Detection, movement and persistence of invertebrate eDNA in groundwater.

Sampling groundwater biodiversity is difficult because of limited access and issues with species identification. Environmental DNA (eDNA) provides a viable alternative to traditional sampling approaches, however limited knowledge of the abundance and fate of DNA in groundwater hinders the interpretation of data from these environments. Groundwater environments are dark and have lower oxygen concentrations and microbial activity than surface waters. Consequently, assumptions about DNA fate in surface ecosystems may not apply to groundwaters. Here, we test the longevity and transport of eDNA in groundwater within a static microcosm and a flow-through mesocosm. A variety of invertebrates were placed within a mesocosm and microcosm to enable DNA shedding, and then removed. DNA persisted for up to 5 weeks after their removal in the static experiment and was detected between 9 and 33 days in the flow-through experiment. Sediments and water both proved important for eDNA detection. Crustacean DNA was detected sporadically and unpredictably, whereas non-crustacean DNA was detected more frequently despite their lower densities. We suggest that detecting crustaceans poses a challenge to utilising eDNA approaches for stygofauna monitoring. This is confounded by the scarcity of sequences for stygofauna in reference databases. Further research is needed before eDNA alone can be routinely employed for stygofauna detection.

Long-term balancing selection for pathogen resistance maintains trans-species polymorphisms in a planktonic crustacean.

Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years.

Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Antiviral Shrimp lncRNA06 Possesses Anti-Tumor Activity by Inducing Apoptosis of Human Gastric Cancer Stem Cells in a Cross-Species Manner.

Virus infection causes the metabolic disorder of host cells, whereas the metabolic disorder of cells is one of the major causes of tumorigenesis, suggesting that antiviral molecules might possess anti-tumor activities by regulating cell metabolism. As the key regulators of gene expression, long non-coding RNAs (lncRNAs) play vital roles in the regulation of cell metabolism. However, the influence of antiviral lncRNAs on tumorigenesis has not been explored. To address this issue, the antiviral and anti-tumor capacities of shrimp lncRNAs were characterized in this study. The results revealed that shrimp lncRNA06, having antiviral activity in shrimp, could suppress the tumorigenesis of human gastric cancer stem cells (GCSCs) via triggering apoptosis of GCSCs in a cross-species manner. Shrimp lncRNA06 could sponge human miR-17-5p to suppress the stemness of GCSCs via the miR-17-5p-p21 axis. At the same time, shrimp lncRNA06 could bind to ATP synthase subunit beta (ATP5F1B) to enhance the stability of the ATP5F1B protein in GCSCs, thus suppressing the tumorigenesis of GCSCs. The in vivo data demonstrated that shrimp lncRNA06 promoted apoptosis and inhibited the stemness of GCSCs through interactions with ATP5F1B and miR-17-5p, leading to the suppression of the tumorigenesis of GCSCs. Therefore, our findings highlighted that antiviral lncRNAs possessed anti-tumor capacities and that antiviral lncRNAs could be the anti-tumor reservoir for the treatment of human cancers.

Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

How Oratosquilla oratoria compound eye response to the polarization of light: In the perspective of vision genes and related proteins.

The special rhabdom structure of the mid-band ommatidium in compound eye contributes to the mantis shrimp being the only animal species known to science that can recognize circularly polarized light (CPL). Although the number of mid-band ommatidium of Oratosquilla oratoria is reduced, the mid-band ommatidium still has orthogonal geometric interleaved rhabdom and short oval distal rhabdom, which may mean that the O. oratoria has weakened circular polarized light vision (CPLV). Here we explored the molecular mechanisms of how O. oratoria response to the polarization of light. Based on the specific expression patterns of vision-related functional genes and proteins, we suggest that the order of light response by O. oratoria compound eye was first natural light, then left-circularly polarized light (LCPL), linearly polarized light, right-circularly polarized light (RCPL) and dark. Meanwhile, we found that the expression levels of vision-related functional genes and proteins in O. oratoria compound eye under RCPL were not significantly different from those in DL, which may imply that O. oratoria cannot respond to RCPL. Furthermore, the response of LCPL is likely facilitated by the differential expression of opsin and microvilli - related functional genes and proteins (arrestin and sodium-coupled neutral amino acid transporter). In conclusion, this study systematically illustrated for the first time how O. oratoria compound eye response to the polarization of light at the genetic level, and it can improve the visual ecological theory behind polarized light vision evolution.

Long noncoding RNA profiling in hepatopancreas of Pacific white shrimp and its role in response to white spot syndrome virus infection.

Long noncoding RNA (lncRNA) is a potential regulator of biological processes, including immunity, reproduction, and development. Although several transcriptome studies have focused on responses of viral infections in several organisms, the role of lncRNAs in viral responses in shrimp is still unclear. Therefore, this work aimed to identify putative lncRNAs and study their role in white spot syndrome virus (WSSV) infection in white shrimp. The hepatopancreas transcriptome from WSSV infected shrimp was analyzed in silico to identify putative lncRNAs. Among 221,347 unigenes of the de novo assembled transcriptome, 44,539 putative lncRNAs were identified, 32 of which were differentially expressed between WSSV-infected and control shrimp. Five candidate lncRNAs were validated for their expressions in shrimp tissues and in response to WSSV infection. Lnc164 was chosen for further investigation of its role in WSSV infection. Knockdown of lnc164 prolonged survival of shrimp when challenged with WSSV, suggesting a role in shrimp immunity. In addition, lnc164 was not directly involved in the control of total hemocytes and viral loads in hemolymph of WSSV-infected shrimp. A set of lnc164-regulated genes was obtained by RNA sequencing among which 251 transcripts were differentially expressed between lnc164 knockdown and control shrimp. Six immune-related genes were validated for their expression profiles. Our work sheds light on lncRNA profiles in L. vannamei in response to WSSV infection and paves the way to a functional study of lnc164 in host antiviral response.

Exosomes drive ferroptosis by stimulating iron accumulation to inhibit bacterial infection in crustaceans.

Ferroptosis, characterized by iron-dependent cell death, has recently emerged as a critical defense mechanism against microbial infections. The present study aims to investigate the involvement of exosomes in the induction of ferroptosis and the inhibition of bacterial infection in crustaceans. Our findings provide compelling evidence for the pivotal role of exosomes in the immune response of crustaceans, wherein they facilitate intracellular iron accumulation and activate the ferroptotic pathways. Using RNA-seq and bioinformatic analysis, we demonstrate that cytochrome P450 (CYP) can effectively trigger ferroptosis. Moreover, by conducting an analysis of exosome cargo proteins, we have identified the participation of six-transmembrane epithelial antigen of prostate 4 in the regulation of hemocyte ferroptotic sensitivity. Subsequent functional investigations unveil that six-transmembrane epithelial antigen of prostate 4 enhances cellular Fe2+ levels, thereby triggering Fenton reactions and accelerating CYP-mediated lipid peroxidation, ultimately culminating in ferroptotic cell death. Additionally, the Fe2+-dependent CYP catalyzes the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid, which activates the peroxisome proliferator-activated receptor. Consequently, the downstream target of peroxisome proliferator-activated receptor, cluster of differentiation 36, promotes intracellular fatty acid accumulation, lipid peroxidation, and ferroptosis. These significant findings shed light on the immune defense mechanisms employed by crustaceans and provide potential strategies for combating bacterial infections in this species.

Comparative transcriptome analysis of eyes reveals the adaptive mechanism of mantis shrimp (oratosquilla oratoria) induced by a dark environment.

The light-dark cycle significantly impacts the growth and development of animals. Mantis shrimps (Oratosquilla oratoria) receive light through their complex photoreceptors. To reveal the adaptive expression mechanism of the mantis shrimp induced in a dark environment, we performed comparative transcriptome analysis with O. oratoria cultured in a light environment (Oo-L) as the control group and O. oratoria cultured in a dark environment (Oo-D) as the experimental group. In the screening of differentially expressed genes (DEGs) between the Oo-L and Oo-D groups, a total of 88 DEGs with |log2FC| > 1 and FDR < 0.05 were identified, of which 78 were upregulated and 10 were downregulated. Then, FBP1 and Pepck were downregulated in the gluconeogenesis pathway, and MKNK2 was upregulated in the MAPK classical pathway, which promoted cell proliferation and differentiation, indicating that the activity of mantis shrimp was slowed and the metabolic rate decreases in the dark environment. As a result, the energy was saved for its growth and development. At the same time, we performed gene set enrichment analysis (GSEA) on all DEGs. In the KEGG pathway analysis, each metabolic pathway in the dark environment showed a slowing trend. GO was enriched in biological processes such as eye development, sensory perception and sensory organ development. The study showed that mantis shrimp slowed down metabolism in the dark, while the role of sensory organs prominent. It provides important information for further understanding the energy metabolism and has great significance to study the physiology of mantis shrimp in dark environment.

Epigenetic Modulations for Prevention of Infectious Diseases in Shrimp Aquaculture.

Aquaculture assumes a pivotal role in meeting the escalating global food demand, and shrimp farming, in particular, holds a significant role in the global economy and food security, providing a rich source of nutrients for human consumption. Nonetheless, the industry faces formidable challenges, primarily attributed to disease outbreaks and the diminishing efficacy of conventional disease management approaches, such as antibiotic usage. Consequently, there is an urgent imperative to explore alternative strategies to ensure the sustainability of the industry. In this context, the field of epigenetics emerges as a promising avenue for combating infectious diseases in shrimp aquaculture. Epigenetic modulations entail chemical alterations in DNA and proteins, orchestrating gene expression patterns without modifying the underlying DNA sequence through DNA methylation, histone modifications, and non-coding RNA molecules. Utilizing epigenetic mechanisms presents an opportunity to enhance immune gene expression and bolster disease resistance in shrimp, thereby contributing to disease management strategies and optimizing shrimp health and productivity. Additionally, the concept of epigenetic inheritability in marine animals holds immense potential for the future of the shrimp farming industry. To this end, this comprehensive review thoroughly explores the dynamics of epigenetic modulations in shrimp aquaculture, with a particular emphasis on its pivotal role in disease management. It conveys the significance of harnessing advantageous epigenetic changes to ensure the long-term viability of shrimp farming while deliberating on the potential consequences of these interventions. Overall, this appraisal highlights the promising trajectory of epigenetic applications, propelling the field toward strengthening sustainability in shrimp aquaculture.

Taxonomy of a New Parasitic Euglenid, Euglenaformis parasitica sp. nov. (Euglenales, Euglenaceae) in Ostracods and Rhabdocoels.

Parasitic euglenids have rarely been studied. We found parasitic euglenids in two species of ostracods (Cyprinotus cassidula, Dolerocypris sinensis) and two species of rhabdocoels (Mesostoma lingua, Microdalyellia armigera) in a rice field. These parasites grew and proliferated inside the host body. These parasites had pellicle strips, one emergent flagellum, and a red stigma, but no chloroplasts, and showed euglenoid movement. Inside the living host, they did not have emergent flagella and moved only by euglenoid movement, but when the host died or the parasites were isolated from the host, they extended their flagella and switched to swimming movement. We conclude that the parasites found in the four hosts that we examined are of the same species, considering the morphological characteristics and identities in the nSSU and nLSU rDNA sequences of those parasites. Molecular phylogenetic analysis showed that the parasite formed a clade with the free-living photoautotrophic species of Euglenaformis, with moderate statistical support. Therefore, the parasite is a secondary osmotroph derived from a photoautotrophic ancestor. Based on the results of morphological observation and molecular phylogenetic analysis, we propose a new species of parasitic euglenid, Euglenaformis parasitica sp. nov.

A new species of the alpheid shrimp genus Athanas Leach, 1814 (Decapoda: Caridea) from the Sea of Japan.

A new species of the alpheid shrimp genus Athanas Leach, 1814 is described based on a holotype from Wakasa Bay, Sea of Japan side of Honshu, Japan, at a depth of 90 m. Athanas exilis n. sp. appears close to A. sydnyensis Anker & Ahyong, 2007, but is distinguished from the latter by some morphological features, including the different shape of the telson, the shorter antennular stylocerite, more strongly produced distal lamella of the antennal scaphocerite, and less developed armature of the merus of the major cheliped. Preliminary genetic analysis using the mitochondrial 16S rRNA gene was performed to compare interspecific genetic divergence within the genus.

Adverse effects of contamination by fipronil and its derivatives on growth, molting, and gene expression in the mysid crustacean, Americamysis bahia, in Japanese estuaries.

Concentrations of fipronil (Fip) and several of its derivatives were detected in samples from four rivers and four estuaries in Japan. LC-MS/MS analysis detected Fip and its derivatives, except for fipronil detrifluoromethylsulfinyl, in almost all samples. The total concentrations of the five compounds were approximately two-fold greater in river water (mean: 21.2, 14.1, and 9.95 ng/L in June, July, and September, respectively) compared to those in estuarine water (mean: 10.3, 8.67, and 6.71 ng/L, respectively). Fipronil, fipronil sulfone (Fip-S), and fipronil sulfide (Fip-Sf) represented more than 70 % of all compounds. This is the first report to demonstrate the contamination of estuarine waters of Japan by these compounds. We further investigated the potentially toxic effects of Fip, Fip-S, and Fip-Sf on the exotic mysid, Americamysis bahia (Crustacea: Mysidae). The lowest effective concentrations of Fip-S (10.9 ng/L) and Fip-Sf (19.2 ng/L) on mysid growth and molting was approximately 12.9- and 7.3-fold lower than Fip (140.3 ng/L), suggesting they had higher toxicity. Quantitative reverse transcription polymerase chain reaction analysis revealed that ecdysone receptor and ultraspiracle gene expression were not affected after 96-h of exposure to Fip, Fip-S, and Fip-Sf, suggesting that these genes may not be involved in the molting disruption induced by Fip, Fip-S, and Fip-Sf. Our findings suggest that environmentally relevant concentrations of Fip and its derivatives can disrupt the growth of A. bahia by promoting molting. However, further studies are required to elucidate its molecular mechanism.

Marine-montane transitions coupled with gill and genetic convergence in extant crustacean.

Marine-terrestrial transition represents an important aspect of organismal evolution that requires numerous morphological and genetic innovations and has been hypothesized to be caused by geological changes. We used talitrid crustaceans with marine-coastal-montane extant species at a global scale to investigate the marine origination and terrestrial adaptation. Using genomic data, we demonstrated that marine ancestors repeatedly colonized montane terrestrial habitats during the Oligocene to Miocene. Biological transitions were well correlated with plate collisions or volcanic island formation, and top-down cladogenesis was observed on the basis of a positive relationship between ancestral habitat elevation and divergence time for montane lineages. We detected convergent variations of convoluted gills and convergent evolution of SMC3 associated with montane transitions. Moreover, using CRISPR-Cas9 mutagenesis, we proposed that SMC3 potentially regulates the development of exites, such as talitrid gills. Our results provide a living model for understanding biological innovations and related genetic regulatory mechanisms associated with marine-terrestrial transitions.

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans.

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Development of a real-time PCR (qPCR) method for the identification of the invasive paddle crab Charybdis japonica (Crustacea, Portunidae).

Crabs can be transported beyond their native range via anthropogenic-mediated means such as aquarium trade, live seafood trade and shipping. Once introduced into new locations, they can establish persisting populations and become invasive, often leading to negative impacts on the recipient environment and native species. Molecular techniques are increasingly being used as complementary tools in biosecurity surveillance and monitoring plans for invasive species. Molecular tools can be particularly useful for early detection, rapid identification and discrimination of closely related species, including when diagnostic morphological characters are absent or challenging, such as early life stages, or when only part of the animal is available. In this study, we developed a species-specific qPCR assay, which targets the cytochrome c oxidase subunit 1 (CO1) region of the Asian paddle crab Charybdis japonica. In Australia, as well as many parts of the world, this species is considered invasive and routine biosecurity surveillance is conducted to reduce the risk of establishment. Through rigorous testing of tissue from target and non-target species we demonstrate that this assay is sensitive enough to detect as little as two copies per reaction and does not cross amplify with other closely related species. Field samples and environmental samples spiked with C. japonica DNA in high and low concentrations indicate that this assay is also a promising tool for detecting trace amounts of C. japonica eDNA in complex substrates, making it a useful complementary tool in marine biosecurity assessments.

CrusTome: a transcriptome database resource for large-scale analyses across Crustacea.

Transcriptomes from nontraditional model organisms often harbor a wealth of unexplored data. Examining these data sets can lead to clarity and novel insights in traditional systems, as well as to discoveries across a multitude of fields. Despite significant advances in DNA sequencing technologies and in their adoption, access to genomic and transcriptomic resources for nontraditional model organisms remains limited. Crustaceans, for example, being among the most numerous, diverse, and widely distributed taxa on the planet, often serve as excellent systems to address ecological, evolutionary, and organismal questions. While they are ubiquitously present across environments, and of economic and food security importance, they remain severely underrepresented in publicly available sequence databases. Here, we present CrusTome, a multispecies, multitissue, transcriptome database of 201 assembled mRNA transcriptomes (189 crustaceans, 30 of which were previously unpublished, and 12 ecdysozoans for phylogenetic context) as an evolving and publicly available resource. This database is suitable for evolutionary, ecological, and functional studies that employ genomic/transcriptomic techniques and data sets. CrusTome is presented in BLAST and DIAMOND formats, providing robust data sets for sequence similarity searches, orthology assignments, phylogenetic inference, etc. and thus allowing for straightforward incorporation into existing custom pipelines for high-throughput analyses. In addition, to illustrate the use and potential of CrusTome, we conducted phylogenetic analyses elucidating the identity and evolution of the cryptochrome/photolyase family of proteins across crustaceans.

The effect of C/N ratio and its frequent addition on commensal and pathogenic bacterial abundances in shrimp Litopeaneus vanname gut in a biofloc system: Ratio and frequent addition interaction matters.

The environmental biotic and abiotic factors form a complicated relationship with the host intestinal microbiota. In our study, we applied different levels of C/N ratio (10, 15, 20) and frequent addition times (once, twice, triple a day) in a factorial experimental design. GC/LC analysis of filtrated biofloc (BF) samples revealed the highest relative fold change for the untargeted bioactive molecules among different treatments, whereas the 16s rRNA analysis revealed the change in the shrimp gut microbiota composition. Based on the available literature on the relationship between the bioactive molecules and the available bacteria in this study, the next bioactive molecules were discussed. Proline was associated with Bacteroidota, Flavobacteriaceae, Gammaproteobacteria, and Flavobacteriales. Plumbagine was associated with Norcardiaceae. Phytosphingosin was associated with Bacteroidota. Phosphocholine compound was associated with Bacteroidota. The monobutyl ether, benzofuran, and piperidone were associated with Micobacteriaceae and Mycobacterium. Generally, C/N 15 and 20 once a day, and C/N 20 triple a day have showed a merit over other treatments in term of low pathogenic and unfavorable bacteria, and high commensal bacterial abundances. The revealed bioactive molecule composition showed the complicity of BF as a source for novel compounds as biosecurity agents in BF system. These molecules could be developed to feed additives upgrading the biosecurity level in aquaculture systems. Other bioactive molecules require future studies to reveal novel molecules in term of aquaculture biosecurity control.

Morphological and phylogenetic study of Acanthosquilla Manning, 1963 (Stomatopoda: Nannosquillidae) mantis shrimps, with description of two new species from the Ryukyu Islands, Japan.

Three species of nannosquillid mantis shrimps, including two new species, Acanthosquilla ryukyuensis n. sp. and Acanthosquilla shoheii n. sp., are described based on specimens collected from the Ryukyu Islands, Japan. The two new species resemble A. derijardi Manning, 1970, but can be distinguished from A. derijardi by the following features: 1) rostral plate anterolateral corner forms almost a right angle; 2) the distal tip of the antennular somite dorsal process reaches or slightly falls short of proximal half of rostral plate; and 3) eighth thoracic somite (= TS8) posterior margin is black medially. Furthermore, A. ryukyuensis n. sp. and A. shoheii n. sp. are easy to identify by the bifurcated lateral tooth of the telson, and by the posterodorsal pattern of the telson, respectively. In this study, molecular analyses based on partial sequences of mitochondrial 12S and 16S ribosomal DNA, cytochrome oxidase subunit I (COI), and the partial nuclear gene of 28S ribosomal DNA recovers these three species of Acanthosquilla and A. multifasciata (Wood-Mason, 1895) (the type species of the genus) in a single clade. The resulting trees also suggest the polyphyly of Nannosquillidae but with low nodal support. Detailed examinations of the morphological and color features and DNA barcoding results allowed us to delineate intraspecific variations and interspecific differences. The number and shape of setae under the dorsal spine of raptorial claw carpus was found to be useful in distinguishing A. shoheii n. sp. from A. derijardi and A. ryukyuensis n. sp., while combinations of the coloration of the rostral plate, posterior margin of TS8 and posterodorsal surface of telson are useful to distinguish the three species.