Gonad Size Measured by Ultrasound to Assign Stage of Maturity in Burbot

Gonad size (diameter and circumference) measured by ultrasound was assessed as a metric to assign stage of maturity in Burbot Lota lota from Lake Roosevelt, Washington. Paired gonad tissue and ultrasound measurements were collected monthly from November 2017 to March 2018. Gonad tissue was processed for histological analysis to confirm stage of maturity. Gonad diameter and circumference were measured by ultrasound. Excised gonad diameter (i.e., true gonad diameter) was measured by digital calipers, and excised gonad circumference (i.e., true gonad circumference) was measured by a measuring tape. All late vitellogenic (stage 6) ovaries measured by ultrasound had a diameter greater than 3.90 cm, suggesting a value of 3.90 cm or greater may be used to characterize females capable of spawning in the current reproductive cycle. One mid-spermatogenic (stage 3) and all ripe (stage 4) testes were too large to be measured and assigned diameter of 5.11 cm, the maximum value capable of being measured by our ultrasound transducer. A value of 5.11 cm or greater may be used to characterize males capable of spawning in the current reproductive cycle. Testis circumference measured by ultrasound is not reported because some testes were wider than the ultrasound transducer and could not be measured. Measurements of testis diameter did not differ between measurement methods (ultrasound vs. true), but ultrasound measurements of ovary diameter and circumference were higher than true measurements. We attributed the difference between measurement methods to flattening of ovary while applying the ultrasound transducer. Gonad diameter and circumference measured by ultrasound were highly correlated with gonadosomatic index and ovarian follicle diameter indicating gonad size measured by ultrasound is an appropriate index of gonad development in Burbot.

Histological study of gonadal tissues of adult Artemia salina (Linnaeus 1758) and immunohistochemistry by Caspase 3 and HSP70 to detect specific apoptosis markers on gonadal tissues after exposure to TBTCl

Background: Several types of research have been recently carried out on the biological effects of TBTs, including investigations of genitals in invertebrates in response to exposure to TBTs in marine water. Aim: The objective of this research was to investigate the acute effects of tributyltin chloride (TBTCl) on gonads in the adult stage of Artemia salina by use normal histology and immunohistochemistry (IHC) (Caspase 3 and HSP70) to see specific apoptosis markers. Methods: After exposure of A. salina to different concentrations of TBTCl (25, 50, 100, 200, and 300 ng.l−1), 50 adult A. salina (25 male and 25 female) were selected randomly from each concentration to histologically study the gonads. The gonad tissue was sectioned (5 μm) and some slides were stained with hematoxylin and eosin and others were stained with IHC avidin–biotin complex, and were examined under a light microscope. Results: The results showed significant differences (p < 0.05) in histological lesions between different concentrations of TBTCl. The histological lesions in the testis and ovary section were undifferentiated cells, degenerating yolk globules, and follicle cells enveloping the oocyte which was then compared with control tissue, and these effects were found to be increased in females more than in males with the highest concentration of TBTCl. Immunohistochemistry (IHC) showed that positive immunostaining was observed in the testis and ovary as brownish deposits to Caspase 3 and HSP70 antibody after exposure to TBTCl, while the testis and ovary section in control tissue had no immunoreactivity to Caspase 3 and HSP70 antibody; these effects were profoundly increased with the highest concentration of TBTCl in females more than in males. Finally, the histological lesions and IHC (Caspase 3 and HSP70) revealed that the apoptosis and immune system stress of A. salina gonad tissue damage in females were more sensitive to TBTCl toxicity as compared to white males. Conclusion: In general, the present study aimed to observe the effects TBTCl on A. salina gonads by using histological sections and IHC (Caspase 3 and HSP70), which were evaluated for the first time and have been proven to possess an important function in apoptosis marker and immune system stress in Artemia. Finally, the specific mechanisms through which TBTCl affects A. salina Caspase 3 and HSP70 expression need further investigation.

Effects of early life stage exposure of largemouth bass to atrazine or a model estrogen (17α-ethinylestradiol)

Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.

An efficient photograph-based quantitative method for assessing castrating trematode parasites in bivalve molluscs

Parasitic castration of bivalves by trematodes is common, and may significantly reduce the reproductive capacity of ecologically important species. Understanding the intensity of infection is desirable, as it can indicate the time that has passed since infection, and influence the host's physiological and reproductive response. In addition, it is useful to know the developmental stage of the trematode, to understand trematode population trends and reproductive success. However, most existing methods (e.g. visually estimating the degree of infection) to assess intensity are approximate only and not reproducible. Here, we present a method to accurately quantify the percentage of bivalve gonad filled with digenean trematode tissue, based on small squashes of gonad tissue rapidly photographed under light microscopy. A maximum of 15 photographs is required to determine the percentage of the whole gonad occupied by trematodes with a minimum of 90% confidence, with smaller mussels requiring fewer. In addition, the stage of trematode infection can be assessed because full sporocysts, spent sporocysts and free cercariae are clearly distinguishable. Although variation exists in the distribution of trematodes in gonad tissue, and thus in the estimate of percentage of the gonad filled with trematodes, this method represents a marked improvement on current coarse assessments of infection which typically focus on binary presence/absence measures. This technique can be used to facilitate a more sophisticated understanding of host–parasite interactions in bivalves, and can inform the conservation and reproductive biology of environmentally crucial species.

Gonadal transcriptomic analysis of yellow catfish (Pelteobagrus fulvidraco): identification of sex-related genes and genetic markers

Yellow catfish ( Pelteobagrus fulvidraco) has been recognized as a vital freshwater aquaculture species in East and Southeast Asia. In addition to its commercial interest, it is also attracted much attention because of its value in studying sex-determination mechanisms. A comprehensive gonadal transcriptome analysis is believed to provide a resource for genome annotation, candidate gene identification, and molecular marker development. Herein, we performed a de novo assembly of yellow catfish gonad transcriptome by high-throughput Illumina sequencing. A total of 82,123 contigs were obtained, ranging from 351 to 21,268 bp, and N50 of 2,329 bp. Unigenes of 21,869 in total were identified. Of these, 229 and 1,188 genes were found to be specifically expressed in XY gonad tissue for 1 yr and 2 yr old yellow catfish, respectively; correspondingly, 51 and 40 genes were identified in XX gonad tissue at those two stages. Gene ontology and KEGG analysis were conducted and classified all contigs into different categories. A large number of unigenes involved in sex determination were identified, as well as microsatellites and SNP variants. The expression patterns of sex-related genes were then validated by quantitative real-time PCR (qRT-PCR) suggesting the high reliability of RNA-Seq results. In this study, the transcriptome of yellow catfish gonad was first sequenced, assembled, and characterized; it provides a valuable genomic resource for better understanding of yellow catfish sex determination as well as development of molecular markers, thereby assisting in the production of monosex yellow catfish for aquaculture.

Differential recovery of δ13C in multiple tissues of white sucker across age classes after the closure of a pulp mill

Techniques to document recovery after the closure of pulp mills that discharge enriching effluents are not well established, but δ13C may be a useful tool. In the 1990s, the muscle tissue of white sucker (Catostomus commersonii) collected downstream of two pulp and paper mills discharging into separate streams (Mattagami and Kapuskasing rivers) was enriched in 13C compared with upstream fish, suggesting uptake of pulp-derived C. The Mattagami River mill was closed in 2006, and analysis of muscle and gonad for δ13C was performed in 2011. As expected, fish captured in 2011 downstream of the operational Kapuskasing mill still showed the influence of the pulp-derived C in muscle and gonad tissue. After the closure of the Mattagami River mill, muscle tissue of white sucker was still enriched in 13C compared with upstream fish, while gonad tissue was not. The patterns observed in the Mattagami River were, however, related to age; the oldest fish showed enrichment of δ13C in both muscle and gonad tissue, suggesting the residual occurrence of pulp-derived C. This study suggests that measurements of stable isotopes in fish across a broad age range may indicate ecosystem improvements. These techniques may also be useful where no data prior to the upgrade or closure are available for comparison.