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.     

海水养殖鱼类仔、稚鱼骨骼发育与畸形发生

骨骼畸形可导致鱼类运动困难、摄食能力降低、生长缓慢、成活率低,因此,畸形鱼苗的出现将增加生产成本、影响养殖的经济效益。在海水鱼类人工育苗阶段,骨骼畸形现象普遍存在,严重制约了海水鱼类养殖产业的可持续发展。本文首先系统总结了卵形鲳鲹、黄尾鰤、尖吻鲈等经济海水鱼类颌骨、脊柱、尾骨的早期发育时序及特点,比较了仔、稚鱼骨骼畸形发生的部位、时间及发生规律。之后针对仔、稚鱼阶段影响骨骼发育的主要因素,从遗传、环境、营养等方面进行了剖析,以期为寻找降低人工养殖条件下海水鱼类仔、稚鱼的骨骼畸形提供启示。     

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.     

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis,Kaup) at early stages of development

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. ‘Minor’ anomalies were predominant, and some of the detected disorders might be a result of non‐/low‐pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stages.     

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.     

The effect of feeding commercial diets on the development of juvenile crucian carp (Carassius carassius,L.). Part 1: Skeletal deformations

The crucian carp (Carassius carassius, L.) is a cyprinid freshwater species, whose wild‐living populations have decreased in recent years due to an increasing competition of invasive species. Several initiatives were launched, attempting to reintroduce this fish back into its native open waters, which requires the use of crucian carp fry grown under controlled conditions. However, the use of popular commercial diets results in severe developmental abnormalities. The aim of this study was to analyse the impact of feeding natural feed (Chironomidae sp. larvae) or two popular commercial diets on the skeletal development of crucian carp juveniles. Whole‐mount staining and histological methods were used in the study. Skeletal tissues of fish fed both commercial diets were demineralized and displayed a variety of deformities, such as abnormal vertebral spines and vertebral fusion, as well as a new type of deformation, which involved the action of dorsal fin pterygiophores upon the spine. Finally, a novel analytical method was proposed, which utilizes LA‐ICP‐MS for the estimation of bone mineralization. The study proved that basic commercial diets are inadequate for the rearing of crucian carp juveniles.     

Early introduction of an inert diet and unenriched Artemia enhances growth and quality of Atlantic cod (Gadus morhua) larvae

The effects of two weaning diets and different weaning protocols on growth, survival, skeletal deformity and gut morphology of Atlantic cod larvae were studied in four groups from 16 to 45 days posthatch (dph). Cod larvae in groups 1 (early weaning with control diet) and 2 (early weaning with experimental diet) were used to evaluate the effects of different polar lipid content of weaning diets on larval and juvenile performance. Cod larvae in groups 2, 3 (early weaning with experimental diet + cofeeding with Artemia) and 4 (earlier weaning with experimental diet and earlier cofeeding with Artemia) were used to evaluate the effects of early introduction of dry diet and Artemia. From 45 to 170 dph, cod juveniles from all four groups were reared using a standard feeding protocol. No significant differences in growth, survival, deformities and gut morphology were found between cod larvae and juveniles from groups 1 and 2. Cod larvae fed on cofeeding regime with Artemia nauplii (groups 3 and 4) were bigger and had lower frequencies of jaw and neck deformities and higher foregut microvillus circumference than cod larvae from group 2. Our results demonstrate the importance of proper weaning protocols in producing better quality cod juveniles.     

Influence of swimbladder inflation failure on mortality,growth and lordotic deformity in Pacific bluefin tuna,Thunnus orientalis, (Temminck & Schlegel) postflexion larvae and juveniles

This study examined the influence of swimbladder inflation (SBI) failure on mortality (Experiment 1), lordotic deformity (Experiment 2) and growth (Experiment 1, 2) in Pacific bluefin tuna, Thunnus orientalis, postflexion larvae and juveniles by producing larvae lacking inflated swimbladders. Experiment 1 was conducted for postflexion larvae to juveniles (from 18 to 30 days‐post‐hatch; dph). Mortality was not significantly different between the fish with (WIS) and without (WOIS) inflated swimbladders. Standard length (SL) and body weight (BW) were significantly smaller in WOIS than WIS. Moreover, the SBI was found in WOIS after postflexion stage. In Experiment 2, two examination trials were conducted on SBI, vertebral deformity and growth for juvenile stage. Lordotic deformities were found neither in the WOIS nor in the WIS. Although SL and BW were significantly smaller in WOIS than WIS at 22 dph, after 37 dph no significant differences were found between them. The results of Experiment 1 and 2 indicate that SBI failure causes growth retardation until juveniles of 30 dph; however, it does not cause growth retardation in juveniles after 37 dph and mortality of postflexion larvae and juveniles. Moreover, SBI failure did not cause lordotic deformity in PBT differently from many other fish.     

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.     

Vertebral deformity in cultured Atlantic cod larvae: ontogeny and effects on mortality

The effects of different egg incubation densities on the incidences of vertebral deformities in Atlantic cod larvae were investigated. Cod eggs were incubated at four different densities, 3, 6, 12 and 48 mL eggs L⁻¹, of water.  When all the eggs hatched, larvae were reared in 30 L glass aquaria. Larval samples were taken at 0, 14, 42 and 56 days post hatch (dph) for deformity analysis. Larval samples were stained using bone and cartilage staining methods to determine vertebral deformity. Incubation densities did not have any significant effects on vertebral deformities in Atlantic cod larvae. However, the incidence of larval vertebral deformity was high at hatch and decreased as the larvae grew older until 42 dph, indicating selective mortality of deformed larvae during this period. Larvae at 56 dph, however, showed an increase in the incidence of vertebral deformity, indicating a possible nutritional or prey-type effect. To our knowledge, no studies have documented the occurrence of variable patterns in vertebral deformities in cod at various developmental stages. Overall, our results suggest that broodstock husbandry, genetics and/or nutrition could play a major role in causing vertebral deformities in Atlantic cod at hatch; however, nutrition and prey type may play a major role during metamorphosis.     

Review of the principal diseases affecting cultured meagre (Argyrosomus regius)

Argyrosomus regius was recently introduced in European aquaculture as a promising species for diversification and with high potential for expansion in the coming years. The reports on pathologies affecting this species are still scarce in the bibliography, however, they can severely affect production and result in major economic losses. Some few reports were made on outbreaks and mortalities associated with the presence of bacteria such as Vibrio anguillarum and Photobacterium damselae subsp. piscicida. Although no viral diseases have been recognized as affecting meagre, it is known that meagre can be an asymptomatic carrier of two genotypes of nodavirus, the striped jack nervous necrosis virus and red‐spotted grouper nervous necrosis virus. Up‐to‐date, parasites affecting meagre are included in the genera Monogenea, Nematoda and Dinoflagelate, but without major mortality outbreaks associated to this type of infections. Osteological deformities have been observed in all life stages particularly affecting the axial system in larval and early juvenile specimens, with a higher incidence in the vertebral column, being affected by vertebral fusions, lordosis and kyphosis. With this review the authors provide an overview of all the pathological and non‐pathological diseases affecting aquaculture produced meagre and provide a comprehensive overview of possible problems for the industry.     

Effect of temperature,hypoxia and disinfection with ozonated seawater during somitogenesis on muscular development of the trunk in larval seven‐band grouper,Epinephelus septemfasciatus (Thunberg)

The cause of abnormal musculature on both sides of the same myotome in artificially cultured seven‐band grouper Epinephelus septemfasciatus (Thunberg) larvae remains unclear. We evaluated the effect of three factors: changes in temperature (between 22 and 24°C), disinfection with ozonated seawater (0.3 mg TROs L^?1, 1 min), and hypoxia (22 or 14% saturation) during the early (20 h post‐fertilization, hpf) and mid‐ (24 hpf) somite stages on trunk muscular development in larval seven‐band grouper. The thermal regimes and disinfection with ozonated seawater had no effect on muscle development (P > 0.05, G‐test). In contrast, exposure to hypoxia during both the early and mid‐somite stages increased the incidence of abnormal musculature in the trunk (P < 0.01, G‐test). These larvae had abnormal musculature on both sides of the trunk. In addition, the number of abnormal myotomes differed among these larvae. Exposure in the early and mid‐somite stages resulted in the highest frequency of abnormalities in the 11th and 13th myotome respectively. Our results suggest that hypoxia during somitogenesis can lead to abnormal muscle development in the trunk of artificially cultured seven‐band grouper larva.     

Lack of essential fatty acids in live feed during larval and post-larval rearing: effect on the performance of juvenile <Emphasis Type="Italic">Solea senegalensis</Emphasis>

Despite the large progress obtained in recent years, Senegalese sole (Solea senegalensis) production of high quality juveniles is still a bottleneck. This paper examines the effect of larval and post-larval lipid nutrition on juvenile performance and quality. Four dietary treatments were tested: A—enriched Artemia spp. (EA); B—non-enriched Artemia spp. (NEA); C—EA during the pelagic larval period and NEA after larval settlement; D—50% EA and 50% NEA. Juvenile fatty acid profile at 60 days after hatching (DAH) clearly reflected the larval and post-larval diet composition. Feeding sole larvae on NEA (poor in lipids and essential fatty acids-EFA) had a negative effect, reducing growth (total length and dry weight) after 30 DAH and decreasing digestive enzyme activity at the end of the rearing period (60 DAH). However, relatively good performance compared to the EFA-richest treatment (A) was obtained when larvae were fed 50% EA and 50% NEA (D) or even EA only during the pelagic larval period followed by NEA after larval settlement (C). Malpigmentation was not affected by the dietary regimes and its incidence was very low. However, skeletal deformities were prevalent, particularly in the caudal complex, independently of diet. The results confirm that Senegalese sole appear to have lower larval EFA requirements than most cultured marine species and potentially even lower requirements during the post-larval stage. The importance of studying the impact of early nutrition on later juvenile stages was clearly highlighted in this study.     

Patchiness of eggs, larvae and juveniles of European hake Merluccius merluccius from the NW Mediterranean

We estimated patchiness for different life stages (egg, larvae and juveniles) of European hake (Merluccius merluccius) from two ichthyoplankton surveys (September and November 1999) and four bottom‐trawl surveys (November 1998, February, June and September 1999). Lloyd's patchiness P = 1 + 1/k, where k is the dispersion parameter of the negative binomial distribution, was calculated. Juveniles showed lower indices of patchiness than early ontogenetic stages. In juveniles, patchiness was highest for smaller size classes (3.5–5 cm TL) settling to their near‐bottom habitat and was very low for those larger than 5 cm up to the 30–35 cm size class (approximately 3 yr of age, 50% mature individuals). Patchiness of settling juveniles was especially high in the June and September surveys, coinciding with peak recruitment to the bottom. Both eggs and yolk sac larvae showed patchiness values <10, increasing in flexion stage larvae from 12 to 20. This pattern is different to that observed in other studies for pelagic species, where patchiness of early‐stage eggs is very high, but coincides with results obtained for other demersal species with pelagic eggs. Factors that may explain these patterns are discussed in light of oceanographic conditions, adult distribution and larval ontogenesis.     

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.     

Effects of two culturing techniques on the growth, survival and larval quality of Dentex dentex Linnaeus, 1758

Common dentex larvae were reared using two culturing techniques, mesocosms and intensive rearing, to determine the principal culture parameters involved in the differences observed in growth, skeletal deformations and survival between the two rearing techniques. In growth, only dry weight of larvae of 40 days post‐hatching (dph) from mesocosms was significantly higher than larvae from intensive rearing. Significant differences were observed in survival at 40 dph (6.58% in mesocosms and 1.58% in intensive rearing) and in the incidence of skeletal deformations, both for percentage of deformed larvae and for some deformation types such as those related to vertebral column and to the caudal complex. Initial larval density and initial prey density and quality are the factors suspected to affect growth and survival performance, while skeletal deformities might also be affected by tank hydrodynamics.     

Skeletal Anomalies of Pacu,Piaractus mesopotamicus,Larvae from a Wild‐caught Broodstock

Skeletal anomalies in fish are frequent, representing a major problem for the aquaculture industry. The anomalies usually arise in the larval phase and worsen, persist, or recover over the ensuing stages of development. In addition to impairing larval quality, they compromise the growth, health, and food conversion of fish, lowering their economic value. This study evaluated the occurrence of skeletal anomalies in pacu larvae during the first 30 d of development. The newly hatched larvae of six breeding pairs taken from the Paranapanema River (offspring A, B, C, D, E, and F) and subjected to induced spawning were reared separately for 30 d, feeding on live prey (Artemia nauplii). Nearly 20 larvae from each breeding pair were killed and stored 1, 2, 4, 6, 8, 10, 13, 16, 19, 22, 25, and 30 days post‐hatching. The larvae were stained to differentiate cartilage and bones and examined under a stereo microscope. A total of 1310 larvae were checked for the presence (1) or absence (0) of anomalies in the head, vertebral column, and fins. Associations between growth parameters (total length [TL] vs. standard length [SL]; SL vs. vertebral column length [VL]) were obtained by regression. Correspondence analysis was used to correlate data on the different parameters evaluated. On completion of observation, larvae from pair E had the highest SL, whereas offspring F were the shortest. All types of skeletal anomalies were detected in the larvae irrespective of the offspring, affecting the normal growth of pacu larvae obtained from wild‐caught breeders.     

Development of the musculature and muscular abnormalities in larval seven-band grouper Epinephelus septemfasciatus

There are a number of reports documenting skeletal development in groupers, but surprisingly little is known about muscle development. We have documented muscle development in the seven-band grouper Epinephelus septemfasciatus (Thunberg), focusing primarily on the cranial muscles from 0 days post-hatching (dph) to 28 dph, using a modified whole mount immunohistochemical staining method. The eye muscles developed first followed by the cheek muscles, which completed development prior to first feeding. The muscles associated with the opercle and the levator internus 1 developed gradually and were not linked to first feeding. Later, between 12 and 16 dph, the muscle composition in the dorsal branchial arches changed to the adult form. We observed a number of cultured larvae that exhibited abnormal musculature in the body and jaw. Our results provide the first documentation of muscle development in larval seven-band grouper. This information can be used to detect abnormalities during development and provide a marker to assess the effect of changes in larval rearing techniques.     

Breeding and larval development of Holothuria mammata,a new target species for aquaculture

Sea cucumber commercial fisheries in the North‐eastern Atlantic and Mediterranean Sea started in Turkey in the 1990s. Due to ineffective management practices, the sea cucumber resource was overexploited. Holothuria mammata is one of the sea cucumber species exploited from this geographical area, showing high potential for its aquaculture. This research was undertaken to develop the breeding, larval and juveniles rearing techniques for development of H. mammata aquaculture. Broodstocks collected from Ria Formosa (Faro, S Portugal) were successfully induced to spawn by thermal stimulation from July to October, yielding up to 6.95 million eggs/female. The eggs and larvae were reared and their development described. H. mammata showed the five typical larval stages of most aspidochirote holoturians. Juveniles stage was reached after 21 days post‐fertilization. This is the first work focused on the aquaculture biotechnology of H. mammata, however, further research is need to improve the survival of juveniles to ensure the future production of this species.