New Polymer Biocomposites Based on Biopoly(Ethylene Terephthalate) and Waste Mollusc Shells.

Currently, scientific studies have are focusing on environmentally friendly solutions, such as the effective use of waste in new green polymeric materials according to circular economy. Waste valorization is the main driving force for upcoming academic research. In this study, the impact of mussel particle size on reinforced biopoly(terphtalate ethylene) (bPET) is investigated. The waste filler was modified using NaOH. The filler content was 10 wt% and the same for all samples. The strength properties of the materials were determined in static tensile, bending and impact tests. The wetting angle was also analyzed for the obtained biocomposites. A low-cycle dynamic test was carried out to determine changes in dissipation energy and to observe the development of relaxation processes. This present study proves that preparation of new biocomposites based on waste mussels is an effective option in waste management.

Morphology and Phylogenetic Position of the Sargassum Nudibranch Scyllaea fulva Quoy & Gaimard, 1824 (Nudibranchia: Scyllaeidae): First Record in Hong Kong.

Scyllaeidae is a small group of nudibranchs comprising three genera (Scyllaea, Notobryon, and Crosslandia) with striking morphological similarities, making their identification challenging based on external features alone. Previous studies have highlighted the significance of central radular teeth in distinguishing Notobryon from Scyllaea and Crosslandia. The genus Scyllaea, commonly known as the sargassum nudibranch, currently consists of only two valid species, Scyllaea pelagica and Scyllaea fulva. These species inhabit seaweed Sargassum spp., feeding on hydroids. During a biodiversity survey conducted in April 2023, seven sargassum nudibranch individuals were collected from the seaweed Sargassum spp. at a depth of 2 meters in Tai She Wan through SCUBA diving. Initially, the specimens were misidentified based on their resemblance to Notobryon wardi and previous local records. However, thorough morphological and molecular examinations confirmed them to be Scyllaea fulva, representing the first record of this species in Hong Kong. Notably, our specimens lacked the blue spots observed in specimens from Thailand and the West Pacific Ocean, as reported in previous studies. Internally, a pineapple-like structure formed a honeycomb pattern on the surface of the masticatory edge of the jaw flap, with the presence of central radular teeth. A Maximum Likelihood tree analysis revealed Crosslandia to be the sister group of Scyllaea. Comparative analysis of intra-specific distances between individuals from the Philippines, French Polynesia, and Hong Kong indicated a close relationship between the Hong Kong specimens and those from the Philippines. Furthermore, we provide a detailed description of the external and internal morphology of Scyllaea fulva in this paper, integrating valuable morphological information for future species identification purposes.

Optimizing mangrove afforestation: Mollusc biodiversity comparisons reveal optimal mudflat-mangrove area ratio.

In recent decades, mangrove wetlands globally have suffered from human activities and climate change, leading to issues like area reduction, degraded ecological functions and declining biodiversity. Restoration efforts, primarily through mangrove afforestation (i.e. mangrove plantation in mudflats), have been widespread, yet they often overlook the significance of unvegetated mudflats. In addition, under the condition that the total area of suitable mudflats is limited, the problem of what is the threshold of mangrove forests and unvegetated mudflats to better protect mangrove biodiversity has not been solved. Therefore, this study conducted a field survey of molluscs in mangrove wetlands in Hainan Island in China and explored the relative importance of mangroves and unvegetated mudflats through taxonomic alpha diversity and functional diversity. The results showed that (1) mollusc abundance of unvegetated mudflats was notably higher than this of mangrove forests, and the species richness, functional richness and functional vulnerability were significantly lower than those of mangrove forests; (2) the abundance and functional vulnerability of molluscs were mainly affected by sediment properties (pH, interstitial water salinity, median diameter, total nitrogen, C/N ratio), while the species richness and functional richness of molluscs were primarily influenced by vegetation structure (plant density); and (3) retaining at least 20% of the unvegetated mudflat area could well protect the biodiversity of mangrove wetlands. To our knowledge, our study is the first to propose the proportion of mangrove forests and unvegetated mudflats on the basis of benthic biodiversity, providing theoretical support and decision-making reference for mangrove protection and restoration.

Genetic Variability in Mediterranean Coastal Ecosystems: Insights into Ostrea spp. (Bivalvia: Ostreidae).

Oysters are sessile, filter-feeding bivalve molluscs widely distributed in estuarine and coastal habitats worldwide. They constitute a valuable resource for fisheries and extensive aquaculture and provide essential ecological services. Yet, their genetic diversity and distribution remain understudied. The variability in shell morphology complicates species classification, which is influenced by environmental and genetic factors. Although molecular phylogenetics research has refined oyster taxonomy and identified approximately 100 extant species, numerous taxonomic uncertainties persist. In the present study, we aimed to document the occurrence of small flat oysters of the genus Ostrea along the Mediterranean coastal areas of Liguria and Sardinia (Italy). Specifically, 16S rRNA sequence data were used to identify Ostrea species. Our findings offer novel insights into the O. stentina species complex and O. neostentina, a new species in the Mediterranean coastal areas of Italy. The study data further our understanding of Ostrea species diversity, distribution, and evolutionary patterns.

Benchmarking sample pooling for epigenomics of natural populations.

DNA methylation (DNAm) is a mechanism for rapid acclimation to environmental conditions. In natural systems, small effect sizes relative to noise necessitates large sampling efforts to detect differences. Large numbers of individually sequenced libraries are costly. Pooling DNA prior to library preparation may be an efficient way to reduce costs and increase sample size, yet there are to date no recommendations in ecological epigenetics research. We test whether pooled and individual libraries yield comparable DNAm signals in a natural system exposed to different pollution levels by generating whole-epigenome data from two invasive molluscs (Corbicula fluminea, Dreissena polymorpha) collected from polluted and unpolluted localities (Italy). DNA of the same individuals were used for pooled and individual epigenomic libraries and sequenced with equivalent resources per individual. We found that pooling effectively captures similar genome-wide and global methylation signals as individual libraries, highlighting that pooled libraries are representative of the global population signal. However, pooled libraries yielded orders of magnitude more data than individual libraries, which was a consequence of higher coverage. We would therefore recommend aiming for a high initial coverage of individual libraries (15×) in future studies. Consequently, we detected many more differentially methylated regions (DMRs) with the pooled libraries and a significantly lower statistical power for regions from individual libraries. Computationally pooled data from the individual libraries produced fewer DMRs and the overlap with wet-lab pooled DMRs was relatively low. We discuss possible causes for discrepancies, list benefits and drawbacks of pooling, and provide recommendations for future epigenomic studies.

Evolution of chitin-synthase in molluscs and their response to ocean acidification.

Chitin-synthase (CHS) is found in most eukaryotes and has a complex evolutionary history. Research into CHS has mainly been in the context of biomineralization of mollusc shells an area of high interest due to the consequences of ocean acidification. Exploration of CHS at the genomic level in molluscs, the evolution of isoforms, their tissue distribution, and response to environmental challenges are largely unknown. Exploiting the extensive molecular resources for mollusc species it is revealed that bivalves possess the largest number of CHS genes (12-22) reported to date in eukaryotes. The evolutionary tree constructed at the class level of molluscs indicates four CHS Type II isoforms (A-D) probably existed in the most recent common ancestor, and Type II-A (Type II-A-1/Type II-A-2) and Type II-C (Type II-C-1/Type II-C-2) underwent further differentiation. Non-specific loss of CHS isoforms occurred at the class level, and in some Type II (B-D groups) isoforms the myosin head domain, which is associated with shell formation, was not preserved and highly species-specific tissue expression of CHS isoforms occurred. These observations strongly support the idea of CHS functional diversification with shell biomineralization being one of several important functions. Analysis of transcriptome data uncovered the species-specific potential of CHS isoforms in shell formation and a species-specific response to ocean acidification (OA). The impact of OA was not CHS isoform-dependent although in Mytilus, Type I-B and Type II-D gene expression was down-regulated in both M. galloprovincialis and M. coruscus. In summary, during CHS evolution the gene family expanded in bivalves generating a large diversity of isoforms with different structures and with a ubiquitous tissue distribution suggesting that chitin is involved in many biological functions. These findings provide insight into CHS evolution in molluscs and lay the foundation for research into their function and response to environmental changes.

Molluscs toxin peptides in cardiovascular disease management.

This letter emphasizes the potential of mollusc toxin peptides as innovative treatments for cardiovascular diseases (CVDs). Given the rising incidence of CVDs and the limitations of current therapies, new approaches are essential. Molluscs produce bioactive peptides that show promising anti-hypertensive, anti-thrombotic, and cardio-protective properties. For example, conotoxins from marine snails inhibit voltage-gated calcium channels, indicating their potential as anti-hypertensive agents. Advancements in peptide engineering can address challenges related to bioavailability and stability. Increased research and collaboration are needed to investigate these peptides' mechanisms, therapeutic potential, and safety, which could lead to groundbreaking CVD treatments.

Understanding the structure and function of mollusc toxins: a gateway to novel biomedical applications.

Mollusc toxins, which are diverse bioactive substances generated by marine invertebrates, present significant potential for biomedical advancements. These structurally diverse toxins, including conotoxins and tetrodotoxin, specifically target physiological processes and serve as blueprints for new therapeutic agents. High-resolution methods have clarified their structures, offering valuable insights into their mechanisms of action. Functional research has broadened our comprehension of biological processes and discovered new drug targets. Additionally, the ecological roles of these toxins highlight their importance in marine ecosystems. Ongoing research into mollusc toxins will deepen our understanding of marine biodiversity and aid in developing innovative treatments.

Continuous variation in the shell colour of the snail Cepaea nemoralis is associated with the colour locus of the supergene.

While the shell of the land snail Cepaea nemoralis is typically classed as yellow, pink, or brown, the reality is that colour variation is continuously distributed. To further understand the origin of the continuous variation, we used crosses of C. nemoralis to compare quantitative measures of the colour with the inferred genotype of the underlying supergene locus. We also used a recently developed linkage map to find quantitative trait loci that may influence colour. The results show that the colour locus of the supergene-at around 31.385 cM on linkage group 11-is involved in determining the quantitative chromatic differences that are perceptible to human vision. We also found some evidence that variation within colour classes may be due to allelic variation at or around the supergene. There are likely other unlinked loci involved in determining colour within classes, but confirmation will require greater statistical power. Although not investigated here, environmental factors, including diet, may also impact upon variation within colour types.

Chemical Treatments on Invasive Bivalve, Corbicula fluminea.

The Asian clam Corbicula fluminea is a native aquatic species in Eastern Asia and Africa but has become one of the ecologically and economically harmful invasive species in aquatic ecosystems in Europe, North America, and South America. Due to their natural characteristics as a hermaphroditic species with a high fecundity and dispersal capacity, Asian clams are extremely difficult to eradicate once they have infiltrated a waterbody. This is an emerging issue for states in the Northeastern United States, as Asian clams expand their range farther North due to climate change. There has been extensive research conducted to develop chemical treatments for reactively controlling invasive mollusc populations and proactively preventing their further spread. However, treatments are mostly targeted toward biofouling bivalves in industrial settings. A comprehensive review of Asian clam chemical treatments used in natural open-water systems was performed to evaluate molluscicides and identify the toxicity ranges of emerging treatments that maximize Asian clam mortality and minimize the negative impact on water quality and non-target species. The potential chemical applications in Asian clam control and management are summarized in this report to assist resource managers and practitioners in invasive Asian clam management.

High heteroplasmy is associated with low mitochondrial copy number and selection against non-synonymous mutations in the snail Cepaea nemoralis.

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Mid-Holocene marine faunas from the Bangkok Clay deposits in Nakhon Nayok, the Central Plain of Thailand.

Based on several field investigations, many molluscan shells and chondrichthyan teeth, together with other invertebrate and actinopterygian remains were found from the marine Bangkok Clay deposits in Ongkharak, Nakhon Nayok, at a depth of ~ 5-7 m below the topsoil surface. Animal macrofossils recovered from these Holocene marine deposits were identified and their chronological context was investigated in order to reconstruct the paleoenvironments of the area at that time. The majority of marine fossils recovered from the site consist of molluscs, with a total of 63 species identified. Other invertebrate species include a stony coral, a mud lobster, barnacles, and a sea urchin. The vertebrates are represented by fish remains, including carcharhinid shark teeth from at least nine species, stingray and trichiurid teeth, and one sciaenid otolith. The molluscan fauna indicates that the paleoenvironments of the area corresponded to intertidal to sublittoral zones, where some areas were mangrove forests and intertidal mudflats. The fish fauna is dominated by the river shark Glyphis, indicating freshwater influences and possibly occasional brackish conditions. The carbon-14 analysis of mollusc and charcoal remains shows that deposition of the marine sediment sequence began during the mid-Holocene, spanning approximately from 8,800 to 5,300 cal yr BP. This study provides in-depth insights into the diversity of fishes, marine molluscs, and other invertebrates from the Bangkok Clay deposits, supporting the existence of a marine transgression onto the Lower Central Plain of Thailand during the mid-Holocene.

Comparative transcriptomics revealed parallel evolution and innovation of photosymbiosis molecular mechanisms in a marine bivalve.

Photosymbioses between heterotrophic hosts and autotrophic symbionts are evolutionarily prevalent and ecologically significant. However, the molecular mechanisms behind such symbioses remain less elucidated, which hinders our understanding of their origin and adaptive evolution. This study compared gene expression patterns in a photosymbiotic bivalve (Fragum sueziense) and a closely related non-symbiotic species (Trigoniocardia granifera) under different light conditions to detect potential molecular pathways involved in mollusc photosymbiosis. We discovered that the presence of algal symbionts greatly impacted host gene expression in symbiont-containing tissues. We found that the host immune functions were suppressed under normal light compared with those in the dark. In addition, we found that cilia in the symbiont-containing tissues play important roles in symbiont regulation or photoreception. Interestingly, many potential photosymbiosis genes could not be annotated or do not exhibit orthologues in T. granifera transcriptomes, indicating unique molecular functions in photosymbiotic bivalves. Overall, we found both novel and known molecular mechanisms involved in animal-algal photosymbiosis within bivalves. Given that many of the molecular pathways are shared among distantly related host lineages, such as molluscs and cnidarians, it indicates that parallel and/or convergent evolution is instrumental in shaping host-symbiont interactions and responses in these organisms.

Histopathological Lesions Caused by a Digenean Trematode in a Pest Apple Snail, Pomacea canaliculata, in Its Native Geographic Distribution Area.

Pomacea canaliculata is one of the most dangerous invasive species. Morphological and molecular analyses have revealed that a digenean species belonging to the family Echinostomatidae parasitizes this snail at two sites in Buenos Aires Province, Argentina, South America. Molecular results confirmed that the species belongs to a genus closely related to Patagifer. Analysis of the 28S rDNA showed that the sequences of the rediae and metacercariae are identical, indicating that the apple snail acts as the first and second intermediate host. The cercariae may encyst as metacercaria inside the redia and also emerge and re-infect the same snail or another snail. The prevalence of digeneans was higher in one of the sampling locations (15.1% vs. 0.72%), probably because the bird species that acts as the definitive host is more abundant in that area. Histopathological examination showed that the parasite quickly invades multiple host organs (gills, intestines, albumen gland, lung, kidney, and mantle border) besides the gonad and digestive gland, as is usual in digeneans. In addition, the partial or total castration of snails was observed in cases of moderate and high infection intensity. In males, there was loss of integrity in testicular tubules, while in females, the replacement of ovarian tissue by rediae was found.

Whole-genome assembly of a novel invertebrate herpesvirus from the gastropod Babylonia areolata.

Molluscan herpesviruses cause disease in species of major importance to aquaculture and are the only known herpesviruses to infect invertebrates, which lack an adaptive immune system. Understanding the evolution of malacoherpesviruses in relation to their hosts will likely require comparative genomic studies on multiple phylogenetic scales. Currently, only two malacoherpesvirus species have genomes that have been fully assembled, which limits the ability to perform comparative genomic studies on this family of viruses. In the present study, we fully assemble a herpesvirus from Illumina and Nanopore sequence data that were previously used to assemble the genome of the gastropod Babylonia areolata. We tentatively assign this novel herpesvirus to the genus Aurivirus within the family Malacoherpesviridae based on a phylogenetic analysis of DNA polymerase. While structurally similar to other malacoherpesvirus genomes, a synteny analysis of the novel herpesvirus with another Aurivirus species indicates that genomic rearrangements might be an important process in the evolution of this genus. We anticipate that future complete assemblies of malacoherpesviruses will be a valuable resource in comparative herpesvirus research.

Semi-purified Antimicrobial Proteins from Oyster Hemolymph Inhibit Pneumococcal Infection.

Pneumococcal infections caused by Streptococcus pneumoniae are a leading cause of morbidity and mortality globally, particularly among children. The ability of S. pneumoniae to form enduring biofilms makes treatment inherently difficult, and options are further limited by emerging antibiotic resistance. The discovery of new antibiotics, particularly those with antibiofilm activity, is therefore increasingly important. Antimicrobial proteins and peptides (AMPs) from marine invertebrates are recognised as promising pharmacological leads. This study determined the in vitro antibacterial activity of hemolymph and unique protein fractions from an Australian oyster (Saccostrea glomerata) against multi-drug-resistant S. pneumoniae. We developed a successful method for hemolymph extraction and separation into 16 fractions by preparative HPLC. The strongest activity was observed in fraction 7: at 42 µg/mL protein, this fraction was bactericidal to S. pneumoniae and inhibited biofilm formation. Proteomic analysis showed that fraction 7 contained relatively high abundance of carbonic anhydrase, cofilin, cystatin B-like, and gelsolin-like proteins, while surrounding fractions, which showed lower or no antibacterial activity, contained these proteins in lower abundance or not at all. This work supports traditional medicinal uses of oysters and contributes to further research and development of novel hemolymph/AMP-based treatments for pneumococcal infections.

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.

An optimized protocol for routine development of cell culture from adult oyster, Crassostrea madrasensis.

Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.

Nematodes associated with terrestrial gastropod molluscs in Belgium and additional characterisation of Pellioditis californica and P. hermaphrodita.

A survey for slug- and snail-associated nematodes was conducted in forests, parks, botanical gardens, and nature reserves at 13 localities in Belgium to uncover more diversity of gastropod mollusc-associated nematodes and to characterise Pellioditis populations found in the country. A total of 319 slugs and snails belonging to nine species were examined. Arion vulgaris was the most commonly found mollusc species in this study (eight locations), and 19.4% of the examined mollusc specimens were found infected by nematodes. The highest prevalence of nematodes was observed in Cornu aspersum (60%) followed by A. vulgaris (34.8%), Limax maximus (28.6%), and Cepaea sp. (20%). Eleven nematode species belonging to eight families were isolated and identified from the mollusc hosts including Alloionema appendiculatum, Angiostoma dentiferum, A. gandavense, Angiostrongylus vasorum, Cosmocerca longicauda, Panagrolaimus cf. subelongatus, Pellioditis californica, P. hermaphrodita, Rhabditis sp., Tetrameres cf. fissispina, and Troglostrongylus cf. brevior.Pellioditis was the most commonly found nematode genus (at nine localities) and C. longicauda and P. californica were reported in Belgium for the first time. Co-infections of more than one nematode species were observed in eight (2.5%) molluscs specimens. Most co-infections consisted of two nematode species. In one A. vulgaris specimen, a co-infection of three nematode species (A. vasorum, P. hermaphrodita, and Tetrameres cf. fissispina) was observed. Four ex vivo cultures of P. californica and six ex vivo cultures of P. hermaphrodita were established from single hermaphrodites, and both species were described based on light microscopy, scanning electron microscopy, and morphometric, morphological, and molecular data.

Molecular and behavioural responses of the mussel Mytilus edulis exposed to a marine heatwave.

Marine heatwaves (MHW) threaten marine organisms and tend to increase in frequency and intensity. We exposed the blue mussel Mytilus edulis to a MHW lasting 23 days, including two 10-d periods of thermal intensity increase of +5 °C (20 °C-25 °C) interspersed by 1 day back to 20 °C, followed by a 4-d recovery period. We investigated behaviour responses of mussels and gene expression changes relative to the circadian rhythm (Per), oxidative stress (SOD), cellular apoptosis (CASP3), energy production (ATPs), and general stress response (hsp70). Results showed that the MHW disturbed the valve activity of mussels. Particularly, mussels increased the number of valve micro-closures, showing a stressful state of organisms. Mussels also decreased Per, CASP3, ATPs, and Hsp70 gene expression. Some behavioural and molecular effects persisted after the MHW, suggesting a limited recovery capacity of individuals. This work highlighted the vulnerability of M. edulis to a realistic MHW.