Occurrence of skeletal deformities and osteological development in red porgy Pagrus pagrus larvae cultured under different rearing techniques

The present study describes the osteological development and the occurrence of skeletal deformities in red porgy Pagrus pagrus larvae in relation to the intensification of the rearing system. Eggs obtained from natural spawning were cultured under two different rearing systems: intensive (100 eggs l^?1) in 2000 l and semi‐intensive (mesocosm) system (5 eggs l^?1) in 40 000 l conico‐cylindrical tanks. Fish samples were periodically collected along the development from hatching to juveniles at 95 days post hatching (dph). Osteological development, meristic counts and the presence of skeletal deformities were evaluated. Despite the external appearance of the juveniles being similar to wild standards, X‐ray studies revealed a high number of fish (semi‐intensive: 37·8%; intensive: 45·5%) with skeletal deformities. Regardless of the rearing system, no significant interaction was found between the per cent of the most common deformities, axial deviations (lordosis and presence of fused vertebrae). Cranial deformities and kyphosis incidences, however, were significantly higher in intensively cultured P. pagrus. Also, the fused vertebrae in these fish were located mainly in the caudal area instead of pre‐haemal area for semi‐intensively reared P. pagrus. Moreover, a significant interaction was found between the total number of vertebrae and the type of rearing system used; fish from the intensive system showing a higher number of fish with an extra vertebrae (10 abdominal + 15 caudal). Present results suggest a relationship among feeding sequence, osteological development and deformity incidence and location in P. pagrus larvae.     

Studies on the appearance of skeletal anomalies in red porgy: effect of culture intensiveness,feeding habits and nutritional quality of live preys

Despite the great interest of red porgy as a new species for Mediterranean aquaculture, its commercial production is constrained by the high incidence of skeletal deformities occurring in this species under culture conditions. Several studies have been conducted to better understand the origin of these anomalies in this species, using different system intensiveness, rotifers enrichment products or rotifers docosahexaenoic acid content. The first study showed that culture intensification increased the number of fish with an extra vertebrae, what was probably related to the different nutritional quality of live preys employed in each treatment, since water temperature, salinity and genetic background were identical for the different batches of fish studied. Total incidence of skeletal abnormalities was higher in the intensive system, particularly cranial abnormalities and kyphosis in the cephalic vertebrae. In both rearing systems the most common skeletal anomalies were vertebral column disorders, lordosis and fused vertebrae, their localization along the column being affected by the culture intensiveness. Rotifer enrichment, predominantly its docosahexaenoic acid content significantly affected deformities occurrence. A marked positive effect of rotifer docosahexaenoic acid content was found on larval survival. X‐ray studies denoted elevated levels of bone abnormalities associated, in both trials, to low docosahexaenoic acid content in live preys. Among different anomalies, the presence of fused vertebrae was the most frequent deformity for both rearing trials. A 50% reduction in the number of deformed fish for each type of deformity was obtained when the larvae were fed higher docosahexaenoic acid levels, denoting the important role of this fatty acid in bone development. Further studies are needed to elucidate the importance of essential fatty acids on the development of bone deformities in fish, since the functions of these fatty acids differ among them and can lead to very different effects in fish metabolism, including bone formation.     

Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density

The teleost zebrafish (Danio rerio), an established model for human skeletal diseases, is reared under controlled conditions with defined parameters for temperature and photoperiod. Studies aimed at defining the proper rearing density have been performed with regard to behavioural and physiological stress response, sex ratio and reproduction. Studies concerning the effect of rearing density on the skeletal phenotype are lacking. This study analyses the response of the skeleton to different rearing densities and describes the skeletal deformities. Wild-type zebrafish were reared up to 30 dpf (days post-fertilization) in a common environment. From 30 to 90 dpf, animals were reared at three different densities: high density (HD), 32 fish l^–1; medium density (MD), 8 fish l^–1 and low density (LD), 2 fish l^–1. Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualize mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that the rearing density significantly influenced the specimens’ average standard length, which decreased with the increase in the rearing density. Differences in meristic counts among the three groups were not observed. Rearing density–independent malformations affected the ribs, neural arches and the spines of the abdominal region, as well as vertebrae of the caudal complex. The HD group showed the highest number of deformities per specimen, the highest number of observed types of deformities and, together with the MD group, the highest frequency of specimens affected by severe deformities. In particular, the HD group showed deformities affecting arches, spines and vertebral centra in the caudal region of the vertebral column. This study provides evidence of an effect of the rearing density on the development of different skeletal phenotypes.     

Use of meristic counts and skeletal anomalies to assess developmental plasticity of farmed rainbow trout (Oncorhynchus mykiss,Walbaum 1792): a preliminary study

Rainbow trout is the longest‐standing intensively‐reared species in Western countries, where it has been subjected by farmers to selection for productive traits for more than a century. One interesting aspect in this context is to try and understand if prolonged artificial selection has in some way altered the phenotypic plasticity of this species. In this preliminary study, we characterized meristic counts and skeletal abnormalities in two different (Italian and French) rainbow trout strains, reared during on‐growing following different approaches (intensive and semi‐intensive), in order to analyze whether there are any differences in the skeletal elements and if so, what. Despite the different genetic origin and rearing methodologies to which the lots analyzed were subjected, the occurrences of malformed and seriously malformed individuals were not significantly different, nor were the mean number of anomalies per specimen. The body region most affected by deformities was the caudal vertebral column in both lots, although the French lot was also characterized by an high occurrence of spinal anomalies in the haemal region. Among the meristic counts, only the number of anal and dorsal rays showed statistically different median values between the two lots.     

Progress in modeling quality in aquaculture: an application of the Self‐Organizing Map to the study of skeletal anomalies and meristic counts in gilthead seabream (Sparus aurata,L. 1758)

One of the most common drawbacks of artificial life conditions imposed by aquaculture is the quite high presence of skeletal anomalies (SAs) in reared fish, which reduce both functional performances and marketing image/commercial value of the reared lots. Thus, skeletal malformations and their incidence are one of the most important factors affecting fish farmer’s production costs, and several efforts have been due to develop appropriate tools in detecting patterns of co‐variation among rearing parameters and fish quality. In this paper we explore the advantages of using Self‐Organized Maps (SOMs) when dealing with the analysis of correlations between the pattern of SA presence and rearing parameters in gilthead seabream (Sparus aurata L.), that is a largely reared fish of high commercial value. SOM, which is one of the best known neural networks with unsupervised learning rules, were applied to develop a model of the occurrence of SAs, both in terms of type and quantity, in seabream lots from different rearing approaches (extensive, semi‐intensive and intensive). The trained SOMs classified lots according to the variation observed in the different weights of SAs, but also allows the detection of a series of correspondence, namely between: (i) the patter of SAs occurrence and the different rearing approach currently used in seabream aquaculture; and (ii) the total SAs incidence and the variability of meristic counts, represent a completely independent dataset. Mesocosms resulted the best rearing approach to produce wild‐like fish, whereas intensive rearing is characterized by the large presence of SA. Globally, results suggested that this approach is reliable to be used for estimate the distance between aquaculture products and the wild‐like phenotype used as quality reference.     

Early development of the skull of Sander lucioperca (L.) (Teleostei: Percidae) relating to growth and mortality

The early osteological development of the skull (chondrocranium and osteocranium) of the pikeperch Sander lucioperca was studied. Specimens were reared at two temperatures, 15·5 and 18·0° C, from hatching until 47 and 43 days after fertilization (DAF), respectively. The skeletal elements characteristic for the different developmental stages were the same at both rearing temperatures, but pikeperch reared at 15·5° C reached the developmental stages later. The formation of the functional complexes, the neurocranium, jaws and suspensorium, branchial basket and hyoid arch, was evaluated chronologically. The focus was on skull development during several functional changes: at hatching, at the shift from endogenous to mixed feeding, the shift to exclusively exogenous feeding and upon reaching the final prey-capture mechanism. Growth in total length differed between fishes reared at the two temperatures, except during a phase of very slow growth from the end of the embryonic stage until the second larval stage. The latter phase, in which most of the bony elements of the viscerocranium started to form, was marked by high mortality. When exogenous feeding began, the growth rates at both temperatures increased distinctly and the first bony elements were formed in the neurocranium. Specimens reared at 18·0° C grew continuously, but those at 15·5° C showed a second period of slow growth and high mortality. Fish reared at 18·0° C reached the successive larval stages distinctly earlier than fish reared at 15·5° C.     

Environmental Conditioning of Skeletal Anomalies Typology and Frequency in Gilthead Seabream (Sparus aurata L., 1758) Juveniles

In this paper, 981 reared juveniles of gilthead seabream (Sparus aurata) were analysed, 721 of which were from a commercial hatchery located in Northern Italy (Venice, Italy) and 260 from the Hellenic Center for Marine Research (Crete, Greece). These individuals were from 4 different egg batches, for a total of 10 different lots. Each egg batch was split into two lots after hatching, and reared with two different methodologies: intensive and semi-intensive. All fish were subjected to processing for skeletal anomaly and meristic count analysis. The aims involved: (1) quantitatively and qualitatively analyzing whether differences in skeletal elements arise between siblings and, if so, what they are; (2) investigating if any skeletal bone tissue/ossification is specifically affected by changing environmental rearing conditions; and (3) contributing to the identification of the best practices for gilthead seabream larval rearing in order to lower the deformity rates, without selections. The results obtained in this study highlighted that: i) in all the semi-intensive lots, the bones having intramembranous ossification showed a consistently lower incidence of anomalies; ii) the same clear pattern was not observed in the skeletal elements whose ossification process requires a cartilaginous precursor. It is thus possible to ameliorate the morphological quality (by reducing the incidence of severe skeletal anomalies and the variability in meristic counts of dermal bones) of reared seabream juveniles by lowering the stocking densities (maximum 16 larvae/L) and increasing the volume of the hatchery rearing tanks (minimum 40 m³). Feeding larvae with a wide variety of live (wild) preys seems further to improve juvenile skeletal quality. Additionally, analysis of the morphological quality of juveniles reared under two different semi-intensive conditions, Mesocosm and Large Volumes, highlighted a somewhat greater capacity of Large Volumes to significantly augment the gap with siblings reared in intensive (conventional) modality.     

Ontogeny of the feeding apparatus of hatchery‐reared white margined sole,Dagetichthys marginatus (Soleidae): implications for cultivation

The aim of this study was to describe the ontogeny of the feeding apparatus of hatchery‐reared Dagetichthys marginatus and to discuss development in relation to generalized early development in (flat)fish and the potential influence of reproduction and/or rearing practices used. Fertilized eggs were obtained from hormone‐induced wild females of the white margined sole, D. marginatus. Subsequent larvae were reared until shortly after metamorphosis between 35 and 45 dah (days‐after‐hatching) when mortalities occurred. At 4 dah a small maxilla and two paired branchial arches appeared. Metamorphosis was completed at 22 dah while the left/migrating eye still remained in its initial position at 31 dah. Based on the likely disadvantages for survival it is assumed that the observed development indicates underdevelopment rather than an evolutionary adaptation. The influence of reproduction and rearing practices used on development and mortalities are discussed.     

Visualizing mineralization in deformed opercular bones of larval gilthead sea bream (Sparus aurata)

During the rearing process of gilthead sea bream (Sparus aurata), abnormal development of the opercular bone is particularly common (Aquaculture 156, 1997, 165). In order to alleviate its occurrence in rearing facilities, it’s crucial to identify the very first physical signs of deviation in normal skeletal development. Nano‐CT‐scanning was tested for its applicability to quantify deviations in bone mineralization levels. Seven opercles were dissected from larvi of 65 days post hatching, randomly sampled at the commercial sea bream hatchery Maricoltura di Rosignano Solvay (Livorno, Italy). The samples were nano‐CT‐scanned and computationally reconstructed. Mineralization intensity was colorcoded using Amira software, resulting in a detailed visualization of opercular morphology and mineralization patterns. In conclusion, nano‐CT‐scanning promises to be a good tool to both describe morphology and detect mineralization levels in the early onset of deformities.     

Rearing environment influences boldness and prey acquisition behavior,and brain and lens development of bull trout

Animals reared in barren captive environments exhibit different developmental trajectories and behaviors than wild counterparts. Hence, the captive phenotypes may influence the success of reintroduction and recovery programs for threatened and endangered species. We collected wild bull trout embryos from the Metolius River Basin, Oregon and reared them in differing environments to better understand how captivity affects the bull trout Salvelinus confluentus phenotype. We compared the boldness and prey acquisition behaviors and development of the brain and eye lens of bull trout reared in conventional barren and more structurally complex captive environments with that of wild fish. Wild fish and captive reared fish from complex habitats exhibited a greater level of boldness and prey acquisition ability, than fish reared in conventional captive environments. In addition, the eye lens of conventionally reared bull trout was larger than complex reared captive fish or same age wild fish. Interestingly, we detected wild fish had a smaller relative cerebellum than either captive reared treatment. Our results suggest that rearing fish in more complex captive environments can create a more wild-like phenotype than conventional rearing practices. A better understanding of the effects of captivity on the development and behavior of bull trout can inform rearing and reintroduction programs though prediction of the performance of released individuals.     

Important disease conditions of newly cultured species in intensive freshwater farms in Greece: first incidence of nodavirus infection in Acipenser sp

We describe here the main pathological conditions of freshwater fish recently introduced for intensive rearing (open ponds and recirculating freshwater systems) in Greece. Sturgeon were susceptible to skeletal abnormalities of the spine (scoliosis and lordosis) of unknown aetiology. Horizontal transmission of nodavirus from infected sea bass to sturgeon was detected for the first time. This caused serious pathology and clinical signs, such as lethargy and imbalance, leading to secondary infections with Aeromonas hydrophila and Trichodina sp. and chronic, but steady, mortality. Sea bass were very susceptible to nodavirus infection, monogenean infections and gas bubble disease. Mullet reared under recirculated and open-flow conditions were very sensitive to Chilodonella sp. infection, whereas catfish were susceptible to infection with Ichthyophthirius sp. leading to secondary infections with A. hydrophila, Saprolegnia sp. and Myxobacteria spp. Tilapia were very susceptible to gas bubble disease and A. hydrophila. This bacterium was associated with management manipulations for all species and fully responsive to corrective hygiene methods.     

Temperature effects on cranial deformities in European sea bass, Dicentrarchus labrax (L.)

The present study investigates the effect of water temperature on the development of deformities during embryonic and larval stages of European sea bass, Dicentrarchus labrax (L.). Two temperature conditions were examined in duplicate by studying the presence of skeletal deformities in a sample of 45 to 51 fish taken from each population at the end of the rearing trials [20–23 mm total length (TL)]. The results indicated that water temperature during the embryonic and larval phase has significant effects on deformation of the branchiostegal rays (P < 0.001), but not of the mouth and the fins (P > 0.05). At 15°C, 27.2–33.4% of the examined fish had branchiostegal rays of abnormal shape and/or orientation, whereas at 20°C this deformity had a frequency of only 4.0–4.1%. The frequency distribution graph of branchiostegal counts demonstrated a significant deviation in the deformed fish from the normal (seven rays on each side of the body) phenotype at both temperatures tested. This deviation was mainly expressed as a lack of one to four rays (56.7% of deformed fish), or the formation of one extra ray (26.7% of deformed fish) (P < 0.001, G‐test). The results are discussed in respect to the possible mechanisms of temperature effects on the development of skeletal deformities.     

Characterization of a partial alpha-amylase clone from red porgy (Pagrus pagrus): expression during larval development

A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.     

Validation of daily increment formation in otoliths for Gymnocypris selincuoensis in the Tibetan Plateau,China

Daily increment validation in fish otolith is fundamental to studies on fish otolith microstructure, age determination and life history traits, and thus is critical for species conservation and fishery management. However, it has never been done for Schizothoracine fish, which is the dominant component of fish fauna in the Tibetan Plateau. This study validated the daily increment formation of Gymnocypris selincuoensis, as a representative of Schizothoracine fish, by monitoring the growth of hatchery‐reared larvae group and wild‐caught post‐yolk‐sac larvae group under controlled experiments. The results from monitoring the hatchery‐reared larvae group showed that sagittae and lapilli were found in yolk‐sac larvae, and formed 5–7 days before hatching, but asterisci were not found until 11 days post‐hatching. The first increment in sagittae and lapilli was formed in the first day after hatching. The daily periodicity of increment formation was examined and confirmed in sagittae and lapilli of both larvae groups. However, sagittae were better for age determination than lapilli for larvae at earlier days. For larval G. selincuoensis older than 50 days, lapilli were the only otolith pair suitable for larvae daily age determination. This study validated the daily increment formation in Schizothoracine fish for the first time has primary implications to other fishes from this subfamily.     

Allometric and ontogenetic larval development of common barbel during rearing under optimal conditions

The rearing of finfish larvae is a key element in their further culture. Improper breeding protocols may result in high mortality rates, body deformation and growth rate decreases in both the larval and fattening periods. These errors can be avoided by thorough exploration of various aspects of early larvae biology, at least in model fish species. In this study, anatomical and morphological developments were analysed using allometric growth patterns of common barbel, Barbus barbus, larvae reared under optimal controlled conditions. Larvae of common barbel, which is a model species for fish of the genus Barbus, were reared for 30 days at 25 °C in the recirculated aquaculture system (RAS). Four periods of the barbel larval development were identified: pre-flexion (0–5 days post hatching – DPH; total length – TL: 9.5 ± 0.3 to 12.3 ± 0.3 mm), flexion (6–11 DPH; TL 12.4 ± 0.3–15.4 ± 0.3 mm), post-flexion (12–21 DPH; TL 16.1 ± 0.5–21.2 ± 0.8 mm) and juvenile (from 22 DPH; TL from 21.4 ± 1.7 mm). The largest changes in barbel growth were observed during the first two periods of their life (pre-flexion and flexion), which resulted in the frequency of noted flexion points (64.3% flexion points) and was also associated with intensive morphometric growth, primarily the head and tail parts of the body. Despite a low degree of growth progress upon hatching (e.g. no eye pigment, no distinct liver or pancreas, no unobstructed alimentary tract), barbel larvae pass through the larval periods very quickly in comparison to other cyprinids and enter the juvenile period (22 days).     

Effects of ambient colour on colour preference and growth of juvenile rainbow trout Oncorhynchus mykiss (Walbaum)

Colour preference of individual juvenile rainbow trout Oncorhynchus mykiss was tested at 1 and 12° C, and also at 12° C after a 42 day growth experiment under white, blue, green, yellow or red ambient colour. All experiments were carried out under controlled laboratory conditions and the preference was assessed by the location of the fish in a preference tank with four chambers. Rainbow trout showed a preference for blue and green at 1° C and for green at 12° C. After the growth experiment the fish reared in blue tanks preferred blue and green but green was the most preferred colour for the fish reared in green, yellow and red tanks. Yellow and especially red chambers were avoided, irrespective of the ambient colour during the growth trial. The final mass of fish reared in the red aquaria was significantly smaller than that of the fish in green tanks. In addition, when the data of the preference tests were correlated with the data of the growth experiment using mean values of the four tested colours, a very good linear relationship was observed between the preference ( i.e. visit frequency in coloured compartments) and growth rate as well as food intake. When considering the results both from the preference and growth trials it is suggested that green is the best environmental colour for rearing juvenile rainbow trout while rearing in a red environment cannot be recommended.     

Detection of Mineralized Structures in Early Stages of Development of Marine Teleostei Using a Modified Alcian Blue-Alizarin Red Double Staining Technique for Bone and Cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 ixm in length can be clearly visualized. The method is suitable for both onto-genic studies during early stages of skeletal development in most marine fishes (e.g., Sporus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.     

Temperature stress induces notochord abnormalities and heat shock proteins expression in larval green sturgeon (Acipenser medirostris Ayres 1854)

The effects of thermal stress on survival, development and heat shock protein (hsp) expression of green sturgeon (GS) yolk‐sac larvae, from hatching through yolk depletion were investigated to provide insight into effects of highly altered natural river hydrographs. Hatched GS larvae were reared at constant water temperatures 18°C (control) through 28°C at 2°C increments. Larval survival significantly decreased at 26–28°C, with 28°C being lethal. Significant proportions of deformed larvae were found at sub‐lethal (20–26°C) and lethal 28°C rearing temperatures, with kyphosis (i.e. backward flexion of notochord) accounting for >99% of morphological deformities. Histological analysis of larvae preparations indicate that elevated water temperature affects notochord cell function and physiology. At rearing temperatures 20–28°C, thermal stress elicited a quick (24 h) and long lasting (yolk‐sac absorption) significant over‐expression of measured heat shock proteins (hsps), all of which are known components of intracellular protein repair and stabilization mechanism. Thermal sensitivity, as indicated by the incidence of abnormalities and expression of different hsps, varied significantly between crosses. Thermally tolerant progeny exhibited a short but rapid hsp72 (size in kDa) over‐expression, and more pronounced hsp60 and hsp90 over‐expression, than less tolerant progeny which exhibited a prolonged hsp72 and hsp78 over‐expression. At environmentally relevant water temperatures bent larvae exhibited spiral swimming, which in the wild would compromise the ability of emerging larvae to forage, avoid predators, and migrate downstream, ultimately compromising survival and recruitment. Before larvae hsp content can be used as a thermal‐stress biomarker for GS, field validation studies are needed.     

The influence of food distribution upon the development of aggressive and competitive behaviour in juvenile chum salmon, Oncorhynchus keta

Groups of juvenile chum salmon were reared on food that was either dispersed throughout, or localized in one area of, the rearing tank. Groups receiving localized food displayed more aggression than those receiving dispersed food. This led to differences in growth, with fish reared on localized food having greater individual growth variability, i.e. growth depensation. However, after several months of rearing in these different feeding/social environments, fish reared on dispersed food were just as aggressive when first exposed to localized food as were fish reared on localized food. Furthermore, in competitive contests between fish of the two rearing histories, those reared on dispersed food were just as likely to become dominant as those reared on localized food. These results suggest that the behavioural development of aggressiveness is not amenable to alteration by manipulation of food distribution.     

High dietary arachidonic acid levels affect the process of eye migration and head shape in pseudoalbino Senegalese sole Solea senegalensis early juveniles

The effect of high dietary levels of arachidonic acid (ARA) on the eye migration and cranial bone remodelling processes in Senegalese sole Solea senegalensis early juveniles (age: 50 days post hatch) was evaluated by means of geometric morphometric analysis and alizarin red staining of cranial skeletal elements. The incidence of normally pigmented fish fed the control diet was 99·1 ± 0·3% (mean ± s.e .), whereas it was only 18·7 ± 7·5% for those fed high levels of ARA (ARA‐H). The frequency of cranial deformities was significantly higher in fish fed ARA‐H (95·1 ± 1·5%) than in those fed the control diet (1·9 ± 1·9%). Cranial deformities were significantly and negatively correlated with the incidence of normally pigmented animals (r² = ?0·88, P < 0·001, n = 16). Thus, fish displaying pigmentary disorders differed in the position of their eyes with regard to the vertebral column and mouth axes, and by the interocular distance and head height, which were shorter than in fish not displaying pigmentary disorders. In addition to changes in the positioning of both eyes, pseudoalbino fish showed some ARA‐induced osteological differences for some of the skeletal elements from the splanchnocranium (e.g. right premaxillary, dentary, angular, lacrimal, ceratohyal and branchiostegal rays) and neurocranium (e.g. sphenotic, left lateral ethmoid and left frontal) by comparison to normally pigmented specimens. Pseudoalbino fish also had teeth in both lower and upper jaws. This is the first study in Pleuronectiformes that describes impaired metamorphic relocation of the ocular side eye, the right eye in the case of S. senegalensis, whereas the left eye migrated into the ocular side almost normally.