Solving the Myxidium rhodei (Myxozoa) puzzle: insights into its phylogeny and host specificity in Cypriniformes.

Myxidium rhodei Léger, 1905 (Cnidaria: Myxozoa) is a kidney-infecting myxosporean that was originally described from the European bitterling Rhodeus amarus. Subsequently, it has been documented based on spore morphology in more than 40 other cypriniform species, with the roach Rutilus rutilus being the most commonly reported host. This study introduces the first comprehensive data assessment of M. rhodei, conducted through morphological, ecological and molecular methods. The morphological and phylogenetic analyses of SSU rDNA sequences of Myxidium isolates obtained from European bitterling and roach did not support parasite conspecificity from these fish. In fact, the roach-infecting isolates represent three distinct parasite species. The first two, M. rutili n. sp. and M. rutilusi n. sp., are closely related cryptic species clustering with other myxosporeans in the freshwater urinary clade, sharing the same tissue tropism. The third one, M. batuevae n. sp., previously assigned to M. cf. rhodei, clustered in the hepatic biliary clade sister to bitterling-infecting M. rhodei. Our examination of diverse cypriniform fishes, coupled with molecular and morphological analyses, allowed us to untangle the cryptic species nature of M. rhodei and discover the existence of novel species. This underscores the largely undiscovered range of myxozoan diversity and highlights the need to incorporate sequence data in diagnosing novel species.

Title: Résoudre le casse-tête de Myxidium rhodei (Myxozoa) : aperçu de sa phylogénie et de sa spécificité d'hôte chez les Cypriniformes. Abstract: Myxidium rhodei Léger, 1905 (Cnidaria : Myxozoa) est un Myxosporea infectant les reins qui a été décrit à l'origine chez la bouvière, Rhodeus amarus. Par la suite, il a été documenté, sur la base de la morphologie des spores, chez plus de 40 autres espèces de cypriniformes, le gardon Rutilus rutilus étant l'hôte le plus fréquemment signalé. Cette étude présente la première évaluation complète des données sur M. rhodei, réalisée par des méthodes morphologiques, écologiques et moléculaires. Les analyse morphologiques et phylogénétiques des séquences d'ADNr SSU des isolats de Myxidium obtenus à partir de bouvières et de gardons européens n'ont pas confirmé la conspécificité du parasite de ces poissons. En fait, les isolats infectant les gardons représentent trois espèces distinctes de parasites. Les deux premières, M. rutili n. sp. et M. rutilusi n. sp., sont des espèces cryptiques étroitement apparentées, regroupées avec d'autres Myxosporea du clade urinaire d'eau douce, partageant le même tropisme tissulaire. La troisième, M. batuevae n. sp., précédemment attribuée à M. cf. rhodei, appartient au clade biliaire hépatique, groupe-frère de M. rhodei infectant la bouvière. Notre examen de divers poissons cypriniformes, couplé à des analyses moléculaires et morphologiques, nous a permis de démêler la nature cryptique des espèces de M. rhodei et de découvrir l'existence de nouvelles espèces. Cela souligne la diversité largement méconnue des Myxozoaires et souligne la nécessité d'incorporer des données de séquence dans le diagnostic de nouvelles espèces.

Quantitative monitoring of diverse fish communities on a large scale combining eDNA metabarcoding and qPCR.

Environmental DNA (eDNA) metabarcoding is an effective method for studying fish communities but allows only an estimation of relative species abundance (density/biomass). Here, we combine metabarcoding with an estimation of the total abundance of eDNA amplified by our universal marker (teleo) using a quantitative (q)PCR approach to infer the absolute abundance of fish species. We carried out a 2850-km eDNA survey within the Danube catchment using a spatial integrative sampling protocol coupled with traditional electrofishing for fish biomass and density estimation. Total fish eDNA concentrations and total fish abundance were highly correlated. The correlation between eDNA concentrations per taxon and absolute specific abundance was of comparable strength when all sites were pooled and remained significant when the sites were considered separately. Furthermore, a nonlinear mixed model showed that species richness was underestimated when the amount of teleo-DNA extracted from a sample was below a threshold of 0.65 × 106 copies of eDNA. This result, combined with the decrease in teleo-DNA concentration by several orders of magnitude with river size, highlights the need to increase sampling effort in large rivers. Our results provide a comprehensive description of longitudinal changes in fish communities and underline our combined metabarcoding/qPCR approach for biomonitoring and bioassessment surveys when a rough estimate of absolute species abundance is sufficient.

Environmental DNA reveals quantitative patterns of fish biodiversity in large rivers despite its downstream transportation.

Despite the ecological and societal importance of large rivers, fish sampling remains costly and limited to specific habitats (e.g., river banks). Using an eDNA metabarcoding approach, we regularly sampled 500 km of a large river (Rhône River). Comparisons with long-term electrofishing surveys demonstrated the ability of eDNA metabarcoding to qualitatively and quantitatively reveal fish assemblage structures (relative species abundance) but eDNA integrated a larger space than the classical sampling location. Combination of a literature review and field data showed that eDNA behaves in the water column like fine particulate organic matter. Its detection distance varied from a few km in a small stream to more than 100 km in a large river. To our knowledge, our results are the first demonstration of the capacity of eDNA metabarcoding to describe longitudinal fish assemblage patterns in a large river, and metabarcoding appears to be a reliable, cost-effective method for future monitoring.

Structure of the seminal pathway in the European chub, Leuciscus cephalus (Cyprinidae); Teleostei.

The testicular efferent duct system of Leuciscus cephalus (Cyprinidae), is described for three phases of testicular development. Testicular main ducts were analyzed by means of conventional histology and transmission electron microscopy. Additional techniques were applied for lectin histochemistry to determine secretory activity, as well as immunohistochemistry for cell proliferation activity and for muscle actin to demonstrate the distribution and amount of contractile cells. The contribution of the main ducts' epithelia and of degenerating spermatocytes to seminal fluid composition was confirmed, with the former being a source of carbohydrates and the latter that of phospholipids. The apical glycocalyx of epithelial cells, which is important in cell recognition and potentially involved in sperm storage, was marked by RCA I, LCA, and WGA lectin. Higher numbers of proliferating epithelial cells were ascertained during spawning phase compared to pre- and postspawning phases. In the ducts' stroma, a large number of cells expressed muscle actin and tropomyosin, indicating the ducts' contractile potential for the transport of seminal fluid towards release. Adjacent to these contractile cells, numerous nerves were found, indicating neuronal control of sperm fluid flow.

Mammalian glucocorticoid metabolites act as androgenic endocrine disruptors in the medaka (Oryzias latipes).

Glucocorticoid metabolites enter the aquatic environment via mammalian excrements. Molecular structures of their C19O3 metabolites strongly resemble the major fish androgen 11-ketotestosterone. Therefore, we tested the hypothesis that the cortisol metabolite 5alpha-androstan-3,11,17-trione acts similarly to 11-ketotestosterone by employing a fish screening assay for endocrine-active substances. After 21 d, both 11-oxygenated compounds had masculinized sex characteristics of the anal fin in female medaka in a dose-dependent manner.