Mitochondrial 16S Research Articles

Metabetaeus lapillicola, a new species of alpheid shrimp (Decapoda: Caridea) from Japan, inhabiting unusual pebble beaches.

The alpheid shrimp genus Metabetaeus Borradaile, 1899 was previously known from two species associated with anchialine pools and caves, M. minutus (Whitelegge, 1897) and M. lohena Banner & Banner, 1960, and one species inhabiting in coral reef, M. mcphersonae Anker, 2010. In this study, a new species of the genus is described based on fourteen specimens collected from Shizuoka and Mie Prefectures, Honshu, central Japan. Metabetaeus lapillicola n. sp. inhabits interstitial spaces among small pebbles deposited in lower intertidal to upper subtidal zone, representing rather unusual habitat not only for Metabetaeus, but even for carideans. It is distinguished from three congeneric species by the presence of a pterygostomial spine on the carapace, greatly unequal chelipeds with the major one being substantially elongate, and the lack of a distinct distolateral spine on the uropodal endopod. Phylogenetic analyses using the mitochondrial 16S rRNA and COI genes support the recognition of the new species. The generic diagnosis of Metabetaeus is emended to accommodate the new species.

Detection of Babesia Spp., Theileria Spp., and Anaplasma Ovis in Ornithodoros Lahorensis from Southern Xinjiang, China.

Ticks are blood-sucking arthropods that have negative economic impacts and can spread a variety of diseases through their bites. There are few reports on soft ticks (Acari: Argasidae) and tick-borne pathogens in southern Xinjiang, China. This investigation supplements the available information for this region and is concerned with an argasid tick, apicomplexan parasites of the Babesia and Theileria genera and a bacterium of the Anaplasma genus. In this study, 330 soft ticks were collected from nine sampling sites in southern Xinjiang between 2020 and 2021. The ticks were identified according to their morphological characteristics and confirmed as Ornithodoros lahorensis using mitochondrial 16S rDNA sequences. Babesia and Theileria were identified at the species level based on two fragments of the 18S rRNA gene, and one set of primers targeting the 16S rRNA gene was used to identify the Anaplasma genus. Among the 330 samples, one Babesia species (Babesia sp.), two Theileria species (T. ovis and T. annulata), and one Anaplasma (A. ovis) species were detected. This study provides fundamental evidence for the occurrence of Babesia, Theileria and Anaplasma spp. in soft ticks. To the best of our knowledge, this is the first report of the detection of Babesia sp. and T. annulata in O. lahorensis. Therefore, the potential threat of soft ticks to livestock and humans should not be ignored.

New Finding of Eggs and Leptocephalus for Muraenesox cinereus off Jeju Island, Korea and Its Relation to Environmental Variables

The daggertooth pike conger, Muraenesox cinereus, is an important demersal fish species in East Asia but the catch amount has declined in recent years. Spawning areas of M. cinereus have not yet been determined; identifying these have serious implications for resource management. Ichthyoplankton surveys are an effective method of distinguishing fish spawning areas and periods. Fish eggs were sampled from the waters adjacent to South Korea in August 2020 and 2022 using a Multiple Opening/Closing Net and Environmental Sensing System or a bongo net. In 2021, M. cinereus eggs were not collected. Three unidentified eggs (2.0–2.2 mm in diameter) were collected from the southeastern sea of Jeju Island at a seawater depth of 20–30 m and temperature of 20–22 °C. Muraenesox cinereus preleptocephali were gathered at 10–20 m depths from Jeju Island’s southernmost and eastern sea areas. The eggs and preleptocephali were identified as M. cinereus by their mitochondrial DNA 16S rRNA sequences. This is a new finding of eggs and leptocephalus of Muraenesox cinereus off Jeju Island, South Korea, which increases our understanding of the recruitment process of M. cinereus to facilitate resource management and species conservation.

A New Conditionally Immortalized Nile Tilapia (Oreochromis niloticus) Heart Cell Line: Establishment and Functional Characterization as a Promising Tool for Tilapia Myocarditis Studies

A new cell line named the tilapia heart cell line (TAH-11) was established from the heart of tilapia (Oreochromis niloticus) by enzymatic digestion and mechanical separation. The TAH-11 cell line has been stably subcultured for over 80 generations and resuscitated after being frozen in liquid nitrogen for six months, with exuberant cell growth. The optimal culture condition of TAH-11 is L-15 medium supplemented with 10% fetal bovine serum at 25 °C. Sequencing of the mitochondrial 18S rRNAs confirmed that TAH-11 cells were derived from the tilapia. TAH-11 was also identified as a myocardial cell line based on the mRNA expression of the troponin I, α-actin and myoglobin genes. Simultaneously, TAH-11 could be successfully transfected with the GFP reporter gene, suggesting that the TAH-11 cell line could be used for exogenous gene expression in vitro. The TAH-11 cells showed susceptibility to CGSIV, which was demonstrated by the presence of a severe cytopathic effect, suggesting that the TAH-11 cell line is an ideal tool for studying host–virus interaction and potential vaccines. In addition, the expression of inflammatory factors in TAH-11 cells can be remarkably induced following Streptococcus agalactiae or Streptococcus iniae. The present data lays a foundation to further explore the mechanism of how Streptococcus spp. causes tilapia myocarditis.

Mare-MAGE curated reference database of fish mitochondrial genes

Biodiversity assessment approaches based on molecular biology techniques such as metabarcoding, RAD-seq, or SnaPshot sequencing are increasingly applied in assessing marine and aquatic ecosystems. Here we present a new reference database for fish meta-barcoding based on mitochondrial genes. The Mare-MAGE database contains quality-checked sequences of the mitochondrial 12S ribosomal RNA and Cytochrome c Oxidase I gene. All sequences were obtained from the National Center for Biotechnology Information- GenBank (NBCI-GenBank), the European Nucleotide Archive (ENA), AquaGene Database and BOLD database, and have undergone intensive processing. They were checked for false annotations and non-target anomalies, according to the Integrated Taxonomic Information System (ITIS) and FishBase. The dataset is compiled in ARB-Home, FASTA and Qiime2 formats, and is publicly available from the Mare-MAGE database website (http://mare-mage.weebly.com/). It includes altogether 231,333 COI and 12S rRNA gene sequences of fish, covering 19,506 species of 4,058 genera and 586 families.

The smallest of its kind: Description of a new cryptic Amnirana species (Amphibia, Anura, Ranidae) from West African rainforests

The distribution of the White-Lipped Frog Amnirana albolabris was long assumed to extend from eastern-central to western African rainforests. However, recent molecular phylogenetic analyses revealed that this taxon likely represents several undescribed species. Because the name-bearing types were collected in Gabon, the distantly related West African populations clearly represent an undescribed species that partly occurs in sympatry with Amnirana fonensis. Based on an integrative taxonomic approach, including molecular, morphological, and acoustic data, we describe the 'albolabris' populations from the Upper Guinean Forest Zone as a new species, and redescribe the morphologically similar A. fonensis on the bases of a larger series of genotyped individuals, including the first known females. We also provide new biological information for A. fonensis, including their advertisement call, habitat, and reproductive data. The new species is sister to A. fonensis and the two species differ by 5.8% in the mitochondrial 16S ribosomal RNA gene. Morphologically, males of the new species are smaller and have relatively smaller humeral glands. Females of the new species seem to have shorter legs than A. fonensis. In comparison to A. fonensis, the advertisement call of the new species has a higher dominant frequency and more pronounced frequency modulation. The two species differ in their distribution and habitat preferences, as revealed by environmental niche modelling. Whereas the new species is restricted to the Upper Guinean forests and thus is a true lowland-rainforest inhabitant, A. fonensis lives predominantly in Guinean montane forests in the forest-savanna mosaic zone.

Genetic diversity of the mantellid frog Blommersia blommersae, and description of a new anuran species from south-eastern Madagascar

ABSTRACT A range-wide assessment of genetic variation in one mitochondrial (16S rRNA) and one nuclear-encoded (Rag-1) gene fragment of the widespread Madagascar frog Blommersia blommersae revealed the presence of up to 12 deep genetic lineages. Many of these differed by genetic distances >3% in the 16S gene. In the Ranomafana area in the southern central east of Madagascar, two mitochondrial lineages differing by 1.2–1.8% in 16S occurred in close syntopy across multiple sites. A phylogeny of representative samples based on multiple mitochondrial genes supported three main mitochondrial clades within B. blommersae, with lineages from the (i) north, (ii) north and northern central east, and (iii) southern central east, respectively. In addition, one lineage from Sainte Luce in the south-east was sister to all other species of Blommersia, and thus clearly not belonging to B. blommersae, to which it has been tentatively assigned previously. Except for the Sainte Luce population, most of the mitochondrial lineages did not show a concordant and consistent differentiation in Rag-1 and no obvious morphological differences were detected among the lineages. We therefore refrain from taxonomic conclusions for them at this time, although indications exist that the populations from the northern central east and southern central east may show certain differences in relative note duration of advertisement calls, which require further study. However, the Sainte Luce individuals were highly divergent in 16S (>10%) and Rag-1, phylogenetically isolated, and characterised by differences in colour pattern. This lineage represents the most evolutionarily divergent species of Blommersia known to date and is herein formally named and described as Blommersia dupreezi sp. nov.

A new species of Gracixalus (Anura, Rhacophoridae) from northwestern Vietnam.

A new species of small tree frog is described from northwestern Vietnam based on morphological differences and molecular divergence. Gracixalustruongisp. nov. is distinguishable from its congeners and other small rhacophorid species on the basis of a combination of the following characters: size relatively small, SVL 32.2-33.1 mm in males, 37.6-39.3 mm in females; head slightly wider than long; vomerine teeth absent; snout round and long RL/SVL 0.17-0.19 in males, 0.16-0.17 in females; spines on upper eyelid absent; supratympanic fold distinct; tympanum distinct; dorsal skin smooth; throat smooth and venter granular; tibiotarsal projection absent; webbing of fingers rudimentary, toes with moderately developed webbing; dorsum moss-green, with an inverse Y-shaped dark green marking extended from interorbital region to posterior region of dorsum; external vocal sac absent in males; males with a nuptial pad on finger I. In the molecular analyses, the new species has no clear sister taxon and is at least 4.5% divergent from other congeners based on a fragment of the mitochondrial 16S rRNA gene.

Genetic differentiation within species exhibiting widespread gene flow; phylogeography of the downstream-inhabiting speciesEphemera orientalis(Insecta: Ephemeroptera)

AbstractElucidating the mechanism of formation of biota on islands will contribute to our understanding of the evolutionary mechanisms that generate biodiversity. In this study, we focus on the evolutionary history of Ephemera orientalis, which is distributed in East Asia. Phylogenetic analyses were performed using the mitochondrial DNA 16S rRNA, cytochrome oxidase subunit I and the nuclear DNA histone H3 regions. We found that these mayflies exhibit genetic differentiation between the Japanese and Korean populations. The Tsushima population, which is located between the Japanese Islands and the Korean Peninsula, was positioned genetically as a continental clade. The populations of the Fuji-Goko Lakes and Lake Ashino-ko in Central Japan were genetically differentiated from the other populations. The genetic structures over a wide area of the Japanese Islands were confirmed and a loose genetic regionality was observed, which was thought to result from secondary contact of the north-eastern and south-western lineages after their long separation by a geohistorical division event. We have gathered important knowledge regarding the evolutionary history of organisms on islands and investigated the colonization and isolation of biological populations within a species with high dispersal ability.

Development of a DNA Metabarcoding Method for the Identification of Insects in Food.

Insects have the potential to become an efficient and reliable food source for humans in the future and could contribute to solving problems with the current food chain. Analytical methods to verify the authenticity of foods are essential for consumer acceptance. We present a DNA metabarcoding method that enables the identification and differentiation of insects in food. The method, developed on Illumina platforms, is targeting a 200 bp mitochondrial 16S rDNA fragment, which we found to be suitable for distinguishing more than 1000 insect species. We designed a novel universal primer pair for a singleplex PCR assay. Individual DNA extracts from reference samples, DNA extracts from model foods and food products commercially available were investigated. In all of the samples investigated, the insect species were correctly identified. The developed DNA metabarcoding method has a high potential to identify and differentiate insect DNA in the context of food authentication in routine analysis.

Analysis of Partial Mitochondrial COI and 16S rRNA Gene Sequences Variation of Pagurus brachiomastus and Pagurus proximus (Decapoda: Paguridae) Populations from the South of Russian Far East

Mitochondrial COI and 16S rRNA gene partial sequences were generated for Pagurus brachiomastus from two locations and P. proximus from single location of the south of Russian Far East. Genetic variation and differentiation of P. brachiomastus Russian and South Korean (GenBank data) populations were estimated and compared on the basis of sequence data on two mitochondrial genes separately. Additionally, P. simulans from South Korea was analyzed. Results show P. brachiomastus from two locations of the south of Russian Far East and South Korea and Korean P. simulans differs at intraspecific level based on molecular markers. Median-Joining analysis indicate presence of two main groups of haplotypes, representing P. proximus and P. brachiomastus + P. simulans, respectively. These data support that P. brachiomastus and P. simulans are conspecific. Mismatch distribution analysis showed population growth for P. brachiomastus at the south of Russian Far East. Sequence pairwise variations of both molecular markers appear differently in two far eastern samples of P. brachiomastus, that can be partially explained by active gene flow through hermit crab migrations between two localities after their isolation.

Development of two new sets of PCR primers for eDNA metabarcoding of brittle stars (Echinodermata, Ophiuroidea)

Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2,100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required.

A new Asian leaf litter toad of the genus Leptobrachella (Amphibia, Anura, Megophryidae) from central south China.

A new species of the Asian leaf litter toad genus Leptobrachella from central south China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and nuclear RAG1 gene sequences indicated the new species as an independent clade in the genus. The new species could be distinguished from its congeners by a combination of the following characters: body of medium size (SVL 29.2-34.2 mm in 15 adult males and 34.4-43.1 mm in seven adult females); distinct black spots present on flanks; toes rudimentary webbed, with wide lateral fringes; ventral belly white with distinct nebulous brown speckling on ventrolateral flanks; skin on dorsum shagreened with fine tiny granules or short ridges; iris copper above, silver below; heels overlapped when thighs are positioned at right angles to the body; tibia-tarsal articulation reaches the middle eye; dorsal surface of tadpole semi-transparent light brown, spots on tail absent, keratodont row formula I: 3+3/2+2: I; call series basically consist of repeated long calls, at dominant frequency (5093 ± 412 Hz).

Diversity, Distribution, and Habitat Association of Anuran Species from Keffa, Southwest Ethiopia

The aim of the current study was to provide a description of the species composition and habitat association of anuran species found in the Keffa area of southwest Ethiopia, among the least studied natural areas in Ethiopia. Data were gathered in both the rainy and dry seasons. Opportunistic observations, audio, and visual encounters were used to perform the survey. To capture different frog species, drift fence and pitfall trap techniques were used. Three habitat types—wetlands, agricultural land, and riverine forests—were explored for the frog species. A total of 3672-person field hours were spent and 5678 individuals of 26 different anuran species from eight different families were recorded. Of these, six species were new records for the Keffa area. Species determination was assessed using morphology and validated with genetic barcoding. The mitochondrial 16S rRNA and COI gene sequence analysis have been used in molecular systematic analyses of the frog species. The Leptopelis ragazzii that was discovered to be present in the earlier study was actually Leptopelis vannutellii, as evidenced by DNA sequencing. The Hyperolius individuals were initially categorized as H. nasutus, but after conducting DNA barcoding, they were found to be H. microps. At least three potential new candidate species (Leptopelis sp. 1, Leptopelis sp. 2, and Phrynobatrachus sp. 1) were discovered. Hyperoliidae was the most abundant family from the recorded anurans, followed by Phrynobatrachidae, while Conrauidae was the least abundant. Most species were found in the wetland habitat, followed by riverine forest, while agricultural land had the least. Wetlands had the highest values for the Shannon diversity index (2.131), followed by agricultural land (1.58) and riverine forests (1.459). The environmental variables revealed that temperature had a considerable negative impact, while precipitation had a non-significantly positive relationship with anuran abundance. The Keffa area is rich in frog species, accounting for 35% of the country’s batrachofauna. However, the habitats have been fragmented and ruined by agricultural expansion, water drainage from the wetlands, and the conversion of horticulture to field crops. We recommend that non-governmental organizations, districts, regional officials, and local people in and around Keffa actively engage in wetland and forest conservation and protection.

Enhanced Expression of Hepatic Genes in Copper-Deficient Rats Detected by the Messenger RNA Differential Display Method

The influence of copper (Cu) status on hepatic gene expression was examined by using the “messenger RNA differential display” technology. This method involves the distribution of mRNA in a two-dimensional array for the rapid identification and cloning of differentially expressed genes. Livers from male Sprague-Dawley rats that had been fed a Cu-deficient (CD) diet (9.4 µmol/kg) or a Cu-adequate (CA) diet (103.9 µmol/kg) for 6 wk were used to supply cytosolic RNA. Cytosolic RNA were reverse-transcribed in the presence of anchor primers and then amplified by polymerase chain reaction with anchor and arbitrary primer sets. The amplified cDNA were then resolved by denaturing polyacrylamide gel electrophoresis. Differences in mRNA expression between the CD and CA rats were identified. DNA fragments were cloned, sequenced and used as probes for Northern blot analysis to confirm that the identified genes were differentially expressed. The analysis of cDNA sequences by computer searches against DNA and protein databases revealed that one cDNA fragment, whose mRNA abundance was enhanced 1.2-fold by copper deficiency, is novel. Four other cDNA fragments were found to have substantial homology with rat ferritin mRNA; rat fetuin mRNA; rat mitochondrial 12S and 16S rRNA, phenylalanine-, valine- and leucine-tRNA genes; rat mitochondrial genes for 16S rRNA, tRNA-leucine and tRNA-valine; and their mRNA abundance was 0.6- to 0.8-fold higher in Cu-deficient rats. Five additional cDNAs detected by this method appeared to represent novel genes because they exhibited no substantial homology to recorded gene and protein sequences deposited in DNA and protein data-bases. These results demonstrate the usefulness of this technology in the detection of genes which were differentially expressed as a result of the deprivation of a single nutrient, dietary copper, in this research project.

Morphological and molecular confirmation of Ornithodoros hasei (Schulze, 1935) (Acari: Argasidae) in Colombia

A large number of tick species are proven vectors for the transmission of bacteria, protozoa, and viruses. Soft ticks (Acari: Argasidae) in South America have been found to be the most frequent carriers of borreliae of the relapsing fever group (RFG); however, there are several information gaps specially on the taxonomy and distribution of some tick species. Here, we used light microscopy, scanning electron microscopy, and PCR amplification of a fragment of the mitochondrial 16S rRNA gene to evaluate 174 larvae of Ornithodoros (Argasidae) collected from three bat species (Eptesicus orinocensis, Molossus rufus and Noctilio albiventris) in the Orinoquia Region of Colombia. The morphological and molecular results confirmed that all the analyzed larvae corresponded to Ornithodoros hasei. Comparisons of mitochondrial 16S rDNA sequences showed low genetic divergence (0% - 0.3%) between larvae of the Department of Arauca in the Orinoquia Region and higher genetic divergence (3.4 - 4.7%) in sequences from other American countries. Our work represents the most recent collection of this species in Colombia and provides a molecular evaluation for the first time. Moreover, a new association of O. hasei with bats such as E. orinocensis is documented. Considering the wide distribution of O. hasei in the American Continent, and its putative role as vector for Borrelia, integrative studies that involve morphological, morphometric, molecular data and experimental crosses are needed to determine if the higher genetic distances are associated with cryptic speciation, as detected in other tick complexes, or represent genetic divergences among geographically different populations of O. hasei.

Ticks in the box: Argas persicus occurrence in nest boxes of secondary cavity-nesting bird species in Italy

Migratory organisms can be vectors of parasitic host organisms that can then be transported along seasonal migratory journeys and spread across continents. Ornithophilic nidicolous ticks (e.g. soft ticks) include species responsible for the transmission of pathogens and bacteria, thus representing a health problem not only for wild species that are directly parasitized, but also for those that share the same environments or reproductive sites with them. In this regard, artificial nests for birds may turn out to be site-sources of parasites. Here, we document the occurrence of different life stages of Argas persicus ticks in nest boxes of wild birds in a natural area (not associated with poultry activities) of central coastal Italy (Maremma Regional Park, Tuscany). Between 2018 and 2022, 168 ticks were collected from nest boxes occupied by different secondary cavity-nesting birds, such as European rollers Coracias garrulus and scops owl Otus scops. Ticks were analysed morphologically, and selected specimens were also identified by mitochondrial ribosomal 16S (16S) subunit gene to ascertain their taxonomic status. All ticks were identified as Argas persicus. This finding not only suggests that this tick species has formed a viable population in this Italian region, but also further confirms the previously doubtful natural origin of the species at country level and sheds new light on its underestimated and little investigated distribution. Possible pathways of introduction and its potential impacts on local avian community are discussed.

Multigene Sequence-Based and Phenotypic Characterization Reveals the Occurrence of a Novel Entomopathogenic Nematode Species, Steinernema anantnagense n. sp.

Three entomopathogenic nematode populations were isolated from agricultural fields in the Anantnag district of Jammu and Kashmir (India). Sequences of multiple gene regions and phenotypic features show that they are conspecific and represent a novel species. Molecular and morphological features provided evidence for placing the new species into the "Kushidai" clade. Within this clade, analysis of sequence data of the internal transcribed spacer (ITS) gene, the D2D3 region of the 28S rRNA gene, the mitochondrial cytochrome oxidase I (mtCOI) gene, and the mitochondrial 12S (mt12S) gene depicted the novel species as a distinctive entity closely related to Steinernema akhursti, S. kushidai, and S. populi. Phylogenetic analyses also show that the new species is a sister species to S. akhursti, and these two species are closely related to S. kushidai and S. populi. Additionally, the new species does not mate or produce fertile progeny with any of the closely related species, reinforcing its uniqueness from a biological species concept standpoint. The new species is further characterized by the third-stage infective juveniles with almost straight bodies (0.7-0.8 mm length), poorly developed stoma and pharynx, and conoid-elongate tail (49-66 µm) with hyaline posterior part. Adult females are characterized by short and conoid tails bearing a short mucron in the first generation and long conoid tails with thin mucron in the second generation. Adult males have ventrally curved spicules in both generations. Moreover, the first-generation male has rounded manubrium, fusiform gubernaculum, conoid and slightly ventrally curved tails with minute mucron, and the second generation has rhomboid manubrium anteriorly ventrad bent, and tails with long and robust mucron. The morphological, morphometrical, molecular, and phylogenetic analyses support the new species status of this nematode, which is hereby described as Steinernema anantnagense n. sp. The bacterial symbiont associated with S. anantnagense n. sp. represents a novel species, closely related to Xenorhabdus japonica. These findings shed light on the diversity of entomopathogenic nematodes and their symbiotic bacteria, providing valuable information for future studies in this field.

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.

A new deep-sea palaemonid shrimp assigned to Periclimenes Costa, 1844 (Decapoda: Caridea) from the West Mariana Ridge, northwestern Pacific

A new palaemonid species, Periclimenes variabilis, is described and illustrated on the basis of four specimens from the Ritto Seamount, West Marina Ridge, northwestern Pacific, at depths of 642-666 m. Specimens of the new species were collected with an unidentified hexacoral of Primnoidae (Cnidaria), an antipatharian octocoral Leiopathes sp. (Cnidaria), and an unidentified crinoid (Echinodermata), suggesting association with those invertebrates. Morphological comparison and molecular phylogenetic analyses using the mitochondrial 16S ribosomal RNA and cytochrome C oxidase subunit I genes indicate that the new species is closest to Periclimenes uniunguiculatus Bruce, 1990 although the possession of more than two spiniform setae on either side of the telson in the new species is consistent with the generic diagnosis of Bathymenes Kou, Li & Bruce, 2016. In reference to the close relationship to the species currently assigned to Periclimenes Costa, 1844 and the unsettled taxonomy of the genus, the new species is provisionally assigned to Periclimenes. Differentiating characters between the new species and closely related taxa are discussed.