A comparative whole genome sequencing analysis identified a candidate locus for lack of operculum in cultivated gilthead seabream (Sparus aurata).

The gilthead seabream (Sparus aurata, Sparidae family) is commonly used for aquaculture. Despite its great economic value, several problems in its cultivation remain. One of the major concerns is the high frequency of morphological abnormalities occurring during the early developmental stages. Partial and/or total lack of operculum is the most frequent anomaly affecting the fish cranial region. The existence of genetic factors that can at least partially determine this defect has been hypothesized. In this work, two DNA pools of highly related fry, one composed of normal-looking (control) fish and the other lacking an operculum (case), were constructed and whole-genome resequencing data produced from the two were compared. The analysis revealed a 1 Mb region on chromosome 2 with higher heterozygosity in the lack of operculum DNA pool than in the control DNA pool, consistent with the enrichment, in the first DNA pool, of one or more haplotypes causing or predisposing to the defect together with other normal haplotypes. A window-based FST analysis between the two DNA pools indicated that the same region had the highest divergence score. This region contained 2921 SNVs, 10 of which, with predicted high impacts (three splice donor and seven stop-gained variants), were detected in novel genes that are homologous to calcium-sensing receptor-like genes, probably involved in bone development. Other studies are needed to clarify the genetic mechanisms involved in predisposing fry to this deformity and then to identify associated markers that could be used in breeding programs to reduce the frequency of this defect in the broodstock.

The Effect of Orally Supplemented Melatonin on Larval Performance and Skeletal Deformities in Farmed Gilthead Seabream (Sparus aurata).

The gilthead seabream larval rearing in continuous light is common in most Mediterranean hatcheries to stimulate larval length growth and increase food consumption. Several studies have shown that continuous light affects larval development and increases the prevalence of skeletal deformities. Melatonin is a crucial pineal neurohormone that displays daily secretion patterns, stimulates cell proliferation and embryonic development in Atlantic salmon and zebrafish, and improves osseointegration in mice and humans. However, no studies have examined the effects of orally supplemented melatonin on skeletal deformities in Sparus aurata larvae. We administered exogenous melatonin to gilthead seabream larvae via enriched rotifers and nauplii of Artemia. Exogenous melatonin induced bone deformities and stimulated parathyroid hormone-related protein-coding gene (PTHrP) mRNA expression. In addition to the melatonin-induced PTHrP high expression level, the recorded non coordinated function of skeletal muscle and bone during growth can be the fountainhead of bone deformities. Both myosin light chain 2 (mlc2) and bone gamma-carboxyglutamate protein-coding gene (bglap) expression levels were significantly affected by melatonin administration in an inverse dose-response manner during the exogenous melatonin administration. This is the first study to report the effect of inducing melatonin bone deformities on Sparus aurata larvae reared under ordinary hatchery conditions.

Recovery of haemal lordosis in Gilthead seabream (Sparus aurata L.).

Haemal lordosis is a frequent abnormality of the vertebral column. It has been recorded to develop in different finfish species, during the hatchery rearing phase. Under certain conditions, this abnormality reaches a high prevalence and severity degree, with significant effects on the external morphology of the fish. We show that haemal lordosis recovers during the on-growing of Gilthead seabream in sea cages. At the end of the hatchery phase, 1700 seabream juveniles were tagged electronically and examined for the presence of haemal lordosis. Subsequently, their morphology was examined periodically up to the end of the on-growing period. We found that the prevalence of fish with a lordotic external morphology decreased during the studied period by approximately 50%. Interestingly, 27% of the recovered fish presented a completely normal vertebral column. Geometric morphometric analysis showed no significant differences in the body shape between the fish with a recovered normal phenotype and the fish that were normal since the beginning of the on-growing period. Our results provide the first evidence for the recovery of lordosis during the growth of fish. A mechanism with multiple levels of remodeling of abnormal bones is suggested.

Morphological and histological characterization of an ectopically mineralized structure in a gilthead sea bream Sparus aurata with opercular deformation.

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro-CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix-rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair.

Gene expression analyses in malformed skeletal structures of gilthead sea bream (Sparus aurata).

The incidence of skeletal anomalies in reared fish has been translated for years in important economic losses for the aquaculture industry. In the present study, we have analysed the gene expression of extracellular matrix components and transcription factors involved in bone development in gilthead sea bream presenting different skeletal anomalies: lordosis (LD), lordosis-scoliosis-kyphosis (LSK) or opercular, dental or jaw malformations in comparison with control (CT) specimens. Results showed a possible link between the presence of LD and LSK and the significant downregulation of genes involved in osteoblasts' maturation and matrix mineralization (collagen type 1-alpha, osteopontin, osteocalcin, matrix Gla protein and tissue non-specific alkaline phosphatase), as well as in bone resorption (cathepsin K and matrix metalloproteinase 9) compared to CT animals. Contrarily, the key osteogenic transcription factor runx2 was upregulated in the malformed vertebra suggesting impaired determination of mesenchymal stem cells towards the osteoblastic lineage. Despite the gene expression patterns of the other malformed structures were not affected in comparison with CT fish, the results of the present study may contribute in the long term to identify potential candidate gene profiles associated with column deformities that may help reducing the incidence of appearance of skeletal anomalies in this important aquaculture species.

Ontogenesis of opercular deformities in gilthead sea bream Sparus aurata: a histological description.

The aim of this study was to characterize histological changes during opercular osteogenesis in farmed gilthead sea bream Sparus aurata larvae from 7 to 69 days post hatching (dph) and compare normal osteogenesis with that of deformed opercles. Mild opercular deformities were first detected in 19 dph larvae by folding of the opercle's distal edge into the gill chamber. Here, the variation in the phenotype and the irregular bone structure at the curled part of the opercles is described and compared with the histology of normal opercles. Results indicated that deformed opercles still undergo bone growth with the addition of new matrix by osteoblasts at the opercular surface, especially at its edges. No significant difference was found in bone thickness between deformed and normal opercles. In addition to differences in bone architecture, differences in collagen fibre thickness between normal and deformed opercles were also found.

Quantitative trait loci for a neurocranium deformity, lack of operculum, in gilthead seabream (Sparus aurata L.).

Lack of operculum, a neurocranial deformity, is the most common external abnormality to be found among industrially produced gilthead seabream (Sparus aurata L.), and this entails significant financial losses. This study conducts, for the first time in this species, a quantitative trait loci (QTL) analysis of the lack of operculum. A total of 142 individuals from a paternal half-sibling family (six full-sibling families) were selected for QTL mapping. They had previously shown a highly significant association with the prevalence of lack of operculum in a segregation analysis. All the fish were genotyped for 106 microsatellite markers using a set of multiplex PCRs (ReMsa1-ReMsa13). A linear regression methodology was used for the QTL analysis. Four QTL were detected for this deformity, two of which (QTLOP1 and QTLOP2) were significant. They were located at LG (linkage group) nine and LG10 respectively. Both QTL showed a large effect (about 27%), and furthermore, the association between lack of operculum and sire allelic segregation observed was statistically significant in the QTLOP1 analysis. These results represent a significant step towards including marker-assisted selection for this deformity in genetic breeding programmes to reduce the incidence of the deformity in the species.

Identification of Quantitative Trait Loci Associated with the Skeletal Deformity LSK complex in Gilthead Seabream (Sparus aurata L.).

Morphological abnormalities, especially skeletal deformities, are some of the most important problems affecting gilthead seabream (Sparus aurata L.) aquaculture industry. In this study, a QTL analysis for LSK complex deformity in gilthead seabream is reported. LSK complex is a severe deformity consisting of a consecutive repetition of three vertebral deformities: lordosis, scoliosis, and kyphosis. Seventy-eight offspring from six breeders from a mass-spawning were analyzed: five full-sibling families, three maternal, and two paternal half-sibling families. They had shown a significant association with the LSK complex prevalence in a previous segregation analysis. Fish were genotyped using a set of multiplex PCRs (ReMsa1-13), which includes 106 microsatellite markers. Two methods were used to perform the QTL analysis: a linear regression with the GridQTL software and a linear mixed model with the Qxpak software. A total of 18 QTL were identified. Four of them (QTLSK3, 6, 12, and 14), located in LG5, 8, 17, and 20, respectively, were the most solid ones. These QTL were significant at genome level and showed an extremely large effect (>35%) with both methods. Markers close to the identified QTL showed a strong association with phenotype. Two of these molecular markers (DId-03-T and Bt-14-F) were considered as potential linked-to-this-deformity markers. The detection of these QTL supposes a critical step in the implementation of marker-assisted selection in this species, which could decrease the incidence of this deformity and other related deformities. The identification of these QTL also represents a major step towards the study of the etiology of skeletal deformities in this species.

Inheritance of skeletal deformities in gilthead seabream (Sparus aurata) - lack of operculum, lordosis, vertebral fusion and LSK complex.

Morphological abnormalities in farmed gilthead seabream (Sparus aurata) are a major problem as it entails significant economic losses. In this study, 3 large scale experiments under different conditions of spawning, offspring handling and breeders phenotype were performed to analyze the inheritance of 4 types of deformities in this species: lack of operculum, lordosis, vertebral fusion, which are 3 of the most important skeletal deformities, and LSK, which is a consecutive repetition of lordosis/scoliosis/kyphosis. In Exp. [1] (mass spawning and fingerling sorting), 900 fish were analyzed at 509 d post-hatching: 846 fish that had been on-grown in a farm and 54 LSK-deformed fish that had been reared separately after being selected during the fingerling sorting process. A total of 89 families were represented. A statistically significant association between 5 of these families (from 6 breeders) and LSK-deformed fish was found. In Exp. [2] (mass spawning and no fingerling sorting), 810 fish were analyzed at 2 ages: 179 and 689 d post-hatching. Significant relationships between 2 of the breeders and 2 of the families with the lack of operculum prevalence of their descendants were found at 689 d but not at 179 d. Heritabilities: 0.09 ± 0.09 at 179 d and 0.17 ± 0.08 at 689 d. Column deformities prevalence was low and no association with family was observed. Family relationships were determined by microsatellites multiplex PCR in both experiments. In Exp. [3] (designed mating), sires suffering from lordosis or lack of operculum or vertebral fusion deformities were mated with non-deformed dams and a mass-spawning mating was considered as a control. After analyzing 11,503 offspring at 159 d post-hatching, a significant relationship between each deformity prevalence and the mating of breeders suffering from the same deformity was observed. In addition, a significant prevalence of lack of operculum in offspring from lordotic matings was observed. Heritabilities ranged from 0.34 to 0.46 for the 3 deformities. The results of the present study suggest that these deformities have a genetic origin. They also suggest that the sorting process is not recommended and that producers should consider these deformities in genetic breeding programs to significantly improve their fish morphological quality and to minimize farmed fish deformities incidence.

A new type of lordosis and vertebral body compression in Gilthead sea bream, Sparus aurata L.: aetiology, anatomy and consequences for survival.

A new type of vertebral malformation is described, consisting of deformed cartilaginous neural and haemal processes and the compression and fusion of vertebral bodies. The malformation is designated as haemal vertebral compression and fusion (haemal VCF). We studied the aetiology of the malformations and described microanatomical histopathological alterations. The malformations were detected during routine quality control in one of six monitored Gilthead sea bream populations. Haemal VCF affected the posterior part of the vertebral column (haemal vertebrae). In 20% of the deformed specimens, haemal VCF was combined with lordosis. At 35 dph (days post-hatching), early anatomical signs of the haemal VCF consisted of abnormal centrum mineralization, malformed cartilaginous neural and haemal processes and developing lordotic alterations. The histological examination of the deformed individuals revealed that haemal VCF is preceded by notochord abnormalities. The frequency of deformed individuals was three times higher at 35 than at 61 dph (50.3% vs. 17.2%, n = 157 and n = 250, respectively). No signs of repair or reversion of malformations have been observed. Thus, the steep decrease in deformities in older animals suggests that haemal VCF is linked to high mortality rates. The results are discussed in respect of the possible causative factors of haemal VCF.

Recovery of opercular anomalies in gilthead sea bream, Sparus aurata L.: morphological and morphometric analysis.

Opercular anomalies are very frequent in reared gilthead sea bream and these can negatively influence the product value. Field observations have suggested that opercular malformations can recover over time. In order to verify this hypothesis, 140-day-old gilthead sea bream with monolateral opercular anomalies were divided into three groups, according to the type and increasing seriousness of the opercular malformations, and another group was composed of fish with bilateral opercular anomalies. All groups were monitored for 16 months. In the group with monolateral anomalies, the opercular recovery process was documented by morphological (stereomicroscope) and morphometric analysis. For the latter analysis, two relevant areas, A and T, were identified in the cephalic region. The ratio (T - A)/T, tending to 1, represents the recovery index (RI) of anatomical integrity and quantifies the recovery level of opercular complex anomalies. Results suggested that the recovery process was considerable over the 16 months of investigation but should not be considered complete. At the end of the study, 61% of the gilthead sea bream population with monolateral opercular defects recovered external integrity, whereas the population with bilateral defects showed a poorer recovery capability.

Changes in the soluble bone proteome of reared white seabream (Diplodus sargus) with skeletal deformities.

One of the main constrains for commercial aquaculture production of white seabream (Diplodus sargus) is the high incidence of skeletal malformations in reared fish. The purpose of this study was to obtain a better understanding of the mechanisms involved in the development of these types of skeletal malformations by comparative proteomic analysis of the vertebral column of normal and deformed fish using 2DE for protein separation and MS for protein identification. We observed a 3.2 and 3.4-fold increase in the expression of two tropomyosin isoforms, one of which (tropomyosin-4) is essential for the motility and polarization cycles of osteoclasts. Furthermore, a 1.6, 1.7 and 1.8-fold increase in three parvalbumin spots was detected, suggesting a cellular response to increased intracellular Ca²(+) levels. These results can be interpreted as signs of increased cellular activity in the bone of white seabream with skeletal deformities coupled to a higher degree of calcium mobilization, which elicits further studies into the use of these proteins as indicators of skeletal metabolic state.

Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions.

The toxicity of dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mainly mediated by the aryl hydrocarbon receptor (AHR), which regulates the multiple target genes including cytochrome P4501A (CYP1A). In general, bony fishes, which possess at least two distinct AHRs are one of the most sensitive vertebrates to TCDD in early life stage. However, the physiological and toxicological roles of piscine multiple AHRs are not fully understood, especially in marine fish. To understand which AHR is responsible for TCDD toxicity in a marine fish species, we characterized the early life stage toxicity related to the expression of AHRs and CYP1A in red seabream (Pagrus major). The embryos at 10h post-fertilization (hpf) were treated with 0-100 microg/L TCDD for 80 min waterborne exposure. TCDD dose-dependently elicited developmental toxicities including mortality, yolk sac edema, retarded body growth, spinal deformity, reduced heart rate, shortened snout, underdeveloped fin, heart, and lower jaw. Intriguingly, hemorrhage and pericardium edema, typical TCDD developmental defects noticed in other fish species, were not found in red seabream until test termination. The EC(egg)50s for yolk sac edema, underdeveloped fin, and spinal deformity were 170, 240, and 340 pg/g, respectively. The LC(egg)50 was 360 pg/g embryo, indicating that this species is one of the most sensitive fishes to TCDD toxicity. The expression levels of rsAHR1, rsAHR2 and CYP1A mRNAs were also determined in different developmental stages. The rsAHR2 mRNA expression dose-dependently increased following TCDD exposure, while rsAHR1 mRNA level was not altered. Level of rsAHR2 mRNA measured by two-step real-time PCR was 30 times higher than rsAHR1 in embryos treated with the highest dose. Temporal patterns of rsAHR2 and CYP1A mRNAs were similar in TCDD-treated embryos, representing a significant positive correlation between rsAHR2 and CYP1A mRNA levels, but not between rsAHR1 and CYP1A. In comparison of temporal trends of TCDD-induced AHRs and CYP1A expression, and developmental toxicities, the highest expression of rsAHR2 and CYP1A mRNA were detected prior to the appearance of maximal incidence of TCDD toxic manifestations. These results suggest that rsAHR2 may be dominantly involved in the transcriptional regulation of CYP1A, and several TCDD defects are dependent on the alteration of rsAHR2 and/or rsAHR2-CYP1A signaling pathway that is controlled through their expression levels.