A rare occurrence of reversal in the four-spotted megrim Lepidorhombus boscii (Risso 1810) (Pleuronectiformes: Scophthalmidae) in the central Adriatic Sea.

One reversal dextral (right-sided) specimen occurred in the sample of 1070 individuals of four-spotted megrim Lepidorhombus boscii caught in the central Adriatic Sea from July 2020 to June 2021. This is the first documented occurrence of reversal in this species in the Adriatic Sea. Besides reverse symmetry, the individual showed similar morphological and morphometric characteristics as non-reversed individuals.

A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis.

Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group-synchronous ovarian developmental pattern with determinate fecundity and support for their batch-spawning strategy.

An hypoxia-tolerant flatfish: consequences of sustained stress on the slender sole Lyopsetta exilis (Pleuronectidae) in the context of a changing ocean.

Slender sole Lyopsetta exilis is an abundant groundfish on the continental shelf and inner waters of British Columbia, Canada, where it reaches a maximum standard length of 44 cm. Benthic image surveys coupled with oxygen measurements in Saanich Inlet document a dense population in bottom conditions near anoxia (0.03 ml l-1 oxygen) where diel migrating zooplankton intersect the bottom; we confirm this species is a planktivore, which limits its depth range to the base of the migration layer. In a comparison with slender sole from a nearby well-oxygenated habitat, several probable effects of living in severe hypoxia emerge: both sexes are significantly smaller in Saanich and the sex ratio is male-skewed. Otoliths from the Saanich fish were difficult to read due to many checks, but both sexes were smaller at age with the largest female (20 cm) from the hypoxia zone registering 17 years. Hypoxia appears to have a direct consequence on growth despite good food supply in this productive basin. Hyperventilation, a low metabolic rate and a very low critical oxygen tension help this fish regulate oxygen uptake in severely hypoxic conditions; it will be particularly resilient as the incidence of hypoxia increases on the continental shelf. Data from small-mesh bottom-trawl surveys over four decades reveal an increase in mean annual catch per unit effort in southern regions of the province, including the outer shelf and the Strait of Georgia. The California Cooperative Oceanic Fisheries Investigations (CalCOFI) ichthyoplankton database records a general decline in fish larvae on the Oregon-California shelf since 1990, but slender sole larvae are increasing there, as they are in the Strait of Georgia. We project that the slender sole will gain relative benefits in the future warming, deoxygenated northeast Pacific Ocean.

Skeletal development of the chondrocranium in the tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae).

This study provides a comprehensive description of chondrocranial development before, during and after larval metamorphosis in the tongue sole Cynoglossus semilaevis, a commercially valuable flatfish in China. Samples were collected at regular intervals ranging from 1 to 23 days post hatching (dph). Based on observations of cleared and double-stained specimens and images from sections stained with safranin O-fast green, major morphological events during early development were described. No cartilaginous structure was visible at hatching. From 2 dph onwards, cartilaginous structures such as the trabecular bar and some elements of the mandibular, hyoid and branchial arches appeared. At this time also, cartilaginous structures of the neurocranium started to form. Hypertrophic chondrocytes could be observed in many splanchnocranium elements at 5 dph. The start of ossification was indicated by alizarin red stain visible at 14 dph. At 17 dph, most of the cartilaginous skeleton was ossified. Soon after, the right eye started to migrate and pass through a slit beneath the dorsal-fin base and above the skull. Metamorphosis was complete at 20 dph, at which time the dorsal-fin base cartilage extended onto the anterior region of the head. Meanwhile, extremities of the hyoid and branchial arch elements remained cartilaginous. At 23 dph, endochondral ossification of the splanchnocranium was nearly complete. Unlike previous observations of other Pleuronectiformes, our study indicates that endochondral ossification of C. semilaevis skull cartilage occurs before metamorphosis.

Structural and functional maturation of skin during metamorphosis in the Atlantic halibut (Hippoglossus hippoglossus).

To establish if the developmental changes in the primary barrier and osmoregulatory capacity of Atlantic halibut skin are modified during metamorphosis, histological, histochemical, gene expression and electrophysiological measurements were made. The morphology of the ocular and abocular skin started to diverge during the metamorphic climax and ocular skin appeared thicker and more stratified. Neutral mucins were the main glycoproteins produced by the goblet cells in skin during metamorphosis. Moreover, the number of goblet cells producing neutral mucins increased during metamorphosis and asymmetry in their abundance was observed between ocular and abocular skin. The increase in goblet cell number and their asymmetric abundance in skin was concomitant with the period that thyroid hormones (THs) increase and suggests that they may be under the control of these hormones. Several mucin transcripts were identified in metamorphosing halibut transcriptomes and Muc18 and Muc5AC were characteristic of the body skin. Na+, K+-ATPase positive (NKA) cells were observed in skin of all metamorphic stages but their number significantly decreased with the onset of metamorphosis. No asymmetry was observed between ocular and abocular skin in NKA cells. The morphological changes observed were linked to modified skin barrier function as revealed by modifications in its electrophysiological properties. However, the maturation of the skin functional characteristics preceded structural maturation and occurred at stage 8 prior to the metamorphic climax. Treatment of Atlantic halibut with the THs disrupter methimazole (MMI) affected the number of goblet cells producing neutral mucins and the NKA cells. The present study reveals that the asymmetric development of the skin in Atlantic halibut is TH sensitive and is associated with metamorphosis and that this barrier's functional properties mature earlier and are independent of metamorphosis.

Effects of organism and substrate size on burial mechanics of English sole, Parophrys vetulus.

Flatfishes use cyclic body undulations to force water into the sediment and fluidize substrate particles, displacing them into the water column. When water velocity decreases, suspended particles settle back onto the fish, hiding it from view. Burial may become more challenging as flatfishes grow because the area to be covered increases exponentially with the second power of length. In addition, particle size is not uniform in naturally occurring substrates, and larger particles require higher water velocities for fluidization. We quantified the effects of organism and particle-size scaling on burial behavior of English sole, Parophrys vetulus We recorded burial events from a size range of individuals (5-32 cm total length, TL), while maintaining constant substrate grain size. Larger fish used lower cycle frequencies and took longer to bury, but overall burial performance was maintained (∼100% coverage). To test the effect of particle size on burial performance, individuals of similar lengths (5.7-8.1 cm TL) were presented with different substrate sizes (0.125-0.710 mm). Particle size did not affect cycle frequency or time to burial, but fish did not achieve 100% coverage with the largest particles because they could not fluidize this substrate. Taken together, these results suggest that both body size and substrate grain size can potentially limit the ability of flatfishes to bury: a very large fish (>150 cm) may move too slowly to fluidize all but the smallest substrate particles and some particles are simply too large for smaller individuals to fluidize.

Comparative performance of growth, vertebral structure and muscle composition in diploid and triploid Paralichthys olivaceus.

Growth, skeletal structure and muscle composition of cold-shock-induced triploid olive flounder Paralichthys olivaceus were investigated. The average values of total length and total weight of triploids were higher than those of diploids from 5 to 11 months posthatch (mph). The growth difference disappeared after 11 mph. The skeletal structure of flounder at 11 mph was observed by X-ray imaging method. There are four kinds of vertebral deformity including vertebrae fusion, one-sided compression, two-sided compression and vertically shifted. The trunk region (V8-18) and tailing end of the vertebral column were the predominant locations of deformity. In general, the frequencies of vertebral deformities in triploids (60.0%) were higher than those in diploids (33.3%, p < 0.05). Both the number of fish with deformed vertebrae and the average frequencies of deformed vertebrae in triploids were significantly higher than those in diploids (p < 0.05). The muscle tissues of diploid and triploid flounder at 11 mph contain the same types of fatty acid and free amino acid profiles. The number of fatty acids with significant higher contents in diploids and triploids was one and ten, respectively (p < 0.05). The contents of free amino acids showed no difference between triploid and diploid fish.

Population specific sperm production in European flounder Platichthys flesus: Adaptation to salinity at spawning.

Marine teleosts inhabiting the brackish Baltic Sea have adapted to the less saline water with activation of spermatozoa at low salinity hypo-osmotic conditions but with shorter longevity and lower swimming speed that affect the fertilization capacity. Aiming to elucidate if the fertilization capacity may be maintained by increasing the number of spermatozoa produced, testis size for the euryhaline flounder Platichthys flesus with external fertilization was assessed along a salinity gradient; with spawning at a salinity of c. 7, 10-18 and 30-35. Fulton's condition factor K = 0.881 ± 0.085 (mean ± S.D.), 0.833 ± 0.096 and 0.851 ± 0.086, for fish spawning at salinities of c. 7, 10-18 and 30-35, respectively, with no difference between areas, i.e. analysed fish were in similar nutritional condition. A general linear model, with testes dry mass as the dependent variable and somatic mass as covariate resulted in a significant difference between areas-populations with larger testes for P. flesus spawning at a salinity of c. 7 but no difference between fish spawning at a salinity of 10-18 and 30-35. The result suggests that adaptation by increasing the number of spermatozoa produced may be a key mechanism for marine teleosts spawning in areas with low salinities to sustain the fertilization capacity as shown here for the euryhaline P. flesus.

Genetic analysis for main length ratio associated with morphological traits in Japanese flounder Paralichthys olivaceus.

The objective of this study was to estimate genetic parameters of main length ratios associated with morphological traits in Paralichthys olivaceus. At 180, 240 and 360 days of age, body length (L1 ), head length (L2 ), body depth (L3 ), caudal peduncle depth (L4 ) and caudal-peduncle length (L5 ) were observed in 4757-5224 individuals from 52 full-sib families. Based on these observed traits, the main length-ratio traits including L3 :L1 , L2 :L1 , L5 :L1 and L4 :L3 were constructed. Three single-trait animal models were used to examine the phenotypic variation of length-ratio traits and were compared using the likelihood ratio test. As a result, the optimal model for all the analysed traits was determined as the model with the fixed, additive genetic and full-sib family effects. Using the optimal model, animal model analysis for single trait showed that heritabilities were estimated to be low, ranging from 0·122 to 0·276. The estimates for all length-ratio traits decreased while the days of age increased except for L3 :L1 . Multivariate animal model analysis indicated that within and among days of age, genetic correlations between length-ratio traits were different in the direction and magnitude. Genetic parameter estimation of main length ratios would be helpful for genetic improvement of body shape in P. olivaceus.

High-density aggregations of rodlet cells in the gonads of Greenland halibut Reinhardtius hippoglossoides, a deep-water marine flatfish.

Large aggregations of rodlet cells in the gonads of male and female Greenland halibut Reinhardtius hippoglossoides are reported for the first time. These rodlet cells were not arranged epithelially but rather were found throughout the connective tissue between oocytes (females) or within lymphatic spaces between testicular lobules (males). The reason for large aggregations of rodlet cells in the gonads and not other tissues of this species is uncertain.

Early growth and development of reciprocal hybrids of the starry flounder Platichthys stellatus and stone flounder Kareius bicoloratus.

Larval growth and development of hybrid flounder were observed and compared with those of their parent species. The reciprocal hybrids of female starry flounder Platichthys stellatus and male stone flounder Kareius bicoloratus (hybrid Sb) and of female K. bicoloratus and male P. stellatus (hybrid Bs) both survived and grew to juveniles. Development was divided into nine stages (A-I). Many of the hybrids' traits were identical and intermediate to those of their parents. The position of the eye, however, was primarily sinistral in both hybrids (80% in Sb and 76% in Bs), a trait possessed by P. stellatus (80%) in the western Pacific Ocean. The daily growth rates of the larvae were similar. In the parent species, development was more rapid in P. stellatus than in K. bicoloratus whereas rate of development was intermediate in both Sb and Bs hybrids. The size at settlement [standard length (LS ) at stage H (mean ± s.d.)] was 9·82 ± 1·47 mm for the hybrid Sb and 9·99 ± 0·90 mm for the hybrid Bs, while the minimum age at metamorphosis (initial age at stage H) was 29 days after hatching (DAH) in both hybrids. In comparison, LS at settlement in parent species was 6·43 ± 0·25 mm for P. stellatus and 12·87 ± 1·29 mm for K. bicoloratus. Minimum age at metamorphosis for the parents was 23 DAH at stage G in P. stellatus and 34 DAH at stage H in K. bicoloratus. Thus, the timing of settlement of hybrids was different from that of their parent species. These traits may occur with high frequency in a natural habitat.

The gustatory stalk of the Remo flounder exemplifies how complex evolutionary novelties may arise.

The appearance of evolutionary novelties is a central issue in biology. Since Darwin's theory, difficulties in explaining how novel intricate body parts arose have often been used by creationists and other deniers to challenge evolution. Here, we describe the gustatory stalk of the Remo flounder (Oncopterus darwinii), an anatomically and functionally complex organ presumably used as a chemoreceptor probe to detect prey buried in the substrate. We demonstrate that the gustatory stalk is derived from the first dorsal-fin ray, which acquired remarkable modifications in its external morphology, integument, skeleton, muscles, and nerves. Such structural innovations are echoed in both functional and ecological specializations. We reveal that the gustatory stalk arose through the gradual accumulation of changes that evolved at different levels of the phylogenetic tree of ray-finned fishes. At least five preconditions arose in nodes preceding Oncopterus darwinii. This finding constitutes an interesting example of how evolution can deeply remodel body parts to perform entirely new functions. In this case, a trivial support structure primitively used for swimming became a sophisticated sensory tool to uncover hidden prey.

A rare occurrence of reversal in the common megrim Lepidorhombus whiffiagonis (Pleuronectiformes: Scophthalmidae) in the northern North Sea.

An adult (231 mm standard length, LS ; 371 mm total length, LT ), dextral (right-sided) common megrim Lepidorhombus whiffiagonis is reported from the northern North Sea. This is the first recorded occurrence of reversal in this species. Other than its reversed asymmetry, the specimen had morphological and meristic characteristics similar to those of non-reversed individuals of the same species.

Sexual dimorphism in scales of marbled flounder Pseudopleuronectes yokohamae (Pleuronectiformes: Pleuronectidae), with comments on the relevance to their spawning behaviour.

Variation of scales on the blind side of Pseudopleuronectes yokohamae in relation to sex, maturity and body size was examined. Immature males often have cycloid scales, while mature males have mostly ctenoid scales. Large females also often have ctenoid scales (but with fewer spines compared with males), and small females have mostly cycloid scales. The number of spines (ctenii) on the blind-side scale increases with body size in both sexes, indicating an ontogenetic change in scale morphology. As P. yokohamae spawn demersal eggs with males positioning themselves above the females on the ocular side, it is hypothesized that ctenoid scales on the blind side in mature males function for maintaining contact with females during spawning.

Morphometric changes in the cultured starry flounder, Platichthys stellatus, in open marine ranching areas.

Hatchery seeds released into open coasts for wildstock enhancement are often a biological pollutant and affect the recipient ecosystem integrity. We studied morphological changes in two hatchery populations of the starry flounder Platichthys stellatus; one released into the open coast from the hatchery (released population) and the other kept in the hatchery (captive population). The released population differed significantly from the captive population 3-36 months after release from the hatchery. Two-way ANOVA comparison revealed that 11 of 15 starry flounders differed significantly in morphological measurements, 10 of 15 differed in pigmentation, and 5 of 15 differed in morphometric ratios between the two populations. Pigmentation on the blind side (a representative sign of captive flounders) also differed between the two populations with an occurrence rate of 22.7% for the former and 39.5% for the latter groups. The released population was more similar to wild populations than to captive populations in terms of morphology; namely, longer and broader heads, a narrower body shape, longer fins, and a shorter and narrower peduncle.

Cardiac remodelling of Atlantic halibut Hippoglossus hippoglossus induced by experimental anaemia with phenylhydrazine.

Phenylhydrazine injections (0.3 mg kg(-1) , followed by a second injection of 0.1 mg kg(-1) 7 days later) induced a reproducible and stable anaemia in Atlantic halibut Hippoglossus hippoglossus, reducing the haematocrit and haemoglobin by 70.0 and 75.5%, respectively, over 3 weeks. There were no changes in blood electrolyte or lactate concentrations, although anaemic fish showed a 37.5 and 33.0% increase in cardiac somatic index and ventricular somatic index, respectively, compared with dimethyl sulphur oxide (DMSO) and saline vehicle controls. Changes in cardiac somatic indices did not correlate with the ratio of ventricular length:height and length:width did correlate with haematocrit and haemoglobin indicating that changes in cardiac shape may occur as a function of anaemic hypoxemia.

Post-release feeding and growth of hatchery-reared Japanese flounder Paralichthys olivaceus: relevance to stocking effectiveness.

The feeding and growth of hatchery-reared (HR) Japanese flounder Paralichthys olivaceus of c. 100 mm total length (L(T) ) released off the coast of Fukushima, Japan, were investigated. From 2 to 15 days after release, the HR P. olivaceus frequently exhibited high empty-stomach frequency (>40%), low stomach-content mass (<1% of body mass), reduced somatic condition from release (c.-10%) and negligible growth. Thereafter, empty-stomach frequency decreased, the stomach-content mass of HR fish increased to 2-8% of body mass, the somatic condition recovered and growth rate increased to 0·5-1·5 mm day(-1) . Prey items were initially mysids, shifting thereafter to fishes such as the Japanese anchovy Engraulis japonica, as observed similarly in wild counterparts. The proportion of mysids decreased with time after release irrespective of size at release, indicating the importance of mysids for adaptation to natural food. Recapture rates at age 1 year, derived from fish market surveys, varied greatly among release years (4-11%). The variation in the recapture rates was largely accounted for by the post-release growth rates (r(2) = 0·5), suggesting a relationship between the post-release growth of HR fish and their survival and subsequent stocking effectiveness.

Early ontogeny of the Atlantic halibut Hippoglossus hippoglossus head.

An ontogenetic sequence of Atlantic halibut Hippoglossus hippoglossus larvae, reared in intensive culture conditions, was cleared and stained and histologically processed to determine normal cranial chondrification for specimens ranging from 0 to 41 days post-hatch (dph). Twenty-six cranial cartilaginous structures were described, at daily intervals post-hatch. The ontogenetic trajectory, composed of alternating steps and thresholds, was interpreted as saltatory. In comparison with other flatfishes, H. hippoglossus exhibits delayed onset of chondrification. From 9 dph onwards, the ontogenetic trajectory resembles more than that of the turbot Psetta maxima than that of the common sole Solea solea or the summer flounder Paralichthys dentatus and winter flounder Pseudopleuronectes americanus. Hippoglossus hippoglossus with the gaping-jaw malformation, common in intensively cultured individuals of this species, were examined histologically. The reason larvae cannot close their mouth, as their yolk-sac resorbs, seems to be related to the fusion of the interhyal to the hyosymplectic and ceratohyal with which it is normally articulated.

Involvement of glycolysis activation in flatfish sexual size dimorphism: Insights from transcriptomic analyses of Platichthys stellatus and Cynoglossus semilaevis.

The starry flounder (Platichthys stellatus), a flatfish cultured at the margins of the North Pacific, displays an obvious female-biased growth advantage, similar to many other fish species. To reveal the molecular mechanism underlying sexual size dimorphism, a comparative transcriptomic analysis of the somatotropic and reproductive axes was conducted. In total, 156, 67, 3434, and 378 differentially expressed genes (DEGs) between female and male samples were obtained in the brain, liver, gonad, and muscle tissues (q < 0.05). These DEGs were significantly enriched for various GO terms, including ion channel activity, protein binding, lipid transporter activity, and glycolytic process. The significantly enriched KEGG pathways included insulin secretion, axon guidance, and glycolysis/gluconeogenesis. In a detailed analysis of DEGs in these significantly enriched pathways, 35 genes showed higher expression levels in female muscle tissues than in male muscle tissues. A protein-protein interaction network further revealed specific interactions involving the glycolysis related-protein enolase (ENO), triosephosphate isomerase (TPI), Bisphosphoglycerate mutase (BPGM), fructose-bisphosphate aldolase (ALDO), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, the role of glycolysis/gluconeogenesis was supported by an analysis of common DEGs between P. stellatus and Chinese tongue sole (Cynoglossus semilaevis). These results indicate that the activation of glycolysis in female muscle tissues contributes to flatfish sexual size dimorphism.

Ultrastructure of the olfactory epithelium in a flatfish, barfin flounder (Verasper moseri).

In this study, we examined the olfactory epithelium (OE) of the barfin flounder by transmission electron microscopy. As in the case of the ordinary teleost, the OE of the barfin flounder had 3 types of olfactory receptor cells (ciliated olfactory receptor cell, microvillous olfactory receptor cell and crypt cell), 3 types of supporting cells (ciliated, microvillous and crypt supporting cells) and basal cells. Each type of OE cells in the barfin flounder had similar ultrastructure to that of the ordinary teleost. Crypt cell is the third type of olfactory receptor cell unique to fish, whose function is unclear. The barfin flounder may be a suitable material to study crypt cells because it has relatively abundant crypt cells in the OE.