Marine Molluscs Research Articles

Cucurbitacins: Nature’s Wonder Molecules

Abstract: Over the past decades, several natural constituents belonging to different classes have been isolated from plants for medicinal purposes. Cucurbitacins is one such type of natural compound. Cucurbitacin is a class of biochemical compounds that some plants, notably members of the pumpkin and gourd family, Cucurbitaceae produce and function as a defense against herbivores. They and their derivatives have been found in many plant families (including Brassicaceae, Cucurbitaceae, Scrophulariaceae, Begoniaceae, Elaeocarpaceae, Datiscaceae, Desfontainiaceae, Polemoniaceae, Primulaceae, Rubiaceae, Sterculiaceae, Rosaceae, and Thymelaeaceae), in some mushrooms (including Russula and Hebeloma) and even in some marine mollusks. They have been isolated from various plant species, chiefly belonging to the Cucurbitaceae family, which comprises around 130 genera and 800 species. Cucurbitacins are a group of tetracyclic triterpenoid substances that are highly oxygenated and contain a cucurbitane skeleton characterized by 9β-methyl−19-norlanosta- 5-ene. Cucurbitacins can be categorized into twelve main groups according to variations in their side-chains. Cucurbitacins A, B, C, D, E, F, I, J, K, L, O, P, Q, R, S, and their glycosides are mainly found in Cucurbitaceae family members. These plants have been used as folk medicines in some countries because of their broad spectrum of crucial pharmacological activities such as anti- inflammatory, anti-cancer, anti-diabetic, and anti-atherosclerotic effects. The present review explores the possibility of a correlation between the chemistry of various Cucurbitacins and the uses of the plants which contain them, thereby opening avenues for further phytochemical, ethnomedicinal, and modern pharmacological research on these important molecules.

Wille La Roche (1872-1942): ship's engineer, shell collector and contributor to Auckland Museum's malacology collection

For much of the 1920s ship’s engineer Willie La Roche (1872 – 1942) obtained specimens of terrestrial, fresh-water and marine molluscs, as well as bulk marine sediment samples, many of which came into the collections of Auckland Museum.

Heritability estimation of high salt tolerance in razor clam (Sinonovacula constricta)

As an important marine mollusk, the growth and development of razor clams (Sinonovacula constricta) are affected by salinity. Genetic selection for salt tolerance is an imperative and effective approach to the improvement of growth and production. To examine whether the tolerance to high salt can be included in razor clam breeding programs, we conducted a genetic analysis of high salt tolerance using three phenotypes and four statistical models. The correlation between tolerance and growth traits was evaluated simultaneously. In this study, we used 9-day experimental stress data of 1005 individuals from 17 full-sib families (the offspring of 17 sires and 17 dams). The survival rate of the families ranged from 0% to 100%, suggesting that the viability of razor clams was different under diverse high salt stress. A positive Pearson and Spearman correlation existed between high salt tolerance and growth traits. A very high genetic correlation (0.886–0.999) was also observed between them, indicating indirect breeding was feasible. Heritabilities of tolerance to high salt were from low to high, with values of 0.55–0.64, 0.11, and 0.047–0.487 for trait TS (test survival), trait DD (day of death), and trait TDS (test-day survival), respectively. The Pearson correlation coefficients of families estimated breeding values between different models ranged from 0.921 to 0.998 which indicated a near-identical ranking of families. This study demonstrated that the salt tolerance of razor clams could be improved genetically through selective breeding. Overall, cross-sectional threshold and linear repeatability models were the most appropriate in estimating heritability for salt tolerance with trait TS and trait TDS in this species. Our study provides important information to guide the effective selection for salt tolerance in S. constricta.

Sclerochronological evidence of pronounced seasonality from the late Pliocene of the southern North Sea basin and its implications

Abstract. Oxygen isotope (δ18O) sclerochronology of benthic marine molluscs provides a means of reconstructing the seasonal range in seafloor temperature, subject to use of an appropriate equation relating shell δ18O to temperature and water δ18O, a reasonably accurate estimation of water δ18O, and due consideration of growth-rate effects. Taking these factors into account, δ18O data from late Pliocene bivalves of the southern North Sea basin (Belgium and the Netherlands) indicate a seasonal seafloor range a little smaller than now in the area. Microgrowth-increment data from Aequipecten opercularis, together with the species composition of the bivalve assemblage and aspects of preservation, suggest a setting below the summer thermocline for all but the latest material investigated. This implies a higher summer temperature at the surface than on the seafloor and consequently a greater seasonal range. A reasonable (3 ∘C) estimate of the difference between maximum seafloor and surface temperature under circumstances of summer stratification points to seasonal surface ranges in excess of the present value (12.4 ∘C nearby). Using a model-derived estimate of water δ18O (0.0 ‰), summer surface temperature was initially in the cool temperate range (<20 ∘C) and then (during the Mid-Piacenzian Warm Period; MPWP) increased into the warm temperate range (>20 ∘C) before reverting to cool temperate values (in conjunction with shallowing and a loss of summer stratification). This pattern is in agreement with biotic-assemblage evidence. Winter temperature was firmly in the cool temperate range (<10 ∘C) throughout, contrary to previous interpretations. Averaging of summer and winter surface temperatures for the MPWP provides a figure for annual sea surface temperature that is 2–3 ∘C higher than the present value (10.9 ∘C nearby) and in close agreement with a figure obtained by averaging alkenone and TEX86 temperatures for the MPWP from the Netherlands. These proxies, however, respectively, underestimate summer temperature and overestimate winter temperature, giving an incomplete picture of seasonality. A higher annual temperature than now is consistent with the notion of global warmth in the MPWP, but a low winter temperature in the southern North Sea basin suggests regional reduction in oceanic heat supply, contrasting with other interpretations of North Atlantic oceanography during the interval. Carbonate clumped isotope (Δ47) and biomineral unit thermometry offer means of checking the δ18O-based temperatures.

Transcriptomic Signatures of Pearl Oyster Pinctada Maxima in Response to Acute Salinity Stress

Acute salinity stress can impact many physiological processes of marine shellfish. The responses ofPinctada maximato salinity stress, especially the osmotic pressure regulation and immune response, are of great significance to health. To investigate the molecular changes in response to acute salinity stress, the pearl oysters were transferred from 30 ppt (C) to 40 ppt (HS) and 20 ppt (LS) for 12 h, and the transcriptome analysis was conducted on the gills. Compared to the control, there were 6613 (3253 up-regulated and 3360 down-regulated) differentially expressed genes (DEGs), 4395 (2180 up-regulated and 2215 down-regulated) DEGs observed in HS and LS, respectively. The related molecular biological processes and potential functions were explored from enrichment analysis. A total of 332 KEGG pathways (including 1514 genes) and 308 KEGG pathways (including 731 genes) were enriched in C vs. HS and C vs. LS, respectively. In addition, there are 1559 DEGs shared by C vs. HS group and C vs. LS group, and the results of the KEGG function annotation showed that 7 DEGs were involved in membrane transport, and 34 DEGs were involved in the immune system. The correlation network for expression of genes shows that the expression of 3 genes was significantly correlated with each other in membrane transport, and there were significant correlations between the expression of 27 genes in immune response. The results of this study will be of great value in understanding the molecular basis of salinity stress adaptation in the pearl oysterP. maxima.

Oral delivery of marine shellfish supramolecule peptides for skin wound healing

Oral administration of peptides/proteins with superior efficacy and fewer side effects is the most advantageous route of administration. In this study, we utilized controllable enzymatic (animal protease) hydrolysis technology to prepare active polypeptide self-assembling supramolecular (APs) from marine shellfish meat to explore the functional mechanism of APs in in vitro and in vivo (oral administration) experiments . In vitro experiments revealed that APs with self-assembly tendency had multifunctional activities. In vivo experiments indicated that oral administration of naturally safe APs could inhibited inflammation, promoted fibroblast proliferation and revascularization, and accelerated the epithelialization process, thus favoring a balanced repair tissue collagen I/III ratio and the promotion of hair follicle regeneration to achieve scarless healing, which was also relevant to "skin-gut" axis. These results showed that APs, as demonstrated in this study, promoted dermal wound healing in mice and may be developed and used to treat skin wounds.

Animal exploitation at the Olympos, southwestern Anatolia: Zooarchaeological analysis

This study presents analysis of animal remains unearthed from 2006 to 2021 excavations at Olympos, an important city of ancient Lycia, southwestern Turkey. Seven faunal assemblages were unearthed from seven distinct areas of the city. Each of them was studied according to their distinct archaeological contexts. The zooarchaeological observation was based on taxonomic identification, species diversity, kill-off patterns, nature of bone modification, including taphonomic and anthropogenic marks, and type of species exploitation at the site. The results demonstrated that the majority of the specimens were consumption residues, comprising mainly of ungulate and carnivore mammals, birds and marine fish and mollusks. Goat remains were the most common in all the assemblages, which is consistent with common animal exploitation patterns in Anatolia. Fish bones mostly represented bonito (Sarda sarda), tuna (Thunnus thynnus) and shark (Carcharhinidae sp.). Among the mollusks, the shells of Triton trumpet, rarely found in the Roman-Byzantine Anatolia, were clearly used as trumpets. As Olympos was an important harbour with a strategic location by the Mediterranean Sea, its faunal remains shed new light on the coastal dietary habbit, animal economy, and cultural contacts in the Roman and Early Byzantine periods in Anatolia.

A Draft Reference Genome Assembly of the Critically Endangered Black Abalone, Haliotis cracherodii.

The once abundant black abalone, Haliotis cracherodii, is a large, long-lived grazing marine mollusk that inhabits the rocky intertidal along the coast of California. The species has experienced dramatic declines since the mid-1980s largely due to the fatal bacterial disease called withering syndrome, leading to the collapse of an economically important fishery and to its inclusion into the IUCN listing as a critically endangered species. In some places impacted by the disease, populations of black abalone have declined by more than 90%, prompting population crashes associated with very little recruitment of new individuals and changes to intertidal communities. Habitats that were dominated by crustose coralline algae and bare rock have become dominated instead by fleshy algae and sessile invertebrates. Here, we present the first high-quality black abalone reference genome, assembled with PacBio HiFi long-reads and assembled with Dovetail Omni-C data to generate a scaffold-level assembly. The black abalone reference genome will be an essential resource in understanding the evolutionary history of this species as well as for exploring its current levels of genetic diversity and establishing future management and restoration plans.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

A multidisciplinary approach to characterise the Early-Middle Holocene palaeoenvironmental evolution of the Sado Valley of Portugal: Implications for late Mesolithic human communities

In this paper we present the results of a multidisciplinary study performed in the Carrasqueira valley, a tributary of the River Sado (SW Portugal), aimed at characterising the Holocene environmental conditions during the late Mesolithic occupation of this valley. Our findings are based on a 13.5 m long sediment core (Arez3) collected on the alluvial plain close to a late Mesolithic shell midden, the Arapouco site. The results of the multiproxy analyses (texture, magnetic susceptibility, organic composition and chemistry, n-alkanes and palynology) point to a greater marine influence between ca. 8850 cal yrs. B.P. (at the core base) and ca. 7450 cal yrs. B.P. (at 750 cm below mean sea level (MSL)) and the existence of an environment similar to the present-day central estuarine basin. At this point in time, sedimentation rates were lower than the rate of sea-level rise, resulting in the formation of a drowned area with intertidal environments developing on the less incised margins. After 7040 cal yrs. B.P. the contribution of organic matter from terrestrial plants and freshwater phytoplankton to the sediment increased, reflecting a change in the sedimentary pattern, with the estuarine environments progressively giving way to freshwater environments. After the Middle Holocene (ca. 6530 cal yrs. B.P.), negative shifts of δ15N to values ~0‰ point to hyper-eutrophication and cyanobacteria bloom episodes under backswamp conditions. According to these results, the estuarine environment prevailed in the area until 7040 cal yrs. B.P. (5090 cal yrs. B.C.; 390 cm below MSL), i.e., during the Mesolithic occupation of the valley, allowing for the occurrence and for the exploitation of marine shellfish and fish by these hunter-gatherer communities at the proximity of the downstream occupation areas.

Pathophysiological Responses to Conotoxin Modulation of Voltage-Gated Ion Currents

Voltage-gated ion channels are plasma membrane proteins that generate electrical signals following a change in the membrane voltage. Since they are involved in several physiological processes, their dysfunction may be responsible for a series of diseases and pain states particularly related to neuronal and muscular systems. It is well established for decades that bioactive peptides isolated from venoms of marine mollusks belonging to the Conus genus, collectively known as conotoxins, can target different types and isoforms of these channels exerting therapeutic effects and pain relief. For this reason, conotoxins are widely used for either therapeutic purposes or studies on ion channel mechanisms of action disclosure. In addition their positive property, however, conotoxins may generate pathological states through similar ion channel modulation. In this narrative review, we provide pieces of evidence on the pathophysiological impacts that different members of conotoxin families exert by targeting the three most important voltage-gated channels, such as sodium, calcium, and potassium, involved in cellular processes.

Genetic diversity and connectivity of the Ostreid herpesvirus 1 populations in France: A first attempt to phylogeographic inference for a marine mollusc disease.

The genetic diversity of viral populations is a key driver of the spatial and temporal diffusion of viruses; yet, studying the diversity of whole genomes from natural populations still remains a challenge. Phylodynamic approaches are commonly used for RNA viruses harboring small genomes but have only rarely been applied to DNA viruses with larger genomes. Here, we used the Pacific oyster mortality syndrome (a disease that affects oyster farms around the world) as a model to study the genetic diversity of its causative agent, the Ostreid herpesvirus 1 (OsHV-1) in the three main French oyster-farming areas. Using ultra-deep sequencing on individual moribund oysters and an innovative combination of bioinformatics tools, we de novo assembled twenty-one OsHV-1 new genomes. Combining quantification of major and minor genetic variations, phylogenetic analysis, and ancestral state reconstruction of discrete traits approaches, we assessed the connectivity of OsHV-1 viral populations between the three oyster-farming areas. Our results suggest that the Marennes-Oléron Bay represents the main source of OsHV-1 diversity, from where the virus has dispersed to other farming areas, a scenario consistent with current practices of oyster transfers in France. We demonstrate that phylodynamic approaches can be applied to aquatic DNA viruses to determine how epidemiological, immunological, and evolutionary processes act and potentially interact to shape their diversity patterns.

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry.

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described-single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)-to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described—single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)—to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Toxins’ classification through Raman spectroscopy with principal component analysis

The method based on the combination of Raman spectroscopy and principal component analysis (PCA) was applied to the set of peptide and protein toxins from animal venoms and to synthetic analogues of peptides. The study demonstrated the possibility of toxin classification according to their primary and secondary structures based on Raman spectroscopy. The method described here allows discrimination of snake venom three-finger toxins from predatory marine mollusks α-conotoxins. Moreover, PCA of the Raman spectra of toxins revealed differences within the group of three-finger toxins and also within the group of conotoxins, related to their spatial structure. In particular, on the basis of the developed technique it is possible to distinguish the disulfide isomers of the same peptide toxin. The results obtained have been confirmed by bioinformatic methods. So, we have proposed a method for the rapid analysis of newly discovered venom-derived protein or peptide toxins by establishing their similarity with other already studied toxins by referring to a particular class. Taking into account a low specimen consumption by Raman spectroscopy, the proposed method could represent a first step in the study of toxins from rare and/or endangered venomous animals. The ability to distinguish configuration of disulfide bonds allows to synthesize the correct isomer of the toxin.

Adaptive divergence and underlying mechanisms in response to salinity gradients between two Crassostrea oysters revealed by phenotypic and transcriptomic analyses.

Comparing the responses of closely related species to environmental changes is an efficient method to explore adaptive divergence, for a better understanding of the adaptive evolution of marine species under rapidly changing climates. Oysters are keystone species thrive in intertidal and estuarine areas where frequent environmental disturbance occurs including fluctuant salinity. The evolutionary divergence of two sister species of sympatric estuarine oysters, Crassostrea hongkongensis and Crassostrea ariakensis, in response to euryhaline habitats on phenotypes and gene expression, and the relative contribution of species effect, environment effect, and their interaction to the divergence were explored. After a 2-month outplanting at high- and low-salinity locations in the same estuary, the high growth rate, percent survival, and high tolerance indicated by physiological parameters suggested that the fitness of C.ariakensis was higher under high-salinity conditions and that of C.hongkongensis was higher under low-salinity conditions. Moreover, a transcriptomic analysis showed the two species exhibited differentiated transcriptional expression in high- and low-salinity habitats, largely caused by the species effect. Several of the important pathways enriched in divergent genes between species were also salinity-responsive pathways. Specifically, the pyruvate and taurine metabolism pathway and several solute carriers may contribute to the hyperosmotic adaptation of C.ariakensis, and some solute carriers may contribute to the hypoosmotic adaptation of C.hongkongensis. Our findings provide insights into the phenotypic and molecular mechanisms underlying salinity adaptation in marine mollusks, which will facilitate the assessment of the adaptive capacity of marine species in the context of climate change and will also provide practical information for marine resource conservation and aquaculture.

Compilation of a database of Holocene nearshore marine mollusk shell geochemistry from the California Current System

Abstract. The shells of marine invertebrates can serve as high-resolution records of oceanographic and atmospheric change through time. In particular, oxygen and carbon isotope analyses of nearshore marine calcifiers that grow by accretion over their lifespans provide seasonal records of environmental and oceanographic conditions. Archaeological shell middens generated by Indigenous communities along the northwest coast of North America contain shells harvested over multiple seasons for millennia. These shell middens, as well as analyses of archival and modern shells, have the potential to provide multi-site, seasonal archives of nearshore conditions throughout the Holocene. A significant volume of oxygen and carbon isotope data from archaeological shells exist, yet they are separately published in archaeological, geochemical, and paleoceanographic journals and have not been comprehensively analyzed to examine oceanographic change over time. Here, we compiled a database of previously published oxygen and carbon isotope data from archaeological, archival, and modern marine mollusks from the California Current System (North American coast of the northeast Pacific, 32 to 55∘ N). This database includes oxygen and carbon isotope data from 598 modern, archaeological, and sub-fossil shells from 8880 years before present (BP) to the present, from which there are 4917 total δ13C and 7366 total δ18O measurements. Shell dating and sampling strategies vary among studies (1–345 samples per shell, mean 44.7 samples per shell) and vary significantly by journal discipline. Data are from various bivalves and gastropod species, with Mytilus spp. being the most commonly analyzed taxon. This novel database can be used to investigate changes in nearshore sea surface conditions including warm–cool oscillations, heat waves, and upwelling intensity, and it provides nearshore calcium carbonate δ13C and δ18O values that can be compared to the vast collections of offshore foraminiferal calcium carbonate δ13C and δ18O data from marine sediment cores. By utilizing previously published geochemical data from midden and museum shells rather than sampling new specimens, future scientific research can reduce or omit the alteration or destruction of culturally valued specimens and sites. The dataset is publicly available through PANGAEA at https://doi.org/10.1594/PANGAEA.941373 (Palmer et al., 2021).

Origin of the tropical–polar biodiversity contrast

AbstractAimThe aim was to investigate the evolutionary origins of the striking biodiversity contrast between high‐ and low‐latitude regions in the present day. Is this a relatively recent phenomenon, causally linked in some way to the greenhouse–icehouse transition and onset of global coolingc. 34 Myr ago, or does it have deeper temporal roots and thus other potential causes?LocationEarly Cenozoic fossil assemblages from two tropical and one polar region, and modern counterparts from various tropical localities and Antarctica.Time periodThe Cretaceous–Palaeogene (K/Pg) mass extinction event, Early Cenozoic (Palaeocene–Eocene) and the present day.Major taxa studiedShelf‐depth marine Mollusca; the four richest modern benthic clades: Imparidentia, Pteriomorphia, Neogastropoda and Littorinimorpha.MethodsThe K/Pg mass extinction and subsequent recovery was compared between two tropical and one polar region at four distinct stratigraphic intervals. Taxa were identified to species level and assigned to principal families within the four largest benthic molluscan clades. Taxon counts were compared between the three regions at each level and also compared with standardized tropical and polar modern faunas.ResultsThe mass extinction was followed by a distinct 25 Myr phase of evolutionary radiation, during which the tropical–polar contrast in the taxonomic composition of all four clades was strongly enhanced; as the global molluscan fauna expanded, it differentiated into distinct low‐ and high‐latitude components.Main conclusionsA marked differentiation of tropical and polar molluscan faunas occurred in the immediate aftermath of the K/Pg mass extinction; it is likely that, at least for the two bivalve clades investigated, this differentiation was initiated well within the Mesozoic era. The greater antiquity of the tropical–polar split suggests that it was not the product of any single controlling factor during the Cenozoic.

Finding the adaptive needles in a population-structured haystack: A case study in a New Zealand mollusc.

Genetic adaptation to future environmental conditions is crucial to help species persist as the climate changes. Genome scans are powerful tools to understand adaptive landscapes, enabling us to correlate genetic diversity with environmental gradients while disentangling neutral from adaptive variation. However, low gene flow can lead to both local adaptation and highly structured populations, and is a major confounding factor for genome scans, resulting in an inflated number of candidate loci.Here, we compared candidate locus detection in a marine mollusc (Onithochiton neglectus), taking advantage of a natural geographical contrast in the levels of genetic structure between its populations. O. neglectus is endemic to New Zealand and distributed throughout an environmental gradient from the subtropical north to the subantarctic south. Due to a brooding developmental mode, populations tend to be locally isolated. However, adult hitchhiking on rafting kelp increases connectivity among southern populations.We applied two genome scans for outliers (Bayescan and PCAdapt) and two genotype–environment association (GEA) tests (BayeScEnv and RDA). To limit issues with false positives, we combined results using the geometric mean of q‐values and performed association tests with random environmental variables. This novel approach is a compromise between stringent and relaxed approaches widely used before, and allowed us to classify candidate loci as low confidence or high confidence.Genome scans for outliers detected a large number of significant outliers in strong and moderately structured populations. No high‐confidence GEA loci were detected in the context of strong population structure. However, 86 high‐confidence loci were associated predominantly with latitudinally varying abiotic factors in the less structured southern populations. This suggests that the degree of connectivity driven by kelp rafting over the southern scale may be insufficient to counteract local adaptation in this species.Our study supports the expectation that genome scans may be prone to errors in highly structured populations. Nonetheless, it also empirically demonstrates that careful statistical controls enable the identification of candidate loci that invite more detailed investigations. Ultimately, genome scans are valuable tools to help guide further research aiming to determine the potential of non‐model species to adapt to future environments.

Scallop IKK1 Responds to Bacterial and Virus-Related Pathogen Stimulation and Interacts With MyD88 Adaptor of Toll-Like Receptor Pathway Signaling.

IKK proteins are key signaling molecules in the innate immune system of animals, and act downstream of pattern recognition receptors. However, research on IKKs in invertebrates, especially marine mollusks, remains scarce. In this study, we cloned CfIKK1 gene from the Zhikong scallop (Chlamys farreri) and studied its function and the signaling it mediates. The open reading frame of CfIKK1 was 2190 bp and encoded 729 amino acids. Phylogenetic analysis showed that CfIKK1 belonged to the invertebrate IKKα/IKKβ family. Quantitative real-time PCR analysis revealed the ubiquitous expression of CfIKK1 mRNA in all scallop tissues and challenge with lipopolysaccharide, peptidoglycan, or poly(I:C) significantly upregulated the expression of CfIKK1. Co-immunoprecipitation assays confirmed the interaction of CfIKK1 with scallop MyD88 (Myeloid differentiation actor 88, the key adaptor of the TLR signaling pathway) via its N-terminal kinase domain. Additionally, CfIKK1 protein could form homodimers and even oligomers, with N-terminal kinase domain and C-terminal scaffold dimerization domain playing key roles in this process. Finally, the results of RNAi experiments showed that when the scallop IKK1 gene was suppressed, the expression of IRF genes also decreased significantly. In conclusion, CfIKK1 could respond to PAMPs challenge and interact with MyD88 protein of scallop TLR signaling, with the formation of CfIKK1 dimers or oligomers. At the same time, the results of RNAi experiments revealed the close regulatory relationship between IKK1 and IRF genes of scallop. Therefore, as a key signal transduction molecule and immune activity regulator, CfIKK1 plays important roles in the innate immune system of scallops.