REPRODUCTIVE AND DEVELOPMENTAL EFFECTS OF SELENOMETHIONINE IN THE FATHEAD MINNOW PIMEPHALES PROMELAS
Selenium (Se) is an essential trace element that undergoes maternal transfer to offspring where it has a high degree of teratogenic potential in egg-laying vertebrates because of the narrow range between nutritional benefit and toxicity. Studying this phenomenon of Se maternal transfer and subsequent toxicities in offspring is difficult in many fish species for logistical and biological reasons. For instance, Se contaminated sites are often located in remote locations which can make sampling efforts problematic, and certain fish species of concern commonly have long reproductive cycles and/or complex life histories which can be difficult to monitor. Thus, there is a need for improved methods to assess the toxicity of Se across diverse species of embryo-larval fish which could potentially aid site-specific risk assessment of Se contamination. Microinjection methodology is a potential surrogate for simulating the maternal transfer of Se and could be utilized to study this phenomena in non-model species which are unable to be spawned in the laboratory, or are difficult to sample from an Se contaminated site when spawning. Therefore, the overall objective of my research was to compare two potential in ovo Se exposure routes, maternal transfer and microinjection, in the fathead minnow Pimephales promelas to determine if early life stage toxicities are comparable between these different exposure routes in a freshwater fish. My thesis research fulfilled this objective by characterizing the effects of dietary Se exposure in P. promelas on fecundity and the maternal transfer of Se to embryos, the subsequent toxicities in embryo-larval offspring, and then used this information to inform a microinjection study with P. promelas embryos to allow for a comparison between the two different in ovo exposure routes. First, a 28-day short-term reproductive assay with P. promelas was performed to determine the dynamics of dietary selenomethionine (SeMet) exposure on maternal transfer and its effects on the F1 generation. Sexually mature P. promelas breeding groups (2 females:3 males) were fed a diet of either control (unspiked) or SeMet-spiked food (Low: 3.88 µg Se/g food dry mass [dm]; Medium: 8.75 µg Se/g food dm; High: 29.6 µg Se/g food dm) and allowed to breed. Fecundity did not decrease in female fish exposed to elevated levels of dietary SeMet and the low treatment (3.88 µg Se/g food dm) produced on average the most embryos per female, suggesting a possible supra-nutritional benefit of SeMet on reproduction. Dietary exposure with SeMet-spiked food rapidly induced the maternal transfer of excess Se and embryo concentrations increased daily until reaching steady-state after approximately 14 days of exposure. In ovo exposure to elevated Se did not affect hatchability of embryos or survival until swim-up in early life stage P. promelas. However, a dose-dependent increase in the frequency of larval fish with any type of morphological abnormality (e.g. edema, skeletal, finfold, craniofacial) present at swim-up was observed at embryo Se concentrations of 28.4 µg Se/g embryo dm. In the ancillary embryo microinjection study, embryos were injected with three doses of SeMet (Low: 9.73 µg Se/g embryo dm; Medium: 13.5 µg Se/g embryo dm; High: 18.9 µg Se/g embryo dm) to simulate maternal transfer and provide a point of comparison for the hatchability, survival and deformity endpoints measured in the preceding maternal transfer study. There were no effects of SeMet microinjection on hatchability up to a concentration of 18.9 µg Se/g embryo dm, however this same embryo Se concentration decreased survival until swim-up. Furthermore, this embryo Se concentration caused a greater increase in the frequency of deformed fathead minnow at swim-up in comparison to the highest embryo Se concentration in the maternal transfer study (28.4 µg Se/g embryo dm), suggesting a more toxic response when the dosage is primarily free SeMet rather than maternally transferred Se which is mainly SeMet incorporated into proteins. With this said, the frequency and type of deformities at embryo Se concentrations in the range of 9.73 – 13.5 µg Se/g embryo dm were similar between the two different exposure routes. The deformities observed in P. promelas as a response to SeMet exposure through both maternal transfer and microinjection followed a dose-dependent trend, and the most common deformities observed were spinal and finfold abnormalities, which were approximately two-fold more common than edema or craniofacial defects. Overall, this thesis research highlights the utility of embryo microinjection as a proxy for studying the maternal transfer of Se and provides an additional line of evidence for potentially extending this methodology to less commonly studied freshwater fish species of concern.
selenium, maternal transfer, fathead minnow, teratogenicity, microinjection
Master of Science (M.Sc.)