Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

Genetic diversity and population structure of Physella acuta (Gastropoda: Physidae) in Thailand using mitochondrial gene markers: COI and 16S rDNA.

Physella acuta is a freshwater snail native to North America. Understanding the phylogeography and genetic structure of P. acuta will help elucidate its evolution. In this study, we used mitochondrial (COI and 16S rDNA) and nuclear (ITS1) markers to identify the species and examine its genetic diversity, population structure, and demographic history of P. acuta in Thailand. Phylogenetic and network analyses of P. acuta in Thailand pertained to clade A, which exhibits a global distribution. Analysis of the genetic structure of the population revealed that the majority of pairwise comparisons showed no genetic dissimilarity. An isolation-by-distance test indicates no significant correlation between genetic and geographical distances among P. acuta populations, suggesting that gene flow is not restricted by distance. Demographic history and haplotype network analyses suggest a population expansion of P. acuta, as evidenced by the star-like structure detected in the median-joining network. Based on these results, we concluded that P. acuta in Thailand showed gene flow and recent population expansion. Our findings provide fundamental insights into the genetic variation of P. acuta in Thailand.

Cellular-resolution gene expression mapping reveals organization in the head ganglia of the gastropod, Berghia stephanieae.

Gastropod molluscs such as Aplysia, Lymnaea, and Tritonia have been important for determining fundamental rules of motor control, learning, and memory because of their large, individually identifiable neurons. Yet only a small number of gastropod neurons have known molecular markers, limiting the ability to establish brain-wide structure-function relations. Here we combine high-throughput, single-cell RNA sequencing with in situ hybridization chain reaction in the nudibranch Berghia stephanieae to identify and visualize the expression of markers for cell types. Broad neuronal classes were characterized by genes associated with neurotransmitters, like acetylcholine, glutamate, serotonin, and GABA, as well as neuropeptides. These classes were subdivided by other genes including transcriptional regulators and unannotated genes. Marker genes expressed by neurons and glia formed discrete, previously unrecognized regions within and between ganglia. This study provides the foundation for understanding the fundamental cellular organization of gastropod nervous systems.

The Families of Non-LTR Transposable Elements within Neritimorpha and Other Gastropoda.

Repeated sequences, especially transposable elements (TEs), are known to be abundant in some members of the important invertebrate class Gastropoda. TEs that do not have long terminal repeated sequences (non-LTR TEs) are frequently the most abundant type but have not been well characterised in any gastropod. Despite this, sequences in draft gastropod genomes are often described as non-LTR TEs, but without identification to family type. This study was conducted to characterise non-LTR TEs in neritimorph snails, using genomic skimming surveys of three species and the recently published draft genome of Theodoxus fluviatilis. Multiple families of non-LTR TEs from the I, Jockey, L1, R2 and RTE superfamilies were found, although there were notably few representatives of the first of these, which is nevertheless abundant in other Gastropoda. Phylogenetic analyses of amino acid sequences of the reverse transcriptase domain from the elements ORF2 regions found considerable interspersion of representatives of the four neritimorph taxa within non-LTR families and sub-families. In contrast, phylogenetic analyses of sequences from the elements' ORF1 region resolved the representatives from individual species as monophyletic. However, using either region, members of the two species of the Neritidae were closely related, suggesting their potential for investigation of phyletic evolution at the family level.

Population genetic structure of Pomacea canaliculata in China based on the COI and ITS1 genes.

Comprehending the phylogeography of invasive organisms enhances our insight into their distribution dynamics, which is instrumental for the development of effective prevention and management strategies. In China, Pomacea canaliculata and Pomacea maculata are the two most widespread and damaging species of the non-native Pomacea spp.. Given this species' rapid spread throughout country, it is urgent to investigate the genetic diversity and structure of its different geographic populations, a task undertaken in the current study using the COI and ITS1 mitochondrial and ribosomal DNA genes, respectively. The result of this study, based on a nationwide systematic survey, a collection of Pomacea spp., and the identification of cryptic species, showed that there is a degree of genetic diversity and differentiation in P. canaliculata, and that all of its variations are mainly due to differences between individuals within different geographical populations. Indeed, this species contains multiple haplotypes, but none of them form a systematic geographical population structure. Furthermore, the COI gene exhibits higher genetic diversity than the ITS1 gene. Our study further clarifies the invasive pathways and dispersal patterns of P. canaliculata in China to provide a theoretical basis.

Transcriptome Analysis of Different Aquaculture Substrates on the Immune Response of Babylonia areolata.

To assess the impact of different substrates in a recirculating water system on the immune response and antioxidant capacity of Babylonia areolata, we conducted a comparative analysis of the transcriptomes and antioxidant performance of the digestive glands in three substrate environments (sand-S group, ceramic granules-C group, and PVC breeding nest-P group). Transcriptome results revealed that the S group and P group exhibited the highest number of differentially expressed genes (DEGs), with a total of 2218 DEGs, including 928 upregulated and 1290 downregulated DEGs. The C group and P group had 1055 DEGs in common, with 316 upregulated and 739 downregulated DEGs. The C group and S group had the fewest DEGs, with 521 in total, including 303 upregulated and 218 downregulated DEGs. GO enrichment analysis showed that in the S vs P group, terms such as catalytic activity, membrane part, and cellular process were enriched with 287, 262, and 180 DEGs, respectively. In the C vs P group, binding, cellular process, and cell part were enriched with 146, 135, and 127 DEGs, respectively. In the C vs S group, catalytic activity, membrane part, and metabolic process were enriched with 90, 83, and 59 DEGs, respectively. Kegg enrichment analysis revealed significant changes in immune-related pathways in the S vs P group, including lysosome, phagosome, and leukocyte transendothelial migration, with 30, 13, and 10 enriched DEGs, respectively. In the C vs P group, phagosome, drug metabolism-other enzymes, and N-Glycan biosynthesis showed significant changes in immune-related pathways, with 9, 6, and 4 enriched DEGs, respectively. In the C vs S group, lysosome, PPAR signaling pathway, and fatty acid degradation exhibited significant changes in immune-related pathways, with 8, 4, and 3 enriched DEGs, respectively. Regarding antioxidant capacity, the S group showed significantly higher total T-AOC than the other experimental groups, while CAT, SOD, POD, and AKP were lower than in the C and P groups. The ACP level in the Sand group was not significantly different from the P group but significantly lower than the C group. In conclusion, substrate environments significantly influence the immune-related genes and key antioxidant enzyme activities in B. areolata.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

The Satellite DNA PcH-Sat, Isolated and Characterized in the Limpet Patella caerulea (Mollusca, Gastropoda), Suggests the Origin from a Nin-SINE Transposable Element.

Satellite DNA (sat-DNA) was previously described as junk and selfish DNA in the cellular economy, without a clear functional role. However, during the last two decades, evidence has been accumulated about the roles of sat-DNA in different cellular functions and its probable involvement in tumorigenesis and adaptation to environmental changes. In molluscs, studies on sat-DNAs have been performed mainly on bivalve species, especially those of economic interest. Conversely, in Gastropoda (which includes about 80% of the currently described molluscs species), studies on sat-DNA have been largely neglected. In this study, we isolated and characterized a sat-DNA, here named PcH-sat, in the limpet Patella caerulea using the restriction enzyme method, particularly HaeIII. Monomeric units of PcH-sat are 179 bp long, AT-rich (58.7%), and with an identity among monomers ranging from 91.6 to 99.8%. Southern blot showed that PcH-sat is conserved in P. depressa and P. ulyssiponensis, while a smeared signal of hybridization was present in the other three investigated limpets (P. ferruginea, P. rustica and P. vulgata). Dot blot showed that PcH-sat represents about 10% of the genome of P. caerulea, 5% of that of P. depressa, and 0.3% of that of P. ulyssiponensis. FISH showed that PcH-sat was mainly localized on pericentromeric regions of chromosome pairs 2 and 4-7 of P. caerulea (2n = 18). A database search showed that PcH-sat contains a large segment (of 118 bp) showing high identity with a homologous trait of the Nin-SINE transposable element (TE) of the patellogastropod Lottia gigantea, supporting the hypothesis that TEs are involved in the rising and tandemization processes of sat-DNAs.

Genetic sex determination in three closely related hydrothermal vent gastropods, including one species with intersex individuals.

Molluscs have undergone many transitions between separate sexes and hermaphroditism, which is of interest in studying the evolution of sex determination and differentiation. Here, we combined multi-locus genotypes obtained from restriction site-associated DNA (RAD) sequencing with anatomical observations of the gonads of three deep-sea hydrothermal vent gastropods of the genus Alviniconcha living in the southwest Pacific. We found that all three species (Alviniconcha boucheti, Alviniconcha strummeri, and Alviniconcha kojimai) share the same male-heterogametic XY sex-determination system but that the gonads of XX A. kojimai individuals are invaded by a variable proportion of male reproductive tissue. The identification of Y-specific RAD loci (found only in A. boucheti) and the phylogenetic analysis of three sex-linked loci shared by all species suggested that X-Y recombination has evolved differently within each species. This situation of three species showing variation in gonadal development around a common sex-determination system provides new insights into the reproductive mode of poorly known deep-sea species and opens up an opportunity to study the evolution of recombination suppression on sex chromosomes and its association with mixed or transitory sexual systems.

Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event.

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

Chromosome-scale Genome Assembly of the Rough Periwinkle Littorina saxatilis.

The intertidal gastropod Littorina saxatilis is a model system to study speciation and local adaptation. The repeated occurrence of distinct ecotypes showing different levels of genetic divergence makes L. saxatilis particularly suited to study different stages of the speciation continuum in the same lineage. A major finding is the presence of several large chromosomal inversions associated with the divergence of ecotypes and, specifically, the species offers a system to study the role of inversions in this divergence. The genome of L. saxatilis is 1.35 Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44 kb), and was quite incomplete, with a BUSCO completeness of 80.1% on the Metazoan dataset. A linkage map of one full-sibling family enabled the placement of 587 Mbp of the genome into 17 linkage groups corresponding to the haploid number of chromosomes, but the fragmented nature of this reference genome limited the understanding of the interplay between divergent selection and gene flow during ecotype formation. Here, we present a newly generated reference genome that is highly contiguous, with a N50 of 67 Mb and 90.4% of the total assembly length placed in 17 super-scaffolds. It is also highly complete with a BUSCO completeness of 94.1% of the Metazoa dataset. This new reference will allow for investigations into the genomic regions implicated in ecotype formation as well as better characterization of the inversions and their role in speciation.

Mitogenomes do not substantially improve phylogenetic resolution in a young non-model adaptive radiation of freshwater gastropods.

BACKGROUND: Species flocks in ancient lakes, and particularly those arising from adaptive radiation, make up the bulk of overall taxonomic and morphological diversity in these insular ecosystems. For these mostly young species assemblages, classical mitochondrial barcoding markers have so far been key to disentangle interspecific relationships. However, with the rise and further development of next-generation sequencing (NGS) methods and mapping tools, genome-wide data have become an increasingly important source of information even for non-model groups. RESULTS: Here, we provide, for the first time, a comprehensive mitogenome dataset of freshwater gastropods endemic to Sulawesi and thus of an ancient lake invertebrate species flock in general. We applied low-coverage whole-genome sequencing for a total of 78 individuals including 27 out of the 28 Tylomelania morphospecies from the Malili lake system as well as selected representatives from Lake Poso and adjacent catchments. Our aim was to assess whether mitogenomes considerably contribute to the phylogenetic resolution within this young species flock. Interestingly, we identified a high number of variable and parsimony-informative sites across the other 'non-traditional' mitochondrial loci. However, although the overall support was very high, the topology obtained was largely congruent with previously published single-locus phylogenies. Several clades remained unresolved and a large number of species was recovered polyphyletic, indicative of both rapid diversification and mitochondrial introgression. CONCLUSIONS: This once again illustrates that, despite the higher number of characters available, mitogenomes behave like a single locus and thus can only make a limited contribution to resolving species boundaries, particularly when introgression events are involved.

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a ß-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

Identification, diversity, and evolution analysis of Commd gene family in Haliotis discus hannai and immune response to biotic and abiotic stresses.

The Commd (Copper Metabolism gene MURR1 Domain) family genes play crucial roles in various biological processes, including copper and sodium transport regulation, NF-κB activity, and cell cycle progression. Their function in Haliotis discus hannai, however, remains unclear. This study focused on identifying and analyzing the Commd genes in H. discus hannai, including their gene structure, phylogenetic relationships, expression profiles, sequence diversity, and alternative splicing. The results revealed significant homology between H. discus hannai's Commd genes and those of other mollusks. Both transcriptome quantitative analysis and qRT-PCR demonstrated the responsiveness of these genes to heat stress and Vibrio parahaemolyticus infection. Notably, alternative splicing analysis revealed that COMMD2, COMMD4, COMMD5, and COMMD7 produce multiple alternative splice variants. Furthermore, sequence diversity analysis uncovered numerous missense mutations, specifically 9 in COMMD5 and 14 in COMMD10. These findings contribute to expanding knowledge on the function and evolution of the Commd gene family and underscore the potential role of COMMD in the innate immune response of H. discus hannai. This research, therefore, offers a novel perspective on the molecular mechanisms underpinning the involvement of Commd genes in innate immunity, paving the way for further explorations in this field.

Molecular characterization, expression and antibacterial function of a macin, HdMac, from Haliotis discus hannai.

Macins are a family of antimicrobial peptides, which play multiple roles in the elimination of invading pathogens. In the present study, a macin was cloned and characterized from Pacific abalone Haliotis discus hannai (Designated as HdMac). Analysis of the conserved domain suggested that HdMac was a new member of the macin family. In non-stimulated abalones, HdMac transcripts were constitutively expressed in all five tested tissues, especially in hemocytes. After Vibrio harveyi stimulation, the expression of HdMac mRNA in hemocytes was significantly up-regulated at 12 hr (P < 0.01). RNAi-mediated knockdown of HdMac transcripts affected the survival rates of abalone against V. harveyi. Moreover, recombinant protein of HdMac (rHdMac) exhibited high antibacterial activities against invading bacteria, especially for Vibrio anguillarum. In addition, rHdMac possessed binding activities towards glucan, lipopolysaccharides (LPS), and peptidoglycan (PGN), but not chitin in vitro. Membrane integrity analysis revealed that rHdMac could increase the membrane permeability of bacteria. Meanwhile, both the phagocytosis and chemotaxis ability of hemocytes could be significantly enhanced by rHdMac. Overall, the results showed that HdMac could function as a versatile molecule involved in immune responses of H. discus hannai.

Gonadal transcriptome analysis of genes related to sex differentiation and sex development in the Pomacea canaliculata.

As an invasive alien animal, Pomacea canaliculata poses a great danger to the ecology and human beings. Recently, there has been a gradual shift towards bio-friendly control. Based on the development of RNA interference and CRISPR technology as molecular regulatory techniques for pest control, it was determined if the knockout of genes related to sex differentiation in P. canaliculata could induce sterility, thereby helping in population control. However, the knowledge of sex differentiation- and development-related genes in P. canaliculata is currently lacking. Here, transcriptomic approaches were used to study the genes expressed in the two genders of P. canaliculata at various developmental stages. Gonad transcriptomes of immature or mature males and females were compared, revealing 12,063 genes with sex-specific expression, of which 6066 were male- and 5997 were female-specific. Among the latter, 581 and 235 genes were up-regulated in immature and mature females, respectively. The sex-specific expressed genes identified included GnRHR2 and TSSK3 in males and ZAR1 and WNT4 in females. Of the genes, six were involved in reproduction: CCNBLIP1, MND1, DMC1, DLC1, MRE11, and E(sev)2B. Compared to immature snail gonads, the expression of HSP90 and CDK1 was markedly reduced in gonadal. It was hypothesized that the two were associated with the development of females. These findings provided new insights into crucial genetic information on sex differentiation and development in P. canaliculata. Additionally, some candidate genes were explored, which can contribute to future studies on controlling P. canaliculata using molecular regulatory techniques.

Genome-Wide Identification and Transcriptome Analysis of the Hsp70 Gene Family in Monodonta labio Reveals Its Role in Response to Nanoplastics Stress.

For marine invertebrates, the disruption of organismal physiology and behavior by nanoplastics (NPs) has been extensively reported. Heat shock proteins (Hsps) are important for redundant protein breakdown, environmental changes, and intracellular protein transport. An exhaustive identification of Hsp70 genes and an experiment where different concentrations of NPs were stressed were performed to study how Hsp70 genes respond to NPs stress in Monodonta labio. Our results identified 15 members of Hsp70 within the genome of M. labio and provided insights into their responses to different concentrations of acute NP stress. Phylogenetic analyses revealed extensive amplification of the Hsp70 genes from the Hsc70 subfamily, with gene duplication events. As a result of NP stress, five of fifteen genes showed significant upregulation or downregulation. Three Hsp70 genes were highly expressed at an NP concentration of 0.1 mg/L, and no genes were downregulated. At 10 mg/L, they showed significant upregulation of two genes and significant downregulation of two genes. At 1 mg/L treatment, three genes were significantly downregulated, and no genes were significantly upregulated. Moreover, a purifying selection was revealed using a selection test conducted on duplicate gene pairs, indicating functional redundancy. This work is the first thorough examination of the Hsp70s in Archaeogastropoda. The findings improve knowledge of Hsp70s in molluscan adaptation to NP stress and intertidal living and offer essential data for the biological study of M. labio.

The genome of the rayed Mediterranean limpet Patella caerulea (Linnaeus, 1758).

Patella caerulea (Linnaeus, 1758) is a mollusc limpet species of the class Gastropoda. Endemic to the Mediterranean Sea, it is considered a keystone species due to its primary role in structuring and regulating the ecological balance of tidal and subtidal habitats. It is currently being used as a bioindicator to assess the environmental quality of coastal marine waters and as a model species to understand adaptation to ocean acidification. Here, we provide a high-quality reference genome assembly and annotation for P. caerulea. We generated ∼30 Gb of Pacific Biosciences high-fidelity data from a single individual and provide a final 749.8 Mb assembly containing 62 contigs, including the mitochondrial genome (14,938 bp). With an N50 of 48.8 Mb and 98% of the assembly contained in the 18 largest contigs, this assembly is near chromosome-scale. Benchmarking Universal Single-Copy Orthologs scores were high (Mollusca, 87.8% complete; Metazoa, 97.2% complete) and similar to metrics observed for other chromosome-level Patella genomes, highlighting a possible bias in the Mollusca database for Patellids. We generated transcriptomic Illumina data from a second individual collected at the same locality and used it together with protein evidence to annotate the genome. A total of 23,938 protein-coding gene models were found. By comparing this annotation with other published Patella annotations, we found that the distribution and median values of exon and gene lengths was comparable with other Patella species despite different annotation approaches. The present high-quality P. caerulea reference genome, available on GenBank (BioProject: PRJNA1045377; assembly: GCA_036850965.1), is an important resource for future ecological and evolutionary studies.

Molluscicidal effect mechanism study on metaldehyde to Pomacea canaliculate at low temperature.

BACKGROUND: Metaldehyde is a molluscicide commonly used to control Pomacea canaliculate. Its efficacy is significantly impacted by water temperature, although the underlying mechanisms have not been fully explored. RESULTS: In this study, we systematically investigated the temperature effect and molecular mechanisms of metaldehyde on P. canaliculata. The molluscicidal effect at various temperatures indicated that metaldehyde's molluscicidal activity significantly decreases with a drop in temperature. The LC50 value was only 458.8176 mg/L at 10 °C, while it surged to a high of 0.8249 mg/L at 25 °C. The impact of low temperature (10 °C) on metaldehyde's molluscicidal activity was analyzed via transcriptomics. The results revealed that the effect of low temperature primarily influences immunity, lipid synthesis, and oxidative stress. The expression of stress and immune-related genes, such as MANF, HSP70, Cldf7, HSP60, and PclaieFc, significantly increased. Furthermore, we studied the function of five target genes using RNA interference (RNAi) and discovered that Cldf7 and HSP70 could notably affect metaldehyde's molluscicidal effect. The mortality of P. canaliculata increased by 36.17% (72 h) after Cldf7 interference and by 48.90% (72 h) after HSP70 interference. CONCLUSION: Our findings demonstrate that low temperature can induce the extensive expression of the Cldf7 and HSP70 genes, resulting in a substantial reduction in metaldehyde's molluscicidal activity. © 2024 Society of Chemical Industry.