John Godwin

This MBIE Smart Idea will use innovative new environmental DNA (eDNA) technologies to test, develop and establish efficient, sensitive and reliable in-the-field surveys of mammalian pests and native species. We will determine if eDNA surveys can rapidly and accurately measure the distributions, densities and movements of pest and other species, from local to landscape scales, using a series of well-controlled, replicated mesocosm experiments. We will then undertake in-the-field experiments in parallel with traditional measures at sites where intensive pest monitoring and control is already undertaken. Finally, we will test our approach with end-users in various real-world management situations, including pest management projects run as part of the Taranaki Mounga Project and rodent control on remote Pacific Islands led by Island Conservation.

California halibut (Paralichthys californicus) are being cultured for stock enhancement purposes at the Hubbs Sea World Research Institute (HSWRI). Recent cohorts (n = 4) have ranged from 69% to 95% male, therefore there is a need to understand culture protocols that produce approximately equal sex ratios. A series of experiments are being planned that will manipulate temperature and other environmental stressors (tank color, light intensity, density, etc.) during the presumed sex determination window in P. californicus, to determine their effect on juvenile sex ratios. Currently, examination of sex in P. californicus is done through visual examination of the gonads. This typically requires fish to be >130mm (total length), which can take approximately 8 months (but may take 6–10 months at higher or lower rearing temperatures). In this proposal, we would evaluate the feasibility of using sex-specific molecular markers expressed during sexual development, to determine juvenile P. californicus sex as early as possible (e.g. at 50–100mm; 3–8 months). This approach has proved successful in closely related Japanese flounder Paralichthys olivaceus and southern flounder Paralichthys lethostigma.

Dr. Donal Manahan, Division Director, Directorate for Biological Sciences (BIO), Division of Integrative Organismal Systems at the National Science Foundation is requesting the assignment of Dr. John Godwin to the position of Program Director in the Neural Systems Cluster Program. This assignment would be for a period of one year beginning on or about August 17, 2020 through August 16, 2021 with the possibility of being extended based on the mutual agreement between the home institution, NSF, and the assignee. Dr. Godwin’s assignment, if approved, will be made under the provisions of Title IV of the Intergovernmental Personnel Act of 1970 (Public Law 91-648). Assignments made under the Act facilitate the beneficial sharing of personnel resources among government and academe by providing for the temporary assignments of personnel to and from the Foundation and State and local government agencies or institutions of higher education in instances where such assignments would be of mutual benefit to the organizations involved.

Our goal is to develop safe, controllable, and effective gene drive technologies that can potentially be applied to eradicate invasive rodent populations on islands. We propose to achieve this by preventing the development of female progeny, thereby reducing population numbers and reproductive capacity. Invasive rodents directly and indirectly cause extinction and endangerment of species on islands globally and represent a major threat to biodiversity. Invasive rodents have these effects by directly preying on native species, out-competing them for resources, and destroying sensitive habitat. Pursuit of this goal is therefore closely aligned with the DARPA-relevant application of maintaining and protecting ecosystem biodiversity. Our proposed research under the Safe Genes program addresses Technical Areas 1 and 3 using three genetic model systems: Escherichia coli, fruit fly, and mouse. Project activities will be in three areas: genetics and reproduction; modeling and risk assessment; and ethics, engagement and communication. Impacts will include findings relevant to mitigating and reversing adverse gene drive effects and public and stakeholder engagement addressing broader concerns of international communities.

For the Sea Grant project “Fishery management implications of environmentally determined sex and biased juvenile sex ratios in southern flounder”, the NC SeaGrant Graduate Fellow will work closely with the PIs on all facets of the proposed research and extension activities as part of his/her dissertation research. The fellow will help coordinate much of the daily and seasonal activities associated with the project to include field sampling and specimen collection; tissue extractions and gene expression/protein analyses; data collection, tabulation, presentation, and storage; statistical analyses; and the training of personnel, including undergraduates, to assist with data collection, tissue analyses, and other aspects of the project. The fellow will work with North Carolina Division of Marine Fisheries (NCDMF) to collect age 0 and age 0-1 juvenile southern flounder specimens from NCDMF P120 and P195 trawl surveys and with PIs to conduct supplemental trawl surveys. This work will entail frequent traveling out to coastal estuarine regions to deploy and retrieve temperature probes and datasondes and to collect fish across varying sampling strata. Fish will be measured and weighed and their tissues sampled. The Fellow will then work in the lab to assess sex of individual animals needed to determine sex ratios within and across sampling regions for age 0 and age 1 fish. A sex biomarker approach will be used that involves gonadal RNA isolation, cDNA synthesis and measurement of three sex-determining genes by quantitative PCR. The Fellow will also take the lead in conducting laboratory studies to test if temperature fluctuations observed in wild flounder nursery habitats is sufficient to masculinize flounder. This will involve maintaining fish at 3 different temperature regimes in recirculating tank system, and growing them out to a size after which sex has been determined. The fellow will monitor the experiment, sample fish periodically, and assess whole body cortisol by radioimmunoassay and the gonadal sex of individuals using the biomarker approach. All data will be compiled and analyzed by the Fellow with help from other personnel and the PIs. The Fellow will present results, assist with annual reports, and contribute to all publications associated with their research. The student will also participate in disseminating results to the science community through presentations at local, regional, and national meetings and to the public by aiding in the production and distribution of a video outlining the findings of the project.

Southern flounder (Paralichthys lethostigma) supports major commercial and recreational fisheries in the southeast US and is the most valuable finfish fishery in NC. However, the most recent assessment indicated that it is overfished, overfishing is still occurring, and the fishery is depleted. Considerable effort is being devoted to managing harvest to rebuild the spawning stock, and substantial effort has also been devoted to understanding how water quality and other nursery habitat variables may affect growth and survival with consequences for recruitment. But evidence from our recent work suggests that another previously unrecognized factor could be having a significant effect on the flounder fishery. Southern flounder (and other Paralichthys species) exhibit environmental sex determination. While genetic males (XY) will always become gonadal males, genetic females (XX) may differentiate into gonadal males (XX males) depending on the temperatures (too high or too low) or other stressors they experience in their nursery habitat during the critical developmental stage (35 – 65 mm). Thus, ideal conditions will produce at most 50% females, with much lower percentages possible if temperature or other variables lead to masculinization of XX females. This phenomenon has important ramifications for two reasons. First, female southern flounder grow faster and reach much larger maximum size than males, so this fishery is heavily dependent on females (few males reach harvestable size). A reduction in the proportion of females translates directly into a reduction in recruitment to the fishery. Second, the models used to estimate fishing mortality and spawning stock biomass rely on an index of year class strength that is based on abundance of age-0 flounder. These fish cannot be sexed macroscopically so the assessment assumes a 1:1 sex ratio. If the juvenile sex ratio is actually biased toward males, year class strength of female flounder will be overestimated, resulting in erroneous model estimates. We have developed, validated and successfully applied physiological biomarker methods that allow us to unambiguously determine the sex of flounder as small as 50 mm. We have preliminary data indicating that the sex ratio of flounder is strongly male-biased (up to 90%) in some years in some nursery habitats (e.g., Neuse) but not in others (e.g., North Pamlico), and that these differences may be due to variation in nursery habitat temperatures. Given the potential importance of such bias, NCDMF personnel have indicated a strong need for better information on juvenile flounder sex ratios. The work we propose will comprehensively evaluate spatial and temporal variation in sex ratios of juvenile flounder across a wide range of NC nursery habitats in relation to variability in temperature and other habitat variables (e.g., oxygen, salinity) occurring during the sex determination period, and determine the temporal stability of flounder sex ratios through late age-0 and early age-1. In the lab we will test the hypothesis that patterns of temperature variation associated with strong male bias and 1:1 sex ratios in the field will reproduce the observed ratios under controlled conditions. We will continue to collaborate and share our results with Tom Wadsworth and other NCDMF biologists and advisory groups involved in flounder management as well as other scientists in NC and elsewhere working on southern flounder and related species, communicate our findings to the broader scientific community through conference presentations and peer-reviewed publications, and share them with the fishing community and the general public through publications such as Coastwatch, NC Cooperative Extension outlets, outreach to K-12 teachers COSEE Southeast programs, and events such as our recent “Creeks to Coast” workshop in Raleigh. Our results will directly contribute to more effective management of the southern flounder fishery, and improve understanding of potential climate change impacts on population dynamics of flounder species. A more sustainable fishery will benefit NC and the region, its recreational and commercial fisheries and economic vitality of coastal communities.

Sexual differentiation of the brain and behavior typically results from an interplay of endogenous and enviromental cues influencing the timing, level and location of gene expression. The relative strength of endogenous and environmental influences on this process varies across species. It is also clear that multiple mechanisms leading to similar patterns of behavioral expression may exist within species. This is suggested by variation in neuroendocrine mechanisms underlying aggression across sexes and seasons and the results of gene knockout studies where the lack of a presumably critical gene product does not abolish or sometimes even affect behavioral expression. We are building a solid understanding of how gonadal steroids influence the sexual differentiation of brain and behavior. Much less well understood are the mechanisms by which environmental influences such as social interactions affect these processes and how these mechanisms overlap and act with or through steroid hormone mediated pathways. This project will explore the role of a recently discovered neuropeptide system, kisspeptin, in mediating socially-controlled sex change. We will use a model system, the bluehead wrasse, that exhibits two interesting and experimentally useful features of its reproductive biology and where the development of the male courtship behavior can be stimulated through both social interactions and steroid hormones. Bluehead wrasses exhibit discrete alternate male mating phenotypes and socially-controlled functional sex and role change. Sex changing females initiate behavioral change immediately and develop fully male behavioral phenotypes within a few days of becoming socially dominant. Importantly, we have shown that even gonadectomized females who cannot develop male morphological characteristics nevertheless develop fully male behavioral phenotypes on attaining dominant status. We have also induced female-to-male sex change and male courtship behavior through gonadal steroid hormone manipulations in nature. The aims of this project are to clone key genes in the kisspeptin signaling system in bluehead wrasses, compare expression of these genes across sexual phenotypes and experimentally induced sex change, and characterize the neuroanatomical locations of expression within the bluehead wrasse brain. Identifying mechanisms by which social interactions influence behavior-controlling mechanisms is of both basic and practical signficance. Aggressive behavior has enormous societal costs and a better understanding of social influences on the neural substrates of this behavior is needed. Also, the increasing importance of finfish aquaculture makes a better understanding of the control of sexual behavior in fishes useful for captive propagation. The bluehead wrasse system presents the opportunity to experimentally dissect gonadal and social influences on these mechanisms in the natural environment.

Southern flounder support critically important recreational and commercial fisheries in the Southeast US, but these fisheries have also suffered substantial recent declines. Southern flounder also show strongly sexually dimorphic growth with females growing much larger than males. Due to this difference in growth and capture size limits, male southern flounder rarely grow large enough to enter the commercial or recreational fisheries and the catch is therefore heavily female-biased. Currently, managers must assume 50:50 sex ratios in Juvenile Abundance Indices (JAI). Departures from this assumed even sex ratio in juveniles could adversely influence estimates of Spawning Stock Biomass (SSB) and mortality (F), hindering efforts to manage and recover these stocks. Importantly, southern flounder also show strong environmental influences on sex determination, particularly with higher temperatures inducing a strong sex ratio skew towards males. Sampling in conjunction with the North Carolina Division of Marine Fisheries in the unusually warm spring/summer of 2012 strongly suggested elevated temperatures in flounder nursery habitats can also strongly skew sex ratios in wild populations and that this can occur without detectable differences in growth. The goal of the proposed studies is to better characterize sex ratios in juvenile flounder. This would benefit management of this economically and ecologically important fishery resource.

This dissertation improvement project would test whether gonadal signals impact the speed and intensity (frequency and duration) of behavioral changes during female-to-male sex change and subordinate-to-dominant male role change. Furthermore, this project would test if gonadal presence affects gene expression of the systems hypothesized to control sex and role change: the vasotocin, kisspeptin, and gonadotropin-releasing hormone systems. The animal model that would be used in this project is the bluehead wrasse, Thalassoma bifasciatum, a diandric (two male phenotypes) protogynous (female-to-male) coral reef fish capable of changing male role and sex.

Rodent pests cause major economic losses and threaten food security and biodiversity worldwide. The problem is particularly acute on islands where most vertebrate extinctions occur. We propose to test an innovative approach based on genetic engineering. This would also support graduate training in the NCSU Genetic Engineering and Society Center.