One becomes three: An integrative morphological and molecular analysis of the windowpane oyster Placuna (Bivalvia: Pectinida) reveals new species.

For decades, many marine animals have been considered to exhibit cosmopolitan or transoceanic distribution. This situation is prevalent in Asia, where many species were collected and named by American or European experts in the 1700s to early 1900s. Using the windowpane oysters Placuna-a small genus of bivalves with five recognized species-we show that careful analysis is required to reassess the validity of these species. Currently, only two species of Placuna (P. placenta and P. ephippium) widely reported in the Indo-Pacific region have been recorded from Chinese coastal waters. Here, we described two new species of Placuna from China. Placuna vitream sp. nov. can be distinguished from P. placenta by its larger ridge angle. Phylogenetic analysis using five gene fragments fully supported that P. vitream sp. nov. is a sister to the specimen from Singapore identified as P. placenta and more distant from other Placuna species with available molecular data. Besides, based on subfossil shells, we describe Placuna aestuaria sp. nov. that differs from its congeneric species by its broad hinge, medium ridge angle, and nearly straight ridges. Finally, we suggest a combination of hinge structure and ridge angle that can be used for identifying Placuna species and preparing a key to this genus. Our findings of two new species expand the diversity of Placuna and prompt reassessment of the many presumably widely distributed marine species in Asia.

Musseling through: Mytilus byssal thread production is unaffected by continuous noise.

Anthropogenic low-frequency noise (ALFN) is a rising pollutant in the world oceans. Despite the ubiquity of ALFN, its effect on marine invertebrates is still poorly understood. Here, we tested how continuous low-frequency noise (CLFN), a substantial component of ALFN, affects the byssal thread production of Mytilus, a cosmopolitan genus of mussels with high ecological and economic importance. The effects of acute CLFN exposure and predator cues on byssogenesis by Mytilus spp. were explored in both the presence and absence of predator cues. While predator effluents increased thread production, CLFN had seemingly no effect on thread counts. Further, trends suggested a synergistic effect of CLFN and predator cues. The behavioral indifference of Mytilus spp. toward CLFN could contribute to the observed prevalence of these animals in inherently disturbed habitats. This would partly explain their success in colonizing and persisting on artificial substrata rife with disturbances.

Doubling the known diversity of a remote island fauna: marine bivalves of the Juan Fernández and Desventuradas oceanic archipelagos (Southeastern Pacific Ocean).

Juan Fernández and Desventuradas are two oceanic archipelagos located in the southeastern Pacific Ocean far off the Chilean coast that received protected status as marine parks in 2016. Remoteness and access difficulty contributed to historically poor biodiversity sampling and limited associated research. This is particularly noticeable for bivalves, with most prior regional publications focused on single taxa or un-illustrated checklists. This study investigates marine bivalves collected between the intertidal and 415 m depth during (1) the 1997 IOC97 expedition aboard the M/V Carlos Porter, with special focus on scuba-collected micro-mollusks of both archipelagos, (2) two expeditions by the R/V Anton Bruun (Cruise 12/1965 and Cruise 17/1966), and (3) Cruise 21 of USNS Eltanin under the United States Antarctic Program, which sampled at Juan Fernández in 1965. Also, relevant historical material of the British H.M.S. Challenger Expedition (1873-1876), the Swedish Pacific Expedition (1916-1917), and by German zoologist Ludwig H. Plate (1893-1895) is critically revised. A total of 48 species are recognized and illustrated, including 19 new species (described herein) and six other potentially new species. The presence of two species mentioned in the literature for the region (Aulacomya atra and Saccella cuneata) could not be confirmed. The genera Verticipronus and Halonympha are reported for the first time from the Eastern Pacific, as are Anadara and Condylocardia from Chilean waters. Lectotypes are designated for Arca (Barbatia) platei and Mytilus algosus. These findings double the number of extant bivalve species known from the Juan Fernández and Desventuradas archipelagos, highlighting the lack of attention these islands groups have received in the past. A high percentage of species endemic to one or both archipelagos are recognized herein, accounting for almost 78% of the total. The newly recognized level of bivalve endemism supports the consideration of Juan Fernández and Desventuradas as two different biogeographic units (Provinces or Ecoregions) of the Eastern Pacific Ocean.

Mitonuclear compatibility is maintained despite relaxed selection on male mitochondrial DNA in bivalves with doubly uniparental inheritance.

Mitonuclear coevolution is common in eukaryotes, but bivalve lineages that have doubly uniparental inheritance (DUI) of mitochondria may be an interesting example. In this system, females transmit mtDNA (F mtDNA) to all offspring, while males transmit a different mtDNA (M mtDNA) solely to their sons. Molecular evolution and functional data suggest oxidative phosphorylation (OXPHOS) genes encoded in M mtDNA evolve under relaxed selection due to their function being limited to sperm only (vs. all other tissues for F mtDNA). This has led to the hypothesis that mitonuclear coevolution is less important for M mtDNA. Here, we use comparative phylogenetics, transcriptomics, and proteomics to understand mitonuclear interactions in DUI bivalves. We found nuclear OXPHOS proteins coevolve and maintain compatibility similarly with both F and M mtDNA OXPHOS proteins. Mitochondrial recombination did not influence mitonuclear compatibility and nuclear-encoded OXPHOS genes were not upregulated in tissues with M mtDNA to offset dysfunction. Our results support that selection maintains mitonuclear compatibility with F and M mtDNA despite relaxed selection on M mtDNA. Strict sperm transmission, lower effective population size, and higher mutation rates may explain the evolution of M mtDNA. Our study highlights that mitonuclear coevolution and compatibility may be broad features of eukaryotes.

The Interactive Effects of the Anti-Sea Lice Pesticide Azamethiphos and Temperature on Oxidative Damage and Antioxidant Responses in the Oyster Ostrea chilensis.

Azamethiphos is used in the salmon industry to treat sea lice and is subsequently discharged into the sea, which may affect non-target species (NTS). A rise in seawater temperature could enhance the sensitivity of NTS. Thus, in the present investigation, the combined effects of azamethiphos (0 µg L-1, 15 µg L-1 and 100 µg L-1) and temperature (12 °C and 15 °C) was assessed over time (7 days) in the gonads and gills of the oyster Ostrea chilensis, assessing its oxidative damage (lipid peroxidation and protein carbonyls) and total antioxidant capacity. Our results indicated that in gonads and gills, lipid peroxidation levels increased over time during exposure to both pesticide concentrations. Protein carbonyl levels in gills increased significantly in all experimental treatments; however, in gonads, only pesticide concentration and exposure time effected a significant increase in protein damage. In both, gill and gonad temperature did not influence oxidative damage levels. Total antioxidant capacity in gonads was influenced only by temperature treatment, whereas in the gills, neither temperature nor azamethiphos concentration influenced defensive responses. In conclusion, our results indicated the time of pesticide exposure (both concentrations) had a greater influence than temperature on the cellular damage in this oyster.

The absence of canonical respiratory complex I subunits in male-type mitogenomes of three Donax species.

Bivalves are an extraordinary class of animals in which species with a doubly uniparental inheritance (DUI) of mitochondrial DNA have been described. DUI is characterized as a mitochondrial homoplasmy of females and heteroplasmy of male individuals where F-type mitogenomes are passed to the progeny with mother egg cells and divergent M-type mitogenomes are inherited with fathers sperm cells. However, in most cases only male individuals retain divergent mitogenome inherited with spermatozoa. Additionally, in many of bivalves, unique mitochondrial features, like additional genes, gene duplication, gene extensions, mitochondrial introns, and recombination, were observed. In this study, we sequenced and assembled male-type mitogenomes of three Donax species. Comparative analysis of mitochondrial sequences revealed a lack of all seven NADH dehydrogenase subunits as well as the presence of three long additional open reading frames lacking identifiable homology to any of the existing genes.

Pulsative venous return from the branchial vessels to the heart of the bivalve Mytilus galloprovincialis supports the constant-volume mechanism.

In bivalves and gastropods, ventricle contraction causes a negative pressure in the auricles and increases venous return from the afferent oblique vein (AOV): the constant-volume (CV) mechanism. The flow in the AOV should be a pulsative flow synchronized with the ventricular contraction. The flow in the heart and adjacent vessels of Mytilus galloprovincialis were measured by magnetic resonance imaging to confirm this hypothesis. Under a regular heartbeat, pulsative flows in the AOV and branchial vessels (BVs) were almost completely synchronized with the flow in the aorta, while filling of the ventricle was in the opposite phase. Flows in the BVs were directed to the posterior direction, and a pair of BVs in the gill axes (the efferent BVs) were connected to the AOV. Based on the images of the whole pathway of the AOV in an oblique slice, we confirmed that haemolymph flow was evoked from the efferent BVs and flow into the ventricle via the auricle was completed in a single heartbeat. Therefore, the walls of the AOV and BVs could resist negative transmural pressure caused by the ventricular contraction. In conclusion, the auricle, the AOV and the BVs, including the gill filaments, act as a suction pump. The pulsative venous return is driven by the negative pressure of the AOV as in the CV mechanism, and the negative pressure in the efferent BVs could draw haemolymph from the sinus via the gill and the afferent BVs. Therefore, Mytilus can start and stop its heartbeat as necessary.

Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis).

BACKGROUND: Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. RESULTS: Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. CONCLUSIONS: We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.

Checklist of the marine malacofauna of Culuccia Peninsula (NW Sardinia, Italy), with notes on relevant species.

Background: Culuccia is a small peninsula of about 3 km2 placed in north-western Sardinia (Italy) at the margin of the Maddalena Archipelago. The marine area surrounding this Peninsula is a Special Area of Conservation, included in the European Natura 2000 Ecological Network of protected areas, but until now, no information on biodiversity of this area is available. In 2021, a research project to study both terrestrial and marine biodiversity of Culuccia has started in order to fill this gap of knowledge. New information: This work provides the first inventory of the marine malacofauna of the coast of Culuccia. Fifteen sites were sampled seasonally for one-year by using different sampling methods and the present study shows the results from approximately 50 scientific SCUBA and free dive surveys, carried out in all main marine habitats of the studied area. In total, 259 species of molluscs were recorded along the coasts of the Culuccia Peninsula (0-25 m depth), belonging to the classes Bivalvia, Gastropoda, Polyplacophora and Scaphopoda. Amongst the four classes recorded, gastropods were the most represented (66.90%; 173 species), followed by bivalves (28.10%; 73 species), polyplacophorans (4.60%; 12 species) and scapophods (0.40%; 1 species). Notes about distribution, conservation status and ecology for some valuable species are provided, together with images of representative species, consisting mainly of in situ photographs. Additionally, the present investigation recorded the presence of four alien species, whose Mediterranean distribution was extended to north-western Sardinia.

Microplastic contamination in different shell length in Tivela mactroides (Born, 1778).

Microplastic identification and distribution throughout oceans has become a great concern due to its substantial uprising and its consequent interactions with marine biota. Microplastics can be absorbed and adsorbed by several marine species owing to their very small size. Among these organisms are bivalves, including ones used as food for humans. In this context, this research aims to understand the absorption scale of microplastics by Tivela mactroides according to their size and consequently life stage. Thus, T. mactroides mussels were collected at Camburi Beach (Brazil) and grouped into 30 different size classes from 06 to 35 mm. Later, 20 specimens from each size class (N = 600) had the soft tissues removed and a pool was performed through digestion with 10 % KOH solution. Results showed the presence of microplastics in all size classes of T. mactroides, evidencing that organisms from 06 mm can already be contaminated by microplastics and a tendency of the number of microplastics particles to increase as shell size increases. Furthermore, the amount of blue filament-type MPs was significantly higher than the other types and colors. Additionally, analyzes performed by Raman spectroscopy showed that the blue filaments have a PET composition. Finally, larger individuals presented larger quantities of MPs, as well as larger filaments. This might suggest that there are differences between the assimilation of the size of MPs in the different size classes, i.e. that size makes a difference.

Ecological structure of diversity-dependent diversification in Phanerozoic marine bivalves.

Rigorous analysis of diversity-dependence-the hypothesis that the rate of proliferation of new species is inversely related to standing diversity-requires consideration of the ecology of the organisms in question. Differences between infaunal marine bivalves (living entirely within the sediment) and epifaunal forms (living partially or completely above the sediment-water interface) predict that these major ecological groups should have different diversity dynamics: epifaunal species may compete more intensely for space and be more susceptible to predation and physical disturbance. By comparing detrended standing diversity with rates of diversification, origination, and extinction in this exceptional fossil record, we find that epifaunal bivalves experienced significant, negative diversity-dependence in origination and net diversification, whereas infaunal forms show little appreciable relationship between diversity and evolutionary rates. This macroevolutionary contrast is robust to the time span over which dynamics are analysed, whether mass-extinction rebounds are included in the analysis, the treatment of stratigraphic ranges that are not maximally resolved, and the details of detrending. We also find that diversity-dependence persists over hundreds of millions of years, even though diversity itself rises nearly exponentially, belying the notion that diversity-dependence must imply equilibrial diversity dynamics.

Methods of quantifying a mass mortality event in freshwater wildlife within the river ecosystem.

This study introduces a comprehensive method for quantifying mass mortality events in freshwater wildlife, exemplified by the ecological disaster in the Odra River in 2022. Our approach integrates field observations, statistical analysis, and ecological assessment to measure the impact of such events on various aquatic species. Key steps include systematic counting of deceased organisms, assessing population declines, and evaluating the ecological repercussions of invasive species. Utilizing the R programming language, we developed a framework that is adaptable to similar ecological crises in different aquatic environments. This methodology facilitates a detailed understanding of the scale and implications of mass mortality events, thereby contributing to effective environmental management and conservation efforts. •The analysis and modeling methods of the disaster are presented in the R programming language.•Exclusively open-source software was used for the analysis.•The analysis includes detailed data on the disaster's impact on various species.

The spatial distribution and physico-chemical characteristic of microplastics in the sediment and cockle (Anadara granosa) from the coastal waters of East Java, Indonesia, and the health hazards associated with cockle consumption.

This study evaluated microplastic (MP) abundances and physico-chemical characteristics in sediments and Anadara granosa along the East Java coast and their health implications. Fibers (74 %) dominated sediment MPs at south coast, while fragments (49-61 %) dominated north coast. Fiber (43-52 %) is the predominant MP in cockle tissues in all locations. Most MP in sediments (31-47 %) and cockle tissues (41-49 %) is black. The majority of microplastics (100-1500 µm) are found in sediment (73-90 %), and cockles (77-79 %). Very weak correlations found between the amount of MP and the length of the cockle shell. However, Spearman correlation shows that as the amount of MP in sediment increases, so does the amount of MP in cockle tissue. Each year, individuals of varying ages consume an average of 20,800 to 156,000 MP items. Cockles contain plasticizer components and microplastic polymers which are classified from II to V regarding of hazard levels, with V being the most hazardous.

Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis)

Background Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. Results Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome ofM. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. Conclusions We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.

A dataset on quantifying a mass mortality event in freshwater wildlife within the Lower Odra River.

In response to the significant ecological disaster in the Odra River during the summer of 2022, a comprehensive data collection process was initiated to quantify the extent of mortality among aquatic species. The dataset focuses on the downstream section of the river, identified as the area with the highest accumulation of deceased organisms. The data collection involved systematic sampling and counting of dead organisms, including fish, bivalves , and aquatic snails. Special attention was given to specific species such as Unionidae mussels, Anodonta anatina, Sinanodonta woodiana, and Viviparus viviparus. Additionally, transects were designated for focused data collection on fish mortality. The dataset provides detailed mortality figures, biomass estimates, and percentage reductions for each species. This comprehensive dataset holds significant potential for reuse by researchers studying the effects of toxins on freshwater ecosystems, the impact of invasive species on native populations, and conservationists aiming to restore the affected areas.

Mollusca collected by Agassiz trawl from the 2016 SO-AntEco (JR15005) expedition to the South Orkney Islands, Antarctica - data.

Background: This dataset contributes to the knowledge of macro- and megafaunal Mollusca associated with a range of benthic habitat types in the South Orkney Islands, Antarctica, an exceptionally diverse region of the Southern Ocean. The information presented is derived from Agassiz trawl samples collected on the archipelago's shelf plateau and slope, within and outside of the South Orkney Islands Southern Shelf Marine Protected Area (SOISS MPA). Sampling was conducted in the framework of the British Antarctic Survey/SCAR "South Orkneys - State of the Antarctic Ecosystem" (SO-AntEco) project aboard RRS James Clark Ross during expedition JR15005 in Austral summer 2016. This dataset is published by the British Antarctic Survey under the licence CC-BY 4.0. We would appreciate it if you could follow the guidelines from the SCAR Data Policy (SCAR 2023) when using the data. If you have any questions regarding this dataset, do not hesitate to contact us via the contact information provided in the metadata or via data-biodiversity-aq@naturalsciences.be. Issues with the dataset can be reported at https://github.com/biodiversity-aq/data-publication/. This dataset is part of the Biodiversity, Evolution and Adaptation Project of the Environmental Change and Evolution Program of the British Antarctic Survey. The cruise report of the expedition is available at https://www.bodc.ac.uk/resources/inventories/cruise_inventory/reports/jr15005.pdf. New information: We report occurrences of Mollusca from individual samples taken with a 2 m-wide Agassiz trawl (AGT) in the framework of the February - March 2016 research expedition JR15005 of RRS James Clark Ross to the SOISS MPA and adjacent shelf and slope areas. Of 78 successful AGT deployments, 44 trawls at depths ranging from 235-2194 m yielded living Mollusca, totalling 2276 individuals, 67 morphospecies and 163 distributional records. One hundred and fifteen empty shells were also collected and recorded in the dataset. Three morphospecies (one Bivalvia and two Gastropoda) were sampled exclusively as empty shells, yielding a total of 70 morphospecies and 2391 specimens represented in the dataset. All specimens were preserved in 96% undenatured ethanol and are stored as vouchers in the collections of the British Antarctic Survey (BAS), Cambridge, United Kingdom. The publication of this dataset aims at increasing the knowledge on the biodiversity, abundance and geographical and bathymetric distribution of larger-sized epi- and shallow infaunal Mollusca of the South Orkney Islands.

Opportunistic consumption of marine pelagic, terrestrial, and chemosynthetic organic matter by macrofauna on the Arctic shelf: a stable isotope approach.

Macrofauna can contribute substantially to the organic matter cycling on the seafloor, yet the role of terrestrial and chemosynthetic organic matter in the diets of microphagous (deposit and suspension) feeders is poorly understood. In the present study, we used stable isotopes of carbon and nitrogen to test the hypothesis that the terrestrial organic matter supplied with river runoff and local chemosynthetic production at methane seeps might be important organic matter sources for macrofaunal consumers on the Laptev Sea shelf. We sampled locations from three habitats with the presumed differences in organic matter supply: "Delta" with terrestrial inputs from the Lena River, "Background" on the northern part of the shelf with pelagic production as the main organic matter source, and "Seep" in the areas with detected methane seepage, where chemosynthetic production might be available. Macrobenthic communities inhabiting each of the habitats were characterized by a distinct isotopic niche, mostly in terms of δ13C values, directly reflecting differences in the origin of organic matter supply, while δ15N values mostly reflected the feeding group (surface deposit/suspension feeders, subsurface deposit feeders, and carnivores). We conclude that both terrestrial and chemosynthetic organic matter sources might be substitutes for pelagic primary production in the benthic food webs on the largely oligotrophic Laptev Sea shelf. Furthermore, species-specific differences in the isotopic niches of species belonging to the same feeding group are discussed, as well as the isotopic niches of the symbiotrophic tubeworm Oligobrachia sp. and the rissoid gastropod Frigidoalvania sp., which are exclusively associated with methane seeps.

Three new species of Axinulus (Bivalvia: Thyasiridae) from the Japan and Kuril-Kamchatka trenches and abyssal zone of the northern Pacific Ocean.

The Thyasiridae is one of the most species-rich families of bivalves in the deep-sea areas of the northern Pacific Ocean. Many thyasirid species form abundant populations in these regions and play an important role in the functioning of deep-sea benthic communities. However, most of these deep-sea thyasirid species have not been identified and many of them are new to science. Based on the material of bivalves collected by eight deep-sea expeditions in the northern Pacific Ocean during the period from 1954 to 2016, three new species of the genus Axinulus (Axinulus krylovae sp. nov., A. alatus sp. nov., and A. cristatus sp. nov.) are described from the Kuril-Kamchatka and Japan trenches, the Bering Sea, and other deep-water regions of the northern Pacific Ocean (3,200-9,583 m depth). The new species are distinguished due to a unique and complex sculpture of the prodissoconch, including tubercles and numerous thin folds of varying length and shape, as well as due to a thickening of the shell in the adductor scar areas, thus rendering the scars raised above the inner surface of the shell. Comparisons with all species of the genus Axinulus are provided.

Validation of the male-specific ORF of the paternally-transmitted mtDNA in Mytilus edulis as a protein-coding gene.

There appears to be an additional set of sex-specific mtDNA-encoded proteins in bivalve species with doubly uniparental mitochondrial inheritance that may be involved in the transmission of the female and male mitogenomes. In the marine mussel Mytilus edulis, the translation of the female-specific open reading frame (F-ORF) was demonstrated but the translation of the male-specific ORF (M-ORF) remains to be shown. Here we validate the male-specific ORF of the paternal mitogenome in M. edulis as a protein-coding gene. The M-ORF protein was detected only in male gonads and localized in sperm mitochondria and acrosome, suggesting that it is involved in a key sperm function in Mytilus edulis.

Playing Peekaboo with a Master Manipulator: Metagenetic Detection and Phylogenetic Analysis of Wolbachia Supergroups in Freshwater Invertebrates.

The infamous "master manipulators"-intracellular bacteria of the genus Wolbachia-infect a broad range of phylogenetically diverse invertebrate hosts in terrestrial ecosystems. Wolbachia has an important impact on the ecology and evolution of their host with documented effects including induced parthenogenesis, male killing, feminization, and cytoplasmic incompatibility. Nonetheless, data on Wolbachia infections in non-terrestrial invertebrates are scarce. Sampling bias and methodological limitations are some of the reasons limiting the detection of these bacteria in aquatic organisms. In this study, we present a new metagenetic method for detecting the co-occurrence of different Wolbachia strains in freshwater invertebrates host species, i.e., freshwater Arthropoda (Crustacea), Mollusca (Bivalvia), and water bears (Tardigrada) by applying NGS primers designed by us and a Python script that allows the identification of Wolbachia target sequences from the microbiome communities. We also compare the results obtained using the commonly applied NGS primers and the Sanger sequencing approach. Finally, we describe three supergroups of Wolbachia: (i) a new supergroup V identified in Crustacea and Bivalvia hosts; (ii) supergroup A identified in Crustacea, Bivalvia, and Eutardigrada hosts, and (iii) supergroup E infection in the Crustacea host microbiome community.