Parasites (Monogenea) of tilapias Oreochromis niloticus and Coptodon rendalli (Cichlidae) in a river spring in Brazil.

In the present study, we examined 30 individuals of introduced African cichlids, Oreochromis niloticus and Coptodon rendalli, collected in a river spring of the Pardo River, Paranapanema River basin, southeastern Brazil. Based on morphological and molecular analyses of the partial LSU rDNA gene, we identified four species of monogeneans, Cichlidogyrus tilapiae, C. thurstonae, C. mbirizei, and Scutogyrus longicornis on the gills of O. niloticus, whereas individuals of C. rendalli were infested only with C. papernastrema. This is the first record of C. mbirizei and C. papernastrema in tilapias from Brazil. The ecological consequences of the introduction of exotic species of tilapia such as O. niloticus and C. rendalli along with their monogenean parasites in a wild environment represented by a river spring are discussed. Our new molecular data on Cichlidogyrus and Scutogyrus contribute to the investigation of the phylogenetic interrelationships of these widely distributed genera of monogeneans since their species composition is still unsettled.

Title: Parasites (Monogenea) des tilapias Oreochromis niloticus et Coptodon rendalli (Cichlidae) dans une source au Brésil. Abstract: Dans la présente étude, nous avons examiné 30 individus de cichlidés africains introduits, Oreochromis niloticus et Coptodon rendalli, collectés dans une source fluviale du fleuve Pardo, bassin du fleuve Paranapanema, dans le sud-est du Brésil. Sur la base d'analyses morphologiques et moléculaires du gène partiel de l'ADNr LSU, nous avons identifié quatre espèces de monogènes, Cichlidogyrus tilapiae, C. thurstonae, C. mbirizei et Scutogyrus longicornis sur les branchies d'O. niloticus, alors que les individus de C. rendalli étaient infestés uniquement par C. papernastrema. Il s'agit du premier signalement de C. mbirizei et C. papernastrema chez des tilapias du Brésil. Les conséquences écologiques de l'introduction d'espèces exotiques de tilapia telles que O. niloticus et C. rendalli ainsi que leurs monogènes parasites dans un environnement sauvage représenté par une source fluviale sont discutées. Nos nouvelles données moléculaires sur Cichlidogyrus et Scutogyrus contribuent à l'étude des interrelations phylogénétiques de ces genres de monogènes largement distribués puisque leur composition spécifique est encore incertaine.

Biological toxicity of sulfamethoxazole in aquatic ecosystem on adult zebrafish (Danio rerio).

This study evaluated the impacts of sulfamethoxazole (SMX) on antioxidant, immune, histopathological dynamic changes, and gut microbiota of zebrafish. SMX was carried out five groups: 0 (C), 3 mg/L (T3), 6 mg/L (T6), 12 mg/L (T12), and 24 mg/L (T24), with 5 replicates per group for an 8-weeks chronic toxicity test. It was found that SMX is considered to have low toxicity to adult zebrafish. SMX with the concentration not higher than 24 mg/L has no obvious inhibitory effect on the growth of fish. Under different concentrations of SMX stress, oxidative damage and immune system disorder were caused to the liver and gill, with the 12 and 24 mg/L concentration being the most significant. At the same time, it also causes varying degrees of pathological changes in both intestinal and liver tissues. As the concentration of SMX increases, the composition and abundance of the gut microbiota in zebrafish significantly decrease.

Effects of different temperatures on Leiocassis longirostris gill structure and intestinal microbial composition.

Fish are poikilothermic vertebrates and their physiological activities are affected by water temperature. In recent years, extreme weather has occurred frequently, and temperature changes have adversely affected the growth of farmed fish. To explore the changes in gill tissue structure caused by changing the water temperature and the relationship between the intestinal microbiota and the Leiocassis longirostris host adaptation mechanism, gill tissue sections and intestinal microbial 16S rRNA amplicon sequencing were conducted under different temperature stress (low temperature 4 °C, normal temperature 26 °C and high temperature 32 °C). The results showed that heat stress and cold stress caused injury and swelling, terminal congestion, cell vacuolation, and necrosis of the gill tissue of L. longirostris. For intestinal microbiota, the abundances of Pseudomonadota and Bacillota increased at the cold stress, while the abundances of Fusobacteriota and Bacteroidota increased at the heat stress. The number of opportunistic bacteria, mainly Aeromonas and Acinetobacter, was the highest under cold stress. In addition, the richness of the intestinal microbiota decreased significantly at heat and cold stresses, while evenness increased. Prediction of intestinal microbiota function showed that most common functions, such as metabolism of cofactors and vitamins, energy metabolism and replication and repair, were decreased significantly at heat stress and cold stress, and phylogenetic relationship analysis revealed significant differences among the groups. In conclusion, the change of temperature altered the gill tissue structure, and affected the structure and homeostasis of the intestinal microbiota, thus affecting the survival time of L. longirostris, and cold stress had a greater effect than heat stress.

Single-Cell RNA Sequencing Profiling Cellular Heterogeneity and Specific Responses of Fish Gills to Microplastics and Nanoplastics.

Fish gills are highly sensitive organs for microplastic (MP) and nanoplastic (NP) invasions, but the cellular heterogeneity of fish gills to MPs and NPs remains largely unknown. We employed single-cell RNA sequencing to investigate the responses of individual cell populations in tilapia Oreochromis niloticus gills to MP and NP exposure at an environmentally relevant concentration. Based on the detected differentially expressed gene (DEG) numbers, the most affected immune cells by MP exposure were macrophages, while the stimulus of NPs primarily targeted T cells. In response to MPs and NPs, H+-ATPase-rich cells exhibited distinct changes as compared with Na+/K+-ATPase-rich cells and pavement cells. Fibroblasts were identified as a potential sensitive cell-type biomarker for MP interaction with O. niloticus gills, as evidenced by the largely reduced cell counts and the mostly detected DEGs among the 12 identified cell populations. The most MP-sensitive fibroblast subpopulation in O. niloticus gills was lipofibroblasts. Cell-cell communications between fibroblasts and H+-ATPase-rich cells, neurons, macrophages, neuroepithelial cells, and Na+/K+-ATPase-rich cells in O. niloticus gills were significantly inhibited by MP exposure. Collectively, our study demonstrated the cellular heterogeneity of O. niloticus gills to MPs and NPs and provided sensitive markers for their toxicological mechanisms at single-cell resolution.

Establishment of a cobia (Rachycentron canadum) gill cell line: A valuable tool for immune response studies.

Cobia (Rachycentron canadum), a commercially important marine fish, has been used to develop a novel gill cell line, designated CG, for the first time. The CG cell line was cultured in Leibovitz's-15 medium with 5% fetal bovine serum (FBS) and successfully sub-cultured more than 110 passages. It underwent verification through sequencing of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Optimal growth rate was achieved when the CG cell line was cultured in a medium supplemented with 5% FBS, 1% Penicillin-Streptomycin (P/S), and 5 parts per thousand (ppt) of coral sea salt water, maintained at a temperature of 27 °C. The addition of 5 ppt of salt in the growth medium suggests that this cell line could be a viable in vitro tool for marine ecosystem toxicological studies or for culturing marine parasitic microorganisms. The CG cell line was also successfully transfected using the pTurbo-GFP plasmids, showing an 18% efficiency, with observable GFP expression. Furthermore, the cell line has been effectively cryopreserved. Gene expression analysis indicated that the CG cell line exhibits responsive regulation of immune gene expression when exposured to various stimulants, highlighting its potential as an in vitro platform for immune response studies. This makes it suitable for exploring dynamic immune signaling pathways and host-pathogen interactions, thereby offering valuable insights for therapeutic development.

Pesticide-induced alterations in zebrafish (Danio rerio) behavior, histology, DNA damage and mRNA expression: An integrated approach.

To assess the impact of glyphosate and 2,4-D herbicides, as well as the insecticide imidacloprid, both individually and in combination, the gills of adult zebrafish were used due to their intimate interaction with chemicals diluted in water. Bioassays were performed exposing the animals to the different pesticides and their mixture for 96 h. The behavior of the fish was analyzed, a histological examination of the gills was carried out, and the genotoxic effects were also analyzed by means of the comet assay (CA) and the change in the expression profiles of genes involved in the pathways of the oxidative stress and cellular apoptosis. The length traveled and the average speed of the control fish, compared to those exposed to the pesticides and mainly those exposed to the mixture, were significantly greater. All the groups exposed individually exhibited a decrease in thigmotaxis time, indicating a reduction in the behavior of protecting themselves from predators. Histological analysis revealed significant differences in the structures of the gill tissues. The quantification of the histological lesions showed mild lesions in the fish exposed to imidacloprid, moderate to severe lesions for glyphosate, and severe lesions in the case of 2,4-D and the mixture of pesticides. The CA revealed the sensitivity of gill cells to DNA damage following exposure to glyphosate, 2,4-D, imidacloprid and the mixture. Finally, both genes involved in the oxidative stress pathway and those related to the cell apoptosis pathway were overexpressed, while the ogg1 gene, involved in DNA repair, was downregulated.

From antioxidant defense to genotoxicity: Deciphering the tissue-specific impact of AgNPs on marine clam Ruditapes philippinarum.

The escalating use of silver nanoparticles (AgNPs) across various sectors for their broad-spectrum antimicrobial capabilities, has raised concern over their potential ecotoxicological effects on aquatic life. This study explores the impact of AgNPs (50 µg/L) on the marine clam Ruditapes philippinarum, with a particular focus on its gills and digestive glands. We adopted an integrated approach that combined in vivo exposure, biochemical assays, and transcriptomic analysis to evaluate the toxicity of AgNPs. The results revealed substantial accumulation of AgNPs in the gills and digestive glands of R. philippinarum, resulting in oxidative stress and DNA damage, with the gills showing more severe oxidative damage. Transcriptomic analysis further highlights an adaptive up-regulation of peroxisome-related genes in the gills responding to AgNP-induxed oxidative stress. Additionally, there was a noteworthy enrichment of differentially expressed genes (DEGs) in key biological processes, including ion binding, NF-kappa B signaling and cytochrome P450-mediated metabolism of xenobiotics. These insights elucidate the toxicological mechanisms of AgNPs to R. philippinarum, emphasizing the gill as a potential sensitive organ for monitoring emerging nanopollutants. Overall, this study significantly advances our understanding of the mechanisms driving nanoparticle-induced stress responses in bivalves and lays the groundwork for future investigations into preventing and treating such pollutants in aquaculture.

Toxicological assessment of bromoxynil and 2-methyl-4-chlorophenoxyacetic acid herbicide in combination on Cirrhinus mrigala using multiple biomarker approach.

The widespread application of herbicides raises concerns about their impact on non-target aquatic organisms. This study aimed to evaluate the toxicity of a commercially available herbicide formulation containing Bromoxynil+MCPA (2-Methyl-4-chlorophenoxyacetic acid) on Cirrhinus mrigala (economically significant fish). A total of 210 juvenile fish were subjected to a triplicate experimental setup, with 70 fish allocated to each replicate, exposed to seven different concentrations of herbicide: 0 mg/L, 0.133 mg/L, 0.266 mg/L, 0.4 mg/L, 0.5 mg/L, 0.66 mg/L, and 0.8 mg/L, respectively, for a duration of 96 h. The median lethal concentration (LC50) was determined to be 0.4 mg/L. Significant hematological alterations were observed, including decreases in RBC counts, hemoglobin, hematocrit, and lymphocyte counts, along with an increase in erythrocyte indices. Biochemical analysis revealed elevated levels of neutrophils, WBCs, bilirubin, urea, creatinine, ALT, AST, ALP, and glucose in treated groups. Morphological abnormalities in erythrocytes and histopathological changes in gills, liver, and kidneys were noted. Pathological alterations in gills, liver and kidneys including epithelial cell uplifting, lamellar fusion, hepatolysis, and renal tubule degeneration were observed. Oxidative stress biomarkers such as TBARS (Thiobarbituric Acid Reactive Substance), ROS (Reactive Oxygen Species), and POD (Peroxides) activity increased, while antioxidant enzymatic activities decreased as toxicant doses increased from low to high concentrations. The study reveals that Bromoxynil+MCPA significantly disrupts physiological and hematobiochemical parameters in Cirrhinus mrigala, which highlights the substantial aquatic risks. In conclusion, the herbicide formulation induced significant alterations in various fish biomarkers, emphasizing their pivotal role in assessing the environmental impact of toxicity. This multi-biomarker approach offers valuable insights regarding the toxicological effects, thereby contributing substantially to the comprehensive evaluation of environmental hazards.

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine.

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63 mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81 mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32 mg/kg diet.

Gills de novo assembly reveals oxidative stress, unfolded protein, and immune response on red cusk-eel (Genypterus chilensis) under thermal stress.

The heat waves on the South Pacific coast could lead to thermal stress in native fish. The red cusk-eel (Genypterus chilensis) is relevant for Chilean artisanal fisheries and aquaculture diversification. This study examined the effect of high-temperature stress in the gills of G. chilensis in control (14 °C) and high-temperature stress (19 °C) conditions. High-temperature stress induces a significant increase in gills cortisol levels. Additionally, oxidative damage was observed in gills (protein carbonylation and lipoperoxidation). RNA-seq data was used to build the first transcriptome assembly of gills in this species (23,656 annotated transcripts). A total of 1138 down-regulated and 1531 up-regulated transcripts were observed in response to high-temperature stress in gills. The enrichment analysis showed immune response and replication enriched processes (on down-regulated transcripts), and processes related to the folding of proteins, endoplasmic reticulum, and transporter activity (on up-regulated transcripts). The present study showed how gills could be affected by high-temperature stress.

Rh proteins and H+ transporters involved in ammonia excretion in Amur Ide (Leuciscus waleckii) under high alkali exposure.

High alkaline environment can lead to respiratory alkalosis and ammonia toxification to freshwater fish. However, the Amur ide (Leuciscus waleckii), which inhabits an extremely alkaline lake in China with titratable alkalinity up to 53.57 mM (pH 9.6) has developed special physiological and molecular mechanisms to adapt to such an environment. Nevertheless, how the Amur ide can maintain acid-base balance and perform ammonia detoxification effectively remains unclear. Therefore, this study was designed to study the ammonia excretion rate (Tamm), total nitrogen accumulation in blood and tissues, including identification, expression, and localization of ammonia-related transporters in gills of both the alkali and freshwater forms of the Amur ide. The results showed that the freshwater form Amur ide does not have a perfect ammonia excretion mechanism exposed to high-alkaline condition. Nevertheless, the alkali form of Amur ide was able to excrete ammonia better than freshwater from Amur ide, which was facilitated by the ionocytes transporters (Rhbg, Rhcg1, Na+/H+ exchanger 2 (NHE2), and V-type H+ ATPase (VHA)) in the gills. Converting ammonia into urea served as an ammonia detoxication strategy to reduced endogenous ammonia accumulation under high-alkaline environment.

The role of the antennal glands and gills in acid-base regulation and ammonia excretion of a marine osmoconforming brachyuran.

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister - an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH4+ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na+/K+-ATPase detection was observed. The V-type H+-ATPase - a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

A biomarker approach to study the effects of polluted Brazilian urban reservoirs in a native fish.

Two of the largest water reservoirs in the Metropolitan Region of São Paulo, Brazil (MRSP), named Billings and Guarapiranga, are facing high levels of anthropic impact. This is evidenced by the presence of contaminants and pollutants, which are deteriorating their water quality. Therefore, this study evaluated antioxidant defense enzymes, lipoperoxidation and genotoxicity, in adult females of a native species, Astyanax altiparanae from the Guarapiranga and Billings reservoirs. The study also aimed to evaluate these biomarkers during two different periods of the year, the rainy (summer) and dry (winter) seasons. The oxidative stress was evaluated by the activity of enzymes such as glutathione peroxidase, glutathione S-transferases, superoxide dismutase, and catalase in the gills and liver, and the occurrence of lipoperoxidation was also evaluated in both organs. The genotoxicity was assessed by performing comet assay, micronucleus, and nuclear abnormality tests on blood samples. The results showed that fish from both reservoirs are subjected to oxidative stress and genotoxic damage, mainly during winter, but fish living in Billings showed greater alterations than fish from Guarapiranga. Likewise, the results of the principal component analysis suggested that caffeine, nitrogenous compounds, and some metals might be triggering these toxic effects in fish.

DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

A remarkable new deep-sea nereidid (Annelida: Nereididae) with gills.

Nereidid polychaetes are well known from shallow marine habitats, but their diversity in the deep sea is poorly known. Here we describe an unusual new nereidid species found at methane seeps off the Pacific coast of Costa Rica. Specimens of Pectinereis strickrotti gen. nov., sp. nov. had been observed dating back to 2009 swimming just above the seafloor at ~1,000 m depth but were not successfully captured until 2018. Male epitokes were collected as well as a fragment of an infaunal female found in a pushcore sample. The specimens were all confirmed as the same species based on mitochondrial COI. Phylogenetic analyses, including one based on available whole mitochondrial genomes for nereidids, revealed no close relative, allowing for the placement of the new species in its own genus within the subfamily Nereidinae. This was supported by the unusual non-reproductive and epitokous morphology, including parapodial cirrostyles as pectinate gills, hooked aciculae, elfin-shoe-shaped ventral cirrophores, and elongate, fusiform dorsal ligules emerging sub-medially to enlarged cirrophores. Additionally, the gill-bearing subfamily Dendronereidinae, generally regarded as a junior synonym of Gymnonereidinae, is reviewed and it is here reinstated and as a monogeneric taxon.

Field application of de novo transcriptomic analysis to evaluate the effects of sublethal freshwater salinization on Gasterosteus aculeatus in urban streams.

Freshwater salinization poses global challenges for aquatic organisms inhabiting urban streams, impacting their physiology and ecology. However, current salinization research predominantly focuses on mortality endpoints in limited model species, overlooking the sublethal effects on a broader spectrum of organisms and the exploration of adaptive mechanisms and pathways under natural field conditions. To address these gaps, we conducted high-throughput sequencing transcriptomic analysis on the gill tissue of the euryhaline fish Gasterosteus aculeatus, investigating its molecular response to salinity stress in the highly urbanized river Boye, Germany. We found that in stream sections with sublethal concentrations of chloride costly osmoregulatory systems were activated, evidenced by the differential expression of genes related to osmoregulation. Our enrichment analysis revealed differentially expressed genes (DEGs) related to transmembrane transport and regulation of transport and other osmoregulation pathways, which aligns with the crucial role of these pathways in maintaining biological homeostasis. Notably, we identified candidate genes involved in increased osmoregulatory activity under salinity stress, including those responsible for moving ions across membranes: ion channels, ion pumps, and ion transporters. Particularly, genes from the solute carrier family SLC, aquaporin AQP1, chloride channel CLC7, ATP-binding cassette transporter ABCE1, and ATPases member ATAD2 exhibited prominent differential expression. These findings provide insights into the potential molecular mechanisms underlying the adaptive response of euryhaline fish to salinity stress and have implications for their conservation and management in the face of freshwater salinization.

A new Cinygmula McDunnough, 1933 species with distinct imaginal frontal fold from eastern Chinese Himalaya (Ephemeroptera: Heptageniidae).

In the family Heptageniidae (Ephemeroptera), Cinygmula hutchinsoni (Traver, 1939) from central Asia is the only species known to possess pointed claws and a distinct frontal fold. Here, a second similar species with the same combination of characters is described. Cinygmula longissima sp. nov. is found in the eastern Himalaya of China, shows stouter imaginal penes, darker wingbases, and narrower nymphal gills than the previous one. This discovery indicates that this species of Heptageniidae are not limited to central Asia but live in high-altitude alpine habitats.

Revision of Thraulus Eaton 1881 (Ephemeroptera: Leptophlebiidae: Choroterpinae).

The genus Thraulus is widespread throughout much of the Eastern Hemisphere. Since Eaton established Thraulus in 1881, 62 species have, at one time or another, been placed in this genus. Thirty-eight of those species were eventually moved to other genera. Any comprehensive study of the remaining species, based on the published literature, is difficult as they were described by many authors, using different criteria, over a period of 142 years. The purpose of this study was to redescribe this genus, based on previously described species and nine new species, and to provide a format for future taxonomic and morphological studies of Thraulus. Redescriptions of most species were based on direct examination of external morphological characters. Descriptions or diagnoses of species, whose types were unavailable for study, were made using the original published description and additional information provided by authors of several of those species. The following species were studied: Thraulus amravati Vasanth, Subramanian & Selvakumar, 2022; T. bellus Eaton, 1881; T. bishopi Peters & Tsui, 1972; T. cuspidatus Vasanth, Subramanian & Selvakumar, 2022; T. demoulini Peters & Tsui, 1973; T. fasciatus (Kimmins, 1956); T. fatuus Kang & Yang, 1994; T. femoratus Li, Liu & Zhou, 2006; T. gopalani Grant & Sivaramakrishnan, 1985; T. jacobusi Isack, Srinivasan, Sivaruban & Barathy, 2022; T. macilentus Kang & Yang, 1994; T. malabarensis Vasanth, Subramanian & Selvakumar, 2022; T. mudumalaiensis Soman, 1991; T. plumeus Selvakumar, Vasanth & Subramanian, 2022; T. semicastaneus (Gillies, 1951); T. thiagarajani Balasubramanian & Muthukatturaja, 2019; T. thraker Jacob, 1988; T. torrentis (Gillies, 1964); T. turbinatus (Ulmer, 1909); T. umbrosus Kang & Yang, 1994; and T. vellimalaiensis Vasanth, Subramanian & Selvakumar, 2022. Nine new species of Thraulus are described: T. connubialis sp. nov., Malaysia; T. cursus sp. nov., Japan; T. eatoni sp. nov., Indonesia; T. ishiwatai sp. nov., Japan; T. madagasikarensis sp. nov., Madagascar; T. nihonensis sp. nov., Japan; T. opifer sp. nov., Australia; T. parentalis sp. nov., Malaysia; and T. petersorum sp. nov., Malaysia. Thraulus can be distinguished from all other genera of Leptophlebiidae by the following combination of characters: In the imagos, 1) upper portion of eyes oval-suboval, major axes diverge anteriorly; 2) vein MA fork of fore wings symmetrical; 3) vein MP fork of fore wings asymmetricala cross vein connects base of MP2 to MP1, MP fork closer to base of wing than Rs fork; 4) strongly oblique cross vein extends between veins R4+5 and MA1 just apical to fork of vein MA; 5) 2 cubital intercalary veins in fore wings; 6) costal projection on hind wings well-developed, bluntly rounded to acutely pointed; 7) claws dissimilarone blunt and pad-like, the other apically hooked; 8) penes long, relatively straight, narrow, parallel, usually contiguous mesally but not fused, apex may have lateral projections; 9) sternum 7 of female with posterior margin straight or shallowly concave or convex mesally; and 10) sternum 9 of females rounded apically. In addition, penile spines occur on most species. In the nymphs, 1) lateral margins of clypeus parallel; 2) width of labrum subequal to width of clypeus; 3) 2 dorsal rows of setae on labrum; 4) venter of labrum with 1 row of short stout setae on either side of midline near anterior margin, rows curve mesally; 5) hypopharynx with small, rounded, posterolateral projections on arms of superlingua; 6) large spine on posterolateral corners of terga 69, 79 or 89; 7) gills 17 dissimilar: gill 1 composed of 1 or 2 subulate lamellae or a dorsal subulate lamella and a ventral fimbriate oval lamella, and gills 27 composed of dorsal and ventral oval lamellae with fimbriate margins. Two species continue to be nomen dubiumT. siewertii (Weyenbergh, 1883) and T. vogleri (Weyenbergh, 1883). Thraulus grandis Gose, 1980 is considered nomen nudum. A review of published phylogenetic studies involving Thraulus is provided. With the species discussed in this paper, along with reports of additional new species to be described, Thraulus has the potential to be included among the more specious genera of Ephemeroptera.