Comparing skeletal development of wild and hatchery-reared Senegalese sole (Solea senegalensis, Kaup 1858): evaluation in larval and postlarval stages

The Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.     

The prevalence of vertebral deformities is increased with higher egg incubation temperatures and triploidy in Atlantic salmon Salmo salar L.

Suboptimal egg incubation temperature is a risk factor for the development of skeletal deformities in teleosts. Triplicate diploid and triploid Atlantic salmon, Salmo salar L., egg batches were incubated at 6, 8 and 10 °C up until first feeding, whereupon fish were reared on a natural temperature before examination for externally visible skeletal deformities (jaw and spine) and radiographed for vertebral deformities and morphology at the parr stage. Increasing incubation temperatures and triploidy increased the number of fish showing one or more deformed vertebrae. Triploids had significantly higher mean vertebrae cranio‐caudal length (L) and dorsal‐ventral height (H) ratio at 6 and 10 °C than diploids, but triploidy had no effect on mean vertebrae centra area. Triploids demonstrated an increase in lower jaw deformities with increased incubation temperature, whereas jaw deformities were rare in diploids. Fish incubated at 10 °C had a significantly lower mean vertebral number than fish incubated at 6 °C, and triploids had lower mean vertebral numbers than diploids. Diploid fish with 58 vertebrae had a significantly higher mean vertebral centra area than fish with 59 vertebrae, but vertebral number did not affect the mean vertebral L/H ratio. The results are discussed with respect to the welfare and production of farmed salmonids.     

Skeletal development and abnormalities of the vertebral column and fins in larval stage of hatchery‐reared American shad,Alosa sapidissima

The present study describes the skeletal development and the occurrence of deformities in American shad, Alosa sapidissima, larvae from hatching to 45 days after hatching (DAH). The ontogeny of the vertebral column started at 16 DAH (days after hatching), with the formation of the posterior neural and haemal arches, and was complete at 28 DAH. In comparison, the vertebral centra started to form at 16 DAH, with ossification being visible in all centra at 38 DAH. The pectorals were the only fins that formed before the onset of feeding (at 2 DAH), with ossification being complete at 45 DAH. The caudal fin formed at 5 DAH, with ossification being complete at 40 DAH. Dorsal and pelvic fin development began at 6 DAH and 20 DAH respectively. The ossification of both the dorsal and pelvic fins was visible at 45 DAH. The anal fin began forming at 14 DAH, and was complete at 30 DAH. The ossification of the anal fin was complete at 45 DAH. Overall, 22 types of skeletal deformities were detected in about 41% of individuals. Most anomalies were detected in the haemal region, while the fewest anomalies were detected in the anal fin. In addition, the frequency of deformities gradually increased with fish age at the different developmental stages. Our results are expected to contribute baseline information on how rearing conditions impact skeletal development, in addition to identifying potential causative factors of skeletal deformities.     

Vertebral deformities in cultured red sea bream, Pagrus major, Temminck and Schlegel

Vertebral deformities were investigated in cultured red sea bream, Pagrus major. In the field, deformities in seedlings were categorized and their incidence was calculated. In the laboratory, the symptoms of major vertebral deformities were examined morphologically using radiographs and by making transparent skeletal specimens. The internal structure of deformed vertebrae was examined histologically. The shortened body condition had the highest incidence (0.9–8.3%) of all deformities in the seedlings. In individuals with the shortened body condition, the ratio of trunk and caudal part length to body height was smaller. These fish had skeletal anomalies in the vertebrae, mainly centrum defects (64.3%) or undersized centrums (25.2%). The specimens with centrum defects had a characteristic anomaly in the vertebrae, with plural pairs of neural and haemal spines on a single centrum. This anomaly was frequently observed in the posterior abdominal vertebrae. The internal skeletal structure of such abnormal centrums was basically the same as that of normal centrums. In all the specimens with undersized centrums, both the centrum length and diameter were shorter than normal except for the first and second centrum, and urostyle.     

Osteological ontogeny and malformations in larval and juvenile golden pompano Trachinotus ovatus (Linnaeus 1758)

The osteological ontogeny and the incidence of deformities occurred in vertebral column and caudal complex were studied from 1 day post hatch (DPH) to 31 DPH in Trachinotus ovatus. Results indicate that the development of both caudal complex and vertebral column initiated between 7 DPH and 9 DPH along with the calcification of haemal arches and neural arches, and occurrence of caudal elements. The last element of caudal complex, uroneural, appeared on 18 DPH. A high number of malformations in the vertebral column and caudal complex were detected in this study. More than 33% experimental fish exhibited at least one type of malformation. Results from this study provide the first reference on the osteological ontogeny and malformation of golden pompano. Such information can be applied to the quality control for fingerling production in golden pompano.     

Osteological development and deformities in hatchery‐reared longtooth grouper (Epinephelus bruneus): Vertebral column,dorsal‐fin supports and caudal‐fin skeleton

Skeletal deformities are one of the serious problems in hatchery‐reared longtooth grouper (Epinephelus bruneus). In this study, seed production of longtooth grouper was carried out in four large hatchery‐grade tanks. Osteological development of the vertebral column, caudal‐fin skeleton and dorsal‐fin supports in larvae and juveniles was described. The vertebral ontogeny started at 4.8 mm total length (TL) with the formation of neural spines 2 and 3, and the vertebral centra began forming at 5.3 mm TL. By 16 mm TL, all vertebral bony elements were formed except for the dorsal ribs. The onsets of the ontogeny of the caudal‐fin skeleton and dorsal‐fin supports occurred at approximately 5.1 mm and 3.9 mm TL, respectively, and the completion of these bony elements occurred at approximately 20 mm TL. The incidence of deformity was examined; some type of deformity was observed in 57%–68% of the specimens. These deformities mainly occurred as three types: lordosis, around vertebrae 1–5; saddleback‐like syndrome, around vertebrae 7–11; and vertebrae fusion, around vertebrae 22–24. The incidence of prehaemal lordosis was significantly higher among individuals with an uninflated swim bladder than among those with an inflated swim bladder.     

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.     

Development of deformities at the vertebral column in Diplodus sargus (L., 1758) early larval stages

One of the bottleneck problems of Diplodus sargus farming is a high incidence of skeletal deformities at the vertebral column. In this study, the pattern of vertebral deformities were determined in three different larval batches from 2 to 30 days after hatching (DAH). During this period, 60 larvae per spawn were observed at 2, 8, 13, 15, 18, 21, 23, 25, 27 and 30 DAH and the different types and frequency of vertebral column malformations were registered. Deformities started from 8 to 13 DAH, when the percentage of deformed larvae rose from approximately 5% to 40%. At 15 DAH, skeletal malformations had frequencies up to 80% of the observed larvae. Serious malformations such as kyphosis, scoliosis and lordosis were observed at 18 DAH but seldom in percentages higher than 15%. Vertebral fusions and compressions especially affected the preurostyle region. Abnormal shape vertebrae were more frequent between vertebrae 15 and 21. Other malformations observed were hypertrophic vertebrae, more common in the trunk and caudal regions, reaching percentages higher than 50% in the former. This study has useful information concerning skeletal malformations at the vertebral column of D. sargus larvae, as it identifies the main deformities observed and the ages of highest incidence.     

Case studies of spinal deformities in ornamental koi,Cyprinus carpio L.

This is a study of vertebral deformities in ornamental koi based on computed radiography and skeletons cleaned by dermestid beetles (Dermestes maculatus). All koi developed gradual onset of swimming abnormalities as adults. Extensive intervertebral osteophyte formation correlated with age of fish and was associated with hindquarter paresis in one koi. Vertebral compression and fusion were the most common spinal deformities occurring at multiple sites, similar to findings in other farmed fish. Site‐specific spinal deformities were thought to develop due to differences in swimming behaviour and rates of vertebral growth. One koi had offspring with spinal deformities. Spinal deformities are significant problems in both European and Australian food fish hatcheries. The heritability of vertebral deformities in farmed fish is reportedly low unless there is concurrent poor husbandry or nutritional deficiencies. The specific aetiologies for vertebral deformities in koi in this study could not be ascertained. Current knowledge on spinal deformities in the better studied European food fish species suggests multifactorial aetiologies. Future research should include prospective longitudinal studies of larger numbers of koi from hatch and consideration of all potential risk factors such as husbandry, nutrition, temperature, photoperiod and genetics.     

Osteological development and anomalies in larval stage of hatchery‐reared yellow catfish Pelteobagrus fulvidraco

Comprehensive knowledge of osteological development of fish not only provides means for understanding its functional development, but also allows early detection of skeletal anomalies. The present study was conducted to determine osteological development and occurrence of anomalies from the first to the 40th day after hatching (DAH) for yellow catfish Pelteobagrus fulvidraco, a commercially important species in China. Vertebral ontogeny started with formation of anterior neural arches at 3 DAH, and completed at 6 DAH. Vertebral centra started to develop at 4 DAH and ossification in all centra was visible at 15 DAH. Caudal fin appeared at 2 DAH and ossification was visible at 26 DAH. The onset of dorsal and anal fins appeared at 4 DAH and their ossifications were visible at 20 DAH. Pectoral fins were present before first feeding and formed entirely at 9 DAH, and their ossifications were visible at 21 DAH. Pelvic fins appeared at 9 DAH, and formed completely at 21 DAH. The ossification of pelvic fins was not finished until the end of the experiment (40 DAH). 24 types of skeletal anomalies were observed. About 20% of individuals showed at least one anomaly. Haemal vertebrae anomalies occurred at the highest frequency, followed by pre‐haemal, caudal vertebrae and anal fin anomalies, and caudal fin had the lowest anomalies frequency. For the first time, our study determined osteological development and anomalies incidence in larval yellow catfish, which help further investigations into rearing conditions leading to appearance of these anomalies to prevent their incidence.     

Effect of dietary vitamin A on Senegalese sole (Solea senegalensis) skeletogenesis and larval quality

The effects of different levels of vitamin A (VA) in Senegalese sole larval performance and development were evaluated by means of a dietary dose–response experiment using enriched Artemia metanauplii as a carrier of this micronutrient. Larvae were fed from 6 to 27 days post hatch (dph) with enriched Artemia containing graded levels of total VA (1.3, 2.1, 4.5 and 12.9 µg VA mg^− 1 DW). The content of VA in live prey directly affected its accumulation in larvae and early juveniles. Retinyl palmitate accumulated during larval ontogeny, whereas retinol showed the opposite trend, decreasing from hatching until 41 dph and then remaining constant until the end of the study.In metamorphic larvae (10 and 15 dph), VA did not affect the number of thyroid follicles or the intensity of the immunoreactive staining of T₃ and T₄. However, at older stages of development (post-metamorphic larvae: 20, 30, 41 and 48 dph), VA decreased the number of thyroid follicles but increased their mean size and enhanced T₃ and T₄ immunoreactive staining. A dietary excess of VA did not affect either larval performance in terms of growth and survival or the maturation of the digestive system. However, the most remarkable impact of this morphogenetic nutrient was detected during skeletal morphogenesis. Dietary VA accelerated the intramembranous ossification of vertebral centrums, which led to the formation of a supranumerary haemal vertebra and a high incidence of fused and compressed vertebrae in fish fed 2.1, 4.5 and 12.9 mg VA mg^− 1 DW. In addition, VA also affected those structures from vertebrae and caudal fin formed by chondral ossification, leading to defects in their shape and fusions with adjacent skeletal elements. In particular, the caudal fin was the region most affected by the dietary treatments. In order of importance, the bones with more developmental anomalies were the modified neural and haemal spines, epural, hypurals and parahypural. The impact of systemic factors such as thyroidal hormones in skeletogenesis should not be neglected since present results revealed that an excess of dietary VA affected the levels of T₃ and T₄, which might have affected bone formation and remodelling, leading to skeletal deformities.     

The external phenotype–skeleton link in post‐hatch farmed Chinook salmon (Oncorhynchus tshawytscha)

Skeletal deformities in farmed fish are a recurrent problem. External malformations are easily recognized, but there is little information on how external malformations relate to malformations of the axial skeleton: the external phenotype–skeleton link. Here, this link is studied in post‐hatch to first‐feed life stages of Chinook salmon (Oncorhynchus tshawytscha) raised at 4, 8 and 12°C. Specimens were whole‐mount‐stained for cartilage and bone, and analysed by histology. In all temperature groups, externally normal specimens can have internal malformations, predominantly fused vertebral centra. Conversely, externally malformed fish usually display internal malformations. Externally curled animals typically have malformed haemal and neural arches. External malformations affecting a single region (tail malformation and bent neck) relate to malformed notochords and early fusion of fused vertebral centra. The frequencies of internal malformations in both externally normal and malformed specimens show a U‐shaped response, with lowest frequency in 8°C specimens. The fused vertebral centra that occur in externally normal specimens represent a malformation that can be contained and could be carried through into harvest size animals. This study highlights the relationship between external phenotype and axial skeleton and may help to set the framework for the early identification of skeletal malformations on fish farms.     

Skeletal development and deformities in cultured larval and juvenile seven-band grouper, Epinephelus septemfasciatus (Thunberg)

The seven‐band grouper, Epinephelus septemfasciatus (Thunberg), is currently recognized as a potential new species for aquaculture in Japan. This study describes normal and abnormal skeletal development of the jaw and vertebrae in cultured larvae and early juveniles of E. septemfasciatus. The ontogenetic stages at which skeletal deformities of jaw and vertebra developed were also described for this species. Osteological observations were made using a clearing and staining method for larvae and soft X‐ray photographs for juveniles. A high incidence of skeletal deformities was observed in the jaws and vertebral column during the larval and juvenile stages. Most of the jaw deformities were explained by an abnormal maxilla curvature. Jaw deformities were visually evident from flexion stage after ossification of the deformed elements. Deformities in the vertebral column (mostly lordosis) were observed from the post‐larval stage and became more evident as growth proceeded. The lordosis generally occurred on the positions of the 8–11th vertebra. These types of deformities are compared with those of other species, and possible causative factors of the skeletal deformities are discussed.
     

Scaling of body‐shape quality in reared gilthead seabream Sparus aurata L. Consumer preference assessment,wild standard and variability in reared phenotype

In this study, a scale of quality for the body shape of Gilthead seabream was developed. For this purpose, a questionnaire survey was carried out to study the preferences of consumers for the body shape of seabream. Furthermore, the variation in body shape in reared seabream was studied under two rearing environments (sea cages vs. tanks) and in comparison with wild‐caught individuals. Following the test–retest analysis of reliability, the 22 of 65 participants in the survey presented a significant correlation between repeated evaluations of the same subjects and were classified as responders of high discrimination efficiency or/and reliability (HDR). Geometric morphometric analysis on the evaluation of HDR responders revealed that compared with the fish of fair body shape, fish with preferred body shape were characterized mainly by a more proximal position of the base of the pelvic fins, the anterior base of the anal fin and the anterior dorsal fin. Of the 45 wild‐caught specimens examined, 26 presented a normal lateral line and no obvious skeletal deformities. The body shape of the normal wild‐caught fish was compared with the shape of reared seabream. Results showed that both the origin of fish (wild vs. reared) and the rearing methodology during the on‐growing phase significantly affected the body shape of seabream (P < 0.001). Compared with the reared fish, the wild‐caught fish were characterized mainly by a less prominent belly, slender trunk, smaller head, shorter caudal peduncle and a more posterior‐ventral position of the upper jaw.     

Association of a lordosis-scoliosis-kyphosis deformity in gilthead seabream (Sparus aurata) with family structure

Skeletal deformities constitute a major problem for aquaculture industry by decreasing the final value of the fish. An analysis of skeletal malformations in 11,640 fish was performed considering families and triplicates per family. Thirtynine different skeletal abnormalities were detected, such as lordosis, vertebral fusion, absence of one or both operculum, bent-jaw, etc. Moreover, a new unusual complex spinal column deformity consisting of a consecutive repetition of lordosis, scoliosis and kyphosis (LSK) from the head to the caudal fin was described. This syndrome was statistically associated with the family structure (Z_{2H–family,LSK}=3.49; p<0.05). The incidence of this deformity was 0.2% in the whole population, and 6.5% within the affected family (2H). The environmental and genetic causes are discussed.     

Towards a classification and an understanding of developmental relationships of vertebral body malformations in Atlantic salmon (Salmo salar L.)

Vertebral column malformations in Atlantic salmon occur under farming conditions and in wild specimens and are commonly diagnosed by X-ray. The literature uses varied terms to describe malformations, and often, different terms refer to similar phenomena or similar terms describe unrelated pathologies. A coherent classification of spine defects is lacking. This shortcoming complicates the comparison of studies, makes it difficult to link particular malformations to particular causes and hampers the assessment of the prevalence of spinal deformities.Expanding available systems to categorise skeletal malformations in different teleost species, we propose 20 types of salmon vertebral column malformations that are repetitively observed under farming conditions. As vertebral column deformities are usually diagnosed by X-ray, the classification of deformity types is based on radiological observations that have been obtained over the past 10 years from more than 5000 specimens. The proposed deformity types should be identifiable independent from the type (analogue, digital) and the quality of radiographs. We propose nine types of compression and fusion-related deformities, four types of abnormal radiotranslucent or radiopaque vertebral bodies, three types of spine curvatures, three types of symmetry deviations and displacement of vertebral bodies and one type of severe multiple malformations. Based on the suggested categories we discuss the relationships between types of vertebral body malformations. Several types of malformations involve the development of heterotopic cartilage, a pathology that is possibly linked to mechanical overload and accelerated growth. Refined categories for vertebral body malformations should help link particular types of malformations to particular causes. It should also help find out which deformities represent final stages and which deformities represent transitory stages.     

A radiological study on the development of vertebral deformities in cultured Atlantic salmon (Salmo salar L.)

This study investigated the development of skeletal deformities in individually tagged Atlantic salmon (n = 805) from the parr stage (Sept 2001, 70 g mean ± 34 g S.D.) until 10 month after transfer to seawater (Feb 2003, 3040 g mean ± 1097 g S.D.). A subgroup of the total population (n = 129) was radiographed as parr (Feb 2002) and again 10 months after transfer to seawater (Feb 2003). Eight percent of the males matured sexually during their first autumn in sea (jack), and were excluded from further analysis. Based on an external examination of each fish (n = 773), 1.8% (n = 14) developed skeletal deformities (0.1% operculum deformities (n = 1), 0.4% jaw deformities (n = 3), 1.3% vertebral deformities (n = 10)) during the experiment. Based on evaluation of radiographs (n = 129), the prevalence of vertebral deformities was 7.0% (n = 9) at the parr stage, and 12.4% (n = 16) 10 months after transfer to seawater. From radiographs, longitudinally compressed vertebral bodies without intervertebral spaces were classified as ankylosis and compression, longitudinally compressed vertebral bodies with intervertebral spaces were classified as compressions, and dislocated vertebral bodies with a normal morphology and without intervertebral spaces were classified as ankylosis and dislocation. Of the fish that developed deformities during the experiment, 8 had ankylosis and compressions, 7 had compressions and 1 had ankylosis and dislocation. Ankylosis and compression developed in the region between vertebrae number 1 and 16, whereas compressions developed in the region between vertebrae number 13 and 49. Most of the individuals with compression 10 months after transfer to seawater had normal vertebral columns as parr, whereas all individuals with compression and ankylosis 10 months after transfer to seawater had deformed vertebral columns as parr.     

The effect of rearing conditions on development of saddleback syndrome and caudal fin deformities in Dentex dentex (L.)

The development of saddleback syndrome and of caudal fin deformities in Dentex dentex (Linnaeus, 1758) was compared under two rearing methods, extensive (E) and semi-extensive (S). The osteological appearance and the meristic characters of the reared fish were compared to that of D. dentex juveniles, collected from the natural environment. All the wild juveniles were normal in respect to their osteological appearance, while the reared specimens presented skeletal deformities resulting from different rearing methods applied. Saddleback syndrome (4.0–4.4%) and severe external deformities of the caudal fin (14.3–15.0%) characterised exclusively the semi-extensive populations, while the extensive populations presented severe abnormalities of the pre-ural centra (25.0–25.6%) with significantly higher frequency than the S reared fish (5.8%). Saddleback syndrome was expressed as a lack of one to all the hard spines of the dorsal fin, accompanied by shape, number and position abnormalities of the related pterygiophores. Caudal fin deformities were mainly characterised by the lack of the upper lepidotrichia or/and dermatotrichia, accompanied by severe deformities of the supporting elements of the upper lobe. The saddleback syndrome and severe caudal fin deformities were anatomically and ontogenetically related to each other, originating at the early larval stage as a result of abnormalities of the primordial marginal finfold and of the posterior tip of the notochord. In respect to the dominant phenotypes, the meristic characters of the normal reared fish were not differentiated from those of the wild, but they presented higher variability.

The results are discussed in view of the aetiology, applications and research targets for the erasure of skeletal malformations.     

Qualitative analysis of cartilaginous jaw element malformation in cultured yellowtail kingfish (Seriola lalandi) larvae

A central problem facing worldwide culture of yellowtail kingfish (Seriola lalandi) is the presence of skeletal malformations, including jaw deformities. This study presents a morphological characterization of normal and abnormal cartilage jaw structures during early larval development. Samples of 70–150 larvae were collected from three cohorts from 2 to 9 days post hatching, anaesthetized and fixed for cartilage staining. Cartilaginous components were defined clearly at four days post hatch (dph) (4.65 ± 0.05 mm total length), and abnormal jaw structures were detectable at this time. Jaw deformities observed included extension of Meckel's cartilage with or without ventral bending of the anterior tip, displacement of ceratohyal and hypohyal cartilage ventrally and below Meckel's cartilage, and shortening and dorsal flexion of the lower jaw. At 4 dph, between 44% and 47% of all larvae examined had jaw abnormalities. The contribution of each deformity to the total number of deformities was variable among the three cohorts examined. To compare shape difference accurately we performed an exploratory, landmark‐based geometric morphometric analysis using seven homologous landmarks. Larvae were classified into three jaw morphology groups. The geometric morphometric approach provides a useful tool to standardize classification of cartilage jaw abnormalities at early developmental larval stages. Early recognition of developing abnormalities is of importance for fish farmers in both improving fish selection efficiency and for evaluating effects of rearing parameters.     

Influence of high temperature on cadmium-induced skeletal deformities in juvenile mosquitofish (<Emphasis Type="Italic">Gambusia affinis</Emphasis>)

The aim of this study was to assess the effect of high temperature on cadmium (Cd)-induced skeletal deformities in juvenile Mosquitofish, Gambusia affinis. For this purpose, 188 juveniles (1 day old) were equally divided into the control group, which was maintained in Cd-free water at 24°C, and three treated groups exposed either to Cd (0.4 mg/l as Cd Cl₂) at 24°C, to high temperature (32°C), or to Cd at 32°C for 30 days. The results showed that Cd exposure at 24°C significantly increased the Cd accumulation (P < 0.0001) in the whole tissues of juveniles as well as the incidence of skeletal deformities (P < 0.01) compared with control animals. Exposure to high temperature also led to a significant increase in the incidence of skeletal deformities (P < 0.01) with respect to the control group. Interestingly, our results showed that the combined exposure to Cd and high temperature led to a more significant increase in Cd accumulation and in the frequency of spinal deformities than exposure to Cd or high temperature alone. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.