Histological study of the gastrointestinal tract in longfin yellowtail (<Emphasis Type="Italic">Seriola rivoliana</Emphasis>) larvae

This work contributes basic knowledge on larval development of Seriola rivoliana. A histological study describes the development of the digestive tract and accessory glands in S. rivoliana larvae reared under laboratory conditions at 24 °C from hatching to 30 days post-hatching (DPH). At hatching (2.6 ± 0.12 mm), larvae had an undifferentiated digestive tract with a closed straight tube and a large yolk sac with an oil globule. The liver and pancreas were observed at 1 and 2 days, and the mouth and anus opened at day 2. Enriched rotifers were visible in their digestive tract. At the beginning of the pre-flexion stage, a mixed nutritional period was observed. At day 3, exogenous feeding began; the digestive tract became differentiated into the buccopharynx, esophagus, an undifferentiated stomach, and the intestines. Zymogen granules were visible in the exocrine pancreas. At day 4, supranuclear vacuoles were present in the posterior intestine, indicating the beginning of intracellular digestion. At day 5, goblet cells were present in the esophagus and became functional at day 7 in the esophagus and intestine. The buccopharynx goblet cells developed at day 15. The presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions during the post-flexion stage occurred at day 20. This was the onset of the juvenile period and the beginning of weaning; however, a long co-feeding phase is recommended. Pyloric caeca were observed at day 30 (13.6 ± 1.6 mm). These results provide valuable information on S. rivoliana larvae biology and digestive physiology, which should be useful to improve cultivation techniques and identify ecological features involved in ontogeny.     

Hatching time effect on the intra‐spawning larval morphology and growth in Northern pike (Esox luciusL.)

Based on the analysis of 11 morphometric variables of body (total length, body area and perimeter, myotome height and eye diameter) and yolk sac (length, height, area, perimeter and volume) of pike larvae, the aim of this study was to evaluate how larval size at hatching and growth of larvae hatched from single egg batches vary according to three hatching times: early, mid and late. Hatching time structures strongly pike larval morphometrics. Early hatched larvae have smaller body sizes at hatch, faster growth and higher yolk use efficiency than late hatched ones. Early hatched larvae seem to be premature and hatch at precocious developmental stage whereas late hatched individuals continue their growth within the egg shell and hatch at larger size but with lower reserves (yolk). A compensatory growth phase was observed for the early hatching pike larvae particularly during the first 5 days post hatch. Consequently, no significant difference in body parameters was recorded from day 10 post hatching whatever the hatching time. The higher growth accomplished by early hatched larvae may be related to a particular metabolic activity that converts more efficiently yolk into body tissues.     

Feeding,somatic condition and survival of sand lance <Emphasis Type="Italic">Ammodytes</Emphasis> sp. larvae in Mutsu Bay,Japan

To clarify the recruitment process of sand lance Ammodytes sp., we investigated larval condition factor, relative gut fullness (%GF), prey abundance and oceanographic structure in Mutsu Bay, Japan, during 1999–2001. Ammodytes sp. larvae, which were collected by horizontal hauls of Motoda nets and a ring net at depths of 1, 10, 20, 30 and 40 m, were mainly distributed at 10–30 m. Larvae at the first feeding time until 12 mm in body length (BL) fed predominantly on copepod nauplii, whereas large larvae with BL of 12.1–14.0 mm fed on a mixture of copepod nauplii, copepodites and appendicularians from late February to April. A path analysis showed that difference in water density between 35- and 5-m depths negatively affected naupliar abundance at 10–30-m depth (standardised path coefficient β = ?0.71, p = 0.005 for 3.3–8.0-mm BL larvae and β = ?0.78, p < 0.001 for 8.1–12.0-mm BL larvae). Naupliar abundance positively affected the %GF of Ammodytes sp. larvae (β = 0.75, p < 0.001 for 3.3–8.0-mm BL larvae and β = 0.66, p < 0.001 for 8.1–12.0-mm BL larvae), whereas it was negatively affected by water temperature (β = ?0.45, p = 0.008 for 3.3–8.0-mm BL larvae and β = ?0.56, p = 0.002 for 8.1–12.0-mm BL larvae), and the temperature effect was weak compared with that of naupliar abundance. In turn, %GF positively affected larval somatic weight (β = 0.91, p < 0.001 for 6.0-mm BL larvae and β = 0.70, p = 0.005 for 10.0-mm BL larvae). The recruitment failure in 1999 was likely caused by a reduced condition factor, which resulted from low naupliar abundance. In contrast, the abundance of nauplii and Oithona similis copepodites was high in 2000 and 2001. It is possible that the higher recruitment success in 2001 was because of the higher water temperatures in Mutsu Bay, sustaining faster growth of the larvae than in 2000 under the high-prey abundance conditions.     

Proteolytic digestive enzyme response in Japanese flounder larvae <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> fed two types of rotifers <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p?<?0.05) at 50 kDa in 1.5- and 3-h incubation whereas L-type treatment had not completely hydrolyzed the proteins after 3 h at the same molecular weight. Larvae fed SS-type had significantly higher (p?<?0.05) trypsin activity at 3, 5, 6, 7 DAH. Contribution of live prey to trypsin fraction in larvae showed significantly higher (p?<?0.05) fraction for SS-type at 5 DAH (2.18?±?0.44%) and 6 DAH (2.04?±?0.29%) and the effect of exogenous trypsin from live prey was relatively low when compared to the total trypsin activity in larvae. This study discusses the differences in ability to digest proteins in Japanese flounder when fed different rotifer morphotypes and highlights the adaptability of this species to alternative rotifer morphotypes during its early developmental stages.     

Molecular endocrine changes of Gh/Igf1 axis in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) exposed to different environmental salinities during larvae to post-larvae stages

The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.     

The efficacy of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> vaccine preparations,administered to tilapia broodstock,in preventing streptococcosis in their offspring,via transfer of maternal immunity

This study aims to evaluate the efficacy of Streptococcus agalactiae vaccine preparations, administered to tilapia broodstock, in preventing streptococcosis, through specific and non-specific immunity being transferred to the offspring. The study was conducted in two phases. The first was the vaccination of the broodstock using a whole-cell vaccine, an extracellular product (ECP) vaccine, and a combination of the two with a ratio of 1:1. The vaccines were administered to the broodstock 2 and 3 weeks before spawning. The second phase was the challenge test for larvae produced by vaccinated broodstock, and larvae from the unvaccinated control broodstock, through immersion in a suspension of 10⁷ cfu mL^?1 pathogenic S. agalactiae for 30 min, at ages 7, 14, 21, and 28 days post-hatching. The parameters evaluated were the broodstock’s blood profile, antibody-lysozyme (in broodstock, eggs, and larvae), and the larvae’s relative percent survival. Treatment with the combined vaccine administered 3 weeks before spawning resulted in the broodstock having significantly better antibody levels, lysozyme activity, and hematology profiles, compared to the other treatments (p?<?0.05). In addition, the larvae produced by broodstock subjected to this treatment, when challenged with the pathogenic S. agalactiae at ages 7, 14, 21, and 28 days, had RPS values of 95.24, 83.33, 72.22, and 56.02%, respectively. It was concluded that the administration of the “whole-cell/ECP” combination vaccine preparation to tilapia broodstock in the 3 weeks before spawning can increase specific and non-specific immunity in the broodstock and protect the larvae from S. agalactiae infection.     

The effect of live feeds bathed with the red seaweed <Emphasis Type="Italic">Asparagopsis armata</Emphasis> on the survival,growth and physiology status of <Emphasis Type="Italic">Sparus aurata</Emphasis> larvae

Larval rearing is affected by a wide range of microorganisms that thrive in larviculture systems. Some seaweed species have metabolites capable of reducing the bacterial load. However, no studies have yet tested whether including seaweed metabolites on larval rearing systems has any effects on the larvae development. This work assessed the development of Sparus aurata larvae fed preys treated with an Asparagopsis armata product. Live prey, Brachionus spp. and Artemia sp., were immersed in a solution containing 0.5% of a commercial extract of A. armata (Ysaline 100, YSA) for 30 min, before being fed to seabream larvae (n = 4 each). In the control, the live feed was immersed in clear water. Larval parameters such as growth, survival, digestive capacity (structural-histology and functional-enzymatic activity), stress level (cortisol content), non-specific immune response (lysozyme activity), anti-bacterial activity (disc-diffusion assay) and microbiota quantification (fish larvae gut and rearing water) were monitored. Fish larvae digestive capacity, stress level and non-specific immune response were not affected by the use of YSA. The number of Vibrionaceae was significantly reduced both in water and larval gut when using YSA. Growth was enhanced for YSA treatment, but higher mortality was also observed, especially until 10 days after hatching (DAH). The mortality peak observed at 8 DAH for both treatments, but higher for YSA, indicates larval higher susceptibility at this development stage, suggesting that lower concentrations of YSA should be used until 10 DAH. The application of YSA after 10 DAH onwards promotes a safer rearing environment.     

Effect of live food transition time on survival,growth and metamorphosis of yellowtail clownfish, <Emphasis Type="Italic">Amphiprion clarkii</Emphasis>, larvae

Amphiprion clarkii, a popular marine ornamental fish, despite having an already established reproduction technology, still presents divergences regarding the feeding protocol used for its larviculture in what concerns the ideal day of transition from rotifers to Artemia. This study aimed to determine the optimum time to start supplying Artemia to larvae. Survival, growth, weight gain and metamorphosis of treatments: (T2) start of Artemia supply at the 2nd day after hatching (DAH); (T4) start at the 4th DAH; and (T6) start at the 6th DAH were evaluated. Survival rates ranged from 60 to 66 %. Fish in T2 had a number of metamorphosed fish statistically higher than T6 and began to metamorphose 1 day sooner than other treatments. The positive results obtained for T2 can be related to the premature ability of this species to capture large live food, which can provide many benefits to the larvae. It is concluded that it is possible to offer Artemia nauplii to yellowtail clownfish at 2 DAH.     

First evidence of viable progeny from three interspecific parents in sturgeon

Polyspermy is the most commonly observed cause of embryonic abnormalities in fertilization, often resulting in death. In sterlet (Acipenser ruthenus), however, polyspermic embryos have high survival (similar to a control group) and morphological development is similar to monospermic larvae. Ploidy of these individuals is n/2n mosaic (whereas the normal state for A. ruthenus is a functional diploid). This study was undertaken to test whether sturgeon eggs can be fertilized by several spermatozoa from different species to produce viable offspring from three interspecific parents: A. ruthenus (2n), A. gueldenstaedtii (4n), and A. baerii (4n). Four trials were performed: (1) and (2) A. baerii eggs were fertilized with a mixture of A. ruthenus and A. gueldenstaedtii sperm; (3) A. gueldenstaedtii eggs were fertilized with a mixture of A. baerii and A. ruthenus sperm; and (4) A. gueldenstaedtii eggs were fertilized with a mixture of A. gueldenstaedtii and A. ruthenus sperm. Fertilized embryos with abnormal cleavage (3, 5, 6, 7, 9, and 10 cells) were collected and kept separately until 14 days post-fertilization. Ploidy level of 25 larvae (hatched from abnormal cleaved embryos) was evaluated by flow cytometry. Forty-four percent of observed hybrids had a mosaic 2n/3n ploidy. Five larva were processed further with microsatellite analysis and demonstrated that three specimens were heterospecific polyspermic larvae, containing alleles from three parents, and two specimens were conspesific polyspermic larvae from two parents. This astonishing phenomenon was emphasized by the fact that it was generated without any significant intervention.     

The effects of Roundup on gametes and early development of common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L)

To determine the effects of Roundup, a commercial formulation of glyphosate, gametes, and embryos of common carp (Cyprinus carpio L) was exposed to wide range of herbicide concentrations (0.0, 0.1, 0.5, 2.0, 5.0, 10.0, 20.0, and 50.0 mg/l). The obtained results showed different effects of Roundup on common carp gametes. Herbicide reduced swelling of eggs (but the effect was not concentration-related), while sperm showed low sensitivity to Roundup (time of spermatozoa motility was reduced in a significant way only at 20 mg/l, and at remaining concentrations, only a slight tendency was observed). During the embryonic development, Roundup caused a decrease of common carp embryonic survival (and the effect was concentration-related); however, it had no effect on development rate. During the embryogenesis, three types of embryo body malformation were observed: yolk sac edema, spine curvature, and shortening of body, but their frequencies were not associated with the presence or concentration of herbicide. However, Roundup affected quality of newly hatched larvae of common carp by increasing their mortality. No effect of herbicide on percentage of deformed larvae was observed but larvae hatched in water with Roundup tended to show more complex anomalies compared to those from the control. Obtained data showed that even low concentrations of this herbicide in waters can significantly reduce egg swelling, survival of embryos, and quality of fish larvae.     

Effects of salinity,stocking density,and algal density on growth and survival of Iwagaki oyster <Emphasis Type="Italic">Crassostrea nippona</Emphasis> larvae

To determine the optimal salinity, stocking density, and algal density for hatchery culture of the Iwagaki oyster Crassostrea nippona larvae, three experiments with salinities of 14, 18, 22, 26, 30, and 34 practical salinity unit (PSU); stocking densities of 0.5, 1, 2, 4, 8, and 12 larvae ml^?1; and algal densities of 10, 20, 40, and 100?×?10³ cells ml^?1 were designed, which included the developmental stages from newly hatched D-larvae to pediveligers. Results showed that larval growth of C. nippona was the fastest at a salinity of 26 PSU, and when salinity was adjusted to a level that was lower or higher than this salinity, survival and growth rate of larvae declined (P <?0.05), resulting both in a decreased mean shell length and a high mortality. Larval growth decreased significantly with increasing stocking density. Larvae reared at 4 larvae ml^?1 had the smallest shell length (198.9 μm) and lowest survival rate (7.9%), whereas larvae reared at 0.5 larvae ml^?1 had the largest shell length (245 μm) and highest survival rate (66.3%) on day 13. And the shell length of larvae reared at 0.5 and 1 larvae ml^?1 was significantly (P?<?0.05) larger than the values in other treatments, except those reared at 2 larvae ml^?1 (P?>?0.05). When feeding the single-algal diet of Isochrysis galbana (clone T-ISO), the shell length of larvae increased markedly as the algal density was increased. Larvae reared at the highest algal density (100?×?10³ cells ml^?1) had the largest mean shell length; however, under the conditions of our experiment, there was no significant difference (P?>?0.05) in growth and survival rates between the treatments at algal densities of 40?×?10³ and 100?×?10³ cells ml^?1. For a large-scale culture, based on the results of this study, a salinity of 26 PSU, stocking density of 0.5–1 larvae ml^?1, and algal density of 40?×?10³ cells ml^?1 are recommended for an early development of C. nippona.     

Identification and characterization of microRNAs in the muscle of <Emphasis Type="Italic">Schizothorax prenanti</Emphasis>

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in the skeletal muscle of Schizothorax prenanti remain unknown. In this study, we analyzed the miRNA profiling in the skeletal muscle of S. prenanti at 30 days post-hatching (dph), 1 year, and 3 years by high-throughput sequencing. Two hundred twenty-nine unique miRNA types aligned to 201 independent pre-miRNA loci according to sequence similarity and 28 novel miRNAs were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in S. prenanti were highly conserved. Eight identified miRNAs validated using stem-loop qRT-PCR were differentially expressed in the process of skeletal muscle development of S. prenanti. This study provide not only an overall insight into the miRNA landscape in the skeletal muscle of S. prenanti but also a basis for further investigation of miRNA roles in skeletal muscle development of S. prenanti.     

Morphology,sex steroid level and gene expression analysis in gonadal sex reversal of triploid female (XXX) rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In non-mammalian vertebrates, estrogens and expressions of cyp19a1 and foxl2 play critical roles in maintaining ovary differentiation and development, while dmrt1 and sox9 are male-specific genes in testicular differentiation and are highly conserved. In order to deeply understand the morphological change, sex steroids level and molecular mechanism of triploid female gonadal reversal in rainbow trout, we studied the ovary morphology, tendency of estradiol-17β (E2) and testosterone (T) levels and the relative expressions of dmrt1, cyp19a1, sox9 and foxl2 in juvenile and adult fish. Our results demonstrated that the development of triploid female gonads in rainbow trout went through arrested development, oocytes dedifferentiation, ovary reconstruction and sex reversal finally. During early gonadal development (154–334 days post-fertilization), the expressions of foxl2 and cyp19a1 increased linearly, while expressions of dmrt1 and sox9 were extremely suppressed, and E2 level was higher, while T level was lower. During the mid-to-late period of triploid female gonadal development (574–964 days post-fertilization), the expressions of dmrt1 and sox9 remained high and were very close to the quantity of diploid male genes, and T levels were even reaching diploid male plasma concentrations, while expressions of cyp19a1 and foxl2 were decreased, leading to decrease in E2 level. We realized that the development model of rainbow trout triploid female gonads was extremely rare, and the regulatory mechanism was very special. Genes involved in gonadal development and endogenous estrogens are pivotal factors in fish natural sex reversal.     

The effects of cadmium and copper on embryonic and larval development of ide Leuciscus idus L.

The effects of Cd and Cu on embryos and larvae of the ide Leuciscus idus were evaluated. The ide is an European cyprinid fish, natural populations of which tend to decrease. The ide is also used as a bioindicator organism to evaluate water quality. However, sensitivity of ide early developmental stages to heavy metal intoxication is not known. Fish were exposed to Cd or Cu (100 μg/L) during embryonic, larval or both developmental periods. Survival of the embryos, time of hatching, size and quality of newly hatched larvae were evaluated at the end of embryonic period. Correctly developed larvae from the control and Cd or Cu-exposed groups were transferred to clean water, Cd or Cu solutions (100 μg/L) immediately after hatching. Larval development was observed, and the larvae were photographed. Time of yolk sac resorption, onset of active feeding and swim bladder inflation were evaluated, and the measurements were done on body and swim bladder size. The results showed that exposure of embryos to Cd and Cu significantly reduced embryonic survival and increased frequency of body malformations and death in newly hatched larvae and delayed hatching. Exposure to Cd and Cu during larval period reduced larval survival, growth and delayed development (yolk utilization, beginning of active feeding and swim bladder inflation). Cadmium was more toxic to the ide embryos and larvae than copper. Exposures to metals during embryonic period alone caused adverse impact on larval performance even when larval development took place in clean water. However, exposure of embryos to Cu reduced toxic impact of metal on larvae in continuous Cu exposure compared to the non-preexposed fish, but no such an effect occurred in case of Cd exposure. The results show that even a short-term exposure to Cd or Cu during early development of ide may adversely affect recruitment of this species. Among the measured endpoints, quality of newly hatched larvae (frequency of body malformations and larvae dead immediately after hatching) and swim bladder size were the most sensitive to intoxication with both metals. Embryos were more sensitive to Cu intoxication than larvae, while in case of Cd, sensitivity of both stages was similar.