Littorina snails and Microphallus trematodes: Diverse consequences of the trematode-induced metabolic shifts.

The intricate relationships between parasites and hosts encompass a wide range of levels, from molecular interactions to population dynamics. Parasites influence not only the physiological processes in the host organism, but also the entire ecosystem, affecting mortality of individuals, the number of offspring through parasitic castration, and matter and energy cycles. Understanding the molecular mechanisms that govern host-parasite relationships and their impact on host physiology and environment remains challenging. In this study, we analyzed how infection with Microphallus trematodes affects the metabolome of two Littorina snail species inhabiting different intertidal zone shore levels. We applied non-targeted GC-MS-based metabolomics to analyze biochemical shifts induced by trematode infection in a host organism. We have identified changes in energy, amino acid, sugar, and lipid metabolism. In particular, we observed intensified amino acid catabolism and nitrogenous catabolites (glutamine, urea) production. These changes primarily correlated with infection and interspecies differences of the hosts rather than shore level. The changes detected in the host metabolism indicate that other aspects of life may have been affected, both within the host organism and at a supra-organismal level. Therefore, we explored changes in microbiota composition, deviations in the host molluscs behavior, and acetylcholinesterase activity (ACE, an enzyme involved in neuromuscular transmission) in relation to infection. Infected snails displayed changes in their microbiome composition. Decreased ACE activity in snails was associated with reduced mobility, but whether it is associated with trematode infection remains unclear. The authors suggest a connection between the identified biochemical changes and the deformation of the shell of molluscs, changes in their behavior, and the associated microbiome. The role of parasitic systems formed by microphallid trematodes and Littorina snails in the nitrogen cycle at the ecosystem level is also assumed.

Measuring ecological quality status in low-diversity Arctic intertidal foraminiferal assemblages using a diversity-based index.

EcoQS assessment of the marine intertidal zone based on its fauna is challenging because the assemblages have a low diversity and consist of stress tolerant species. The new approach we propose is to pool foraminiferal diversity (effective number of species exp(H'bc)) across the whole intertidal zone including the salt marsh and tidal flat. In seven fjordheads studied in northern Fennoscandia, polycyclic aromatic hydrocarbon (PAH) concentrations indicated low levels of pollution (EcoQSPAH Excellent to Moderate). Jadammina or Balticammina dominated the salt marsh, Elphidium albiumbilicatum, Elphidium williamsoni, Elphidium clavatum, and Buccella frigida occurred in the tidal flat. Ovammina opaca thrived in both belts. While foraminiferal test abnormalities are often proposed to measure pollution impacts, we did not detect any correlation with PAHs. EcoQS based on foraminiferal diversity (EcoQSforam Excellent to Good) matched EcoQS based on PAHs suggesting that pooled foraminiferal diversity reliably measures intertidal EcoQS.

First insights into the gut microbiomes and the diet of the Littorina snail ecotypes, a recently emerged marine evolutionary model.

Microbes can play a prominent role in the evolution of their hosts, facilitating adaptation to various environments and promoting ecological divergence. The Wave and Crab ecotypes of the intertidal snail Littorina saxatilis is an evolutionary model of rapid and repeated adaptation to environmental gradients. While patterns of genomic divergence of the Littorina ecotypes along the shore gradients have been extensively studied, their microbiomes have been so far overlooked. The aim of the present study is to start filling this gap by comparing gut microbiome composition of the Wave and Crab ecotypes using metabarcoding approach. Since Littorina snails are micro-grazers feeding on the intertidal biofilm, we also compare biofilm composition (i.e. typical snail diet) in the crab and wave habitats. In the results, we found that bacterial and eukaryotic biofilm composition varies between the typical habitats of the ecotypes. Further, the snail gut bacteriome was different from outer environments, being dominated by Gammaproteobacteria, Fusobacteria, Bacteroidia and Alphaproteobacteria. There were clear differences in the gut bacterial communities between the Crab and the Wave ecotypes as well as between the Wave ecotype snails from the low and high shores. These differences were both observed in the abundances and in the presence of different bacteria, as well as at different taxonomic level, from bacterial OTU's to families. Altogether, our first insights show that Littorina snails and their associated bacteria are a promising marine system to study co-evolution of the microbes and their hosts, which can help us to predict the future for wild species in the face of rapidly changing marine environments.

Data on RNA-seq analysis of the oviducts of five closely related species genus Littorina (Mollusca, Caenogastropoda): L. saxatilis, L. arcana, L. compressa, L. obtusata, L. fabalis.

In the evolution of invertebrates, the transition from egg-layers to brooders occurred many times. However, the molecular mechanisms underlying this transition are still not well understood. Recently diverged species genus Littorina (Mollusca, Gastropoda, Caenogastropoda, Littorinimorpha): Littorina saxatilis, L. arcana, L. compressa, L. obtusata and L. fabalis might be a fruitful model for elucidation of these mechanisms. All five species sympatrically inhabit an intertidal zone. Only L. saxatilis is ovoviviparous while the other four species form clutches. Although in L. saxatilis jelly gland of the pallial oviduct function as a brood pouch, it is not deeply modified at the morphological level in comparison to egg-laying relatives. Comparative analysis of transcriptomic profiles of the pallial oviducts of these closely related species might help to uncover the molecular mechanisms of the egg-laying to brooding transition. Unraveling of the mechanisms underlying this transition in L. saxatilis is important not only in aspects of reproduction biology and strategy, but also in a broader view as an example of relatively fast evolutionary transformations. We generated an RNA-seq dataset (224 104 446 clean reads) for oviducts of five species genus Littorina. Libraries of all five species were sequenced using Illumina HiSeq 2500; additional reads for L. arcana were obtained using Illumina NovaSeq 6000. Transcriptomic profiles were analyzed in pooled samples (of three individuals) with two biological replicates for each species (each biological replicate was prepared and sequenced as a separate library). The transcriptome was assembled de novo and annotated with five assembles corresponding to each species. The raw data were uploaded to the SRA database, the BioProject IDs are PRJNA662103 ("obtusata" group) and PRJNA707549 ("saxatilis" group).

Divergence together with microbes: A comparative study of the associated microbiomes in the closely related Littorina species.

Any multicellular organism during its life is involved in relatively stable interactions with microorganisms. The organism and its microbiome make up a holobiont, possessing a unique set of characteristics and evolving as a whole system. This study aimed to evaluate the degree of the conservativeness of microbiomes associated with intertidal gastropods. We studied the composition and the geographic and phylogenetic variability of the gut and body surface microbiomes of five closely related sympatric Littorina (Neritrema) spp. and a more distant species, L. littorea, from the sister subgenus Littorina (Littorina). Although snail-associated microbiomes included many lineages (207-603), they were dominated by a small number of OTUs of the genera Psychromonas, Vibrio, and Psychrilyobacter. The geographic variability was greater than the interspecific differences at the same collection site. While the microbiomes of the six Littorina spp. did not differ at the high taxonomic level, the OTU composition differed between groups of cryptic species and subgenera. A few species-specific OTUs were detected within the collection sites; notably, such OTUs never dominated microbiomes. We conclude that the composition of the high-rank taxa of the associated microbiome ("scaffolding enterotype") is more evolutionarily conserved than the composition of the low-rank individual OTUs, which may be site- and / or species-specific.

Species-Specific Proteins in the Oviducts of Snail Sibling Species: Proteotranscriptomic Study of Littorina fabalis and L. obtusata.

Genus Littorina subgenus Neritrema (Mollusca, Caenogastropoda) includes the "obtusata" group of closely related species (Littorina obtusata and L. fabalis). The anatomy of the adult reproductive system (pallial oviduct) is the only reliable feature used for species identification in females of these species. Reproductive system anatomy and reproduction-associated proteins often diverge between sibling species. Despite being of high evolutionary interest, the molecular basis of this divergence remains poorly understood. We performed proteotranscriptomic comparison of oviducts of L. obtusata and L. fabalis by RNA-seq on Illumina HiSeq 2500 and two-dimensional protein electrophoresis (2D DIGE) with MS/MS identification of the species-specific proteins. The interspecies differences in the oviduct were associated with (1) metabolic proteins reflecting overall physiological differences between L. obtusata and L. fabalis, (2) receptor proteins, and (3) transcripts related to transposable elements (TEs). Various receptors identified may recognize a wide variety of ligands from pathogen-associated molecular patterns to specific carbohydrates on the sperm surface. Therefore, these may participate in immune defense as well as in sperm storage and regulation. Species-specificity of multiple TE sequences (coding for reverse transcriptase and ribonuclease H) may indicate the important role of these genomic elements in the Littorina species divergence, which has not been reported previously.

Linking ecology, morphology, and metabolism: Niche differentiation in sympatric populations of closely related species of the genus Littorina (Neritrema).

Divergence of ecological niches in phylogenetically closely related species indicates the importance of ecology in speciation, especially for sympatric species are considered. Such ecological diversification provides an advantage of alleviating interspecies competition and promotes more efficient exploitation of environmental resources, thus being a basis for ecological speciation. We analyzed a group of closely related species from the subgenus Neritrema (genus Littorina, Caenogastropoda) from the gravel-bouldery shores. In two distant sites at the Barents and Norwegian Sea, we examined the patterns of snail distribution during low tide (quantitative sampling stratified by intertidal level, presence of macrophytes, macrophyte species, and position on them), shell shape and its variability (geometric morphometrics), and metabolic characteristics (metabolomic profiling). The studied species diversified microbiotopes, which imply an important role of ecological specification in the recent evolution of this group. The only exception to this trend was the species pair L. arcana / L. saxatilis, which is specifically discussed. The ecological divergence was accompanied by differences in shell shape and metabolomic characteristics. Significant differences were found between L. obtusata versus L. fabalis and L. saxatilis / L. arcana versus L. compressa both in shell morphology and in metabolomes. L. saxatilis demonstrated a clear variability depending on intertidal level which corresponds to a shift in conditions within the occupied microhabitat. Interestingly, the differences between L. arcana (inhabiting the upper intertidal level) and L. compressa (inhabiting the lower one) were analogous to those between the upper and lower fractions of L. saxatilis. No significant level-dependent changes were found between the upper and lower fractions of L. obtusata, most probably due to habitat amelioration by fucoid macroalgae. All these results are discussed in the contexts of the role of ecology in speciation, ecological niche dynamics and conservatism, and evolutionary history of the Neritrema species.

Premating barriers in young sympatric snail species.

Sympatric coexistence of recently diverged species raises the question of barriers restricting the gene flow between them. Reproductive isolation may be implemented at several levels, and the weakening of some, e.g. premating, barriers may require the strengthening of the others, e.g. postcopulatory ones. We analysed mating patterns and shell size of mates in recently diverged closely related species of the subgenus Littorina Neritrema (Littorinidae, Caenogastropoda) in order to assess the role of premating reproductive barriers between them. We compared mating frequencies observed in the wild with those expected based on relative densities using partial canonical correspondence analysis. We introduced the fidelity index (FI) to estimate the relative accuracy of mating with conspecific females and precopulatory isolation index (IPC) to characterize the strength of premating barriers. The species under study, with the exception of L. arcana, clearly demonstrated preferential mating with conspecifics. According to FI and IPC, L. fabalis and L. compressa appeared reliably isolated from their closest relatives within Neritrema. Individuals of these two species tend to be smaller than those of the others, highlighting the importance of shell size changes in gastropod species divergence. L. arcana males were often found in pairs with L. saxatilis females, and no interspecific size differences were revealed in this sibling species pair. We discuss the lack of discriminative mate choice in the sympatric populations of L. arcana and L. saxatilis, and possible additional mechanisms restricting gene flow between them.

Genetic and morphological variation of metacercariae of Microphallus piriformes (Trematoda, Microphallidae): Effects of paraxenia and geographic location.

Host organism offers an environment for a parasite, and this environment is heterogenous within the host, variable among individual as well as between the hosts, and changing during the host's lifetime. This heterogeneity may act as a prerequisite for parasite species divergence. Intraspecific variability related to a certain type of heterogeneity may indicate an initial stage of speciation, and thus poses an evolutionary importance. Here we analyzed genetic and morphologic variation of trematode metacercariae of Microphallus piriformes (Trematoda, Microphallidae). Genetic variability of trematodes was assessed from sequences of cytochrome c oxidase subunit 1 (COI) and internal transcribed spacer region (ITS-1). Morphological variation of metacercarial body shape was for the first time analyzed using geometric morphometrics. Parasites from the White Sea and the Barents Sea coasts demonstrated partial genetic divergence (according to COI sequence analysis) and had significantly different body shape. Neither genetic nor morphological variation of metacercariae was related to intermediate host species. We discuss possible causes of the observed genetic divergence of parasite populations in different geographic regions.

Proteomic similarity of the Littorinid snails in the evolutionary context.

BACKGROUND: The introduction of DNA-based molecular markers made a revolution in biological systematics. However, in cases of very recent divergence events, the neutral divergence may be too slow, and the analysis of adaptive part of the genome is more informative to reconstruct the recent evolutionary history of young species. The advantage of proteomics is its ability to reflect the biochemical machinery of life. It may help both to identify rapidly evolving genes and to interpret their functions. METHODS: Here we applied a comparative gel-based proteomic analysis to several species from the gastropod family Littorinidae. Proteomes were clustered to assess differences related to species, geographic location, sex and body part, using data on presence/absence of proteins in samples and data on protein occurrence frequency in samples of different species. Cluster support was assessed using multiscale bootstrap resampling and the stability of clustering-using cluster-wise index of cluster stability. Taxon-specific protein markers were derived using IndVal method. Proteomic trees were compared to consensus phylogenetic tree (based on neutral genetic markers) using estimates of the Robinson-Foulds distance, the Fowlkes-Mallows index and cophenetic correlation. RESULTS: Overall, the DNA-based phylogenetic tree and the proteomic similarity tree had consistent topologies. Further, we observed some interesting deviations of the proteomic littorinid tree from the neutral expectations. (1) There were signs of molecular parallelism in two Littoraria species that phylogenetically are quite distant, but live in similar habitats. (2) Proteome divergence was unexpectedly high between very closely related Littorina fabalis and L. obtusata, possibly reflecting their ecology-driven divergence. (3) Conservative house-keeping proteins were usually identified as markers for cryptic species groups ("saxatilis" and "obtusata" groups in the Littorina genus) and for genera (Littoraria and Echinolittorina species pairs), while metabolic enzymes and stress-related proteins (both potentially adaptively important) were often identified as markers supporting species branches. (4) In all five Littorina species British populations were separated from the European mainland populations, possibly reflecting their recent phylogeographic history. Altogether our study shows that proteomic data, when interpreted in the context of DNA-based phylogeny, can bring additional information on the evolutionary history of species.

Hydrocarbon molecular markers in the Holocene bottom sediments of the Barents Sea as indicators of natural and anthropogenic impacts.

The recent intensification of energy resource exploration and human activities in the Barents Sea (BS) requires a more thorough assessment of the natural and anthropogenic impact of hydrocarbons on the environment. We analyzed a wide set of sensitive indicators, including hydrocarbon molecular markers and organic matter (OM) maturity parameters in the Holocene sediments from three regions of the BS: the Kola-Kanin Monocline (KKM), the Svalbard shelf, and the Shtokman gas-condensate field (GCF). An increase in pyrogenic polycyclic aromatic hydrocarbons toward the core surface traces the intensification of anthropogenic contamination in the KKM region during last century. An input of highly mature OM from the eroded coal rocks of Barentsburg were confirmed by comparison of biomarker distribution in sediments and coals. An increase in biogenic hopanes and hopenes content down-core, and a crude-oil stage of OM maturity in surface sediments of the Shtokman GCF attests to hydrocarbons migration from subsurface strata.

Unexpected Levels of Biological Activity during the Polar Night Offer New Perspectives on a Warming Arctic.

The current understanding of Arctic ecosystems is deeply rooted in the classical view of a bottom-up controlled system with strong physical forcing and seasonality in primary-production regimes. Consequently, the Arctic polar night is commonly disregarded as a time of year when biological activities are reduced to a minimum due to a reduced food supply. Here, based upon a multidisciplinary ecosystem-scale study from the polar night at 79°N, we present an entirely different view. Instead of an ecosystem that has entered a resting state, we document a system with high activity levels and biological interactions across most trophic levels. In some habitats, biological diversity and presence of juvenile stages were elevated in winter months compared to the more productive and sunlit periods. Ultimately, our results suggest a different perspective regarding ecosystem function that will be of importance for future environmental management and decision making, especially at a time when Arctic regions are experiencing accelerated environmental change [1].

Micro-spatial distribution of two sibling periwinkle species across the intertidal indicates hybrdization.

Populations of periwinkles Littorina saxatilis (Olivi 1792) and L. arcana Hannaford Ellis, 1978 are well suited for microevolutionary studies, being at the same time closely related and intraspecifically diverse. The divergence between these two sibling species, sympatric over large parts of their distribution areas, is small, the only morphological difference being the pallial gland complex structure in females. Molecular identification is possible with the use of a RAPD nuclear marker (cloned A2.8 DNA fragment) typical for L. arcana. However, in some individuals from sympatric populations molecular and morphological criteria suggest conflicting species affiliation, which may be explained either by hybridization or by shared ancestral polymorphism. We tested the hybridization hypotheses examining the micro-spatial distribution of these two species across the intertidal zone in two distant sites at the Barents Sea. We found that (a) the frequency of putative hybrids in sympatric populations was proportional to the frequency of L. arcana; (b) L. saxatilis bearing A2.8 DNA fragment were almost absent in the lower part of the intertidal zone, where L. arcana was absent too; (c) there was a close positive correlation between the distribution of potential parent molluscs and putative hybrids. Moreover, logistic regression models showed a good agreement between the distribution of putative hybrid frequencies and that of parental species frequencies. All our observations taken together support the hypothesis of hybridization between L. saxatilis and L. arcana. Elucidating the mechanisms that support the species status of these sympatric populations is necessary.

Population structure and growth rates at biogeographic extremes: a case study of the common cockle, Cerastoderma edule (L.) in the Barents Sea.

A descriptive study of population structure, growth rates and shell morphometry was conducted on nine intertidal populations of the infaunal bivalve Cerastoderma edule in the Murmansk coast of the Barents Sea. Year-to-year population dynamics was analyzed during 2002-2006 on a tidal flat Dalniy Plaj (eastern Murmansk coast). The region constitutes the northern extremes of C. edule range where populations occupied the middle to low intertidal zone and were characterized by low densities. The distribution of age groups and unstable age structure across years in the cockle populations suggest irregular recruitment. Growth rates and shell morphometry showed little variation across the populations studied, and there were no gradient changes from the west to the east parts of Murmansk coast.