Method for the molecular and quantitative identification of oocytes and their developmental stage in teleost fish gonads.

Fish display diverse reproductive strategies and their gametogenesis is influenced by numerous genetic, physiological and environmental factors. The analysis of 5S rRNA expression levels in gonads has been proposed as useful method for the molecular identification of the presence of oocytes in fish tissues. The present method provides an easy and unbiased approach to analyse the expression of tRNAs and 5S rRNA in teleost gonads and stablish the presence and developmental stage of oocytes. Total RNA extracted from gonads is analysed through capillary electrophoresis in a Bioanalyzer 2100 (Agilent Technologies) using Small RNA Assays. Electropherograms allow quantifying the concentrations of tRNAs, 5S rRNA and 5.8S rRNA per sample and calculate their tRNA/5.8S rRNA and 5S/5.8S rRNA indices. Both indices clearly differentiate ovaries from testes and can be used to identify testes that present oocytes due to exposure to environmental xenoestrogens. The tRNA/5.8S and 5S/5.8S indices show the highest values in ovaries in previtellogenic stage, values decreasing as they advance towards maturity.•Detailed molecular method to sex fish and quantitatively identify the maturity stage of females.•tRNA levels in gonads can help in the study of teleost reproduction (female fecundity assessment, molecular gonad sexing) and environmental health assessment.

High production of transfer RNAs identifies the presence of developing oocytes in ovaries and intersex testes of teleost fish.

5S rRNA is highly transcribed in fish oocytes and this transcription levels can be used to identify the presence of oocytes in the intersex testes of fish exposed to xenoestrogens. Similar to 5S rRNA, tRNAs are transcribed by RNA polymerase III (Pol-III) in eukaryotes, so this study focuses in the analysis of the levels of expression of tRNAs in the gonads (ovaries and testes) of eight teleost species as a possible new oocyte molecular marker. Total RNA extracted from gonads of six commercial teleost species in the Biscay Bay, from the pollution sentinel species thicklip grey mullet (Chelon labrosus) known present intersex testes in response to xenoestrogens in Gernika estuary and from the laboratory model species Danio rerio were analysed through capillary electrophoresis. Bioanalyzer electropherograms were used to quantify the concentrations of tRNAs, 5S and 5.8S rRNA. All studied ovaries expressed significantly higher levels of tRNAs and 5S rRNA than testes. A tRNA to 5.8S rRNA index was calculated which differentiates ovaries from testes, and identifies some intersex testes in between testes and ovaries in mullets. The tRNA/5.8S ratio was highest in ovaries in previtellogenic stage, decreasing towards maturity. Thus, strong oocyte expression of tRNAs is an additional proof of high activity levels of Pol-III during early stages of oocyte development in teleost ovaries. Incidentally, we observed that miRNA concentrations were always higher in testes than ovaries. The indexing approach developed in the present study could have multiple applications in teleost reproduction research and in the development of early molecular markers of intersex condition.

Reproductive biology of mud crabs (Scylla olivacea) collected from Paikgachha, Khulna, Bangladesh.

OBJECTIVE: This study was carried out to estimate the sex ratio, maturity size, gonadosomatic index (GSI), and peak breeding season of mud crabs. MATERIALS AND METHODS: Samples were collected randomly from the estuary and river of the study area. Sampling was carried out monthly from April to September at every full moon during one high tide. A total number of 240 specimens were sampled, where 53 individuals were hermaphrodite. The crabs were shifted alive to the biology and histology lab for detailed biological study. Sex was determined. Male and female sex ratio and breeding season were also investigated. RESULTS: The male:female ratio was 1:0.96 and the ovarian development was categorized into five stages based on internal observations, viz. immature (stage I), underdeveloped (stage II), early developed (stage III), late developed (stage IV), and mature (stage V). The maturity percentages were 37%, 19%, 13%, 11%, and 20%, respectively. 50% maturation was estimated at 82.36 mm internal carapace width (ICW). The highest mean GSI value was 7.97 ± 3.03. The mature stage was found in all the working periods. This shows that females have activated ovaries in all the working months, and the species are continuous breeders. A higher frequency of vitellogenic ovary and higher GSI value were found in September. The maximum GSI value was found in the size group 70-79 mm. CONCLUSION: The study shows that the capture from the wild sources of mud crabs without any regulation can threaten the population structure. The capture of female mud crabs should be more than 82.36 mm ICW, which will help conserve and protect young crabs.

SCHeMA EU Project Summer School Report (Bilbao June 16 - 17, 2016).

This conference report describes the training activities that took place in the frame of the Integrated in Situ Chemical MApping probe (SCHeMA) summer school organized from the 14th to the 16th of June 2016 in Bilbao (Spain).