, and hydrolase activities, while the downregulated proteins were involved in lipid metabolism, actin binding, and translation processes. Additionally, upregulated proteins were predicted to be involved in lysosome, oxidative phosphorylation, and metabolic pathways, while downregulated proteins were implicated in focal adhesion, regulation of actin cytoskeleton, protein digestion and absorption pathways. This study showed that Y. ruckeri infection can alter protein abundance involved in serine-type carboxypeptidase, cysteine and aspartic-type endopeptidases, metallopeptidases, antioxidant defense, calcium ion binding, glycolytic and carbohydrate metabolic processes in the proteome of the intestinal mucosa of rainbow trout.

Abstract Background Whirling disease (WD), caused by the myxozoan parasite Myxobolus cerebralis, is responsible for high mortalities in rainbow trout hatcheries and natural populations. To elucidate how resistant and susceptible rainbow trout strains respond to early invasion, a well-established model of WD was used to demonstrate the kinetics of local and systemic immune responses in two rainbow trout strains, the susceptible American Trout Lodge (TL) and the more resistant German Hofer strain (HO). Methods Parasite load and cellular immune responses were compared across several time points after M. cerebralis exposure to elucidate the kinetics of immune cells in resistant and susceptible rainbow trout in response to early invasion. In the course of the 20 days following exposure, leukocyte kinetics was monitored by flow cytometry in the caudal fin (CF), head kidney (HK) and spleen (SP). For the analysis of the leukocyte composition, cells were stained using a set of monoclonal antibodies with known specificity for distinct subpopulations of rainbow trout leukocytes. Results Experiments indicated general increases of CF, HK and SP myeloid cells, while decreases of B cells and T cells in the SP and HK were observed at several time points in the TL strain. On the other hand, in the HO strain, increases of T cells were dominant in CF, HK and SP at multiple time points. The differences between HO and TL were most distinct at 2, 4, 12 and 48 hours post-exposure (hpe) as well as at 4 days post-exposure (dpe), with the vast majority of innate immune response cells having higher values in the susceptible TL strain. Alteration of the leukocyte populations with augmented local cellular responses and excessive immune reactions likely lead to subsequent host tissue damage and supports parasite invasion and development in TL. Conclusions The findings of this study highlight the significance of effective local and systemic immune reaction and indicate proper activation of T lymphocytes critical for host resistance during M. cerebralis infection. The present study provides insights into the cellular basis of protective immune responses against M. cerebralis and can help us to elucidate the mechanisms underlying the variation in resistance to WD.

Tetracapsuloides bryosalmonae, a myxozoan endoparasite, causes proliferative kidney disease in salmonids. The life cycle of T. bryosalmonae occurs between invertebrate bryozoan and vertebrate fish hosts. T. bryosalmonae develops in the body cavity of colonial bryozoan and spores are released from mature spore sacs into the water likely through the vestibular pore and infect fish by attaching to their gills. However, very little is known about the transcriptome of this important parasite, which hampers studies into the molecular mechanisms of host-parasite interactions and understanding the parasite biology. In order to circumvent this limitation, we performed de novo transcriptome assembly on the sacs of T. bryosalmonae, collected from infected bryozoan Fredericella sultana. A total of 111.5 million filtered paired-end reads was obtained and assembled into 25,908 contigs corresponding to putative transcripts that were functionally annotated. More than 50% of the assembled transcripts (13,071 contigs) had a significant hit in NCBI non-redundant database. Based on Gene ontology annotation, the most highly scored categories of molecular function of the contigs were related to binding and catalytic activities in T. bryosalmonae. This study provides a global overview of the T. bryosalmonae transcriptome that will be a valuable resource for identifying virulence factors, gene discovery, genome annotation, and vaccine development applications. This data is accessible via NCBI BioProject (PRJNA680464).

At present, chemical Si/Al sources are mainly used as precursor materials for the manufacturing of zeolites. Such precursor materials are quite expensive for commercial synthesis. Here, we have reported the synthesis of Ca-based zeolite from incense stick ash waste by the alkali-treatment method for the first time. Incense stick ash (ISA) was used as a precursor material for the synthesis of low Si zeolites by the alkali-treatment method. The as-synthesized zeolites were characterized by various instruments like particle size analyzer (PSA), Fourier transform infrared (FTIR), X-ray diffraction (XRD), field emission scanning electron microscope (FESEM), electron diffraction spectroscopy (EDS), transmission electron microscopy (TEM), and X-ray fluorescence (XRF). FTIR and XRD helped in the identification of the microstructure and crystalline nature of the zeolites and also confirmed the synthesis of Ca-based zeolite with two thetas at 25.7°. The microscopic analysis by FESEM and TEM exhibited that the size of synthesized Ca-rich zeolites varies from 200 to 700 nm and they are aggregated and cuboidal in shape. Additionally, structural, electronic, and density of states’ characteristics of gismondine (Ca2Al4Si4O16·9H2O) structures were evaluated by computational simulations (first principle, density functional theorem). The structural optimization of structures was carried out in the first stage under the lowest condition of total energy and forces acting on atoms for the lattice constant, as well as the available experimental and theoretical findings. The present research approach predicted the transformation of ISA waste into a value-added mineral, i.e., zeolite, which was further used for the removal of both heavy metals and alkali metals from fly ash-based wastewater using inductively coupled plasma-optical emission spectroscopy (ICP-OES).

Bryozoans are aquatic invertebrate moss animals that are found worldwide. Fredericella sultana is a freshwater bryozoan and is the most common primary host of myxozoan parasite, Tetracapsuloides bryosalmonae. However, limited genomic resources are available for this bryozoan, which hampers investigations into the molecular mechanisms of host-parasite interactions. To better understand these interactions, there is a need to build a transcriptome dataset of F. sultana, for functional genomics analysis by large-scale RNA sequencing. Total RNA was extracted from zooids of F. sultana cultivated under controlled laboratory conditions. cDNA libraries were prepared and were analyzed by the Illumina paired-ends sequencing. The sequencing data were used for de novo transcriptome assembly and functional annotation. Approximately 118 million clean reads were obtained, and assembled into 85,544 contigs with an average length of 852 bp, an N50 of 1,085 bp, and an average GC content 51.4%. A total of 23,978 (28%) contigs were annotated using BLASTX analysis. Of these transcripts, 4,400 contigs had highest similarity to brachiopod species Lingula anatina. Based on Gene ontology (GO) annotation, the most highly scored categories of biological process were categorized into cellular process (27%), metabolic process (24%), and biological regulation (8%) in the transcriptome of F. sultana. This study gives first insights into the transcriptome of F. sultana and provides comprehensive genetic resources for the species. We believe that the transcriptome of F. sultana will serve as a useful genomic dataset to accelerate research of functional genomics and will help facilitate whole genome sequencing and annotation. Candidate genes potentially involved in growth, proteolysis, and stress/immunity-response were identified, and are worthy of further investigation.

Myxosporeans are well-known parasites infecting food fishes in fresh and marine water around the globe. Grass carp (Ctenopharyngodon idella), a freshwater food fish commonly cultured in India with has significant economic importance. Herein, the study focuses on the description of a new myxosporean species, Myxobolus grassi sp. nov. from the gills as primary site and liver as secondary site of infection in grass carp. Both organs (gill and liver) were infected concurrently in the host and the prevalence of grass carp infection was 4.05% in gill filaments and liver, respectively. Identification of species was based on the morphological and morphometric features of the myxospore as well as 18S rDNA sequence data. A smear from gill and liver exhibited hundreds of morphologically similar myxospores. BLAST search revealed 98% sequence similarity and 0.03 genetic distance with M. catlae (KM029967) infecting gill lamellae of mrigal carp (Cirrhinus cirrhosus) from India and 98–84% sequence similarity with other myxobolids in India, China, Japan, Malaysia, Turkey and Hungary. Phylogenetically, it clustered with other myxobolids infecting gills and related organs (i.e., vital organ) of Indian cyprinid carp species. On the basis of myxospore morphology and 18S sequence, we propose M. grassi sp. nov.

Abstract Background The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is increasingly being used for genome editing experiments. It is a system to add, delete and/or replace parts of a gene in situ in a time- and cost-efficient manner. The genome of many organisms has been edited using this system. We tested the CRISPR/Cas9 system in Aphanomyces invadans, an oomycete, which is the causative agent of epizootic ulcerative syndrome (EUS) in many fish species. Extracellular proteases produced by this oomycete are believed to play a role in EUS virulence. Methods We designed three single guide-RNAs (gRNA) to target A. invadans serine protease gene. These gRNAs were individually combined with the Cas9 to form ribo-nucleo-protein (RNP) complex. A. invadans protoplasts were then transfected with RNP complexes. After the transfection, the target gene was amplified and subjected to sequencing. Zoospores of A. invadans were also transfected with the RNP complex. Three groups of dwarf gourami (Trichogaster lalius) were then experimentally inoculated with (i) non-treated A. invadans zoospores; (ii) RNP-treated A. invadans zoospores; and (iii) autoclaved pond water as negative control, to investigate the effect of edited serine protease gene on the virulence of A. invadans in vivo. Results Fluorescence microscopy showed sub-cellular localization of RNP complex in A. invadans protoplasts and zoospores. Sequencing results from the protoplast DNA revealed a point mutation in the target gene. A matching mutation was also detected in zoospores after similar treatment with the same RNP complex. In vivo results showed that the CRISPR/Cas9-treated A. invadans zoospores did not produce EUS clinical signs in the fish. These results were then confirmed by histopathological staining of the muscle sections using Gomori’s methenamine silver nitrate and hematoxylin and eosin stains. Conclusions Results obtained in this study indicate that the RNP complex caused effective mutation in the target gene. This hindered the production of serine protease, which ultimately impeded the manifestation of EUS in the fish. Our methods thus establish a promising approach for functional genomics studies in A. invadans and provide novel avenues to develop effective strategies to control this pathogen.

Abstract Background Most of the studies on fish diseases focus on single infections, although in nature co-infections occur more often. The two freshwater myxozoan parasites of salmonids, having high economic and ecologic relevance are Tetracapsuloides bryosalmonae (Malacosporea), the etiological agent of proliferative kidney disease, and Myxobolus cerebralis (Myxosporea), the etiological agent of whirling disease. The present study aims to investigate immune modulation in rainbow trouts (Oncorhynchus mykiss) during single and co-infections by these parasites. Methods Fish were initially infected with T. bryosalmonae (one group) and M. cerebralis (another group) separately. At 30 days post-exposure (dpe), both the single species infected groups were co-infected, respectively, with the other parasite. Posterior kidney and cartilage cranium samples were collected at 30, 60, 90 and 120 dpe and RT-qPCR was performed on them to assess the transcription of suppressors of cytokine signaling (SOCS) -1 and -3, Janus kinase-1 (JAK-1) and signal transducer and activator of transcription-3 (STAT-3) genes. Results Kidney samples from the T. bryosalmonae-infected group showed upregulation of all immune genes tested between 60–120 dpe. Crania from the single M. cerebralis-infected group and the M. cerebralis and T. bryosalmonae co-infected group exhibited upregulation of SOCS-1 and JAK-1 between 60–120 dpe and SOCS-3 at 120 dpe. However, only in the single M. cerebralis-infected group, was a statistically significant expression of STAT-3 observed at 30 and 60 dpe. Conclusions The results of this study indicate that both T. bryosalmonae and M. cerebralis induce overexpression of SOCS-1 and SOCS-3 genes and modulate the host immune response during the development of parasite to cause immunosuppression.

Abstract Background The myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), is responsible for considerable losses in farmed and wild fish populations in Europe and North America. Recently, T. bryosalmonae was detected in many European countries, and strategy to control the disease in the wild and farmed fish population is yet to be developed. Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technology that does not require any thermal cycling, and lateral flow dipstick (LFD) is a rapid, cost-effective, and easy-to-handle assay that enables stable detection. Results In this study, we developed and optimized a rapid and sensitive RPA assay combined with an LFD for the detection of T. bryosalmonae. The PKD-RPA assay was specific to T. bryosalmonae, as no cross-reaction or false positive signals were observed with any of the other tested DNAs. The developed PKD-RPA assay was ten times more sensitive than an existing diagnostic polymerase chain reaction (PCR) assay for this parasite. The estimated time to perform PKD-RPA assay is 25 min compared to 4 h for PKD-PCR assay. Conclusions A novel PKD-RPA assay for the detection of T. bryosalmonae was developed. The assay offers considerable advantages including speed, sensitivity, specificity and visual detection. Applying the PKD-RPA assay combined with an LFD enhances the surveillance and early detection of T. bryosalmonae in salmonids.

Abstract Ichthyophthirius multifiliis is a ciliated protozoan parasite recognized as one of the most pathogenic diseases of wild and cultured freshwater fish. Fish skin mucus plays a significant role against invading pathogens. However, the protein-based modulation against infection with I. multifiliis, of host fish at this barrier is unknown. Thus, we investigated the skin mucus proteome of common carp using a shotgun proteomic approach at days 1 and 9 after I. multifiliis exposure. We identified 25 differentially expressed proteins in infected carp skin mucus. Upregulated proteins were mainly involved in metabolism, whereas downregulated proteins were mainly structural. This is the first proteomic analysis of infected common carp skin mucus, and it provides novel information about proteome alteration caused by I. multifiliis. Furthermore, we identified novel proteins with yet unknown function in common carp following penetrating injuries such as olfactomedin 4, lumican, dermatopontin, papilin and I cytoskeletal 18. This analysis, therefore, represents a key for the search for potential biomarkers, which can help in a better understanding and monitoring of interactions between carp and I. multifiliis. This proteomic study not only provides information on the protein-level pathways involved in fish-ciliate interactions but also could represent a complementary system for studying tissue repair.

Tetracapsuloides bryosalmonae, a myxozoan endoparasite often causes chronic infection in brown trout. Antiparasite immunity mediated by antibodies and B cells is known as an important determinant of host survival and parasite proliferation during chronic infections. Accordingly, studying their time course during proliferative kidney disease (PKD) might be helpful in improving our understanding of its chronic nature. Therefore, we conducted this study to examine parasite specific serum antibody and B-cell-mediated response in laboratory-infected brown trout at different time points. Brown trout were exposed to the spores of T. bryosalmonae, derived from infected bryozoans. Samples were collected at different time points and processed for indirect ELISA, histopathology, and qRT-PCR. T. bryosalmonae specific antibody was detected at 4 weeks post exposure (wpe) and it persisted until 17 wpe. Additionally, the expressions of C4A, CD34, CD79A, BLNK, CD74, BCL7, and CD22 were differentially regulated in the important immune organs, kidney and spleen. To our knowledge, this is the first study addressing anti-T. bryosalmonae antibody response in brown trout at different time points. The results from this study provide valuable insights into the processes leading to changes in B cell development, inflammation and antibody production during the course of PKD in brown trout.

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.

Abstract Background Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated. Results BLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively. Conclusions The identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

Abstract Background Myxozoan parasites pose emerging health issues for wild and farmed salmonid fish. Rainbow trout (Oncorhynchus mykiss) is a particularly susceptible species to Tetracapsuloides bryosalmonae (Malacosporea), the etiological agent of Proliferative Kidney Disease (PKD), and to Myxobolus cerebralis (Myxosporea), the etiological agent of Whirling Disease (WD). The objective of this study was to investigate the impact of myxozoan co-infections on the pathogenesis of PKD and WD in the rainbow trout. Methods Two groups of rainbow trout (96 fish each) were primarily infected with T. bryosalmonae and triactinomyxons of M. cerebralis; after 30 days half of the fish in each group were co-infected with these parasites vice versa and remaining half was continued as single infection. Mortalities and clinical signs were recorded at different time points. Histopathology and immunohistochemistry were performed to assess the extent of each infection and estimate the parasite burden between groups. Results Fish firstly infected with M. cerebralis and co-infected with T. bryosalmonae exhibited exacerbated pathological changes of both parasitic diseases and elicited a higher mortality rate. A higher kidney swelling index (grade 4) appeared together with more severe cartilage destruction and displacement, when compared to the pathological changes in fish upon single infections with T. bryosalmonae or M. cerebralis. Conversely, fish firstly infected with T. bryosalmonae and co-infected with M. cerebralis also exhibited typical pathological changes of both parasitic diseases, but with a lower mortality rate, similar as caused by the single T. bryosalmonae or M. cerebralis infection. WD clinical signs were milder, without skeletal deformities, while kidney swelling index was similar to single infection with T. bryosalmonae (grade 2 to 3). Conclusions In this study, a co-infection with myxozoan parasites was for the first time successfully achieved in the laboratory under controlled conditions. The impact of co-infections in concurrent myxozoan infections mainly depends on the primary pathogen infecting the host, which could alter the outcomes of the secondary pathogen infection. The primary M. cerebralis infection followed by T. bryosalmonae had a much more serious impact and elicited a synergistic interaction. Contrasting results were instead seen in rainbow trout primarily infected with T. bryosalmonae and then co-infected with M. cerebralis.

Abstract Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) of salmonids. There is little information regarding the proteomics of Y. ruckeri. Herein, we perform whole protein identification and quantification of biotype 1 and biotype 2 strains of Y. ruckeri grown under standard culture conditions using a shotgun proteomic approach. Proteins were extracted, digested and peptides were separated by a nano liquid chromatography system and analyzed with a high-resolution hybrid triple quadrupole time of flight mass spectrometer coupled via a nano ESI interface. SWATH-MS technology and sophisticated statistical analyses were used to identify proteome differences among virulent and avirulent strains. GO annotation, subcellular localization, virulence proteins and antibiotic resistance ontology were predicted using bioinformatic tools. A total of 1395 proteins were identified in the whole cell of Y. ruckeri. These included proteases, chaperones, cell division proteins, outer membrane proteins, lipoproteins, receptors, ion binding proteins, transporters and catalytic proteins. In virulent strains, a total of 16 proteins were upregulated including anti-sigma regulatory factor, arginine deiminase, phosphate-binding protein PstS and superoxide dismutase Cu–Zu. Additionally, several virulence proteins were predicted such as Clp and Lon pro-teases, TolB, PPIases, PstS, PhoP and LuxR family transcriptional regulators. These putative virulence proteins might be used for development of novel targets for treatment of ERM in fish. Our study represents one of the first global proteomic reference profiles of Y. ruckeri and this data can be accessed via ProteomeXchange with identifier PXD005439. These proteomic profiles elucidate proteomic mechanisms, pathogenicity, host-interactions, antibiotic resistance ontology and localization of Y. ruckeri proteins.

Abstract Fredericella sultana is an invertebrate host of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonids. The bryozoan produces seed-like statoblasts to facilitate its persistence during unfavourable conditions. Statoblasts from infected bryozoans can harbor T. bryosalmonae and give rise to infected bryozoan colonies when conditions improve. We aimed in the present study to evaluate the integrity and viability of T. bryosalmonae-infected statoblasts after a range of harsh treatment conditions. We tested if statoblasts could survive ingestion by either brown trout or common carp. After ingestion, the fish faeces was collected at different time points. We also tested physical stressors: statoblasts collected from infected colonies were desiccated at room temperature, or frozen with and without Bryozoan Medium C (BMC). After treatments, statoblasts were assessed for physical integrity before being incubated on BMC to allow them to hatch. After 4 weeks, hatched and unhatched statoblasts were tested by PCR for the presence of the parasite. We found that statoblasts ingested by brown trout and those frozen in BMC were completely broken. In contrast, statoblasts ingested by common carp and those subjected to dry freezing were able to survive and hatch. T. bryosalmonae was detected by PCR in both hatched and unhatched infected statoblasts, but neither from broken nor uninfected statoblasts. Our results confirmed for the first time the ability of infected statoblasts to survive passage through a fish, and freezing. These findings suggest potential pathways for both persistence and spread of T. bryosalmonae-infected statoblasts in natural aquatic systems.

Globally, parasites are increasingly being recognized as catastrophic agents in both aquaculture sector and in the wild aquatic habitats leading to an estimated annual loss between 1.05 billion and 9.58 billion USD. The currently available therapeutic and control measures are accompanied by many limitations. Hence, vaccines are recommended as the “only green and effective solution” to address these concerns and protect fish from pathogens. However, vaccine development warrants a better understanding of host–parasite interaction and parasite biology. Currently, only one commercial parasite vaccine is available against the ectoparasite sea lice. Additionally, only a few trials have reported potential vaccine candidates against endoparasites. Transcriptome, genome, and proteomic data at present are available only for a limited number of aquatic parasites. Omics-based interventions can be significant in the identification of suitable vaccine candidates, finally leading to the development of multivalent vaccines for significant protection against parasitic infections in fish. The present review highlights the progress in the immunobiology of pathogenic parasites and the prospects of vaccine development. Finally, an approach for developing a multivalent vaccine for parasitic diseases is presented. Data sources to prepare this review included Pubmed, google scholar, official reports, and websites.

Tetracapsuloides bryosalmonae is a myxozoan parasite responsible for proliferative kidney disease (PKD) in a wide range of salmonids. PKD, characterized by high mortality and morbidity, is well known for affecting aquaculture operations and wild salmonid populations across Europe and North America. The life cycle of T. bryosalmonae revolves around freshwater bryozoan and salmonid fish hosts. In recent years, T. bryosalmonae has been reported among wild salmonids from the European countries where it has not been reported previously. T. bryosalmonae is believed to be a possible reason for the diminishing wild salmonid populations in the natural water bodies of many European countries. Climate crisis driven rising water temperature can further accelerate the distribution of T. bryosalmonae. Expansion of the geographical distribution of T. bryosalmonae may further advocate the decline of wild salmonid populations, especially brown trout (Salmo trutta) in their habitats. Mathematical models are used to understand the pattern and distribution of T. bryosalmonae among the host in the natural water bodies. The present manuscript not only summarizes the incidences of T. bryosalmonae among the wild salmonid populations, but also discusses the contemporary understanding about the development of T. bryosalmonae in its hosts and the influences of various factors in the spread of the disease in the wild.