Complete mitochondrial genome and phylogenetic analysis of Dollfustrema vaneyi (Trematoda: Bucephalidae).

BACKGROUND: The Bucephalidae is a large family of digenean trematodes but most previous analyses of its phylogenetic position have relied on a single mitochondrial gene or morphological features. Mitochondrial genomes (mitogenomes) remain unavailable for the entire family. To address this, we sequenced the complete mitogenome of Dollfustrema vaneyi and analyzed the phylogenetic relationships with other trematodes. RESULTS: The circular genome of Dollfustrema vaneyi spanned 14,959 bp and contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a major non-coding region. We used concatenated amino acid and nucleotide sequences of all 36 genes for phylogenetic analyses, conducted using MrBayes, IQ-TREE and PhyloBayes. We identified pronounced topological instability across different analyses. The addition of recently sequenced two mitogenomes for the Aspidogastrea subclass along with the use of a site-heterogeneous model stabilized the topology, particularly the positions of Azygiidae and Bucephalidae. The stabilized results indicated that Azygiidae was the closest lineage to Bucephalidae in the available dataset, and together, they clustered at the base of the Plagiorchiida. CONCLUSIONS: Our study provides the first comprehensive description and annotation of the mitochondrial genome for the Bucephalidae family. The results indicate a close phylogenetic relationship between Azygiidae and Bucephalidae, and reveal their basal placement within the order Plagiorchiida. Furthermore, the inclusion of Aspidogastrea mitogenomes and the site-heterogeneous model significantly improved the topological stability. These data will provide key molecular resources for future taxonomic and phylogenetic studies of the family Bucephalidae.

Immune response of Rhinogobio ventralis to Ichthyophthirius multifiliis infection: Insights from histopathological and real-time gene expression analyses.

Ichthyophthirius multifiliis is a parasite that poses a considerable threat to aquaculture and the ornamental fish industry, but with limited effective treatment options available. This study employed RT-qPCR to detect and analyze the expression changes of partial toll-like receptor (TLR) genes (TLR1 and TLR21), adapter protein and signal transduction molecule genes (MyD88, TRIF, NF-κB, IRAK4, and IRF3), and cytokines (IL-6, IL-8, IL-13, CXC-α and CXCR1), as well as complement C3, in the skin, gill, fin, liver, head kidney and spleen of Rhinogobio ventralis under different infection conditions. Additionally, tissue sections and scanning electron microscopy were utilized to observe the pathological changes in the gills and fins of R. ventralis after infection with I. multifiliis. The expression patterns of TLR-related DEGs (differentially expressed genes) in diseased wild fish were analyzed, revealing upregulation of TLR1, TLR21, MyD88, NF-κB, IRAK4, TRIF, IRF3, IL-6, IL-8, IL-13, CXC-α, CXCR1, and C3 genes in various tissues, indicating that these genes may be involved in the immune response of R. ventralis to I. multifiliis infection. To further analyze the gene expression of sampled from the field, an artificial infection model of R. ventralis was established under laboratory conditions, with additional sampling from the skin and fins. These genes continued to show varying degrees of upregulation, but the results were not entirely consistent with those from Wudongde samples, which may be due to the more complex environment in the wild or differences in the degree of I. multifiliis infection in wild fish. The infection of I. multifiliis caused severe damage to the gills and fins of R. ventralis, characterized by extensive secretions on the gill and fin surfaces, with the presence of attached I. multifiliis trophonts, including damage and loss of gill filaments, swollen gill lamellae, and deformed gill plates, as well as cell proliferation and necrosis of gill epithelial cells. This study sheds light on the role of the TLR signaling pathway in resisting I. multifiliis infection and its associated histopathological changes in R. ventralis, providing valuable insights for the prevention and treatment of I. multifiliis infection in R. ventralis.

Potential effects of host competence and schooling behavior on parasite transmission in a host-pathogen system: a test of the dilution effect.

High species diversity in a community may reduce the risk of infectious disease, termed the dilution effect. However, the generality of the dilution effect in different disease systems remains controversial as both host competence and behaviors of hosts may play roles in dilution or amplification of disease. Using the goldfish (Carassius auratus)-monogenean ectoparasite (Gyrodactylus kobayashii) system, effects of host competence and schooling behavior on parasite transmission were investigated while holding focal host density constant. Following competency tests of 12 fish species as potential hosts for the parasite, infection by G. kobayashii was determined on fins of goldfish mixed with each of three different species based on their level of host competence, including Prussian carp, Carassius gibelio (low competence), grass carp, Ctenopharyngodon idellus (non-competent), swordtail, Xiphophorus helleri (non-competent), and the four species combined. Compared with mean abundance (85.8 ± 25.1) on goldfish in the control group, the mean abundance on goldfish decreased significantly when paired with 10 Prussian carp (30.0 ± 16.5), but did not differ significantly when paired with 10 swordtail (70.0 ± 22.2), 10 grass carp (116.1 ± 33.2), or the multi-species of three Prussian carp, four grass carp and three swordtail (75.9 ± 30.8) during the 11-day experiment. The parasite was also found on the Prussian carp in the Prussian carp group and the multi-species group at a mean abundance of 7.1 and 10.9, respectively. Video recording showed that the school of goldfish mixed well with the Prussian carp, while they maintained separation from the grass carp and swordtail when mixed together. The distance between goldfish increased, and swimming speed and contact time decreased with the additional of other fish species for all groups. The results suggested that the presence of a low-competence host in sufficient numbers was a necessary condition for a dilution effect due to encounter reduction, and the dilution effect may also be enhanced by changes in schooling behavior of goldfish in the presence of low competence hosts. However, the presence of non-competent hosts did not result in any dilution effect owing to the specialist nature of the parasites and the lack of mixing with schools of goldfish.

Drivers of interlineage variability in mitogenomic evolutionary rates in Platyhelminthes.

Studies of forces driving interlineage variability in the evolutionary rates (both sequence and architecture) of mitochondrial genomes often produce contradictory results. Flatworms (Platyhelminthes) exhibit the fastest-evolving mitogenomic sequences among all bilaterian phyla. To test the effects of multiple factors previously associated with different aspects of mitogenomic evolution, we used mitogenomes of 223 flatworm species, phylogenetic multilevel regression models, and causal inference. Thermic host environment (endothermic vs. ectothermic) had nonsignificant impacts on both sequence evolution and mitogenomic size. Mitogenomic gene order rearrangements (GORR) were mostly positively correlated with mitogenomic size (R2 ≈ 20-30%). Longevity was not (negatively) correlated with sequence evolution in flatworms. The predominantly free-living "turbellaria" exhibited much shorter branches and faster-evolving mitogenomic architecture than parasitic Neodermata. As a result, "parasitism" had a strong explanatory power on the branch length variability (>90%), and there was a negative correlation between GORR and branch length. However, the stem branch of Neodermata comprised 63.6% of the total average branch length. This evolutionary period was also marked by a high rate of gene order rearrangements in the ancestral Neodermata. We discuss how this period of rapid evolution deep in the evolutionary history may have decoupled sequence evolution rates from longevity and GORR, and overestimated the explanatory power of "parasitism". This study shows that impacts of variables often vary across lineages, and stresses the importance accounting for the episodic nature of evolutionary patterns in studies of mitogenomic evolution.

Infection with Clonorchis sinensis (Cobbold, 1875) Metacercariae in Fish from the East Lake of Wuhan: Freshwater Fish in Urban Lakes May Act as Infection Sources of Liver Fluke.

The liver fluke disease caused by Clonorchis sinensis is one of the most serious food-borne parasitic diseases in China. Many freshwater fish and shrimps can be infected with C. sinensis metacercariae as the second intermediate hosts in endemic regions. Owing to the lack of infected humans and the good administration of pet dogs and cats in cities of non-endemic regions, few fish are expected to be infected with C. sinensis metacercariae in urban lakes. To determine the infection of C. sinensis metacercariae in freshwater fish and shrimps in urban lakes, a total of 18 fish species and one shrimp species were investigated in the East Lake of Wuhan City. Metacercariae were isolated by artificial digestive juice and identified using morphology and rDNA-ITS2 sequences. Five species of fish, Pseudorasbora parva, Ctenogobius giurinus, Squalidus argentatus, Hemiculter leuciclus, and Rhodeus spp., were infected with C. sinensis metacercariae. The overall prevalence of C. sinensis was 32.5%. The highest prevalence was found in P. parva with 57.9%, while S. argentatus exhibited the highest mean abundance (13.9). Apart from the C. sinensis metacercariae, four species of other trematode metacercariae were also identified across twelve fish species in total. Owing to the consumption of undercooked fish and feeding cats with small fish caught by anglers, there is a potential risk that the small fish infected with C. sinensis metacercariae may act as an infection source to spread liver fluke. Given the complete life cycle of C. sinensis, stray cats and rats were inferred to act as the important final hosts of C. sinensis in urban lakes in non-endemic areas.

Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) on bitterling fishes (Acheilognathinae) from China.

Three new species of Gyrodactylus are described from three species of bitterling in Donghu Lake, China: Gyrodactylus ocellorhodei n. sp. from Rhodeus ocellatus; G. sinenorhodei n. sp. from Rhodeus sinensis; and G. acheilorhodei n. sp. from Acheilognathus macropterus. All the three new species showed similar opisthaptor morphology, especially the marginal hooks: all had a slender and perpendicular sickle shaft, and flat sickle base with distinct heel and inner arch which was different from the G. rhodei-group species parasitic on bitterling. Multivariate analyses based on hamulus and marginal hooks suggested that these three new species cannot be completely distinguished, despite some morphology divergence observed in certain less reliable morphometric features, such as hamulus root length, ventral bar total length and process shape. These three new species shared an identical 18S ribosomal RNA gene sequence, while the variation in the Internal Transcribed Spacers (ITS1-ITS2) sequence among them (8.4-11.2%, K2P) far exceeded the 1% ITS sequence difference that had been suggested as a threshold for species delimitation of Gyrodactylus. Phylogenetic analysis based on ITS1-ITS2 showed that all these sequenced Gyrodactylus spp. parasitic on the subfamily Acheilognathinae host formed a monophyletic group. However, a clear differentiation (18.9-20.9%, K2P of ITS1-ITS2) could be found between the subgroup from China (G. ocellorhodei n. sp., G. sinenorhodei n. sp. and G. acheilorhodei n. sp.) and that from Europe (G. rhodei).

Comprehensive molecular characterisation of the complete mitogenome of Ergasilus tumidus and phylogenetic relationships of Copepoda inferred from mitogenomes.

Although parasitic copepods of the genus Ergasilus von Nordmann, 1832 are globally distributed parasites of fish, their phylogenetic relationships with other Copepoda are not clear, and the characteristics of their mitochondrial genomes (mitogenomes) are not thoroughly understood. The objective of this study was to address these knowledge gaps by sequencing the complete mitogenome of Ergasilus tumidus Markevich, 1940. The complete mitogenome (GenBank Acc. No. OQ596537) was 14,431 bp long and it comprised 13 protein-coding genes (PCGs), 22 tRNAs, two tRNAs, and two control regions (CRs). Phylogenetic analyses, conducted using concatenated nucleotide and amino acid sequences of 13 protein-coding genes, produced two partially incongruent topologies. While the order Calanoida was consistently resolved as the sister lineage to the other three orders, topological instability was observed in the relationships of the orders Cyclopoida, Siphonostomatoida and Harpacticoida. Siphonostomatoida clustered with Cyclopoida in the nucleotide-based phylogeny, but with Harpacticoida in the amino acid-based phylogeny. The latter topology conforms to the widely accepted relationships, but we speculate that the former topology is more likely to be the correct one. Our study provides a complete mitogenome sequence of E. tumidus, which helps us better understand the molecular evolution of the genus Ergasilus. Additionally, we suggest a different perspective on the controversial phylogenetic relationships among Siphonostomatoida, Cyclopoida and Harpacticoida, diverging from previously accepted views.

Genome assembly of a symbiotic balantidia (Balantidium ctenopharyngodoni) in fish hindgut.

Balantidium ctenopharyngodoni is identified as the sole ciliate species that exclusively resides within the hindgut of grass carp with high prevalence and intensity. In this study, the successful cultivation of B. ctenopharyngodoni enabled us to collect enough cells for genome sequencing. Consequently, we acquired a high-quality genome assembly spanning 68.66 Mb, encompassing a total of 22,334 nanochromosomes. Furthermore, we predicted 29,348 protein-coding genes, and 95.5% of them was supported by the RNA-seq data. The trend of GC content in the subtelomeric regions of single-gene chromosomes was similar to other ciliates containing nanochromosomes. A large number of genes encoding carbohydrate-binding modules with affinities for starch and peptidoglycans was identified. The identification of mitochondrion-related organelles (MROs) within genome indicates its well-suited adaptation to the anaerobic conditions in the hindgut environment. In summary, our results will offer resources for understanding the genetic basis and molecular adaptations of balantidia to hindgut of herbivorous fish.

Immune response and apoptosis of gibel carp (Carassius auratus gibelio) gills to Chilodonella hexasticha infection: Insights from histopathological and multi-omics analyses.

Gibel carp (Carassius auratus gibelio) is an important economically farmed fish in China. Chilodonella hexasticha parasitizes on the gills and fins of host fish, causing disruption to their normal respiration and movement, ultimately resulting in death of the fish. In this study, a combination of histopathological, immunohistochemical, transferase dUTP nick end labeling (TUNEL), multi-omics, and molecular approaches were employed to identify the immune reaction and cell apoptosis in gill tissue in response to C. hexasticha infection. Significant lamellae fusion, hyperplasia, hyperemia, necrosis, and desquamation of infected gibel carp gills were observed. In total, the expression of 3619 genes was higher, and 3143 lower, for gills in the infected group compared to the control group. Furthermore, 76 metabolites were significantly increased and 105 were significantly decreased in the infected group compared with the control group. From the qRT-PCR analysis results, immune system-related genes encoding IL-8, CXCL8a, and CXC11 were significantly up-regulated in infected gibel carp, while ZAP70 was significantly down-regulated. Immunohistochemical results also showed the down-regulated ZAP70 in the infected group. Apoptosis-related genes encoding CASP3 and Mcl-1b were up-regulated in response to C. hexasticha infection. These genes indicate the activation of CASP family-related apoptosis and Bim-mediated mitochondrial apoptotic pathways. TUNEL assays also revealed severe apoptosis in the infected group. Based on this study's results, it can be concluded that C. hexasticha infection leads to histopathological changes in the gills of infected fish, and induces both a significant immune response and apoptosis.

Geography, phylogeny and host switch drive the coevolution of parasitic Gyrodactylus flatworms and their hosts.

BACKGROUND: Gyrodactylus is a lineage of monogenean flatworm ectoparasites exhibiting many features that make them a suitable model to study the host-parasite coevolutionary dynamics. Previous coevolutionary studies of this lineage mainly relied on low-power datasets (a small number of samples and a single molecular marker) and (now) outdated algorithms. METHODS: To investigate the coevolutionary relationship of gyrodactylids and their fish hosts in high resolution, we used complete mitogenomes (including two newly sequenced Gyrodactylus species), a large number of species in the single-gene dataset, and four different coevolutionary algorithms. RESULTS: The overall coevolutionary fit between the parasites and hosts was consistently significant. Multiple indicators confirmed that gyrodactylids are generally highly host-specific parasites, but several species could parasitize either multiple (more than 5) or phylogenetically distant fish hosts. The molecular dating results indicated that gyrodactylids tend to evolve towards high host specificity. Speciation by host switch was identified as a more important speciation mode than co-speciation. Assuming that the ancestral host belonged to Cypriniformes, we inferred four major host switch events to non-Cypriniformes hosts (mostly Salmoniformes), all of which occurred deep in the evolutionary history. Despite their relative rarity, these events had strong macroevolutionary consequences for gyrodactylid diversity. For example, in our dataset, 57.28% of all studied gyrodactylids parasitized only non-Cypriniformes hosts, which implies that the evolutionary history of more than half of all included lineages could be traced back to these major host switch events. The geographical co-occurrence of fishes and gyrodactylids determined the host use by these gyrodactylids, and geography accounted for most of the phylogenetic signal in host use. CONCLUSIONS: Our findings suggest that the coevolution of Gyrodactylus flatworms and their hosts is largely driven by geography, phylogeny, and host switches.

Strong mitonuclear discordance in the phylogeny of Neodermata and evolutionary rates of Polyopisthocotylea.

The genomic evolution of Polyopisthocotylea remains poorly understood in comparison to the remaining three classes of Neodermata: Monopisthocotylea, Cestoda, and Trematoda. Moreover, the evolutionary sequence of major events in the phylogeny of Neodermata remains unresolved. Herein we sequenced the mitogenome and transcriptome of the polyopisthocotylean Diplorchis sp., and conducted comparative evolutionary analyses using nuclear (nDNA) and mitochondrial (mtDNA) genomic datasets of Neodermata. We found strong mitonuclear discordance in the phylogeny of Neodermata. Polyopisthocotylea exhibited striking mitonuclear discordance in relative evolutionary rates: the fastest-evolving mtDNA in Neodermata and a comparatively slowly-evolving nDNA genome. This was largely attributable to its very long stem branch in mtDNA topologies, not exhibited by the nDNA data. We found indications that the fast evolution of mitochondrial genomes of Polyopisthocotylea may be driven both by relaxed purifying selection pressures and elevated levels of directional selection. We identified mitochondria-associated genes encoded in the nuclear genome: they exhibited unique evolutionary rates, but not correlated with the evolutionary rate of mtDNA, and there is no evidence for compensatory evolution (they evolved slower than the rest of the genome). Finally, there appears to exist an exceptionally large (≈6.3 kb) nuclear mitochondrial DNA segment (numt) in the nuclear genome of newly sequenced Diplorchis sp. A 3'-end segment of the 16S rRNA gene encoded by the numt was expressed, suggesting that this gene acquired novel, regulatory functions after the transposition to the nuclear genome. In conclusion, Polyopisthocotylea appears to be the lineage with the fastest-evolving mtDNA sequences among all of Bilateria, but most of the substitutions were accumulated deep in the evolutionary history of this lineage. As the nuclear genome does not exhibit a similar pattern, the circumstances underpinning this evolutionary phenomenon remain a mystery.

Investigation of Ichthyophthirius multifiliis infection in fish from natural water bodies in the Lhasa and Nagqu regions of Tibet.

This study aimed to examine the prevalence of Ichthyophthirius multifiliis in fish inhabiting natural water bodies in the Lhasa and Nagqu regions of Tibet in September 2020 and August 2021. The results showed that Schizopygopsis selincuoensis had the highest prevalence of I. multifiliis at 33.73% (56/166), followed by Triplophysa tibetana at 30.00% (6/20), Triplophysa brevicauda at 27.91% (12/43) and Schizopygopsis thermalis at 23.66% (31/131). No infection with I. multifiliis was observed in exotic fish species. In addition, the prevalence of I. multifiliis in Boqu Zangbo (river), Selincuo Lake and Cuona Lake in the Nagqu region was found to be significantly higher than that in Lalu Wetland and Chabalang Wetland in the Lhasa region (P < 0.05). The study revealed a significantly lower prevalence in Lhasa River than in Cuona Lake (P < 0.05). Notably, our findings revealed instances of I. multifiliis infections even in saline water bodies, thereby emphasizing the potential threat that this parasite poses to the preservation of indigenous fish resources in Tibet. Consequently, immediate and effective countermeasures are imperative. This study represents the first systematic investigation of I. multifiliis infection in natural water bodies in Tibet.

Interactions between Balantidium ctenopharyngodoni and microbiota reveal its low pathogenicity in the hindgut of grass carp.

BACKGROUND: Hosts, parasites, and microbiota interact with each other, forming a complex ecosystem. Alterations to the microbial structure have been observed in various enteric parasitic infections (e.g. parasitic protists and helminths). Interestingly, some parasites are associated with healthy gut microbiota linked to the intestinal eubiosis state. So the changes in bacteria and metabolites induced by parasite infection may offer benefits to the host, including protection from other parasitesand promotion of intestinal health. The only ciliate known to inhabit the hindgut of grass carp, Balantidium ctenopharyngodoni, does not cause obvious damage to the intestinal mucosa. To date, its impact on intestinal microbiota composition remains unknown. In this study, we investigated the microbial composition in the hindgut of grass carp infected with B. ctenopharyngodoni, as well as the changes of metabolites in intestinal contents resulting from infection. RESULTS: Colonization by B. ctenopharyngodoni was associated with an increase in bacterial diversity, a higher relative abundance of Clostridium, and a lower abundance of Enterobacteriaceae. The family Aeromonadaceae and the genus Citrobacter had significantly lower relative abundance in infected fish. Additionally, grass carp infected with B. ctenopharyngodoni exhibited a significant increase in creatine content in the hindgut. This suggested that the presence of B. ctenopharyngodoni may improve intestinal health through changes in microbiota and metabolites. CONCLUSIONS: We found that grass carp infected with B. ctenopharyngodoni exhibit a healthy microbiota with an increased bacterial diversity. The results suggested that B. ctenopharyngodoni reshaped the composition of hindgut microbiota similarly to other protists with low pathogenicity. The shifts in the microbiota and metabolites during the colonization and proliferation of B. ctenopharyngodoni indicated that it may provide positive effects in the hindgut of grass carp.

Alterations of gut microbiota and short-chain fatty acids induced by Balantidium polyvacuolum in the hindgut of Xenocyprinae fishes providing new insights into the relationship among protozoa, gut microbiota and host.

Introduction: Parasitic ciliates are protozoans with a global distribution. Along with the gut microbiota, they have formed a micro-ecosystem that affects the host's nutrition, metabolism, and immunity. The interactions and relationships among the three components of this microecosystem (protozoa, gut microbiota, and host) remain only partially understood. Xenocypris fish and the unique ciliate Balantidium polyvacuolum in its hindgut are good materials to study the interplay. Methods: In this study, 16S rRNA gene amplicon sequencing and short-chain fatty acids (SCFAs) identification were used. Network was also constructed to understand their relationships. Results: We found that the gut microbiota of B. polyvacuolum-infected X. davidi and X. argentea had higher diversity, richness, and evenness than uninfected ones. B. polyvacuolum could lead to an increase of Fusobacterium and Chloroflexi in both X. davidi and X. argentea, while significantly increase the abundance of genera Romboutsia and Clostridium in X. argentea. Besides, B. polyvacuolum could significantly increase the content of total SCFAs and acetic acid in X. davidi and increase the concentrations of propionic, isobutyric and butanoic acids in X. argentea. Furthermore, correlation analyses showed that B. polyvacuolum may alter SCFAs by affecting key SCFAs-producing bacteria such as Clostridium and Cetobacterium. Discussion: This study greatly expands our understanding of relationships among B. polyvacuolum, gut microbiota and host Xenocypris fish, which sheds new insights into the mechanism of interaction among protozoa, gut microbiota and host.

Mitogenomic evolutionary rates in bilateria are influenced by parasitic lifestyle and locomotory capacity.

The evidence that parasitic animals exhibit elevated mitogenomic evolutionary rates is inconsistent and limited to Arthropoda. Similarly, the evidence that mitogenomic evolution is faster in species with low locomotory capacity is limited to a handful of animal lineages. We hypothesised that these two variables are associated and that locomotory capacity is a major underlying factor driving the elevated rates in parasites. Here, we study the evolutionary rates of mitogenomes of 10,906 bilaterian species classified according to their locomotory capacity and parasitic/free-living life history. In Bilateria, evolutionary rates were by far the highest in endoparasites, much lower in ectoparasites with reduced locomotory capacity and free-living lineages with low locomotory capacity, followed by parasitoids, ectoparasites with high locomotory capacity, and finally micropredatory and free-living lineages. The life history categorisation (parasitism) explained ≈45%, locomotory capacity categorisation explained ≈39%, and together they explained ≈56% of the total variability in evolutionary rates of mitochondrial protein-coding genes in Bilateria. Our findings suggest that these two variables play major roles in calibrating the mitogenomic molecular clock in bilaterian animals.

The energy metabolism of Balantidium polyvacuolum inhabiting the hindgut of Xenocypris davidi.

Anaerobic parasitic ciliates are a specialized group of ciliates that are adapted to anoxic and oxygen-depleted habitats. Among them, Balantidium polyvacuolum, which inhabits the hindgut of Xenocyprinae fishes, has received very limited scientific attention, so the molecular mechanism of its adaptation to the digestive tract microenvironment is still unclear. In this study, transmission electron microscopy (TEM) and single-cell transcriptome analysis were used to uncover the metabolism of B. polyvacuolum. Starch granules, endosymbiotic bacteria, and multiple specialized mitochondrion-related organelles (MROs) of various shapes were observed. The MROs may have completely lost the electron transport chain (ETC) complexes I, III, IV, and V and only retained succinate dehydrogenase subunit A (SDHA) of complex II. The tricarboxylic acid (TCA) cycle was also incomplete. It can be inferred that the hypoxic intestinal environment has led to the specialization of the mitochondria in B. polyvacuolum. Moreover, carbohydrate-active enzymes (CAZymes), including carbohydrate esterases, enzymes with a carbohydrate-binding module, glycoside hydrolases, and glycosyltransferases, were identified, which may constitute evidence that B. polyvacuolum is able to digest carbohydrates and starch. These findings can improve our knowledge of the energy metabolism and adaptive mechanisms of B. polyvacuolum.

Comparative Transcriptomics of Chilodonella hexasticha and C. uncinata Provide New Insights into Adaptations to a Parasitic Lifestyle and Mdivi-1 as a Potential Agent for Chilodonellosis Control.

Chilodonella hexasticha is a harmful parasitic ciliate that can cause severe damage to fish and high mortalities worldwide. Its congeneric species, C. uncinata, is a facultative parasite that not only can be free-living but also can parasitize on fish gills and fins. In this study, single-cell transcriptomes of these two species were assembled and characterized. Numerous enzymes related to energy metabolism and parasitic adaption were identified through annotation in the Non-Redundant (NR), Clusters of Orthologous Genes (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The expression of isocitrate dehydrogenase (IDH), cytochrome c oxidase subunit 1 (Cox1) and ATP synthase F1, delta subunit (ATP5D) was up-regulated in C. hexasticha compared with C. uncinata. The oxidative phosphorylation process was also enriched in C. hexasticha. The main mitochondrial metabolic pathways in C. hexasticha were depicted and enzymes related to energy metabolism pathways were compared between these two species. More importantly, mitochondrial division inhibitor 1 (mdivi-1) proved to be very effective in killing both C. hexasticha and C. uncinata, which could be a novel drug for Chilodonellosis control. This study can help us better understand the energy metabolisms of C. hexasticha and C. uncinata and provide new insight into novel targets for chilodonellosis control. Meanwhile, the transcriptome data can also facilitate genomic studies of these two species in the future.

Fate of high-risk antibiotic resistance genes in large-scale aquaculture sediments: Geographical differentiation and corresponding drivers.

Antibiotic resistance genes (ARGs), emerging environmental contaminants, have become challenges of public health security. However, the distribution and drivers of ARGs, especially high-risk ARGs, in large-scale aquaculture sediments remain unknown. Here, we collected sediment samples from 40 crayfish ponds in seven main crayfish culture provinces in China and then investigated the distribution and risk of ARGs based on high-throughput sequencing and quantitative PCR techniques. Our results suggested that aquaculture sediment was potential reservoir of ARGs and the abundance of aadA-02 was the highest. High-risk ARG (floR) was also prevalent in the sediment and was the most abundant in Jiangsu Province, where opportunistic pathogens were also enriched. The abundance of floR was positively correlated with different environmental factors, such as total phosphorus in water and total carbon in sediment. In addition, Mycobacterium sp., opportunistic pathogenic bacteria, might be potential host for floR. Furthermore, the potential propagation pathway of ARGs was from sediment to crayfish gut, and Bacteroidetes and Proteobacteria might be the main bacterial groups responsible for the proliferation of ARGs. Generally, our results illustrate that pond sediment may be an ARG reservoir of aquatic animals. Meanwhile, our study helps develop valuable strategies for accessing risks and managing ARGs.

Evolutionary Rates, Divergence Rates, and Performance of Individual Mitochondrial Genes Based on Phylogenetic Analysis of Copepoda.

Copepoda is a large and diverse group of crustaceans, which is widely distributed worldwide. It encompasses roughly 9 orders, whose phylogeny remains unresolved. We sequenced the complete mitochondrial genome (mitogenome) of Sinergasilus major (Markevich, 1940) and used it to explore the phylogeny and mitogenomic evolution of Copepoda. The mitogenome of S. major (14,588 bp) encodes the standard 37 genes as well as a putative control region, and molecular features are highly conserved compared to other Copepoda mitogenomes. Comparative analyses indicated that the nad2 gene has relatively high nucleotide diversity and evolutionary rate, as well as the largest amount of phylogenetic information. These results indicate that nad2 may be a better marker to investigate phylogenetic relationships among closely related species in Copepoda than the commonly used cox1 gene. The sister-group relationship of Siphonostomatoida and Cyclopoida was recovered with strong support in our study. The only topological ambiguity was found within Cyclopoida, which might be caused by the rapid evolution and sparse taxon sampling of this lineage. More taxa and genes should be used to reconstruct the Copepoda phylogeny in the future.