Assessing microplastic exposure of large marine filter-feeders.

Large filter-feeding animals are potential sentinels for understanding the extent of microplastic pollution, as their mode of foraging and prey mean they are continuously sampling the environment. However, there is considerable uncertainty about the total and mode of exposure (environmental vs trophic). Here, we explore microplastic exposure and ingestion by baleen whales feeding year-round in coastal Auckland waters, New Zealand. Plastic and DNA were extracted concurrently from whale scat, with 32 ± 24 (mean ± SD, n = 21) microplastics per 6 g scat sample detected. Using a novel stochastic simulation modeling incorporating new and previously published DNA diet information, we extrapolate this to total microplastic exposure levels of 24,028 (95% CI: 2119, 69,270) microplastics per mouthful of prey, or 3,408,002 microplastics (95% CI: 295,810, 10,031,370) per day, substantially higher than previous estimates for large filter-feeding animals. Critically, we find that the total exposure is four orders of magnitude more than expected from microplastic measurements of local coastal surface waters. This suggests that trophic transfer, rather than environmental exposure, is the predominant mode of exposure of large filter feeders for microplastic pollution. Measuring plastic concentration from the environment alone significantly underestimates exposure levels, an important consideration for future risk assessment studies.

Transcriptomics profiling and steroid production in mummichog (Fundulus heteroclitus) testes after treatment with 5α-dihydrotestosterone.

5α-Dihydrotestosterone (DHT) is a potent androgen in mammals with multiple roles; however the physiological actions of DHT in male fishes are not well known. To address this knowledge gap, male mummichog (Fundulus heteroclitus) were continuously exposed to 0, 5, and 50 µg/L DHT for 21 days. Following exposure, testes were separated for histology, ex vivo incubation to measure steroidogenic capacity, and gene expression analyses (real-time PCR and microarray). DHT significantly decreased ex vivo 11-ketotestosterone (11KT) production in males exposed to 50 µg/L DHT but not 5 µg/L DHT, and DHT exposure did not affect ex vivo testosterone production. Histological examination revealed that the amount of interlobular and connective tissue present in the testes was increased in the 50 µg/L DHT treatment. Despite reductions in the production of 11KT, DHT did not affect the expression of targeted genes in the steroidogenic pathway such as steroidogenic acute regulatory protein (star), P450 side chain cleavage (cyp11a1) and 11ß-hydroxysteroid dehydrogenase (hsd11b3). Microarray analysis in the testes of individuals from control and 50 µg/L DHT revealed that males exposed to 50 µg/L DHT showed regulated transcriptional sub-networks that were related to immunity, regulation of blood flow, lipids and xenobiotic clearance, suggesting that DHT may be involved in the physiological regulation of these processes in the fish testes. A second objective of this study was to determine the feasibility of measuring mRNA levels in tissues used for ex vivo steroid production by comparing RNA integrity and transcript levels in testes of both immediately flash frozen tissue and incubated tissue. There was no significant difference in RNA quality between the two time points, indicating RNA integrity can remain intact for at least 18 h in ex vivo assays, thereby providing a viable option for researchers assessing multi-level biological reproductive endpoints when limited tissue is available. While the gene expression levels of actb, efla, rps12, rps18, star, and hsd11b3 remained unchanged, esr2a (esrba), esr2b (esrbb) and cyp11a1 were significantly lower in incubated tissue compared to flash frozen tissue. Therefore caution must be used as the steady-state levels of select genes may change over time. This study improves our understanding of DHT action in the teleostean testis and generates new hypotheses regarding cell processes that are regulated by this underexplored and potent androgen.

The effects of 17-α-ethinylestradiol (EE2) on molecular signaling cascades in mummichog (Fundulus heteroclitus).

Exposures to ≤10 ng/L of 17-α-ethinylestradiol (EE2) will reduce or shut down egg production in freshwater fish models, while mummichog (Fundulus heteroclitus), an estuarine species, are able to produce eggs at EE2 concentrations >3000 ng/L. The objective of this study was to gain mechanistic insight into how mummichog are able to produce eggs during exposures to high EE2. Mummichog were exposed to 0, 50 or 250 ng/L of EE2 for 14 d. There were no changes in gonadosomatic index, liversomatic index, gonad development, or plasma estradiol levels after exposure to EE2. However, testosterone significantly decreased with EE2 exposures (50, 250 ng/L). Microarray analysis in the liver revealed that cell processes associated with lipids were affected by EE2 at the transcriptome level. Based on the transcriptomics data, we hypothesize that mummichog are able to maintain lipid transport and uptake into the ovary and this may be associated with apolipoproteins, facilitating normal oocyte development. Novel gene regulatory networks for protein modification targets were also constructed to learn more about the potential roles of estrogens in the teleost liver. Although post-translational modifications (PTMs) are important regulatory mechanisms, the roles of PTMs in protein regulation in fish and the susceptibility of PTMs to aquatic pollutants are largely unexplored and may offer novel insight into mechanisms of endocrine disruption.