Freshwater trematodes differ from marine trematodes in patterns connected with division of labor.

Background: Prior research suggests that trematode rediae, a developmental stage of trematode parasites that reproduce clonally within a snail host, show evidence of division of labor (DOL). Single-species infections often have two morphologically distinct groups: small rediae, the 'soldiers', are active, aggressive, and do not appear to reproduce; large rediae, the 'reproductives', are larger, sluggish, and full of offspring. Most data supporting DOL come from trematodes infecting marine snails, while data from freshwater trematodes are more limited and generally do not supported DOL. The shorter lifespan typical of freshwater snails may partially explain this difference: defending a short-lived host at the expense of reproduction likely provides few advantages. Here, we present data from sixty-one colonies spanning twenty species of freshwater trematode exploring morphological and behavioral patterns commonly reported from marine trematodes believed to have DOL. Methods: Trematode rediae were obtained from sixty-one infected snails collected in central Vermont, USA. A portion of the COI gene was sequenced to make tentative species identifications ('COI species'). Samples of rediae were photographed, observed, and measured to look for DOL-associated patterns including a bimodal size distribution, absence of embryos in small rediae, and pronounced appendages and enlarged pharynges (mouthparts) in small rediae. Additional rediae were used to compare activity levels and likelihood to attack heterospecific trematodes in large vs. small rediae. Results: Many of the tests for DOL-associated patterns showed mixed results, even among colonies of the same COI species. However, we note a few consistent patterns. First, small rediae of most colonies appeared capable of reproduction, and we saw no indication (admittedly based on a small sample size and possibly insufficient attack trial methodology) that small rediae were more active or aggressive. This differs from patterns reported from most marine trematodes. Second, the small rediae of most colonies had larger pharynges relative to their body size than large rediae, consistent with marine trematodes. We also observed that colonies of three sampled COI species appear to produce a group of large rediae that have distinctly large pharynges. Conclusions: We conclude that these freshwater species likely do not have a group of specialized non-reproductive soldiers because small rediae of at least some colonies in almost every species do appear to produce embryos. We cannot rule out the possibility that small rediae act as a temporary soldier caste. We are intrigued by the presence of rediae with enlarged pharynges in some species and propose that they may serve an adaptive role, possibly similar to the defensive role of small 'soldier' rediae of marine trematodes. Large-pharynx rediae have been documented in other species previously, and we encourage future efforts to study these large-pharynx rediae.

Morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae from Chinese pipistrelle bats and viviparid snails in Thailand.

This study aimed to describe the morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae isolated in Thailand. Adult flukes were isolated from the Chinese pipistrelle bat (Hypsugo sp.), and cercariae were detected in the viviparid snail (Filopaludina martensi martensi) from Chiang Mai province. The morphological characteristics were observed and described using conventional methods, and the molecular characteristics with internal transcribed spacer 2 (ITS2) and 28S rDNA gene sequences. The adult flukes were fusiform, 0.84-0.98 mm in length, and 0.37-0.49 mm in width, and were distinguishable from other species by the presence of longitudinal uterine coils. The cercariae were nonvirgulate xiphidiocercariae, with the oral sucker bigger than the acetabulum, the tail without fin fold, a body size of 117.5-138.3 × 48.3-52.2 µm, and a tail size of 100.7-103.7 × 15.0-18.9 µm. Molecular studies revealed that the adults and cercariae shared 99.3% (ITS2) and 99.6% (28S rDNA) homology with each other. They were phylogenetically close to P. longiforme with an identity of 94.5% for ITS2 and 98.7% for 28S rDNA. This study provides new information on the natural definitive host and first intermediate host of P. longiforme in Thailand. The discovery of its cercarial stage in Filopaludina snails highlights the importance of monitoring the associated second intermediate host and prevention and control of this potentially zoonotic trematode.

Two new genera and species of the valvatiform hydrobiid snails (Caenogastropoda: Truncatelloidea: Hydrobiidae) from Morocco.

Stygobiont and crenobiont minute gastropods representing the family Hydrobiidae (Caenogastropoda: Truncatelloidea), characterized by the valvatoid low-spired shell, were collected from one spring and four wells in Bouregreg region, NW Morocco. The shells were photographed and measured; shell biometry is illustrated with principal component analysis. Penes were illustrated and described. Mitochondrial cytochrome oxidase subunit 1 (COI) and 16S rRNA, as well as nuclear 18S rRNA sequences were used to infer the phylogeny. The snails represented two genera, both new to science. Their closest relatives were taxa from the Iberian Peninsula, the rough molecular estimate of the time of divergence between these Moroccan and Iberian genera coincided with the Pliocene Flooding, which restored the Strait of Gibraltar to connect the Mediterranean Basin with Atlantic Ocean.

Comparative Mitogenome Analyses of Fifteen Ramshorn Snails and Insights into the Phylogeny of Planorbidae (Gastropoda: Hygrophila).

Ramshorn snails from the family Planorbidae are important freshwater snails due to their low trophic level, and some of them act as intermediate hosts for zoonotic trematodes. There are about 250 species from 40 genera of Planorbidae, but only 14 species from 5 genera (Anisus, Biomphalaria, Bulinus, Gyraulus, and Planorbella) have sequenced complete mitochondrial genomes (mitogenomes). In this study, we sequenced and assembled a high-quality mitogenome of a ramshorn snail, Polypylis sp. TS-2018, which represented the first mitogenome of the genus. The mitogenome of Polypylis sp. TS-2018 is 13,749 bp in length, which is shorter than that of most gastropods. It contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA). We compared mitogenome characteristics, selection pressure, and gene rearrangement among all of the available mitogenomes of ramshorn snails. We found that the nonsynonymous and synonymous substitution rates (Ka/Ks) of most PCGs indicated purifying and negative selection, except for atp8 of Anisus, Biomphalaria, and Gyraulus, which indicated positive selection. We observed that transpositions and reverse transpositions occurred on 10 tRNAs and rrnS, which resulted in six gene arrangement types. We reconstructed the phylogenetic trees using the sequences of PCGs and rRNAs and strongly supported the monophyly of each genus, as well as three tribes in Planorbidae. Both the gene rearrangement and phylogenetic results suggested that Polypylis had a close relationship with Anisus and Gyraulus, while Bulinus was the sister group to all of the other genera. Our results provide useful data for further investigation of species identification, population genetics, and phylogenetics among ramshorn snails.

Diverse pathways to speciation revealed by marine snails.

Speciation is a key evolutionary process that is not yet fully understood. Combining population genomic and ecological data from multiple diverging pairs of marine snails (Littorina) supports the search for speciation mechanisms. Placing pairs on a one-dimensional speciation continuum, from undifferentiated populations to species, obscured the complexity of speciation. Adding multiple axes helped to describe either speciation routes or reproductive isolation in the snails. Divergent ecological selection repeatedly generated barriers between ecotypes, but appeared less important in completing speciation while genetic incompatibilities played a key role. Chromosomal inversions contributed to genomic barriers, but with variable impact. A multidimensional (hypercube) approach supported framing of questions and identification of knowledge gaps and can be useful to understand speciation in many other systems.

Many roads to success: alternative routes to building an economic shell in land snails.

Land snails exhibit an extraordinary variety of shell shapes. The way shells are constructed underlies biological and mechanical constraints that vary across gastropod clades. Here, we quantify shell geometry of the two largest groups, Stylommatophora and Cyclophoroidea, to assess the potential causes for variation in shell shape and its relative frequency. Based on micro-computed tomography scans, we estimate material efficiency through 2D and 3D generalizations of the isoperimetric ratio, quantifying the ratios between area and perimeter of whorl cross-sections (2D) and shell volume and surface (3D), respectively. We find that stylommatophorans optimize material usage through whorl overlap, which may have promoted the diversification of flat-shelled species. Cyclophoroids are bound to a circular cross-section because of their operculum; flat shells are comparatively rare. Both groups show similar solutions for tall shells, where local geometry has a smaller effect because of the double overlap between previous and current whorls. Our results suggest that material efficiency is a driving factor in the selection of shell geometry. Essentially, the evolutionary success of Stylommatophora likely roots in their higher flexibility to produce an economic shell.

Insights into the genome of the 'Loco' Concholepas concholepas (Gastropoda: Muricidae) from low-coverage short-read sequencing: genome size, ploidy, transposable elements, nuclear RNA gene operon, mitochondrial genome, and phylogenetic placement in the family Muricidae.

BACKGROUND: The Peruvian 'chanque' or Chilean 'loco' Concholepas concholepas is an economically, ecologically, and culturally important muricid gastropod heavily exploited by artisanal fisheries in the temperate southeastern Pacific Ocean. In this study, we have profited from a set of bioinformatics tools to recover important biological information of C. concholepas from low-coverage short-read NGS datasets. Specifically, we calculated the size of the nuclear genome, ploidy, and estimated transposable elements content using an in silico k-mer approach, we discovered, annotated, and quantified those transposable elements, we assembled and annotated the 45S rDNA RNA operon and mitochondrial genome, and we confirmed the phylogenetic position of C. concholepas within the muricid subfamily Rapaninae based on translated protein coding genes. RESULTS: Using a k-mer approach, the haploid genome size estimated for the predicted diploid genome of C. concholepas varied between 1.83 Gbp (with kmer = 24) and 2.32 Gbp (with kmer = 36). Between half and two thirds of the nuclear genome of C. concholepas was composed of transposable elements. The most common transposable elements were classified as Long Interspersed Nuclear Elements and Short Interspersed Nuclear Elements, which were more abundant than DNA transposons, simple repeats, and Long Terminal Repeats. Less abundant repeat elements included Helitron mobile elements, 45S rRNA DNA, and Satellite DNA, among a few others.The 45S rRNA DNA operon of C. concholepas that encodes for the ssrRNA, 5.8S rRNA, and lsrRNA genes was assembled into a single contig 8,090 bp long. The assembled mitochondrial genome of C. concholepas is 15,449 bp long and encodes 13 protein coding genes, two ribosomal genes, and 22 transfer RNAs. CONCLUSION: The information gained by this study will inform the assembly of a high quality nuclear genome for C. concholepas and will support bioprospecting and biomonitoring using environmental DNA to advance development of conservation and management plans in this overexploited marine snail.

Genome assembly and population genomic data of a pulmonate snail Ellobium chinense.

Ellobium chinense is an airbreathing, pulmonate gastropod species that inhabits saltmarshes in estuaries of the northwestern Pacific. Due to a rapid population decline and their unique ecological niche in estuarine ecosystems, this species has attracted special attention regarding their conservation and the genomic basis of adaptation to frequently changing environments. Here we report a draft genome assembly of E. chinense with a total size of 949.470 Mb and a scaffold N50 of 1.465 Mb. Comparative genomic analysis revealed that the GO terms enriched among four gastropod species are related to signal transduction involved in maintaining electrochemical gradients across the cell membrane. Population genomic analysis using the MSMC model for 14 re-sequenced individuals revealed a drastic decline in Korean and Japanese populations during the last glacial period, while the southern Chinese population retained a much larger effective population size (Ne). These contrasting demographic changes might be attributed to multiple environmental factors during the glacial-interglacial cycles. This study provides valuable genomic resources for understanding adaptation and historical demographic responses to climate change.

Evolutionary paths to new phenotypes.

Ecological model systems inform on innovative traits in plants and animals.

The genetic basis of a recent transition to live-bearing in marine snails.

Key innovations are fundamental to biological diversification, but their genetic basis is poorly understood. A recent transition from egg-laying to live-bearing in marine snails (Littorina spp.) provides the opportunity to study the genetic architecture of an innovation that has evolved repeatedly across animals. Individuals do not cluster by reproductive mode in a genome-wide phylogeny, but local genealogical analysis revealed numerous small genomic regions where all live-bearers carry the same core haplotype. Candidate regions show evidence for live-bearer-specific positive selection and are enriched for genes that are differentially expressed between egg-laying and live-bearing reproductive systems. Ages of selective sweeps suggest that live-bearer-specific alleles accumulated over more than 200,000 generations. Our results suggest that new functions evolve through the recruitment of many alleles rather than in a single evolutionary step.

Effects of blood mercury accumulation on DNA methylation levels in the Khorat snail-eating turtle (Malayemys khoratensis).

Mercury (Hg) has adverse effects on humans and wildlife. Hg exposure can cause significant alterations in DNA methylation, an epigenetic modification that causes various illnesses. Hg accumulation in the blood of the Khorat snail-eating turtle (Malayemys khoratensis) from northeastern Thailand was previously reported. Thus, this study aimed to assess total mercury (THg) levels in M. khoratensis blood and to examine the impact of these concentrations on DNA methylation (5-methylcytosine, 5-mC) levels. We divided turtles based on morphological characteristics into two groups, normal and deformed, and then the levels of each variable in both groups were assessed. The deformed group presented higher mean THg concentration and DNA methylation levels compared to the normal group; however, the differences were not significant. Additionally, we found no correlation between DNA methylation levels and THg concentrations in both groups. This study is the first attempt to investigate the relationship between mercury accumulation and DNA methylation in the blood of deformed freshwater turtles.

Local and regional geographic variation in inducible defenses.

Invasive predators can cause substantial evolutionary change in native prey populations. Although invasions by predators typically occur over large scales, their distributions are usually characterized by substantial spatiotemporal heterogeneity that can lead to patchiness in the response of native prey species. Our ability to understand how local variation shapes patterns of inducible defense expression has thus far been limited by insufficient replication of populations within regions. Here, we examined local and regional variation in the inducible defenses of 12 native marine snail (Littorina obtusata) populations within two geographic regions in the Gulf of Maine that are characterized by vastly different contact histories with the invasive predatory green crab (Carcinus maenas). When exposed in the field to waterborne risk cues from the green crab for 90 days, snails expressed plastic increases in shell thickness that reduced their vulnerability to this shell-crushing predator. Despite significant differences in contact history with this invasive predator, snail populations from both regions produced similar levels of shell thickness and shell thickness plasticity in response to risk cues. Such phenotypic similarity emerged even though there were substantial geographic differences in the shell thickness of juvenile snails at the beginning of the experiment, and we suggest that it may reflect the effects of warming ocean temperatures and countergradient variation. Consistent with plasticity theory, a trend in our results suggests that southern snail populations, which have a longer contact history with the green crab, paid less in the form of reduced tissue mass for thicker shells than northern populations.

Coupling DNA barcodes and exon-capture to resolve the phylogeny of Turridae (Gastropoda, Conoidea).

Taxon sampling in most phylogenomic studies is often based on known taxa and/or morphospecies, thus ignoring undescribed diversity and/or cryptic lineages. The family Turridae is a group of venomous snails within the hyperdiverse superfamily Conoidea that includes many undescribed and cryptic species. Therefore 'traditional' taxon sampling could constitute a strong risk of undersampling or oversampling Turridae lineages. To minimize potential biases, we establish a robust sampling strategy, from species delimitation to phylogenomics. More than 3,000 cox-1 "barcode" sequences were used to propose 201 primary species hypotheses, nearly half of them corresponding to species potentially new to science, including several cryptic species. A 110-taxa exon-capture tree, including species representatives of the diversity uncovered with the cox-1 dataset, was build using up to 4,178 loci. Our results show the polyphyly of the genus Gemmula, that is split into up to 10 separate lineages, of which half would not have been detected if the sampling strategy was based only on described species. Our results strongly suggest that the use of blind, exploratory and intensive barcode sampling is necessary to avoid sampling biases in phylogenomic studies.

Out of the ancient lake: Multiple riverine colonizations and diversification of the freshwater snails in the genus Semisulcospira around Lake Biwa.

Ancient lakes are a hotspot of biodiversity. Freshwater species often experience spectacular species radiation after colonizing lakes from riverine habitats. Therefore, the relationship between the fauna of the ancient lakes and the surrounding riverine system has a special significance in understanding their origin and evolutionary history. The study of ancient lake species often focused on the lake colonization of riverine species. In contrast, far less attention has been placed on the reverse direction: the riverine colonization of the lake species, despite its importance in disentangling their complex evolutionary history. The freshwater snails in the genus Semisulcospira involve endemic groups that radiated in the ancient Lake Biwa. Using genetics and fossil records, we inferred that the ancestors of these lake-endemic Semisulcospira snails historically colonized the riverine habitats at least three times during the Middle Pleistocene. Each colonization resulted in the formation of a new lineage that was genetically and morphologically distinct from other lineages. Further, one of these colonizations was followed by hybridization with a cosmopolitan riverine species, which potentially facilitated the population persistence of the colonizers in the new environment. Despite their complex histories, all these colonizers were currently grouped within a single species, Semisulcospira kurodai, suggesting cryptic diversity in this species. This study highlights the significance of the riverine colonizations of the lake species to fully understand the diversification history of freshwater fauna in and around the ancient lakes.

Systematics and Distributions of Upper Bathyal Species in Bathyancistrolepis, a Deep-Sea Whelk Genus Endemic to the Northwest Pacific (Gastropoda: Buccinidae).

The deep-sea buccinid snail genus Bathyancistrolepis is redefined based on the reconstruction of a molecular phylogeny and morphological examination of shell and radular characters. This genus is distinguished from other genera of the subfamily Parancistrolepidinae with a combination of shell traits, including (1) a low spire, (2) sharp, carinate spiral cords or keels and (3) a long, curved siphonal canal, but not with a difference in radular morphology as suggested by previous authors. Three allopatric or parapatric species are recognized in the upper bathyal (447-2057 m) waters around Japan and Taiwan: B. tokoyodaensis from off Hokkaido to Sagami Bay in the Northwest Pacific, B. trochoidea off Kumano-nada to Miyazaki in the Northwest Pacific and along Nansei Islands in the East China Sea, and B. taiwanensis sp. nov. in the South China Sea. These species bear large paucispiral protoconchs that are indicative of benthic early development without a pelagic larval period, and hence low dispersal capability. Seafloor topography seems to have acted as a barrier for their dispersal; the range of B. tokoyodaensis supports the previous finding that Izu Peninsula delimits westward distribution of bathyal gastropod species of boreal origins.

VECTOR COMPATIBILITY OF NEW MEXICO GALBA SPECIES WITH THE CANINE SCHISTOSOME HETEROBILHARZIA AMERICANA, INCLUDING THE FIRST REPORT OF GALBA SCHIRAZENSIS AS A COMPATIBLE HOST.

The indigenous North American mammalian schistosome Heterobilharzia americana has recently attracted attention for causing outbreaks in dogs in states outside of its southeastern U.S. distribution. Although H. americana has yet to be reported in New Mexico, we examined 2 New Mexico isolates of Galba snails to determine their susceptibility to experimental infection with an isolate of H. americana from Utah. One of the Galba isolates from the Rio Grande bosque in the Albuquerque suburb of Corrales was identified as Galba humilis, and like specimens of the same taxon from Utah, proved susceptible to H. americana (27.6% of exposed surviving snails positive). The second Galba isolate sourced from the northern mountains of New Mexico, which surprisingly was revealed to be Galba schirazensis based on cytochrome c oxidase 1, 16S rRNA, and the internal transcribed spacer 2 markers, was also susceptible to H. americana (56.3% of exposed surviving field-derived snails and 46.4% first generation [F1] snails positive). This is the first report of the latter snail being a compatible snail host for H. americana. As G. schirazensis has a wide, albeit spotty, distribution and is considered an invasive species, it provides yet another opportunity for H. americana to expand its known range, potentially including the state of New Mexico as well.

Covariation between microeukaryotes and bacteria associated with Planorbidae snails.

Background: Microbial communities associated with macroorganisms might affect host physiology and homeostasis. Bacteria are well studied in this context, but the diversity of microeukaryotes, as well as covariations with bacterial communities, remains almost unknown. Methods: To study microeukaryotic communities associated with Planorbidae snails, we developed a blocking primer to reduce amplification of host DNA during metabarcoding analyses. Analyses of alpha and beta diversities were computed to describe microeukaryotes and bacteria using metabarcoding of 18S and 16S rRNA genes, respectively. Results: Only three phyla (Amoebozoa, Opisthokonta and Alveolata) were dominant for microeukaryotes. Bacteria were more diverse with five dominant phyla (Proteobacteria, Bacteroidetes, Tenericutes, Planctomycetes and Actinobacteria). The composition of microeukaryotes and bacteria were correlated for the Biomphalaria glabrata species, but not for Planorbarius metidjensis. Network analysis highlighted clusters of covarying taxa. Among them, several links might reflect top-down control of bacterial populations by microeukaryotes, but also possible competition between microeukaryotes having opposite distributions (Lobosa and Ichthyosporea). The role of these taxa remains unknown, but we believe that the blocking primer developed herein offers new possibilities to study the hidden diversity of microeukaryotes within snail microbiota, and to shed light on their underestimated interactions with bacteria and hosts.

DNA barcoding indicates multiple invasions of the freshwater snail Melanoides tuberculata sensu lato in Florida.

Melanoides tuberculata sensu lato (Thiaridae) are polymorphic female-clonal snails of Asian and African origins that have invaded freshwaters worldwide, including those in Florida. Although the snails have been documented in Florida for at least 70 years, no studies have investigated whether the observed distribution is due to a single introduction or multiple independent invasions. Here, cytochrome oxidase I was used to measure genetic diversity within and among sites in Florida and compare genetic diversity between Florida and other regions of the world. We also examined the relationship between shell morphology and haplotype diversity to determine if shell morphs can serve as a proxy for haplotypes. In total, we recovered 8 haplotypes randomly distributed across populations in Florida. Phylogenetic reconstruction supported the hypothesis of multiple invasions by diverse representatives of the M. tuberculata species complex. In contrast, shell morphology was not found to be a useful phylogeographic character, with divergent haplotypes represented by similar shell forms. These results suggest that the observed invasion patterns in Florida are best explained by serial introductions, and that shell morphology cannot be used to predict haplotypes or reconstruct invasion history of Melanoides tuberculata s.l. and that extensive taxonomic revisions are needed to investigate invasion dynamics.

A qPCR assay for the rapid and specific detection of Shining ram's-horn snail (Segmentina nitida) eDNA from Stodmarsh National Nature Reserve, UK.

Segmentina nitida Müller 1774 is a freshwater snail which was formerly widespread throughout England and south Wales. Since the 1840s it has seen a rapid decline in its range which has been attributed to deteriorating water quality due to nutrient enrichment, lowering of water tables and over-management of the ditches in which it resides. S. nitida has therefore been identified as a UK Biodiversity Action Plan (UKBAP) priority species which recommends further research for its conservation. Here we have developed a Taqman based qPCR eDNA assay for the detection of S. nitida at the Stodmarsh National Nature Reserve and compared the results with a manual survey of the ditches at this location. 32 ditches were surveyed in November 2020 (22 at Stodmarsh) and February 2021 (10 outside the known range of S.nitida). Our eDNA analysis exhibited an observed percentage agreement of 84% with a kappa coefficient of agreement between manual and eDNA surveys of 0.56 (95% CI 0.22 to 0.92). Three ditches determined to be negative for S. nitida by eDNA analysis were manual survey positive, and a further two ditches that were negative by manual survey were positive by eDNA analysis revealing the potential for improved overall detection rates using a combination of manual and eDNA methodologies. eDNA analysis could therefore augment manual survey techniques for S. nitida as a relatively quick and inexpensive tool for collecting presence and distribution data that could be used to inform manual surveys and management of ditches.

Overcoming the congenitally disadvantageous mutation through adaptation to environmental UV exposure in land snails.

Congenital fitness-disadvantageous mutations are not maintained in the population; they are purged from the population through processes such as purifying selection. However, these mutations could persist in the population as polymorphisms when it is advantageous for the individuals carrying them in adapting to a specific external environment. We tested this hypothesis using the dimorphic land snail Euhadra peliomphala simodae in Japan; these snails have dark or bright coloured shells. The survival rate of dark snails at hatching was lower than that of the bright ones, as observed in the F1 progenies produced through crossing. Dark snails have a congenital fitness-disadvantageous mutation; however, they also have protection against ultraviolet radiation. They have a higher survival rate than the bright snails in a UV environment, as observed using the UV exposure experiments and UV transmittance measurements. This is a good example of a congenitally disadvantageous mutation that is advantageous for adapting to the external environment. These results explain the maintenance of polymorphism and highlight the genotypic and phenotypic diversity in the wild population.