Cryptic species diversity and molecular diagnosis of Channa orientalis; an endemic freshwater fish of Sri Lanka.

Fish genetic resources and diversity are very important aspects of environmental management and fisheries and are vital for making decisions on their commercial exploitation as well as conservation. The snakehead fishes in the world have significant economic importance as food and ornamental fish. A clear understanding of species' taxonomic status and genetic diversity is important for the utilization and implementation of conservation and management practices. Channa orientalis is a snakehead endemic to Sri Lanka that is heavily utilized in the ornamental fish export trade. Its genetic diversity has not yet been fully understood and it is difficult to distinguish it from closely resembling species. Therefore, we examined the genetic diversity of C. orientalis and developed a DNA-based marker that permits accurate, low cost, and reliable identification of C. orientalis. Determination of genetic diversity was mainly carried out through genetic analysis of the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene. The development of the DNA-based marker for the identification of C. orientalis was done through Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis. Our analyses confirmed the presence of two distinct genetically divergent and geographically separated lineages of C. orientalis in Sri Lanka. The fast cost-effective gel-based PCR-RFLP marker method developed by us was successful in diagnosing C. orientalis from its closely resembling species. Thus, we believe our findings on the cryptic diversity and diagnostic methods will have important implications for the conservation and management of this endemic species.

Combined antisense knockdown of type 1 and type 2 iodothyronine deiodinases disrupts embryonic development in zebrafish (Danio rerio).

Thyroid hormones (THs) are important regulators of gene expression during vertebrate development. In teleosts, early embryos rely on the maternal TH deposit in the egg yolk, consisting predominantly of T(4). Activation of T(4) to T(3) by iodothyronine deiodinases (Ds) may therefore be an important factor in determining T(3)-dependent development. In zebrafish, both Ds capable of T(3) production, D1 and D2, are first expressed very early during embryonic development. We sought to determine their relative importance for zebrafish embryonic development by inhibiting their expression via antisense oligonucleotides against D1 and D2, and by a combined knockdown of both deiodinases. The impact of these treatments on the rate of embryonic development was estimated via three morphological indices: otic vesicle length, head-trunk angle and pigmentation index. Knockdown of D1 alone seemed not to affect developmental progression. In contrast, D2 knockdown resulted in a clear developmental delay in all parameters scored, suggesting that D2 is the major contributor to TH activation in developing zebrafish embryos. Importantly, combined knockdown of D1 and D2 caused not only a more pronounced developmental delay than D2 knockdown alone but also the appearance of dysmorphologies in a substantial minority of treated embryos. This shows that although D1 may not be essential in euthyroid conditions, it may be crucial under depleted thyroid status as is the case when T(3) production by D2 is inhibited. These results indicate that zebrafish embryos are dependent on T(4) uptake and its subsequent activation to T(3), and suggest that substantial inhibition of embryonic T(4) to T(3) conversion reduces intracellular T(3) availability below the threshold level necessary for normal development.

Type 2 iodothyronine deiodinase is essential for thyroid hormone-dependent embryonic development and pigmentation in zebrafish.

Despite the known importance of thyroid hormones (THs) in vertebrate growth and development, the role of tissue-specific TH activation in early embryogenesis remains unclear. We therefore examined the function of type 2 iodothyronine deiodinase (D2), one of the two tissue-specific enzymes catalyzing the conversion of T4 to T3, in developing zebrafish embryos (Danio rerio). Microinjection of early embryos with antisense oligonucleotides targeting either the D2 translation start site or the splice junction between the first exon and intron induced delays in development and pigmentation, as determined through the measurement of otic vesicle length, head-trunk angle, and pigmentation index at 31 h after fertilization. The antisense-induced delays in developmental progression and pigmentation were reversible through treatment with T3, suggesting that these phenotypic effects may be due to the depletion of intracellular T3 levels. Additional evidence for this hypothesis was provided by quantitative RT-PCR analysis of TH receptor-beta expression in D2 knockdown embryos, revealing a significant down-regulation of this T3-induced transcript that could be reversed by T3 treatment. Tyrosinase expression was also down-regulated in D2 knockdown embryos to a greater degree than could be predicted by the observed delay in developmental progression, suggesting that reduced D2 activity and resultant low intracellular T3 availability may directly influence pigmentation in zebrafish. These data indicate that TH activation by D2 is essential for embryonic development and pigmentation in zebrafish.

The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (Danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors.

The importance of thyroid hormones (TH) for embryonic development has long been shown in many vertebrates, but is not yet established in pre-hatch teleost models despite the presence of TH, TH receptors and iodothyronine deiodinases. Lack of data about the dynamics of TH metabolism in embryonic stages of fish does not allow to speculate about the involvement and/or role of TH in fish embryonic development. We therefore set up an experiment to examine the effect of 3,5,3'-triiodothyronine (T(3)) on zebrafish (Danio rerio) embryonic development and on the expression of some thyroid hormone-regulated genes as measured by real-time PCR. Maternally derived thyroxine (T(4)) and T(3) were detected throughout embryonic development and total levels remained stable. Thyroid hormone receptor (TR) alpha and beta mRNA were found to be present in early embryos. After an initial fall, TRalpha mRNA levels in the control group increased gradually from 12h post fertilization (HPF) onwards. TRbeta mRNA levels of control embryos were relatively stable during embryonic development, but increased around the hatching period. We also quantified type I (D1) and type II (D2) deiodinase mRNA expression in zebrafish embryos. D1 mRNA levels in the control group gradually increased during development while D2 levels were kept at a low and stable level until hatching. At 75 HPF, a fivefold increase of D2 expression was observed compared to embryonic levels. Exogenous T(3) added to the water (5nM) was taken up by the embryos, causing increased pigmentation and accelerated hatching. T(3) treatment significantly up regulated TRalpha mRNA levels at 48 HPF, while D2 mRNA was significantly down regulated at 75 HPF. Neither TRbeta nor D1 mRNA levels seemed responsive to the treatment. Taken together, these data show that during embryonic development zebrafish already have the necessary regulatory machinery for TH activation and signaling, and that T(3) treatment at that stage indeed influence embryonic development.

The influence of stress on thyroid hormone production and peripheral deiodination in the Nile tilapia (Oreochromis niloticus).

The existence of an interaction between the adrenal/interrenal axis and the thyroidal axis has since long been established in vertebrates, including fish. However, in contrast to mammals, birds and amphibians, no effort was made in fish to expand these studies beyond the level of measuring plasma thyroid hormones. We therefore set out to examine the acute effects of a single dose of dexamethasone (DEX) on plasma thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)) levels, as well as on the activity and mRNA expression of the different iodothyronine deiodinases in liver, gills, kidney and brain in Nile tilapia. To take into account the effect of handling stress, this treatment was compared both to a non-treated and to a saline injected group. In general, the observed changes were acute (3 and 6h) while values had returned to control levels by 24h post-injection. Only DEX administration caused an acute drop in circulating T(3) levels compared to non-treated animals, while none of the treatments affected plasma T(4) levels. This indicates that the DEX induced decrease in plasma T(3) levels was not due to a lowered thyroidal hormone production and secretion. DEX injection provoked a decrease in peripheral T(3) production capacity via a decrease in hepatic outer ring deiodination activity (both D1 and D2), whereas T(3) clearance increased by induction of the inner ring deiodinating D3 pathway in liver and in gills. Deiodination activities in kidney and brain were not affected. Effects of saline injection were only observed in liver, where D1 activity decreased and D3 activity increased as in the DEX group, but to a lesser extent. Real-time PCR showed that the changes in hepatic D3 were clearly regulated at the pretranslational level, while this was not confirmed for the other changes. Our results show that both handling stress and DEX injection acutely disturb peripheral deiodination activity in Nile tilapia. However, the effects of the long acting glucocorticoid analogue are more pronounced and result in a decrease in circulating T(3) availability.