ABSTRACTGiant clams are key denizens of coral reef ecosystems, forming holobionts through symbiotic relationships with algae of the family Symbiodiniaceae, as in reef‐building corals. In this study, we performed a tissue‐specific microbiome analysis of the boring giant clam, Tridacna crocea and evaluated the impact of dark‐induced bleaching on its outer mantle bacterial community. Using 16S rRNA metabarcoding, Endozoicomonas was identified as the dominant bacterial genus in most tissues, particularly in gills, implying an important contribution to the giant clam holobiont. In contrast, in the outer mantle, where algal symbionts reside, the microbiome exhibited greater diversity, with a significant presence of carotenoid‐producing bacteria such as Rubritalea (Rubritaleaceae) and Muricauda (Flavobacteriaceae). These bacteria may protect symbiotic algae from light and thermal stresses, potentially enhancing holobiont resilience in coral reef environments. Although dark‐induced bleaching significantly reduced algal cell density, bacterial diversity remained largely unaffected, suggesting a robust bacterial association, independent of algal dynamics. This study highlights the potential ecological significance of Endozoicomonas and carotenoid‐producing bacteria in sustaining giant clam holobiont health and provides insights into microbial mechanisms that support stress tolerance in coral reef organisms.

Analysis of molecular identity and function of putative serotonin receptors in the Giant clam (Tridacna crocea) and the potential role of 5-HT1D-like receptor in reproduction

The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

Sex is an integral part of the evolution of species and often takes the form of intersex and sexual change in mollusks. As a major member of the deep-sea coral reef ecosystem, Tridacna crocea has complex gonadal development mechanisms and reproductive strategies, and the regulation model of spermatogenesis needs to be further explored. In this study, gonadal transcripts of T. crocea in resting, spermatogonia and sperm stage were evaluated. According to the statistics of bioinformatics analysis, a total of 10,260 DEGs were identified, including 3021 genes shared by three developmental stages. Through dynamic expression analysis, we identified the expression patterns and functional classification of genes that regulate spermatogenesis. These candidate genes were involved in regulatory mechanisms such as nucleotide binding, cell cycle, sexual reproduction and DNA replication. Subsequently, based on functional screening, PPI analysis was performed on the target gene cluster to learn the network relationships among candidate genes and other genes closely related to candidate genes. The results showed that in addition to candidate genes (zmcm6, gins2, mcm9, blm, mcm4, psmc3ip, haus, ccnb3, spice, rad1, rad17, ncapg, bub1b, lin9, tipin, and cks), several genes that have been shown to be associated with gonadal development were present in the network (such as fanci, aurka, kif20a and nek2), suggesting the reliability of the results. Finally, the expression levels of candidate genes between individuals at different developmental stages were compared with transcriptional expression profiles, which further proved that these candidate genes were related to T. crocea gonad development traits. These results make up for the lack of research on T. crocea gametogenesis, spermatogenesis and gonad development, and provide a reference for the research on Tridacna reproductive mechanism.

Tridacna crocea is an ecologically important marine bivalve inhabiting tropical coral reef waters. High quality and available genomic resources will help us understand the population structure and genetic diversity of giant clams. This study reports a high-quality chromosome-scale T. crocea genome sequence of 1.30 Gb, with a scaffold N50 and contig N50 of 56.38 Mb and 1.29 Mb, respectively, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences cover 71.60% of the total length, and a total of 25,440 protein-coding genes were annotated. A total of 1,963 non-coding RNA (ncRNA) were determined in the T. crocea genome, including 62 micro RNA (miRNA), 58 small nuclear RNA (snRNA), 83 ribosomal RNA (rRNA), and 1,760 transfer RNA (tRNA). Phylogenetic analysis revealed that giant clams diverged from oyster about 505.7 Mya during the evolution of bivalves. The genome assembly presented here provides valuable genomic resources to enhance our understanding of the genetic diversity and population structure of giant clams.

An overview over the international trade in live giant clams (Bivalvia: Tridacninae) from 2001 to 2019 was made based on worldwide import data from the CITES Trade Database. A distinction was made between trade records of wild-caught clams and maricultured ones. A general decline in the trade of wild giant clams was observed. Since these bivalves naturally live attached to rocky substrate in coral reefs and their forceful removal is harmful to surrounding corals, this trend helped to prevent damage to their natural environment. Possible reasons behind this decline are lower population numbers, trade restrictions, and a decrease in demand. Trade in wild-caught Tridacna crocea decreased, whereas that of T. maxima increased, taking over the former's position as most popular species. From 2015 onwards, over 50% of the source was made up by aquaculture, up to 2019. Aquaculture of live giant clams can be profitable, but there are still obstacles to overcome in order to ensure stable production numbers and long-term profit. With more research and support from governments, aquaculture can help to take pressure off wild populations of giant clams that are already suffering from exploitation for the sea-food market and souvenir industry, and also from habitat degradation due to climate change and other anthropogenic disturbances.

Megabenthos is a group of fauna that inhabits the benthic zone, which is the bottom or substrate of aquatic environments (both infauna and epifauna), with a body size exceeding 10 mm (1 cm). This study aims to analyze the abundance of megabenthos in the coral reef ecosystem, examine the relationship between megabenthos abundance and the physical and chemical parameters of the aquatic environment in the coral reef ecosystem, and analyze the relationship between megabenthos abundance and coral life forms in the waters of Gelasa Island. This research was conducted in September 2022 in the waters of Gelasa Island. Megabenthos data were collected using the Bentos Belt Transect (BBT) method, while coral data were collected using the Underwater Photo Transect (UPT) method. The study identified 10 species of megabenthos in the coral reef ecosystem of Gelasa Island. The species found at the research site include Diadema setosum, Diadema antillarium, Echinothrix calamaris, Drupella cornus, Drupella rugosa, Trochus conus, Tectus pyramis, Tridacna squamosa, Tridacna crocea, and Acanthaster planci. The percentage of live coral cover at the research site ranged from 26.60% to 71.00%. Megabenthos abundance was analyzed using Principal Component Analysis (PCA) to examine the relationship between megabenthos and the physical and chemical parameters of the aquatic environment and the relationship between megabenthos and coral life forms.

This research aims to find out the biodiversity and distribution patterns of clams and the relationship between clam abundance and coral reef conditions in the Waters of Kayu Angin Bira Island, Kepulauan Seribu. The research was conducted between April and July 2022, with field data collection taking place in September 2022 at four chosen stations. These stations were selected based on variations in water depth and were situated in the South, West, North, and East regions of the waters surrounding Kayu Angin Bira Island. The data collection was conducted using survey methods. Coral reef data collection using LIT (Line Intercept Transect) method and clam observation using belt transect. The results showed that in the waters of Bira Kayu Angin Island, 25 individuals of 2 species of bivalve class were found, namely Tridacna squamosa, Tridacna crocea. The highest relative abundance of clams is found at the eastern station which is 20% and the lowest relative abundance is found at the southern, western and northern stations which is only 8%. Low diversity index of Tridacninae Clams was discovered at the four stations, which ranged from 0.451 - 0.662 and the uniformity index at the four stations ranged from 0.650 - 0.954, indicates high population uniformity. Based on Morisita distribution index, which ranges from 0.4 - 0.74, the spatial distribution of Tridacninae clams could be assigned as uniform. The relationship between clams and coral reefs is 0.7284, shows that clams have a strong relationship with the condition of coral reefs.

Ocean ecosystems are highly effective in the recycling of energy and matter. Carbon fixation is almost recycled because net carbon burial in terrestrial systems and export to the ocean via rivers. Heterotrophs efficiently reprocess organic matter because they depend on the energy in organic matter. Withal, heterotrophs cannot use total organic energy because some is shunted into metabolites like ammonium, and under anoxic conditions into reduced substances such as sulphide. These reduced inorganic compounds are used by chemo (litho) autotrophs to obtain energy for inorganic carbon fixation. Host - associated microbial symbionts are critical to the conversion of inorganic carbon into organic biomass (Beinart, R.A., 2019). In the world’s oceans, Boring clams belongs to family Teredinidae, (Shipworms) with habitat of eating wood, assisted by cellulases from the intracellular symbiotic gammaproteobacteria that inhabit their gills. Other shipworms (Kuphus polythalamius) also relying on gill-dwelling gammaproteobacteria for sulphur oxidation (Altamia et al., 2020) and Methane Oxidation. The Symbionts of the gills Teredinibacter turnerae T7901 and similar strains are among the greatest sources of Biosynthetic Gene Clusters (BGCs), with content equivalent to well-known commercial manufacturers such as Streptomyces spp. This implies that shipworms might be a good source of new compounds for drug discovery (Altamia et al., 2020).

Many examples of mimicry have been reported in coral reef fishes of which the most well known is the mimicry of the bluestreak cleaner wrasse, Labroides dimidiatus by the false cleanerfish, Aspidontus taeniatus. To examine the effect of protective and aggressive mimicry of A. taeniatus, mortality and feeding behavior were compared with those of the non-mimic lance blenny, Aspidontus dussumieri, by field observations on the coral reefs of Miyako Island, Okinawa, southern Japan. Survival rate of A. taeniatus was more than twice higher than that of A. dussumieri, but the detected differences were not significant, and the effect of protective mimicry could not be determined. The benthic foods common to both species (the tubeworm, Spirobranchus giganteus, and the boring clam, Tridacna crocea) were very scarce in the study sites, and the feeding behavior of the two species was clearly different: A. dussumieri pecked at the bottom substrate, whereas A. taeniatus fed on fish fins and eggs of damselfish, regardless of body size. Our findings are the first documented evidence of the effect of aggressive mimicry on biting fish fins in relation to the availability of other foods not only in small but also in large A. taeniatus.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10164-022-00769-8.

Various studies on population genetics in the Indo-Pacific that include the Coral Triangle region have revealed phylogeographic patterns in marine species populations. However, little is known about the population structure and connectivity pattern among the coral reefs in four seas surrounding Malaysia: the Strait of Malacca (Andaman Sea), the South China Sea, the Sulu Sea, and the Sulawesi Sea. In view of this, we examined the genetic population structure of two invertebrate species: the blue sea star (Linckia laevigata) and the boring clam (Tridacna crocea) from seven localities across Malaysia, including Pulau Layang-Layang in the Spratly Islands. Additional samples of L. laevigata were obtained from the Davao Gulf, the Philippines. The analyses were based on partial mitochondrial cytochrome oxidase I sequences of L. laevigata and T. crocea. Populations of L. laevigata and T. crocea showed departure from neutrality, indicating selection or population expansion. However, a mismatch analysis suggested population expansion. Lack of genetic population structure in L. laevigata (ΦST = − 0.001, P = 0.399) implies high connectivity among the sampled reef sites. There was subtle genetic structuring in populations of T. crocea (ΦST = 0.032, P = 0.009), but AMOVA did not detect any structure in the a priori groupings. The genetic population structure and scale in connectivity are most likely attributed to the life history traits of the sea star and giant clam species, and also to the geological history and seasonal current patterns of the region.

P-element-induced wimpy testis (Piwi) is a key gene involved in germ cell development in a diverse range of organisms. However, in giant clams, the function of Piwi remains unclear. In the present study, we isolated the full-length cDNA of Piwi ortholog (Tc-Piwi1) and analyzed its expression patterns in the gonads of adult and juvenile Tridacna crocea. The results of qPCR showed that the transcript of Tc-Piwi1 was mainly expressed in gonad tissue. In addition, the relative expression level of Tc-Piwi1 increased with the proliferation of male and female germ cells during the adult gonad development stage, suggesting that Tc-Piwi1 might be involved in gametogenesis. In situ hybridization revealed that Tc-Piwi1 RNA was located in female and male germ cells and strongly expressed in male germ cells in the early stage. Furthermore, immunohistochemical experiments further confirmed that Tc-Piwi1 was mainly located in primordial germ cells (PGCs), germ stem cells (GSCs), and female and male germ cells of early development, so it could be used as a marker gene of T. crocea germ cells. Whole-mount in situ hybridization suggested that Tc-Piwi1 was of maternal origin and located in two clusters of cells in the trochophore-larvae stage, implying that these cells might be putative PGCs during the embryo development. Finally, Tc-Piwi1 was used as a molecular marker to elucidate the gonadal formation, sex differentiation, and gonadal maturation process of juvenile T. crocea for the first time in the Tridacna family. Collectively, all these results revealed that Tc-Piwi1 was involved in germline formation and sex differentiation in T. crocea.

Giant clams are marine bivalves that inhabit Indo-Pacific coral reefs. The boring giant clam, Tridacna crocea, exhibits bright and conspicuous mantle coloration based on the specialized cells (iridocytes) that generate structural colors. In order to illustrate the coloring mechanism of individual iridocytes, the reflection spectra curve of iridocytes was obtained by a micro-hyperspectral imager. TEM images were obtained to show the inner nanostructures of iridocytes. FDTD simulation was conducted to analyze the relationship between the color of iridocytes and the unique lamellar structure. We found that the laminae in the regular arrangement within cells govern the coloration of individual iridocytes. With the gradual increase of lamellar thickness and spacing, the color of the structure varies from bright violet to orange-red, forming a full visible spectrum. This study provides a new understanding of the various colors produced by individual iridocytes.

Abstract. Kusnadi A, Kurnianto D, Madduppa H, Zamani NP, Ibrahim PS, HernawanU E, Utami RT, Triandiza T. 2022. Genetic diversity and population structure of the boring giant clam (Tridacna crocea) in Kei Islands, Maluku, Indonesia. Biodiversitas 23: 1273-1282. Giant clams (Tridacninae) are ecologically important species in the coral reef ecosystems. They provide valuable functionsto traditional fisheries in Kei Islands, Maluku. However, their population isunder great pressure due to anthropogenic threats, such as overfishing and habitat degradation. To provide important data for devising effective conservation management strategies for giant clams, we investigated genetic diversity and population structure of the boring giant clams Tridacna croceain Kei Islands based on partial mitochondrial COI gene sequence. Tissue samples were collected from six sites: Kur, Dullah Laut, Tanimbar Kei, Dar, Labetawi, and Difur. We sequenced 477 base pairs of COI gene and identified 42 haplotypes and 52 polymorphic sites. Analysis of genetic diversity showed Dullah Laut and Dar had the highest genetic diversity. Population structure and genetic distance analysis showed unstructured populations with high genetic closeness among sites. This finding was also confirmed by the mixture pattern of the haplotype network. Further analysis using Bayesian models on gene flow revealed high genetic exchange among sites and that Dullah Population predominantly served as a source site for the other sites. This indicated a high probability of successful larval dispersal among sites. Based on these findings, we predict that the boring giant clams likely form a single population in Kei Islands. Our study warrants conservation priority for Dullah population as the main source of gene flow.

We present a new record and information on the distribution of the IUCN listed Tridacna crocea Lamarck, 1819 in the Philippines. The new record in Patnanungan Island extends the known distribution of this species by 80 km east of the nearest previously known occurrence. The collected specimens are found in shallow water at a depth of 3 m, exhibit a relatively small size, and showed the characteristic of completely burrowing its valves in coral substrates. DNA barcoding was also done, and the constructed phylogenetic tree demonstrated that the giant clams created a monophyletic group. &amp;nbsp;Tridacna crocea has a wide distribution and is relatively abundant throughout the Philippine reefs. We recommend updating the population status and stock assessment of giant clams in the country for local regulation and conservation management.

According to the RGB law display, the polymorphism of the giant clam mantle color pattern is through four iridocytes. The boring giant clam (Tridacna crocea) exhibits diverse mantle colors, including blue, green, purple, gold, and orange. In order to evaluate the genetic laws driving these mantle color patterns, a complete diallel cross between two color strains [blue strain (only blue iridocyte) and the yellow-green strain (yellow and green iridocytes)] was performed. Using a single-to-single mating system, two intra-strain crosses (BB and YY) and two reciprocal inter-strain crosses (BY and YB) were produced in triplicates. Higher fertilization rate and hatching rate were observed in all experimental groups, suggesting that there was no sperm–egg recognition barrier between the two strains. In the grow-out stage, the size of the reciprocal hybrids was larger than that of the two pure strains with a degree of heterosis. In addition, compared with the two pure strains, the hybrids have higher larval metamorphosis rate and higher survival rate. At 1 year of age, the mantle color pattern of pure strains showed 100% stable inheritance, while the reciprocal hybrids exhibited colorful patterns (a combination of blue, yellow, and green), suggesting that there was a genetic recombination of the mantle colors during the stable expression period. These results provide a theoretical basis for the formation of the mantle color of giant clam and its genetic segregation law, as well as provide guidance for genetic breeding of giant clams.

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules – in particular proteins – that are essential for molluscan shell formation.

The evaluation of culture performance and mantle coloration of two boring giant clam (Tridacna crocea) strains

Batu Bilis Island Kelarik Village is surrounded by shallow coral reefs with a fairly bright level of water brightness, making it a habitat for important economic biota in the form of clam shells (Tridacnidae). The purpose of this study is to identify the types of clam shells and determine the value of shellfish. Closing data is carried out on 9 (nine) points. Using the Benthos Belt Transect method, with an observation area of 140 m². The results were found 2 (two) types of clams, namely Hole Clams (Tridacna crocea) and Small Clams (Tridacna maxima). The highest density of Hole Clams is at point 9 (nine) with a value of 1.142.9 individual/ha, then the lowest density at point 7 (seven) 142,86 individual/ha. The highest density of Small Clams is at point 7 (seven) with a density value of 928,57 individual/ha and the lowest density at point 8 (eight) with a value of 71,43 individual/ha. The quality of physical-chemical parameters in Batu Bilis waters is still in the normal range for the life of clams shells such as temperature 29,4⁰C, salinity 29,5‰, current speed 0,08 m/s and brightness 100%.

In spite of being a protected species in the Philippines, the Tridacna crocea or crocus clam, the smallest among eight known giant clam species in the country, is getting rare in most reef areas of Palawan. However, a high density of this species has recently been noted in Rita Island, Ulugan Bay. A total of 44 photos with known dimensions having a total area of 13.26 m2 suitable habitats with clams were analyzed for size structure and density of T. crocea. In total, 215 T. crocea were noted with 84.91 (±25.6) mm average shell length (±sd). The average density (±sd) was 16.22 (±15.75) individual (ind)∙m-2 but the clams had occurred up to 17 individuals in 0.28 m2 or 59.91 ind∙m-2. This high density of T. crocea could be due to suitable environment and the absence of exploitation within the vicinity of the island resort. The importance of other resorts in biodiversity conservation may be investigated.