Morphological, histological and phylogenetic identification of three species of Myxobolus (Myxosporea: Myxobolidae) infecting different carp lineages in China.

The global cultivation of common carp Cyprinus carpio is developed primarily for either food or recreational purposes and myxosporean infections causing significant economic losses are regularly troublesome for carp farmers. However, most myxosporean species are poorly characterized, making it difficult to correctly elaborate on their parasitism mode and determine pathogenicity. Here, based on an integrative approach, we supplemented fundamental information on three Myxobolus species infecting different carp lineages in China. Myxobolus elliptoides Wu et Chen, 1987; Nine new species of Myxosporida from freshwater fish of Wuhu lake, Hubei, China. Acta Hydrobiologica Sinica, 11, 161 formed yellowish pseudocysts in the anal fin of common carp and were observed with ellipsoidal myxospores and two polar capsules of distinct dimensions. The dermal tissue within the fin ray was regarded as the type site for growing plasmodia of M. elliptoides. Myxobolus basilamellaris Lom et Molnar, 1983; Myxobolus basilamellaris sp. n. (myxozoa, Myxosporea), a parasite of the gills of common carp (Cyprinus carpio L.). Folia Parasitologica, 30, 1 aggregated ellipsoidal myxospores at the base of the gill filaments of koi carp juvenile, causing significant tissue damage. Myxobolus artus Achmerov, 1960, enwrapped in diffuse pseudocysts, was observed throughout the trunk muscle of mirror carp. Its oblate myxospores were asymmetrical and contained two unequal-sized polar capsules. According to the localization of large plasmodia, inter- and intramuscular preferences were displayed by M. artus. For precise species identification, we provided the SSU rDNA sequences for each species of Myxobolus. Among them, M. elliptoides was molecularly characterized for the first time, showing the highest 94.21% identity to Thelohanellus sinensis (KY469292). Phylogenetically, the affinity of both M. artus and M. basilamellaris to their conspecific species derived from different carp lineages was highly supported. Moreover, the intermixed cluster of Myxobolus species, including M. elliptoides, with those of Thelohanellus species provided evidence for querying the monophyletic evolution history of these taxa. This work updates the appreciation of the concerned species and enhances our understanding of the parasite fauna of common carp.

Sub-acute toxicity of the herbicide glufosinate-ammonium exposure in adult red swamp crayfish (Procambarus clarkii).

Glufosinateammonium (GLA) is one of the most widely used agricultural herbicides. It is frequently detected in surface waters near farmland and may pose a risk to non-target aquatic species. This study aimed to explore the toxicity of subacute GLA exposure in crayfish. Adult red swamp crayfish were exposed to GLA (0, 1, 10, and 100 mg/L) for 21 days. Bioaccumulation, oxidative stress, nonspecific immunity, and the expression of genes encoding xenobiotic detoxification-related enzymes were examined. The results showed GLA accumulation and hepatopancreatic histopathological changes (dilation of hepatic tubules and vacuolation of hepatocytes) in the exposed crayfish. GLA exposure induced ROS production, inhibited glutathione expression, and catalase activity in the crayfish hepatopancreas, as well as inhibited immunoenzyme expression (acid phosphatase, alkaline phosphatase, and lysozyme) in the hemolymph. In addition, the total hemocyte number decreased, and the proportion of hemocyte subsets changed significantly. Superoxide dismutase first increased and then decreased with increasing GLA dosage. GLA promoted the expression of biotransformation enzymes (cypb5, gst) in the hepatopancreas. Our results suggest that subacute GLA exposure caused structural damage to the hepatopancreatic tissue and decreased antioxidant capacity and non-specific immunity in crayfish. These findings provide insight into the toxicity of herbicides on non-target organisms.

Histopathological evaluation of the infection elicited by three species of Myxobolus (Cnidaria: Myxosporea), with identification of Myxobolusxiaoganensis n. sp. isolated from the silver carp Hypophthalmichthys molitrix.

Several parasites infecting the Asian carp species have been broadly spread along with the global fish trade. However, the diversity of specific parasite groups and their pattern of parasitism remain insufficiently elucidated even in their native regions. Here, we conducted a holistic identification and histological analysis of three Myxobolus species. The oblate myxospores of the first isolates were found infecting the spleen of silver carp Hypophthalmichthys molitrix, measuring 6.4 ± 0.4 (5.4-7.1) µm in length, 8.2 ± 0.4 (7.5-9.0) in width, and 5.9 ± 0.3 (5.2-6.2) in thickness. They were morphologically distinct from other congeners and regarded as a novel species, Myxobolus xiaoganensis n. sp. For the second isolates, we associated the formation of round plasmodia on the gill raker of silver carp with Myxobolus allotypica Chen, 1998. The third isolates, encapsulated in the intestinal serosa membrane of the grass carp Ctenopharyngodon idella, was proved to be conspecific to Myxobolus huasaensis Chen, 1998. While M. xiaoganensis n. sp. and M. huasaensis exhibited distinct origins, with genetic differences exceeding 4% from other congeners, M. allotypica displayed complete genetic congruence with an item available in Genbank. Histologically, myxospores of M. xiaoganensis n. sp. were scattered in the spleen, and the branchial and intestinal infections of M. allotypica and M. huasaensis were determined, respectively. Phylogenetic analysis demonstrated a non-monophyletic origin of both the Thelohanellus and Myxobolus genera, with a remarkable association of host affinity with myxosporean clustering.

Integrated morphological and transcriptome profiles reveal a highly-developed extrusome system associated to virulence in the notorious fish parasite, Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is an obligate parasitic ciliate that causes severe economic damage in aquaculture. The parasite contains numerous extrusive organelles (extrusomes) that assist in its pathogenesis and reproduction. However, the structure of these extrusomes and the molecular profiles involved in exocytosis remain unclear. In the present study, through comparative ultrastructural observations across the life cycle of I. multifiliis, we demonstrated that all three of its life stages (theront, trophont, and tomont) exhibited an abundance of extrusomes. In addition, two different types of extrusomes were identified according to their unique structures. Type I extrusomes (mucocysts) are crystalline, oval-shaped, 0.7-1.4 × 0.6-1.1 µm, and distributed as "rosettes" below the trophont membrane. Type II extrusomes, 2.0-3.0 × 0.2-0.3 µm, are rod-shaped with tubular cores and identified as toxicysts, the aggregation of which in the anterior part of the theront and cortex of the trophont revealed their potential roles in I. multifiliis invasion. This was confirmed by our transcriptome investigations of the three stages of I. multifiliis, which revealed that a set of genes involved in proteolysis and DNA/protein biogenesis was highly expressed in the theront and trophont. Furthermore, to map the molecular mechanisms of extrusome release, we characterized 25 Rab family genes in I. multifiliis and determined their expression profiles across the life cycle, reflecting the distribution patterns of the two extrusomes. Collectively, our data revealed that a highly developed extrusome system could play a potential role in the virulence of I. multifiliis, which facilitates a better understanding of the parasite's development.

Identification and pathogenicity of multidrug-resistant Elizabethkingia miricola isolated from farmed American bullfrogs Rana catesbeiana in China with in vitro screening of herbal antimicrobial agents.

OBJECTIVE: In 2021, an outbreak of an infectious disease characterized by torticollis, cataracts, and neurological disorders caused massive mortality in farmed American bullfrogs Rana catesbeiana in Hubei province, China. We identified the causal agent in this outbreak, characterized its pathogenicity, and screened candidate antimicrobial agents for future disease control. METHODS: Bacterium was isolated from the diseased American bullfrogs and identified based on biochemical tests, sequence analyses (16S ribosomal RNA; DNA gyrase subunit B), and experimental challenge. Furthermore, antibiotic sensitivity of the isolated strain was detected with Kirby-Bauer paper diffusion method, and the antibacterial activity of 60 traditional Chinese herbal extracts against the isolated strain was evaluated by agar disc diffusion and broth dilution assays. RESULT: We identified Elizabathkingia miricola strain FB210601 as the causative agent of this disease. The isolated E. miricola strain FB210601 exhibited extensive antibiotic resistance to all tested quinolones, ß-lactam antibiotics, and aminoglycosides. Eight herbal extracts exhibited excellent antimicrobial activity against E. miricola FB210601, especially Caesalpinia sappan and Rhus chinensis, with minimal inhibitory concentrations less than 0.2 mg/mL. Additionally, the combined effects of two-component herbal mixtures containing C. sappan or R. chinensis were greater than those of the individual extracts. CONCLUSION: Our results provide a reference for understanding the pathogenesis of Elizabethkingia infection in frogs. Furthermore, this study will aid in the application of herbal extracts for protection against infections caused by multidrug-resistant Elizabathkingia in the future.

Identification of Myxobolus distalisensis n. sp. (Cnidaria: Myxozoa) infecting yellow catfish Tachysurus fulvidraco (Richardson), with a supplement description of M. voremkhai (Akhmerov, 1960) Landsberg and Lom, 1991.

With growing scale of intensive fish cultivation, the risk of parasite infection in commercial fish is increased. Precisely identifying and characterizing the parasites that infect the farmed fish is critical to understanding the dynamics of their communities. Here, two species of Myxobolus were identified in farmed yellow catfish Tachysurus fulvidraco (Richardson) in China. Myxobolus distalisensis n. sp. developed plasmodia in the gill filaments, with oval to elliptical myxospores measuring 11.3 ± 0.6 (10.4-12.6) × 8.1 ± 0.3 (7.5-8.6) × 5.5 ± 0.2 (5.2-5.8) µm. Two pyriform polar capsules of equal size were measured 5.3 ± 0.4 (4.5-6.3) × 2.7 ± 0.1 (2.3-3) µm. Myxobolus voremkhai (Akhmerov, 1960) Landsberg and Lom, 1991 developed plasmodia in the gill arch and had a myxospore morphology similar to the conspecific isolates described in previous studies. The consensus sequences of M. distalisensis was remarkably distinct from those deposited in the GenBank, with exception of whereas M. voremkhai showing 99.84% identity. The genetic data on both isolates differed considerably from each other, revealing only 86.96% molecular identity. Histologically, M. distalisensis resided in the filament cartilage, and the aggressive proliferation of the sporogenic stages led to lytic cartilage corrosion. In contrast, plasmodia of M. voremkhai grossly observed at the base of the gill filament were embedded by the connective tissue in the gills arch. Phylogenetically, both isolates were separately placed in different subclades, indicating difference in their evolutionary history. Besides, the taxon under the family Myxobolidae was demonstrated non-monophyletic origins, and parasite radiation largely followed their host affinity.

Mangiferin and Taurine Ameliorate MSRV Infection by Suppressing NF-κB Signaling.

The emergence or reemergence of viruses pose a substantial threat and challenge to the world population, livestock, and wildlife. However, the landscape of antiviral agents either for human or animal viral diseases is still underdeveloped. The far tougher actuality is the case that there are no approved antiviral drugs in the aquaculture industry, although there are diverse viral pathogens. In this study, using a novel epithelial cell line derived from the brain of Micropterus salmoides (MSBr), inflammation and oxidative stress were found to implicate the major pathophysiology of M. salmoides rhabdovirus (MSRV) through transcriptome analysis and biochemical tests. Elevated levels of proinflammatory cytokines (interleukin-1ß [IL-1ß], IL-6, IL-8, tumor necrosis factor alpha [TNF-α], and gamma interferon [IFN-γ]) and accumulated contents of reactive oxygen species (ROS) as well as biomarkers of oxidative damage (protein carbonyl and 8-OHdG) were observed after MSRV infection in the MSBr cells. Mangiferin or taurine dampened MSRV-induced inflammation and rescued the oxidative stress and, thus, inhibited the replication of MSRV in the MSBr cells with 50% effective concentration (EC50) values of 6.77 µg/mL and 8.02 µg/mL, respectively. Further, mangiferin or taurine hampered the activation of NF-κB1 and the NF-κB1 promoter as well as the increase of phosphorylated NF-κB (p65) protein level induced by MSRV infection, indicating their antiviral mechanism by suppressing NF-κB signaling. These findings exemplify a practice approach, aiming to dampen and redirect inflammatory responses, to develop broad-spectrum antivirals. IMPORTANCE Aquaculture now provides almost half of all fish for human food in 2021 and plays a significant role in eliminating hunger, promoting health, and reducing poverty. There are diverse viral pathogens that decrease production in aquaculture. We developed a novel epithelial cell line derived from the brain of Micropterus salmoides, which can be used for virus isolation, gene expressing, and drug screening. In this study, we focus on M. salmoides rhabdovirus (MSRV) and revealed its pathophysiology of inflammation and oxidative stress. Aiming to dampen and redirect inflammatory responses, mangiferin or taurine exhibited their antiviral capability by suppressing NF-κB signaling. Our findings exemplify a practice approach to develop broad-spectrum antivirals by dampening and redirecting inflammatory responses.

Hepatopancreas Proteomic Analysis Reveals Key Proteins and Pathways in Regulatory of Ovary Maturation of Macrobrachium nipponense.

A TMT-based (Tandem Mass Tag) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was employed to explore differentially expressed proteins (DEPs) and KEGG pathways in hepatopancreas of 5 ovary stages. In total, 17,999 peptides were detected, among which 3395 proteins were identified. Further analysis revealed 26, 24, 37, and 308 DEPs in HE-I versus HE-II, HE-II versus HE-Ⅲ, HE-Ⅲ versus HE-Ⅳ, and HE-Ⅳ versus HE-Ⅴ, respectively (HE-I, HE-II, HE-III, HE-IV, and HE-V means hepatopancreas sampled from ovary stage I to V.). Gene ontology (GO) analysis indicated that DEPs were significantly enriched in "catalytic activity", "metabolic process", and "cell" of 4 comparison groups in turn. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results showed that in hepatopancreas, as the ovaries developed to maturation, carbohydrate metabolism, lipid metabolism, amino acid metabolism, and lysosome played important roles in turn. The mRNA expression of 15 selected DEPs were consistent with proteome results by qPCR analysis. Further mRNA expression investigation results suggested 4 proteins (fatty acid-binding protein, NPC intracellular cholesterol transporter 1, Serine hydroxymethyltransferase, and Crustapin) were involved in ovary maturation. These results enhance the understanding of the regulatory role of hepatopancreas in M. nipponense ovary maturation and provide new insights for understanding the crustacean regulation mechanisms.

Myxobolus shuifuensis sp. n. (Myxozoa: Myxobolidae) infecting the exotic mrigal Cirrhinus mrigala feral in China.

Myxobolus species represents a group of cosmopolitan metazoan parasites commonly harbored in the farmed and wild fish populations. Here, a species of Myxobolus is found in the kidney of an exotic mrigal Cirrhinus mrigala feral in the Yangtze River and utilized for an integrative characterization. Ellipsoidal myxospores are measured at 15.68 ± 0.8 (13.93-17.11) × 11.42 ± 0.54 (10.34-12.3) × 7.94 ± 0.35 (7.58-8.5) µm in dimension. The polar capsules are pyriform, and unequal in size. The morphological and morphometric characteristics of the present isolates are distinct from those of other congeners. Molecularly, the pairwise comparison based on the SSU rDNA sequence indicates that the present amplicon does not match any sequences available in the GenBank database and shares the highest similarity of 92.12% to Myxobolus pavlovskii (MG520369). Accordingly, we propose a name Myxobolus shuifuensis sp. n. for the present isolates. Phylogenetical trees indicate an apparent host-associated phylogenetic pattern. M. shuifuensis sp. n. groups loosely with other Myxobolus species isolated from Cirrhinus fish. Insead, it forms a sister clade to some myxosporeans belonging to the Thelohanellus genus. This result underpins the species identification and provides evidence for challenging the taxonomic separation among both morphologically comparable genera.

Effect of dietary Bacillus subtilis supplement on Cd toxicokinetics and Cd-induced immune and antioxidant impairment of Procambarus clarkii.

Cadmium (Cd), a non-biodegradable contaminant in freshwater ecosystems, can pose a serious threat to aquatic animals at high levels. In this study, the Cd toxicokinetics and the immune and antioxidant defense were explored in Procambarus clarkii exposed to different levels of Cd (0, 0.1, 1.0 mg Cd/L) or treated with 1.0 mg Cd/L and dietary Bacillus subtilis supplementation (1 × 107 cfu/g). Results from the 21-day uptake and depuration experiment revealed that Cd exposure elicited a dose- and time-dependent uptake in all crayfish tissues, and the rank order of Cd concentration was gill > hepatopancreas > exoskeleton > muscle. The one-compartment model demonstrated that gills had the highest uptake rate (ku) value after Cd aqueous exposure and the ku and elimination rate (kd) values in gill, hepatopancreas, and exoskeleton of the group with 1.0 mg Cd/L were higher than those of the group at alow Cd concentration (0.1 mg Cd/L). However, B. subtilis could decrease Cd ku and increase Cd kd in hepatopancreas, resulting in the reduction of bioconcentration factors (BCF), steady-state concentrations (Css), and biological half-life (Tb1/2). A positive correlation was found between aqueous Cd concentration and the severity of hepatopancreas histopathological injury, while B. subtilis could ameliorate the pathological damage in the high Cd group. Similarly, aqueous exposure to Cd elevated malonaldehyde (MDA) content and suppressed the activities of lysozyme (LZM), acid phosphatase (ACP) in hepatopancreas and alkaline phosphatase (AKP) in hemolymph. The activities of superoxide dismutase (SOD) and catalase (CAT) in hepatopancreas were also inhibited. Nevertheless, they were all recovered with the dietary addition of B. subtilis. In conclusion, our results indicated that exposure to Cd significantly increased Cd accumulation and toxic damages in crayfish hepatopancreas, while dietary administration of B. subtilis to crayfish significantly decreased Cd accumulation and improved the immune and antioxidant defense, leading to the prevention in toxic effects of Cd.

Transcriptomic Insights into the Diversity and Evolution of Myxozoa (Cnidaria, Endocnidozoa) Toxin-like Proteins.

Myxozoa is a speciose group of endoparasitic cnidarians that can cause severe ecological and economic effects. Their cnidarian affinity is affirmed by genetic relatedness and the presence of nematocysts, historically called "polar capsules". Previous studies have revealed the presence of toxin-like proteins in myxozoans; however, the diversity and evolution of venom in Myxozoa are not fully understood. Here, we performed a comparative analysis using the newly sequenced transcriptomes of five Myxobolidae species as well as some public datasets. Toxin mining revealed that myxozoans have lost most of their toxin families, while most species retained Kunitz, M12B, and CRISP, which may play a role in endoparasitism. The venom composition of Endocnidozoa (Myxozoa + Polypodium) differs from that of free-living cnidarians and may be influenced by ecological and environmental factors. Phylogenetic analyses showed that toxin families of myxozoans and free-living cnidarians were clustered into different clades. Selection analyses showed that purifying selection was the dominant evolutionary pressure in toxins, while they were still influenced by episodic adaptive selection. This suggests that the potency or specificity of a particular toxin or species might increase. Overall, our findings provide a more comprehensive framework for understanding the diversity and evolution of Myxozoa venoms.

The Microbiome Structure of a Rice-Crayfish Integrated Breeding Model and Its Association with Crayfish Growth and Water Quality.

The rice-crayfish (RC) integrated breeding model is an important and special agricultural ecosystem that provides a unique ecological environment for exploring the microbial diversity, composition, and functional capacity. To date, little is known about the effect of the breeding model on microbiome assembly and breeding model-specific microbiome composition and the association of the microbiome with water quality and crayfish growth. In the present study, we assessed the taxonomic shifts in gut and water microbiomes and their associations with water quality and crayfish growth in the RC and crayfish monoculture (CM) breeding models across six time points of rice growth, including seedling (a), tillering and jointing (b), blooming (c), filling (d), fruiting (e), and rotting of rice residues (f). Dominant bacterial phyla, such as Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, were detected in both gut and water microbiomes across breeding models. Notably, the diversity and structure of the gut and water microbiomes significantly (P < 0.001) differed between the RC and CM models, with higher microbial diversity being noted in the RC model than in the CM model. The taxa enriched in the RC model included Bacillus sp., Streptomyces sp., Lactobacillus sp., Prevotella sp., Rhodobacter sp., Bifidobacterium sp., Akkermansia sp., and Lactococcus sp., some of which are potentially beneficial to animals. However, opportunistic pathogens, such as Citrobacter sp. and Aeromonas sp., were depleted in the RC model. Furthermore, in the RC model, the enriched taxa that formed complex cooccurrence networks showed a significant positive correlation with water quality and crayfish growth, whereas the depleted taxa showed a significant negative correlation with water quality and crayfish growth. These results suggest that the RC model has a better microbiome composition and that RC model-specific microbes could play important roles in improving crayfish growth and water quality. IMPORTANCE The present study comprehensively compared two different breeding models in terms of their microbiome composition and the associations of the microbiomes with crayfish growth and water quality. RC model-specific microbiome composition was identified; these microbes were found to have a positive association with water quality and crayfish growth. These results provide valuable information for guiding microbial isolation and culture and for potentially harnessing the power of the microbiome to improve crayfish production and health and water quality.

A myxozoan genome reveals mosaic evolution in a parasitic cnidarian.

BACKGROUND: Parasite evolution has been conceptualized as a process of genetic loss and simplification. Contrary to this model, there is evidence of expansion and conservation of gene families related to essential functions of parasitism in some parasite genomes, reminiscent of widespread mosaic evolution-where subregions of a genome have different rates of evolutionary change. We found evidence of mosaic genome evolution in the cnidarian Myxobolus honghuensis, a myxozoan parasite of fish, with extremely simple morphology. RESULTS: We compared M. honghuensis with other myxozoans and free-living cnidarians, and determined that it has a relatively larger myxozoan genome (206 Mb), which is less reduced and less compact due to gene retention, large introns, transposon insertion, but not polyploidy. Relative to other metazoans, the M. honghuensis genome is depleted of neural genes and has only the simplest animal immune components. Conversely, it has relatively more genes involved in stress resistance, tissue invasion, energy metabolism, and cellular processes compared to other myxozoans and free-living cnidarians. We postulate that the expansion of these gene families is the result of evolutionary adaptations to endoparasitism. M. honghuensis retains genes found in free-living Cnidaria, including a reduced nervous system, myogenic components, ANTP class Homeobox genes, and components of the Wnt and Hedgehog pathways. CONCLUSIONS: Our analyses suggest that the M. honghuensis genome evolved as a mosaic of conservative, divergent, depleted, and enhanced genes and pathways. These findings illustrate that myxozoans are not as genetically simple as previously regarded, and the evolution of some myxozoans is driven by both genomic streamlining and expansion.

Quantitative Insights into the Contribution of Nematocysts to the Adaptive Success of Cnidarians Based on Proteomic Analysis.

Nematocysts are secretory organelles in cnidarians that play important roles in predation, defense, locomotion, and host invasion. However, the extent to which nematocysts contribute to adaptation and the mechanisms underlying nematocyst evolution are unclear. Here, we investigated the role of the nematocyst in cnidarian evolution based on eight nematocyst proteomes and 110 cnidarian transcriptomes/genomes. We detected extensive species-specific adaptive mutations in nematocyst proteins (NEMs) and evidence for decentralized evolution, in which most evolutionary events involved non-core NEMs, reflecting the rapid diversification of NEMs in cnidarians. Moreover, there was a 33-55 million year macroevolutionary lag between nematocyst evolution and the main phases of cnidarian diversification, suggesting that the nematocyst can act as a driving force in evolution. Quantitative analysis revealed an excess of adaptive changes in NEMs and enrichment for positively selected conserved NEMs. Together, these findings suggest that nematocysts may be key to the adaptive success of cnidarians and provide a reference for quantitative analyses of the roles of phenotypic novelties in adaptation.

New considerations on the phylogeny of Sessilida (Protista: Ciliophora: Peritrichia) based on multiple-gene information, with emphasis on colonial taxa.

The subclass Peritrichia, containing two orders Sessilida and Mobilida, is a major group of ciliates with worldwide distribution and high species diversity. Several studies have investigated the phylogeny of peritrichs; however, the evolutionary relationships and classification of some families and genera within the Sessilida remain unclear. In the present study, we isolated and identified 22 peritrich populations representing four families and six genera and obtained 64 rDNA sequences to perform phylogenetic analyses and assess their systematic relationships. Ancestral character reconstruction was also carried out to infer evolutionary routes within the Sessilida. The results indicate: (1) family Vaginicolidae is monophyletic and acquisition of the typical peritrich lorica represents a single evolutionary divergence; (2) core epistylidids evolved from a Zoothamnium-like ancestor and experienced spasmoneme loss during evolution; (3) Campanella clusters with species in the basal clade and shows stable morphological differences with other epistylidids, supporting its assignment to a separate family; (4) the structure of the peristomial lip may be a genus-level character rather than a diagnostic character for discriminating Epistylididae and Operculariidae, thus a redefinition of Operculariidae should be carried out when more species have been investigated; (5) some characters, such as lifestyle (solitary or colonial), spasmoneme and living habit (sessile or free-swimming), evolved repeatedly among sessilids indicating that species with non-contractile stalks or that are free-swimming have multiple evolutionary routes and might derive from any sessilid lineage without a lorica. The close phylogenetic relationships of some morphologically distinct sessilids imply that the diagnoses of some genera and families should be improved.

Health risk assessment and bioaccumulation of heavy metals in Procambarus clarkii from six provinces of China.

Contamination with heavy metals in wild red swamp crayfish (Procambarus clarkii) from 7 different geographical areas in six provinces of China (Hubei, Hunan, Jiangxi, Anhui, Jiangsu, and Shandong) was evaluated. Concentrations of chromium (Cr), arsenic (As), lead (Pb), cadmium (Cd), and mercury (Hg) in the abdominal muscle, gonad, and hepatopancreas were determined by inductively coupled plasma mass spectrometry (ICP-MS) and atomic fluorescence spectrometer (AFS). Except for the Cd content in the hepatopancreas, the contents of selected heavy metals in three different tissues were significantly lower than the proposed limits provided by United States Environmental Protection Agency (USEPA). The maximum accumulations of Cd and Pb were in the hepatopancreas, while the maximum accumulation of As was in the gonad, and the maximum accumulations of Hg and Cr were in the abdominal muscle. The highest contents of Cr, Hg, and Pb were all detected in Dongting Lake, Hunan, which was consistent with the trend of the metal pollution index (MPI). Risk value of the target hazard quotient (THQ) was below 1.0, suggesting that the intake of selected heavy metals through crayfish consumption would not pose a significant health risk to consumers.

Fins infestation induced by Myxobolus xiantaoensis in yellow catfish Tachysurus fulvidraco Richardson, 1846: Some pathophysiological and molecular insights.

The myxozoan parasite Myxobolus xiantaoensis is a fin pathogen of commercially important yellow catfish Tachysurus fulvidraco Richardson, 1846, in the freshwater ponds of China. In the present work, four geographical isolates of M. xiantaoensis were sampled from the fins of yellow catfish. It was found that the spores of four isolates exhibited few markable differences in morphometrics. The small subunit ribosomal DNA (SSU rDNA) sequences of four isolates were conspecific to the SSU rDNA sequence of M. xiantaoensis. No genetic level variation was observed, even in the characteristically more variable internal transcribed spacer (ITS) region. This absence of variability suggests high gene flow as a result of panmixia in the parasitic populations. ITS phylogeny placed four isolates of M. xiantaoensis in a clade together with myxozoans species infecting Siluriformes. The M. xiantaoensis infection inflicted severe hemorrhages on epidermis of ray-fins, which grew into inflammatory epithelial hyperplasia and lytic cartilage signs. The histochemical analysis of infected fins biopsies is characterized by damage of collagen components of cartilage, resulting in weakness, breaks, and missing fin rays. These tissue sections also had a remarkable inflammatory response around the fin cartilage, with the absence of mature spores and chondrocytes. These results indicate that the fin cartilage damage appeared before the development of tissue inflammation and the parasitic infestation of the fins. The present four geographical isolates of M. xiantaoensis were identified by a holistic approach of species characterization based on biological, morphological, and molecular evidence. These four isolates showed some morphological and genetic variations but within the intraspecific range.

Transcriptome analysis of goldfish (Carassius auratus) in response to Gyrodactylus kobayashii infection.

Gyrodactylid monogeneans are widespread parasites of teleost fishes, and infection with these parasites results in high host morbidity and mortality in aquaculture. To comprehensively elucidate the immune mechanisms against Gyrodactylus kobayashii, the transcriptome profiles of goldfish (Carassius auratus) skin after challenge with G. kobayashii were first investigated using next-generation sequencing. Approximately 21 million clean reads per library were obtained, and the average percentage of these clean reads mapped to the reference genome was 82.25%. A total of 556 differentially expressed genes (DEGs), including 344 upregulated and 212 downregulated genes, were identified, and 380 DEGs were successfully annotated and assigned to 95 signaling pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, 14 pathways associated with immune response were identified mainly including mTOR signaling pathway, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, toll-like receptor signaling pathway, and phagosome. Twelve genes were selected and validated using qRT-PCR. A similar trend of these genes between RNA-Seq and qRT-PCR was observed, indicating that RNA-Seq data was reliable. Besides, the ALP activity and NO content in serum were significantly higher in the infected goldfish compared with the non-infected goldfish. In summary, this study provides better understandings of immune defense mechanisms of goldfish against G. kobayashii, which will support future molecular research on gyrodactylids and facilitate the prevention and treatment of gyrodactylosis in aquaculture.

Molecular and light microscopy evidence for the transfer of Myxobolus honghuensis from Carassius auratus gibelio broodfish to progeny.

Myxozoans usually have a complex life cycle involving indirect transmission between vertebrate and invertebrate hosts. The vertical transmission of these parasites in vertebrate hosts has not been documented so far. Here, we assessed whether the myxozoan parasite Myxobolus honghuensis is vertically transmitted in naturally infected allogynogenetic gibel carp Carassius auratus gibelio (Bloch). M. honghuensis infection of broodfish, fertilized eggs and laboratory-cultured progeny was monitored in 2018 and 2019. The presporogonic stage was microscopically observed in the pharynx of broodfish and their progeny. In situ hybridization confirmed the presence of M. honghuensis presporogonic stage in the pharynx of broodfish and progeny. Nested PCR results showed that M. honghuensis was present in tissues and eggs of broodfish, fertilized eggs and their corresponding progeny. The sequences obtained from broodfish and progeny showed 98.0-99.8% similarity with ITS-5.8S rDNA of M. honghuensis. This study provides molecular and light microscopy evidence for the transfer of M. honghuensis from broodfish to progeny via the eggs, but it is insufficient to assert that M. honghuensis can transmit vertically in naturally infected allogynogenetic gibel carp. This is the first record about vertical transfer of myxozoan in the vertebrate host.

CCPRD: A Novel Analytical Framework for the Comprehensive Proteomic Reference Database Construction of NonModel Organisms.

Protein reference databases are a critical part of producing efficient proteomic analyses. However, the method for constructing clean, efficient, and comprehensive protein reference databases of nonmodel organisms is lacking. Existing methods either do not have contamination control procedures, or these methods rely on a three-frame and/or six-frame translation that sharply increases the search space and the need for computational resources. Herein, we propose a framework for constructing a customized comprehensive proteomic reference database (CCPRD) from draft genomes and deep sequencing transcriptomes. Its effectiveness is demonstrated by incorporating the proteomes of nematocysts from endoparasitic cnidarian: myxozoans. By applying customized contamination removal procedures, contaminations in omic data were successfully identified and removed. This is an effective method that does not result in overdecontamination. This can be shown by comparing the CCPRD MS results with an artificially contaminated database and another database with removed contaminations in genomes and transcriptomes added back. CCPRD outperformed traditional frame-based methods by identifying 35.2-50.7% more peptides and 35.8-43.8% more proteins, with a maximum of 84.6% in size reduction. A BUSCO analysis showed that the CCPRD maintained a relatively high level of completeness compared to traditional methods. These results confirm the superiority of the CCPRD over existing methods in peptide and protein identification numbers, database size, and completeness. By providing a general framework for generating the reference database, the CCPRD, which does not need a high-quality genome, can potentially be applied to nonmodel organisms and significantly contribute to proteomic research.