Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.

Sciadonus alphacrucis Melo, Gomes, Møller & Nielsen, 2022 is a rare deep-sea species, previously known from only two specimens collected off São Paulo State, southeastern Brazil, in the western South Atlantic. Herein, we report a new specimen of S. alphacrucis collected on the continental slope off Santa Catarina State, southern Brazil, thereby extending its known distribution by 420 km. Additionally, we provide the new meristic and morphometric data, the molecular identification using sequences of the cytochrome c oxidase subunit I (COI), an updated distribution map, and a discussion of troglomorphic traits.

BACKGROUND: The Northeast India, being part of two global biodiversity hotspot namely the Indo-Burma and Eastern Himalayan Hotspots supports a wide variety of rich aquatic biodiversity including fishes. The family Danionidae is a widely diverse group inhabiting the upper colder stretches of river although few are abundant in the lower stretches. The persisting similarity in the morphological appearance and body colouration within the members of this family seeks an integrated method to identify the species correctly.
RESULTS: In the present study, the mt-DNA barcode was generated for correct identification and confirmation of the species. A total of nine mitochondrial cytochrome c oxidase subunit I gene sequences were generated for each species under the study. The pairwise distance values ranged from 0.09 to 9.11% within species and 9.06-32.71% between species. A neighbour-joining tree was constructed based on the Kimura 2 parameter model. Two major groups were observed where Danioninae formed a sister group to the Chedrinae and Rasborinae.
CONCLUSIONS: The present study is a preliminary work to document and identify the species under the family Danionidae from Brahmaputra basin, Assam, using molecular tools and establish the phylogenetic relationship.

Pimelodus is the most speciose genus of the family Pimelodidae, and is amply distributed in the Neotropical region. The species-level taxonomy and phylogenetic relationships within this genus are still poorly resolved, however. These taxonomic problems and the general lack of data have generated major uncertainties with regard to the identification of specimens from different localities. In the present study, we applied a single-locus species delimitation approach to identify the MOTUs found within the genus Pimelodus and provide sound evidence for the evaluation of the species richness of this genus in the different river basins of the Neotropical region. The study was based on the analysis of sequences of the mitochondrial COI gene of 13 nominal species, which resulted in the identification of 24 consensus MOTUs. Only six nominal species were recovered as well-defined molecular entities by both the traditional barcoding analysis and the molecular delimitation methods, while the other seven presented cryptic diversity or persistent taxonomic uncertainties. The lineages identified from the Parnaíba ecoregions, Amazonas Estuary and Coastal Drainages may represent a much greater diversity of Pimelodus species than that recognized currently, although a more detailed study of this diversity will be necessary to provide a more definitive classification of the genus.

OBJECTIVE: Myiases are infestations of human and animal tissues by fly larvae. These conditions are widespread in tropical countries and travelers in those areas are at risk of becoming infested. Although Cordylobia anthropophaga (Blanchard & Berenger-Feraud, 1872) is one of the most common myiasis-causing species, few high-quality images and molecular sequences are available for this fly. We present a case of C. anthropophaga infestation in an Italian patient returning from Senegal, with the aim of increasing both visual and molecular data for this species.
METHODS: After removal, the larva was determined following standardized morphological keys and photographed under a digital microscope. Molecular characterization of the Cytochrome c oxidase subunit I (COI) was performed using universal primers.
RESULTS: The general appearance, the structural organization of the cephalic region, of the cephaloskeleton, and of the posterior tracheal spiracles suggested that the causative agent of the myiasis was a third instar larva of C. anthropophaga. The morphological data are further supported by the molecular data: the COI sequence showed high levels of identity with the already published verified COI sequences of C. anthropophaga.
CONCLUSIONS: We provide high-quality morphological and molecular data useful for the identification of larvae of C. anthropophaga. We highlight that myiasis might be common in Senegal and better data about its prevalence in travelers and in the endemic countries are needed to understand the burden of this condition.

Pinna nobilis, commonly known as the noble pen shell, is a marine bivalve endemic to the Mediterranean Sea. Unfortunately, due to a multifactorial disease that began affecting its populations in 2016, the species is currently facing the threat of extinction. To gain insights into the evolutionary history of P. nobilis before the mass mortality event (MME), and to obtain a comprehensive understanding of how evolutionary processes led to the adaptation of the species into the Mediterranean Sea, phylogenetic and phylogeographic analyses were carried out. The dataset analysed includes 469 sequences of COI gene fragment both from GenBank and the present study (100). The analysis performed evidenced that P. nobilis diverged about 2.5 mya, after the entrance of its ancestor into the Mediterranean Sea following the Zanclean flood (5.33 mya). Moreover, our results suggest that the starting point of colonisation was the central part of the western Mediterranean basin, with the eastern basin being populated subsequently. From a conservational viewpoint, these results provide important hints for present and future restocking plans, helping to reconstruct the pre-existing genetic variability in sites where the species became extinct.

Zooplankton are key components of estuarine trophic networks. However, routine monitoring is hindered by the difficulty of morphology-based identification. DNA-based methods allow us to circumvent some of these hurdles, providing precise species identifications regardless of the taxonomic expertise of the investigator or the developmental stage of the specimens. However, the process is dependent on the completeness of the reference libraries. In this study, we sought to evaluate the potential of DNA metabarcoding to assess the seasonal (summer, autumn, and early spring) and spatial dynamics of zooplankton (four locations spanning ca. 6 km) in the Lima estuary (NW Portugal). Two genetic markers were used: the cytochrome c oxidase subunit I and the V4 hypervariable region of the ribosomal 18S rRNA genes. Overall, 327 species were recovered, and both markers displayed minute overlap (7% were detected with both markers). Species richness, composition, and taxonomic distinctness were majorly influenced by the season, with a declining tendency from summer (highest number of exclusive species, n = 74) to spring. Second to season, the taxa composition was influenced by spatial variation where the most downstream site displayed the highest number of exclusive species, n = 53. A total of 16 non-indigenous species were detected using metabarcoding, but only one (Austrominus modestus) has been documented out in the estuary. In conclusion, both the seasonal and spatial gradients influenced the recovered richness, composition, and taxonomic distinctness, confirming the great aptitude of DNA metabarcoding for providing higher density monitoring and shedding new light on the composition and dynamics of complex zooplankton communities.

Glyptothoa sagara gen. and sp. nov. is described from the host fish Glyptophidium macropus Alcock, 1894 (Ophidiidae), at depths 300 to 650 metres from the southwest coast of India. The mitochondrial cytochrome c oxidase subunit I (COI) gene of the species was sequenced and compared with other closely related branchial cymothoid genera. Both morphological and molecular data corroborate the inclusion of this parasitic isopod as a new genus, and we describe Glyptothoa sagara gen. and sp. nov. The following combinations of characters characterise the genus: cephalon immersed in pereonite 1; dorsum vaulted; all coxae visible in dorsal view; coxae shorter than or as long as pereonites; pereonites 4-7 slightly decrease in width towards one side, slightly asymmetrical, lateral margins slightly constricted, in hunched side; relatively wide pleon, with large lateral gaps between pleonites; antennula narrowly separated by rostrum, slender, shorter than antenna; antenna with 13 articles, buccal cone obscuring antennal bases; brood pouch arising from coxae 1-4, 6; oostegite 1 bilobed; pleopods rami all simple, without proximomedial lamellar lobe, without folds or thickened ridges. The adult life stages, such as females (ovigerous and non-ovigerous), males and transitional stage of the new species are described. The species is currently known only from the type locality and the type host. The ecological remarks of the newly described taxon are also provided. The following species are transferred from Elthusa Schioedte and Meinert, 1884: Glyptothoa myripristae (Bruce, 1990) comb. nov., Glyptothoa propinqua (Richardson, 1904) comb. nov. and Glyptothoa caudata (Schioedte and Meinert, 1884) comb. nov.

Variations of the radula and shell microstructures in 33 species of Japanese chiton were investigated along with molecular phylogenetic trees. The molecular phylogenetic trees indicated that Chitonida was composed of four clades, of which two clades formed Acanthochitonina and corresponded to Mopalioidea and Cryptoplacoidea, respectively, and the other clades formed Chitonina. In the radula, the shapes of the central and centro-lateral teeth and the petaloid process varied greatly among species or genera and were useful for the identification of particular species or genera. The presence of accessory and petaloid processes and the cusp shape were relatively conserved and useful for recognizing particular genera or even suborders. In the valves, four to six shell layers were found at the section, but the ventral mesostracum was not observed in Acanthochitonina. The shell microstructures in the ventral sublayer of the tegmentum varied at suborder, but those in the other layers were almost constant. The megalaesthete chamber type varied at superfamily and was helpful to identify particular families or superfamilies. The characteristics of the shell layers and shell microstructures appear to be a synapomorphy shared by the members of Acanthochitonina. The classification within Chitonina needs to be reexamined because the variations of the cusp shape and megalaesthete chamber type were relatively large and did not correspond to the current classification. Callochiton formed a sister group with Chitonida and would be equally closely related to Chitonina and Acanthochitonina because of possessing a mosaic of characteristics from both.

Pelvic spine polymorphism occurs in several species in the stickleback family (Gasterosteidae). Given the similar phenotypic polymorphisms in multiple stickleback species, we sought to determine the extent of parallelism in the ecological correlates of pelvic spine reduction. Based on a metabarcoding analysis of brook stickleback gut contents in two polymorphic populations, we found that significant diet differences were associated with pelvic spine reduction, but we found no clear or consistent trend supporting a tendency for benthic feeding in pelvic-reduced brook sticklebacks. These results contrast with those found in threespine sticklebacks where pelvic spine reduction is often associated with a benthic diet. Hence, we found non-parallel consequences of spine polymorphism across species. Furthermore, a difference in gill raker morphology has been frequently observed between ecomorphs with different diets in many fish species. However, we found no evidence of any difference in gill raker morphology associated with pelvic spine polymorphism in brook sticklebacks.