Trophic Dynamics and Feeding Ecology of Skipjack Tuna (Katsuwonus pelamis) off Eastern and Western Taiwan.

The skipjack tuna (Katsuwonus pelamis) is a mesopredator fish species with seasonal abundance in waters off Taiwan. Regional ecological and life-history information has been historically lacking for this species. In recent years, stable isotope analysis (SIA) of carbon and nitrogen has been used to assess predator feeding ecology and broader ecosystem trophic dynamics. This study evaluated comparative skipjack feeding ecology in distinct regions off Taiwan, combining traditional stomach content analysis with SIA of individuals off western (n = 43; 2020) and eastern (n = 347; 2012-2014 and n = 167; 2020) Taiwan. The stomach content analysis showed the most important prey to be ponyfish (Photopectoralis bindus) in western Taiwan and epipelagic squids (Myopsina spp.) and carangids (Decapterus macrosoma;) in eastern Taiwan from 2012 to 2014 and epipelagic carangids (Decapterus spp.) and flying fishes (Cheilopogon spp.) in eastern Taiwan in 2020, suggesting that the skipjack tuna is a generalist predator across regions. In contrast, time-integrated diet estimates from Bayesian mixing models indicated the importance of cephalopods and crustaceans as prey, potentially demonstrating more mesopelagic feeding in less productive waters during skipjack migrations outside the study regions. Skipjack off western Taiwan had a slightly higher estimated trophic position than in the waters off eastern Taiwan, potentially driven by the varying nutrient-driven pelagic food web structures. Skipjack SI values increased with body size off eastern Taiwan but not in western waters, suggesting that opportunistic predation can still result in different predator-prey size dynamics between regions.

Phylogenomics illuminates the evolution of bobtail and bottletail squid (order Sepiolida).

Bobtail and bottletail squid are small cephalopods with striking anti-predatory defensive mechanisms, bioluminescence, and complex morphology; that inhabit nektobenthic and pelagic environments around the world's oceans. Yet, the evolution and diversification of these animals remain unclear. Here, we used shallow genome sequencing of thirty-two bobtail and bottletail squids to estimate their evolutionary relationships and divergence time. Our phylogenetic analyses show that each of Sepiadariidae, Sepiolidae, and the three subfamilies of the Sepiolidae are monophyletic. We found that the ancestor of the Sepiolinae very likely possessed a bilobed light organ with bacteriogenic luminescence. Sepiolinae forms a sister group to Rossinae and Heteroteuthinae, and split into Indo-Pacific and Atlantic-Mediterranean lineages. The origin of these lineages coincides with the end of the Tethys Sea and the separation of these regions during the Eocene and the beginning of the Oligocene. We demonstrated that sepiolids radiated after the Late Cretaceous and that major biogeographic events might have shaped their distribution and speciation.

Phylogenomic analyses recover a clade of large-bodied decapodiform cephalopods.

Phylogenetic relationships among the squids and cuttlefishes (Cephalopoda:Decapodiformes) have resisted clarification for decades, despite multiple analyses of morphological, molecular and combined data sets. More recently, analyses of complete mitochondrial genomes and hundreds of nuclear loci have yielded similarly ambiguous results. In this study, we re-evaluate hypotheses of decapodiform relationships by increasing taxonomic breadth and utilizing higher-quality genome and transcriptome data for several taxa. We also employ analytical approaches to (1) identify contamination in transcriptome data, (2) better assess model adequacy, and (3) account for potential biases. Using this larger data set, we consistently recover a clade comprising Myopsida (closed-eye squid), Sepiida (cuttlefishes), and Oegopsida (open-eye squid) that is sister to a Sepiolida (bobtail and bottletail squid) clade. Idiosepiida (pygmy squid) is consistently recovered as the sister group to all sampled decapodiform lineages. Further, a weighted Shimodaira-Hasegawa test applied to one of our larger data matrices rejects all alternatives to these ordinal-level relationships. At present, available nuclear genome-scale data support nested clades of relatively large-bodied decapodiform cephalopods to the exclusion of pygmy squids, but improved taxon sampling and additional genomic data will be needed to test these novel hypotheses rigorously.

Revisiting the phylogeny of the genus Lolliguncula Steenstrup 1881 improves understanding of their biogeography and proves the validity of Lolliguncula argus Brakoniecki & Roper, 1985.

The biogeography of American loliginid squids has been improved in recent years, but certain key taxa have been missing. Given that the most accurate phylogenies and estimates of divergence times of common ancestors depend heavily on good taxonomic coverage we have reanalyzed the genus Lolliguncula in light of new samples that increase the geographic and taxonomic coverage. New sequences were produced using standard methods to update an existing dataset for COI, 16S and Rhodopsin markers. Data was analyzed using various species delimitation methods, rigorous phylogenetic analyses and estimates of divergence times between clades. Within Lolliguncula we recover five monophyletic lineages that relate to the known species L. argus, L. diomedeae, L. panamensis, L. brevis North Atlantic and L. brevis South Atlantic. Except when using low divergence thresholds in ABGD, species delimitation methods only identify four of these lineages as distinct species, grouping L. argus and L. diomedeae as a single species. However, considering the reciprocal monophyly, recent divergence time estimate and morphological diagnoses we refrain from synonymizing L. argus within L. diomedeae, considering them very recently diverged species. The biogeography of the American loliginids is discussed, wherein basal cladogenesis in both Lolliguncula and Doryteuthis occur between the Atlantic and Pacific about 45 mya, with subsequent speciation around 20 mya associated with seafloor changes during the formation of the Caribbean. The recent speciation between L. argus and L. diomedeae is associated to oceanic environmental changes associated with glaciation, deep sea cooling and tropical upwelling.

Long-term changes in habitat and trophic level of Southern Ocean squid in relation to environmental conditions.

Long-term studies of pelagic nekton in the Southern Ocean and their responses to ongoing environmental change are rare. Using stable isotope ratios measured in squid beaks recovered from diet samples of wandering albatrosses Diomedea exulans, we assessed decadal variation (from 1976 to 2016) in the habitat (δ13C) and trophic level (δ15N) of five important Southern Ocean squid species in relation to indices of environmental conditions-Southern Oscillation Index (SOI) and Southern Annular Mode (SAM). Based on δ13C values, corrected for the Suess effect, habitat had changed over the last 50 years for Taonius sp. B (Voss), Gonatus antarcticus, Galiteuthis glacialis and Histioteuthis atlantica but not Moroteuthopsis longimana. By comparison, mean δ15N values were similar across decades for all five species, suggesting minimal changes in trophic levels. Both SAM and SOI have increased in strength and frequency over the study period but, of the five species, only in Taonius sp. B (Voss) did these indices correlate with, δ13C and δ15N values, indicating direct relationships between environmental conditions, habitat and trophic level. The five cephalopod species therefore changed their habitats with changing environmental conditions over the last 50 years but maintained similar trophic levels. Hence, cephalopods are likely to remain important prey for top predators in Southern Ocean food webs, despite ongoing climate change.

Phylogenetic relationships and cryptic species in the genus Sthenoteuthis (Cephalopoda: Ommastrephidae) in the South China Sea.

Large squids of the genus Sthenoteuthis are commercially relevant species that include two truly oceanic squids. They are large nektonic predators being widely distributed throughout tropical and subtropical waters of the Atlantic and Indo-Pacific Ocean. The present study investigates different morphs varying in size at maturity, and assesses the genetic divergence in Sthenotheutis in relation to geographic patterns in the South China Sea. We obtained sequences using a mitochondrial (cytochrome c oxidase subunit I) and a nuclear (Histone H3) gene marker from 111 individuals in 23 locations of the South China Sea. In combination with sequences available in public databases, we performed tests on DNA taxonomy, mostly based on molecular phylogenies. Our results suggest that the genus Sthenoteuthis includes at least three species. The Indo-Pacific purpleback squid Sthenoteuthis oualaniensis contains at least two genetically distinct lineages that can be considered separate species, a dwarf species and a medium-sized species, separated by both the mitochondrial marker and the more conserved nuclear marker. We also assessed whether the few cases of mitonuclear discordance could be the result of genetic introgression and past hybridization or incongruence lineage sorting. The medium-sized species is more widely distributed and dominant in the South China Sea than the dwarf species. The medium-sized species inhabits the whole South China Sea, whereas the dwarf species is restricted to the equatorial waters of the South China Sea. The medium-sized species has two further genetic clades, one distributed in the East Pacific Ocean and the other in the South China Sea. This high level of genetic differentiation is in agreement with the discriminant analysis on the morphological measurements, clearly separating the dwarf and medium-sized species, indicating the presence of a complex of pseudo-cryptic species in S. oualaniensis, clearly identifiable by differences in DNA sequences and in body size, and statistically differentiated in their body measurements.

The grass squid Pickfordiateuthis pulchella is a paedomorphic loliginid.

The wide disparity in adult body size observed both within and among animal taxa has long attracted widespread interest, with several general rules having been proposed to explain trends in body size evolution. Adult body size disparity among the cephalopod mollusks is remarkable, with adult body sizes ranging from a few centimeters to several meters. Some of the smallest cephalopods are found within Pickfordiateuthis, a group comprising three described species of squid found in the western Atlantic and tropical eastern Pacific. Pickfordiateuthis pulchella, the type species of the genus, was initially proposed to be closely related to the loliginid squids (Loliginidae), with subsequent descriptions of additional species supporting a placement within Loliginidae. Pickfordiateuthis is remarkable in that all species reach sexual maturity at about one-fifth to one-tenth the size seen in most loliginid species. To date, no phylogenetic analyses have included representatives of Pickfordiateuthis. To infer the phylogenetic position of Pickfordiateuthis and explore its implications for body size evolution, we collected specimens of Pickfordiateuthis pulchella from Brazilian waters and sequenced regions of two loci-the mitochondrial large ribosomal subunit (rrnL a.k.a. 16S) gene and the nuclear gene rhodopsin. Maximum likelihood and Bayesian analyses of these sequences support a placement of Pickfordiateuthis pulchella as sister to a clade comprising the Western Hemisphere loliginid genera Doryteuthis and Lolliguncula. Analyses of body size evolution within Loliginidae suggest that a shift to a smaller body size optimum occurred along the lineage leading to P. pulchella, with some evidence of shifts toward larger sizes in the ancestors of Loligo and Sepioteuthis; these inferences seem to be robust to phylogenetic uncertainty and incomplete taxon sampling. The small size and juvenile-like morphological traits seen in adult Pickfordiateuthis (e.g., sepiolid-like fins and biserial sucker arrangement in the tentacles) may be due to paedomorphosis.

The Characteristics and Expression Profile of Transferrin in the Accessory Nidamental Gland of the Bigfin Reef Squid during Bacteria Transmission.

The accessory nidamental gland (ANG) is a female reproductive organ found in most squid and cuttlefish that contains a consortium of bacteria. These symbiotic bacteria are transmitted from the marine environment and selected by the host through an unknown mechanism. In animals, a common antimicrobial mechanism of innate immunity is iron sequestration, which is based on the development of transferrin (TF)-like proteins. To understand this mechanism of host-microbe interaction, we attempted to characterize the role of transferrin in bigfin reef squid (Sepioteuthis lessoniana) during bacterial transmission. qPCR analysis showed that Tf was exclusively expressed in the outer layer of ANG,and this was confirmed by in situ hybridization, which showed that Tf was localized in the outer epithelial cell layer of the ANG. Western blot analysis indicated that TF is a soluble glycoprotein. Immunohistochemical staining also showed that TF is localized in the outer epithelial cell layer of the ANG and that it is mainly expressed in the outer layer during ANG growth. These results suggest that robust Tf mRNA and TF protein expression in the outer layer of the ANG plays an important role in microbe selection by the host during bacterial transmission.

New bobtail squid (Sepiolidae: Sepiolinae) from the Ryukyu islands revealed by molecular and morphological analysis.

Bobtail squid are emerging models for host-microbe interactions, behavior, and development, yet their species diversity and distribution remain poorly characterized. Here, we combine mitochondrial and transcriptome sequences with morphological analysis to describe three species of bobtail squid (Sepiolidae: Sepiolinae) from the Ryukyu archipelago, and compare them with related taxa. One Ryukyuan type was previously unknown, and is described here as Euprymna brenneri sp. nov. Another Ryukyuan type is morphologically indistinguishable from Sepiola parva Sasaki, 1913. Molecular analyses, however, place this taxon within the genus Euprymna Steenstrup, 1887, and additional morphological investigation led to formal rediagnosis of Euprymna and reassignment of this species as Euprymna parva comb. nov. While no adults from the third Ryukyuan type were found, sequences from hatchlings suggest a close relationship with E. pardalota Reid, 2011, known from Australia and East Timor. The broadly sampled transcriptomes reported here provide a foundation for future phylogenetic and comparative studies.

Predatory behaviour and taphonomy of a Jurassic belemnoid coleoid (Diplobelida, Cephalopoda).

We describe four complete specimens of the early squid-like cephalopod Clarkeiteuthis conocauda from the Toarcian Posidonienschiefer (Jurassic) each preserved with the bony fish Leptolepis bronni in its arms. Based on the arrangement of prey and predator, we suggest that the cephalopods caught and killed the fishes while still in well-oxygenated waters and then descended into oxygen-depleted water layers (distraction sinking) where the cephalopod suffocated. This explains the exceptional preservation, for which the Posidonienschiefer is famed. This association raises the question for the hunting behaviour of belemnoid Coleoidea. Using the proportions of soft and skeletal body parts of diplobelids and belemnitids, we estimated their body mass and buoyancy and determined the centres of mass and buoyancy. These two points were very close to each other in belemnitids, implying a low hydrodynamic stability (when ignoring the fins), while in diplobelids, the distance between those centres was greater. This suggests that diplobelids usually assumed an oblique to vertical orientation of the body axis while belemnitids could effortlessly achieve a horizontal orientation of their body. Presuming larger fins were attached to the bigger belemnitid rostra, belemnitids were better swimmers and perhaps pursuit predators while diplobelids rather ambushed their prey.

Potential use of stable isotope and fatty acid analyses for traceability of geographic origins of jumbo squid (Dosidicus gigas).

RATIONALE: Squid is an important seafood resource for Asian and European countries. With the continuous development of processed squid products, an effective traceability system has become increasingly prominent. Here, we attempt to trace the fishery products of the main target species, jumbo squid (Dosidicus gigas), by using biochemical tracers. METHODS: Carbon and nitrogen isotope ratios (δ13 C and δ15 N values) and fatty acid profiles were identified in squid from three harvest locations in the eastern Pacific Ocean by isotope ratio mass spectrometry and gas chromatography/mass spectrometry, respectively. Comparative analysis was used to evaluate the geographic variations in tracers and to identify the suitable discriminatory variables among origins. RESULTS: Significant spatial variations were found in isotopic values and fatty acid profiles in squid muscle tissues, possibly because of different food availability and/or oceanographic conditions that each group experiences at a given location. The stepwise discriminant analysis indicated that δ15 N, C16:1n7, C17:1n7, C18:2n6, C20:1 and C20:4n6 were effective variables at differentiating origin. CONCLUSIONS: Combined use of stable isotope ratios and fatty acid analyses could trace geographic origins of jumbo squid. This study provides an alternative approach for improving authenticity evaluation of commercial squid products.

Assessing the utility of transcriptome data for inferring phylogenetic relationships among coleoid cephalopods.

Historically, deep-level relationships within the molluscan class Cephalopoda (squids, cuttlefishes, octopods and their relatives) have remained elusive due in part to the considerable morphological diversity of extant taxa, a limited fossil record for species that lack a calcareous shell and difficulties in sampling open ocean taxa. Many conflicts identified by morphologists in the early 1900s remain unresolved today in spite of advances in morphological, molecular and analytical methods. In this study we assess the utility of transcriptome data for resolving cephalopod phylogeny, with special focus on the orders of Decapodiformes (open-eye squids, bobtail squids, cuttlefishes and relatives). To do so, we took new and previously published transcriptome data and used a unique cephalopod core ortholog set to generate a dataset that was subjected to an array of filtering and analytical methods to assess the impacts of: taxon sampling, ortholog number, compositional and rate heterogeneity and incongruence across loci. Analyses indicated that datasets that maximized taxonomic coverage but included fewer orthologs were less stable than datasets that sacrificed taxon sampling to increase the number of orthologs. Clades recovered irrespective of dataset, filtering or analytical method included Octopodiformes (Vampyroteuthis infernalis + octopods), Decapodiformes (squids, cuttlefishes and their relatives), and orders Oegopsida (open-eyed squids) and Myopsida (e.g., loliginid squids). Ordinal-level relationships within Decapodiformes were the most susceptible to dataset perturbation, further emphasizing the challenges associated with uncovering relationships at deep nodes in the cephalopod tree of life.

Updated molecular phylogeny of the squid family Ommastrephidae: Insights into the evolution of spawning strategies.

Two types of spawning strategy have been described for ommastrephid squids: coastal and oceanic. It has been suggested that ancestral ommastrephids inhabited coastal waters and expanded their distribution into the open ocean during global changes in ocean circulation in the Oligocene. This hypothesis could explain the different reproductive strategies in oceanic squids, but has never been tested in a phylogenetic context. In the present study, we assess the coastal-to-open-ocean hypothesis through inferring the evolution of reproductive traits (spawning type) of ommastrephid squids using the phylogenetic comparative method to estimate ancestral states and divergence times. This analysis was performed using a robust molecular phylogeny with three mitochondrial genes (COI, CYTB and 16S) and two nuclear genes (RHO and 18S) for nearly all species of ommastrephid squid. Our results support dividing the Ommastrephidae into the three traditional subfamilies, plus the monotypic subfamily Todaropsinae as proposed previously. Divergence times were found to be older than those suggested. Our analyses strongly suggest that early ommastrephid squids spawned in coastal areas, with some species subsequently switching to spawn in oceanic areas, supporting previous non-tested hypotheses. We found evidence of gradual evolution change of spawning type in ommastrephid squids estimated to have occurred since the Cretaceous.

Molecular phylogeny of Loliginidae inferred from mitochondrial DNA sequence variation.

Loliginidae includes many economically important species in trophic systems worldwide. Here, we investigated genetic relationships and diversity in this family. Sequence comparisons and phylogenetic analyses revealed considerable variations between mitochondrial 16 S rRNA gene and cytochrome coxidase subunit I gene among nine Loliginid species. We identified three similar non-coding regions in eight Loliginid species, but not in Sepioteuthislessoniana. We detected a single extended termination-associated sequence and three conserved sequence blocks among these eight species. Our results suggest that Loliginidae forms a major lineage, with S. lessoniana located at the most basal position and forming an individual clade as sister to the remaining species. Loligobeka, Loliolusjaponica, Loliolusuyii, Loligochinensis, Loligoedulis, Loligoduvauceli, Loligobleekeri, and Loligoopalescensare clustered into a monophyletic group. We identified repetitive elements and repeat numbers in the control regions.

Seafood Identification in Multispecies Products: Assessment of 16SrRNA, cytb, and COI Universal Primers' Efficiency as a Preliminary Analytical Step for Setting up Metabarcoding Next-Generation Sequencing Techniques.

Few studies applying NGS have been conducted in the food inspection field, particularly on multispecies seafood products. A preliminary study screening the performance and the potential application in NGS analysis of 14 "universal primers" amplifying 16SrRNA, cytb, and COI genes in fish and cephalopods was performed. Species used in surimi preparation were chosen as target. An in silico analysis was conducted to test primers' coverage capacity by assessing mismatches (number and position) with the target sequences. The 9 pairs showing the best coverage capacity were tested in PCR on DNA samples of 53 collected species to assess their amplification performance (amplification rate and amplicon concentration). The results confirm that primers designed for the 16SrRNA gene amplification are the most suitable for NGS analysis also for identification of multispecies seafood products. In particular, the primer pair of Chapela et al. (2002) is the best candidate.

Whole mitochondrial genome of the Ram's Horn Squid shines light on the phylogenetic position of the monotypic order Spirulida (Haeckel, 1896).

The phylogenetic position of the only known species within the order Spirulida, the Ram's Horn Squid, Spirula spirula, may be the key to resolving relationships within Decapodiformes (squids and cuttlefishes). Spirula spirula possesses several unique features including an internal calcareous chambered shell unlike the familiar cuttlebone of Sepiidae (cuttlefishes). The shell is reduced to a gladius or absent in other decapod clades. To resolve decapodiform phylogenetic relationships we sequenced the mitochondrial genome of S. spirula and Sepiadarium austrinum and analysed these along with other mitochondrial genomes. Sequence analyses found that S. spirula and Sepiidae, the only two extant phragmocone bearing groups, were not sister taxa. Rather, in most analyses S. spirula was placed within a clade containing Bathyteuthoidea and Oegopsida either as the sister taxon to Bathyteuthoidea+Oegopsida or the sister taxon to Bathyteuthoidea only, depending upon the analysis method. Sepiidae was the sister taxon to a clade containing all remaining decapods. Spirulid mitochondrial gene order was identical to that of Octopodiformes, which we recognize as close to that of ancestral molluscs. The phylogenetic position of Idiosepiidae differed among analysis methods of molecular sequence data. However, gene order analysis resolved a highly supported monophyletic relationship containing Idiosepiidae and Sepiolida.

Identification of four squid species by quantitative real-time polymerase chain reaction.

Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control.

Complete mitochondrial genome of the Loliolus (Nipponololigo) uyii.

In this study, we determined the complete mitochondrial genome of the little squid (Loliolus (Nipponololigo) uyii). The genome was 17,134 bp in length and contained 13 protein-coding genes,22 transfer RNA genes, 2 ribosomal RNA genes and 3 main non-coding regions. The composition and order of genes were similar to most other invertebrates. The overall base composition of L. uyii is A 39.98%, C 18.65 %, T 32.39% and G 8.98%, with a high A + T bias of 72.37%. All of the three control regions (CR) contain termination-associated sequences and conserved sequence blocks. These mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the Loliginidae.

Complete mitochondrial genome of Argentine shortfin squid (Illex argentines).

In this study, we determined the complete mitochondrial genome of the Illex argentinus. The genome was 20,278 bp in length and contained 18 protein-coding genes, 23 transfer RNA genes, 2 ribosomal RNA genes and 3 main non-coding regions. The composition and order of genes were different to some other invertebrates. The overall base composition of I. argentinus was A 39.23%, C 17.50%, T 33.71% and G 9.56%, with a highly A + T bias of 72.94%. All of the three control regions (CR) contained termination-associated sequences and conserved sequence blocks. This mitogenome sequence data would play an important role in the taxonomic resolution and phylogeography of the Ommastrephidae.

The complete mitochondrial genome of the cryptic "lineage A" big-fin reef squid, Sepioteuthis lessoniana (Cephalopoda: Loliginidae) in Indo-West Pacific.

In this study, the complete mitogenome sequence of the cryptic "lineage A" big-fin reef squid, Sepioteuthis lessoniana (Cephalopoda: Loliginidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome consists of 16,605 bp, which includes 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs genes. The overall base composition of "lineage A" S. lessoniana is 37.5% for A, 17.4% for C, 9.1% for G, and 35.9% for T and shows 87% identities to "lineage C" S. lessoniana. It is also noticed by its high T + A content (73.4%), two non-coding regions with TA tandem repeats. The complete mitogenome of the cryptic "lineage A" S. lessoniana provides essential and important DNA molecular data for further phylogeography and evolutionary analysis for big-fin reef squid species complex.