Thyroid gland development in Senegalese sole (Solea senegalensis Kaup 1858) during early life stages: A histochemical and immunohistochemical approach

A key to success in the culture of marine fish species is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system (pituitary, thyroid, interrenal glands) during early ontogeny of marine fish. Particularly, the thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), influence numerous metabolic processes, such as growth, differentiation, metamorphosis, reproduction, respiration, migratory behaviour, central nervous system activity, seasonal adaptation, etc. Therefore the aim of this study was to describe the development of the thyroid gland and the ontogeny appearance of the thyroid hormones in Solea senegalensis larvae by means of histological and immunohistochemical techniques. The first thyroid follicle was present at 4 days-post-hatch (dph) coinciding with first feeding. During metamorphosis (12–20 dph) the follicles increased in both number and size, and by 30 dph presented the same characteristics as that seen in adult fish. Tissue immunostaining of both thyroid hormones decreased during the endogenous larvae development to nearly undetectable levels at the completion of yolk-sac absorption. During larvae exogenous phase, T₃ and T₄ immunostaining was first detected by 6 dph and an increase of specific staining for both hormones was detected between 12 and 20 dph, during metamorphosis phase.     

Thyroid hormone deficiency in abnormal larvae of the Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   In large-scale rearing of juveniles of the Japanese flounder Paralichthys olivaceus, a certain morphological abnormality occurred spontaneously in 4% of the fish. These fish showed a slight but clearly different appearance from any developmental stage of this species, and did not settle when all the other juveniles in the same tank completed metamorphosis and had settled. From comparisons of external and internal structures between the normal and the abnormal fish, the abnormality was attributed to unbalanced progress of metamorphosis, mainly due to metamorphic stasis. The thyroid of the abnormal fish was apparently activated morphologically. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal fish. After 14 days of T4 treatment (0.1 p.p.m) of the abnormal fish, all the abnormal characteristics disappeared, and the fish recovered to normal, suggesting normal responsiveness to thyroid hormones in peripheral tissues, whereas thiourea treatment (30 p.p.m., 14 days) further delayed metamorphosis. These results suggest that these abnormal fish were suffering from thyroid hormone deficiency, and were unable to secrete a sufficient amount of thyroid hormone to complete metamorphosis.     

Selenium supplementation changes glutathione peroxidase activity and thyroid hormone production in Senegalese sole (Solea senegalensis) larvae

Dietary selenium (Se) influences the glutathione peroxidase (GPx) activity and the thyroid hormone system in vertebrates, but these relations are poorly described in fish. The present study aimed to evaluate the effect of Se supplementation on Senegalese sole larvae, focusing on GPx activity and thyroid status. Two groups of larvae were fed on either control (C) or Se‐enriched live feed, from 2 days after hatch (DAH) until 34 DAH. Senegalese sole larvae fed Se‐enriched live feed significantly increased their whole‐body Se, compared to larvae from C group. The activity of GPx was higher in larvae from Se group, suggesting that the levels of this mineral were suboptimal in the control diet. TH levels were also higher in larvae fed Se‐enriched live feed, suggesting that Se may have a positive influence on the TH production in early life stages of marine fish larvae.     

Iodine and selenium supplementation increased survival and changed thyroid hormone status in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system

To test how iodine and both iodine and selenium supplementation affected the thyroid status as well as growth and survival in Senegalese sole, larvae were reared in a recirculation system from 15 to 34 DAH. Sets of three tanks were assigned to each of the following three diets: control (C), iodine (I) and iodine and selenium (I?+?Se). Samples were collected at 15, 27 and 34 DAH to determine dry weight, iodine and selenium levels, GPx and ORD activities, thyroid hormone levels and thyroid follicles histology. At 34 DAH, fish from the control (C) treatment suffered from hyperplasia of the thyroid follicles (goitre), whereas iodine-treated larvae did not (I and I?+?Se). Lower survival rates in the C groups were probably a consequence of the hyperplasia. Moreover, there was an improvement in thyroid hormone status in I- and I?+?Se-treated larvae, showing that further supplementation of live feed with iodine can be crucial for fish at early life stages, as it seems to sustain normal larval development, when reared in a recirculation system. Selenium did not affect the results. Together with previous results, this indicates selenium supplement is more important at younger life stages.     

The thyroid gland and thyroid hormones in sheepshead minnow (<Emphasis Type="Italic">Cyprinodon variegatus</Emphasis>) during early development and metamorphosis

The sheepshead minnow is widely used in ecotoxicological studies that only recently have begun to focus on disruption of the thyroid axis by xenobiotics and endocrine disrupting compounds. However, reference levels of the thyroid prohormone thyroxine (T4) and biologically active hormone 3,5,3′-triiodothyronine (T3) and their developmental patterns are unknown. This study set out to describe the ontogeny and morphology of the thyroid gland in sheepshead minnow, and to correlate these with whole-body concentrations of thyroid hormones during early development and metamorphosis. Eggs were collected by natural spawning in our laboratory. T4 and T3 were extracted from embryos, larvae and juveniles and an enzyme-linked immunoassay was used to measure whole-body hormone levels. Length and body mass, hatching success, gross morphology, thyroid hormone levels and histology were measured. The onset of metamorphosis at 12-day post-hatching coincided with surges in whole-body T4 and T3 concentrations. Thyroid follicles were first observed in pre-metamorphic larvae at hatching and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Follicle size and thyrocyte epithelial cell heights varied during development, indicating fluctuations in thyroid hormone synthesis activity. The increase in the whole-body T3/T4 ratio was indicative of an increase in outer ring deiodination activity. This study establishes a baseline for thyroid hormones in sheepshead minnows, which will be useful for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.     

Development of thyroid gland and changes in thyroid hormone levels in Leptocephali of Japanese Eel (Anguilla japonica)

Development of the thyroid gland of the Japanese eel (Anguilla japonica) was studied with the use of tank-reared fish. A single thyroid follicle was first found in larvae at 29 days post hatching (dph), total length (TL) 12 mm. Until reaching 25 mm in TL (100 dph), leptocephali had one or two follicles per individual. The inner colloid of the follicles was weakly immuno-positive against the anti-thyroxine (T₄) antibody. The number of thyroid follicles and the immunoreactivity later increased as the larvae grew. Thyroid hormones (TH) T₄ and triiodothyronine (T₃) were not detected in premetamorphic larvae by radioimmunoassays, but became detectable during metamorphosis. The maximum level of T₄ was seen in fish at the end of metamorphosis and in just-metamorphosed juveniles, whereas T₃ reached the highest level during metamorphosis and declined toward the end of metamorphosis. The results indicated that the thyroid gland first became active during metamorphosis in the development of eel.     

鳙、团头鲂和短盖巨脂鲤早期发育阶段甲状腺激素含量变化研究

鳙、团头鲂和短盖巨脂鲤胚胎与仔鱼体内三碘甲腺原氨酸和四状腺素含量的变化，采用放射免疫测定法进行测定，研究发现胚胎阶段的Ｔ３和Ｔ４含较稳定，出膜后仔鱼阶段的Ｔ３和Ｔ４含量逐渐下降。     

Thyroid hormone modulation of ovarian recrudescence of air-breathing catfish Clarias gariepinus

In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T₄) ‘overdose’ were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T₄ treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol–17β levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.     

Effect of dietary taurine levels on the conjugated bile acid composition and growth of juvenile Korean rockfish Sebastes schlegeli (Hilgendorf)

This study was conducted to investigate the effects of different taurine levels on the conjugated bile acid composition and growth of juvenile rockfish. Five taurine level diets were made by the supplementation of taurine (0%, 0.5%, 1.0%, 1.5% and 2.0%) to a basal diet. Fish meal washed with 70% ethanol to exclude taurine was used for the sole protein source. Feeding experiments were accomplished at 15°C using juvenile fish (initial BW: 13.5 g). Fish were fed the experimental diets for 4 months. At the end of experiments, fish were weighed and stored at ?80°C for analysis of free amino acids and conjugated bile acids contents. The growth performance and feed efficiency of Korean rockfish were improved by taurine supplementation in the experimental diets. The taurine contents of the whole body increased with the increase in the taurine supplementation. The primary conjugated bile acids in gall bladder were taurocholic acid (83.5–165.6 mg mL^?1) and taurochenodeoxycholic acid (6.3–20.0 mg mL^?1). Total bile acids increased with increases in dietary taurine. Taurocholic acid content was more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to make conjugated bile acid in Korean rockfish. Dietary taurine levels improved the conjugated bile acid composition in juvenile Korean rockfish.     

Iodine‐enriched rotifers and Artemia prevent goitre in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system

To study the effect of dietary supplementation of iodine in Solea senegalensis, larvae were randomly distributed in six tanks. Larvae in three tanks were given rotifers and Artemia enriched with iodine in addition to Rich Advance or Super Selco from 2 days after hatch (DAH) until 31 DAH. Larvae in a second set of three tanks were fed control rotifers and Artemia, enriched only with Rich Advance or Super Selco. Samples were collected at 2, 5, 10, 15 and 31 DAH to determine dry weight, total length, myotome height and thyroid status. Larvae fed the iodine‐enriched diet had significantly higher weight at 31 DAH and higher levels of whole body iodine concentration, compared to control larvae. At 31 DAH, larvae from the control treatment showed typical goitrous thyroid follicles. Thyroid cells of larvae from this treatment appeared columnar or afollicular, with the colloid partly or completely depleted, representative of hyperplasia (goitre). The lower growth rate in fish larvae from the control treatment was possibly a consequence of the hyperplasia, and the iodine enrichment prevented Senegalese sole larvae from developing goitre. This study demonstrates the importance of iodine enrichment of live feed for fish reared in a recirculation system.     

Survival of larval summer flounder Paralichthys dentatus on formulated diets and failure of thyroid hormone treatment to improve performance

Summer flounder is a new aquaculture species, the culture of which could be made simpler by the replacement, to the greatest extent possible, of live feed with formulated diets. Previous work has shown that development of the summer flounder stomach can be accelerated by treatment of the larvae with thyroid hormone. Two series of experiments were conducted. In the first experiment, 2-week feeding trials were conducted beginning with fish (which were not treated with thyroid hormone) 14, 21, 28, 35, or 42 days after hatch (DAH) to determine when they could begin to utilize commercially available formulated diets. In the second experiment, two groups of sibling larvae, one treated with thyroid hormone, the other not, were placed in similar 2-week feeding trials at 25, 30 or 35 DAH to test whether survival of thyroid hormone-treated larvae would surpass those of untreated larvae. The first series of experiments demonstrated that the larvae could not effectively utilize the formulated diets until 35 DAH. The second series of experiments indicated that thyroid hormone-treated larvae do not exhibit significantly greater survival than untreated larvae.     

Intrafollicular thyroid hormone staining in whole-mount zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos for the detection of thyroid hormone synthesis disruption

Endocrine-disrupting chemicals are known to impact multiple hormonal axes of vertebrates, among which the thyroid system is crucial for multiple developmental and physiological processes. Thus, the present study focused on the semi-quantitative visualization of intrafollicular triiodothyronine (T3) and thyroxin (T4) in zebrafish embryos as a potential test system for the detection of disrupted thyroid hormone synthesis. To this end, an antibody-based fluorescence double-staining protocol for whole-mount zebrafish embryos and larvae was adapted to simultaneously detect intrafollicular T3 and T4. During normal development until 10 days post-fertilization (dpf), the number of thyroid follicles increased along the ventral aorta. Concentrations of T4 and T3, measured by fluorescence intensity, increased until 6 dpf, but decreased thereafter. Exposure of zebrafish embryos to propylthiouracil (PTU), a known inhibitor of TH synthesis, resulted in a significant decrease in the number of follicles that stained for T3, whereas a trend for increase in follicles that stained for T4 was observed. In contrast, fluorescence intensity for both thyroid hormones decreased significantly after exposure to PTU. Overall, the zebrafish embryo appears to be suitable for the simultaneous visualization and detection of changing intrafollicular TH contents during normal development and after PTU treatment.     

大黄鱼仔稚鱼不同发育阶段矢耳石形态发育和微结构特征

对人工培育大黄鱼(Larimichthys crocea)的生长发育与矢耳石形态及微结构特征进行研究,结果表明:(1)大黄鱼矢耳石上的轮纹是每日形成的,第1日轮在孵化后第2天形成,与其初次摄食相对应。(2)大黄鱼卵黄囊期和前弯曲期仔鱼的耳石形态为圆形,进入弯曲期耳石长轴迅速伸长,在后弯曲期耳石形态变为椭圆形。进入稚鱼期,矢耳石开始形成次生核。随后次生核数量逐渐增加,在孵化后47～78 d的个体中,次生核数量稳定在5～7个,耳石近似盾形。(3)根据耳石日轮宽度推算的大黄鱼稚鱼在其仔鱼期生长率(b)与第1个次生核的形成时间(tsp1)之间存在明显的线性关系,表明生长较快的个体形成次生核的时间较早,进入稚鱼期所需的时间更短。以上结论表明,大黄鱼矢耳石可以反演其早期生活史阶段的生长发育特征。     

Ontogeny of thyroid tissue and tissue thyroid hormone clearance in rainbow trout embryos reared at two temperatures

The development of the thyroid tissue and clearance of thyroid hormones was studied in rainbow trout embryos reared at 8.5 and 5.5 °C, in order to determine if these events are affected by temperature in the same manner as growth rate. Although the change in growth rate was found to be predictable using the degree-day model, the pattern of whole body thyroid hormone clearance was not similarly related to rearing temperature. Further, although the thyroid follicles appeared at different times in embryos reared at the two temperatures, the differences could not be predicted using the degree-day model. These results suggest that the development of the thyroid tissue and the clearance of yolk hormones are not directly related to rearing temperature. Immunohistochemical staining of the thyroid tissue indicated two phases of thyroid activity, one associated with hormonogenesis, and the other a later phase of hormone release. However, there was no increase in 'whole body' thyroid hormone level associated with the time of appearance of colloid in the follicles. Further, evidence was presented to suggest that the extraction method used did not remove thyroid hormone associated with the colloid in the follicles. There was also evidence of sporadic increases in 'whole body' thyroid hormone content, after the development of phase 2 thyroid follicles which might indicate pulsatile release of T4 and T3.     

Effects of thyroxine and thiourea on the metamorphosis of coral trout grouper Plectropomus leopardus

The contribution of the thyroid hormone to the metamorphosis of the coral trout Plectropomus leopardus was examined. Juveniles, aged 35 days, were exposed to thyroxine (T4, 0.1 p.p.m) and thiourea (TU; inhibitor of thyroid hormone synthesis, 30 p.p.m) for 13 days. All the fish in the T4-treated group had completed metamorphosis (second metamorphosis, pigmentation and resorption of fin spines) within three days after the beginning of the experiment (day 3), whereas it took 13 days for this to occur in the control fish. Moreover, the fish in the TU group had not completed metamorphosis even by day 13. Settling behavior was significantly stimulated by T4 treatment, as was the resorption of the dorsal and pelvic fin spines, the reduction of serration on the spines, and the transition of body color into an opaque reddish hue. Fish in the TU group had longer dorsal and pelvic spines, retarded pigmentation and an abnormal black coloration. These results suggest that thyroid hormone accelerates most changes during the second metamorphosis in the coral trout.     

Free Amino Acids in Pacu,Piaractus mesopotamicus,Eggs and Larvae

Free amino acids (FAA) are used principally as substrate in protein synthesis and the source of energy in aerobic catabolism. In marine fish, embryo and larvae FAA are used to maintain body fluid osmolality during fish early development. However, there is essentially no information about FAA concentrations in early ontogeny of freshwater neotropical species in comparison to marine fishes. Therefore, the aim of this study was to evaluate the FAA concentrations in pacu, Piaractus mesopotamicus, eggs and larvae. Broodstock fish were induced to spawn and ovulated females were stripped of their eggs and immediately sampled for analysis. Larvae were sampled right after hatching (HL) and after the completion of the yolk‐sac absorption (YSA). The wet weight of the HL and YSA larvae amounted to 0.5 ± 0.1 mg and 1.1 ± 0.3 mg, respectively. HL larvae showed higher levels of most of the indispensable amino acids (IAA) in comparison to eggs and YSA larvae. Exceptions were observed with His and Trp that showed higher or similar levels, respectively, in YSA larvae. The FAA Orn, Tau, Glu, Gln, Gly, and Tyr increased concentrations in both larval stages while that of Tau was found in higher concentration in all analyzed stages. Also, the concentrations of Asn, Ala, Pro, Ser, and Asp were higher in HL larvae. Both larval stages displayed a rise in total free IAA/total free DAA (dispensable amino acids) ratio. The authors conclude that the highest level of FAA in HL pacu larvae is indicative of active proteolysis of yolk reserves and a probable catabolism regulation of some FAA through spare‐effect. In addition, Tau is one of the major FAA occurring during pacu ontogeny and may be performing regulation on body fluid osmolality regulation .     

三丁基锡对褐菖鲉胚胎卵黄囊吸收的影响

鱼类胚胎期的营养对各种器官的发育至关重要,环境条件对营养吸收的影响会增加畸形率和死亡率。研究三丁基锡(tributyltin,TBT)对鱼类胚胎期营养吸收的影响有助于深入认识TBT对鱼类胚胎发育的毒性。本实验以海洋经济鱼类褐菖鲉(Sebastiscus marmoratus)为对象,研究环境水平TBT(0.01、0.1、1、10 ng.L-1)对胚胎卵黄囊吸收的影响。通过油红O染色,发现TBT暴露引起褐菖鲉胚胎血管系统中脂类染色信号减少,而卵黄中的脂类染色正常,说明TBT可能影响了胚胎正常血液循环,从而影响了正常的营养运输。另外,TBT对Na+,K+-ATP酶、碱性磷酸酶活性的抑制以及对甲状腺激素受体α和糖皮质激素受体基因表达的影响可能和其对卵黄囊的吸收抑制有关。TBT对鱼类胚胎卵黄吸收的抑制将影响鱼类的生长和发育,增加畸形率和死亡率,最终导致鱼类种群数量减少。     

云纹石斑鱼胚胎发育及仔、稚、幼鱼形态观察

对云纹石斑鱼Epinephelus moara胚胎发育及仔、稚、幼鱼形态进行了观察与研究,详细描述了各发育期的形态特征和发育时间。结果表明:1)在水温22±0.2℃、盐度30、溶氧7.8mg/L、pH8.25的条件下,云纹石斑鱼的受精卵历时38h 17min开始孵化出膜。胚胎发育可分为受精卵、卵裂、原肠、神经胚和器官形成及出膜6个阶段,受精、胚盘形成、2细胞等28个时期。2)在水温23±1℃,盐度30±3,DO≥5mg/L,pH8.0±0.5的海水中,培育至5d,卵黄囊完全消失,成为后期仔鱼;培育至27d,发育最快的云纹石斑鱼结束仔鱼期,进入稚鱼期;培育至65d,发育最快的稚鱼完成变态,成为幼鱼。胚后发育过程主要是根据卵黄囊、鳍膜、鳞片、体色及第1腹鳍棘与第2背鳍棘相对长度的变化分为仔鱼、稚鱼和幼鱼3个时期。其中仔鱼期又根据其卵黄囊的有无划分为前期仔鱼和后期仔鱼。     

High abundance of larval rockfish over Cobb Seamount, an isolated seamount in the Northeast Pacific

The larval fish community in the region of Cobb Seamount (500 km west of Oregon) is dominated by myctophid species commonly encountered in the subarctic North Pacific. However, during a survey in June 1992, the ichthyoplankton community within 30 km of the seamount summit was almost completely dominated by larvae of various rockfish ( Sebastes ) species. Given their very small size (and hence very young age) and the fact that they occurred only rarely in samples collected > 30 km from the seamount summit, we conclude that these Sebastes larvae were produced locally over Cobb Seamount. Previous studies have shown that the Cobb fish fauna is dominated by various Sebastes spp. and that, unlike other fish present on the seamount, the rockfish populations may be self-recruiting. We suggest that a persistent clockwise (i.e. downwelling) eddy, consistent with a stratified Taylor cone, plays a critical role in retaining larval rockfish over Cobb Seamount and may contribute to the process of self-recruitment. The key to the success of rockfish on Cobb and other shallow Northeast Pacific seamounts seems to be linked to their viviparous life history.