Anne Boettcher

Short-term exposure to a synthetic estrogen disrupts mating dynamics in a pipefish

Sexual selection is responsible for the evolution of some of the most elaborate traits occurring in nature, many of which play a vital role in competition over access to mates and individual reproductive fitness. Because expression of these traits is typically regulated by sex-steroids there is a significant potential for their expression to be affected by the presence of certain pollutants, such as endocrine disrupting compounds. Endocrine disruptors have been shown to alter primary sexual traits and impact reproduction, but few studies have investigated how these compounds affect secondary sexual trait expression and how that may, in turn, impact mating dynamics. In this study we examine how short-term exposure to a synthetic estrogen impacts secondary sexual trait expression and mating dynamics in the Gulf pipefish, a species displaying sex-role reversal. Our results show that only 10days of exposure to 17α-ethinylestradiol results in adult male pipefish developing female-like secondary sexual traits. While these males are capable of reproduction, females discriminate against exposed males in mate choice trials. In natural populations, this type of discrimination would reduce male mating opportunities, thus potentially reducing their long-term reproductive success. Importantly, the effects of these compounds on mating dynamics and mating opportunity would not be observed using the current standard methods of assessing environmental contamination. However, disrupting these processes could have profound effects on the viability of exposed populations.

Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791).

For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.

Population structure and reproductive characteristics of the gulf pipefish,Syngnathus scovelli, in Mobile Bay, Alabama

The gulf pipefish,Syngnathus scovelli, is the dominant syngnathid found in coastal regions of the Gulf of Mexico and is the only species in this region known to occur in both freshwater and saltwater habitats. Relatively little is known about the population and reproductive cycles ofS. scovelli, particularly for those found in low salinity environments. The focus of the current study was to collect population structure and reproductive data forS. scovelli from a low salinity habitat, Meaher Park, located at the mouth of Mobile Bay, Alabama. Sampling was conducted twice a month from January 2003 to January 2004. Environmental parameters, as well as population and reproductive parameters, were collected. Water temperature was the primary abiotic factor associated with both the appearance ofS. scovelli and their breeding cycle. Based on gonadosomatic (GSI) and brood pouch somatic indices (BPSI), females and males were reproductively active throughout the summer. Peaks in male GSI and BPSI were consistent with the subsequent appearance of a large number of juveniles in early fall. These event coincided with the higher temperatures seen during late summer and early fall. Sex ratios (male : female 1 : 1.64) and operational sex ratios (1 : 4.09) indicated that this was a female biased population. Although there was no difference in the average size of males and females in this population, the largest individuals collected were female. The female-biased sex ratio supports previous genetic analyses suggesting thatS. scovelli exhibits true sex-role reversal and that operational sex ratios are a reliable indicator of the intensity of mating competition and sex roles.

A quantitative real‐time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009 ) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real‐time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species‐specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.

Liquid culture and growth quantification of the seagrass pathogen, Labyrinthula spp.

Symptoms characteristic of wasting disease, thought to result from infection by protozoan pathogens (i.e. Labyrinthula spp.), are a common phenomenon affecting seagrass species worldwide. However relatively little is known about factors that control the survival and success of Labyrinthula in part due to the difficulty associated with quantifying the growth of this organism. Here we describe a simple and inexpensive method for measuring growth of Labyrinthula in liquid culture that takes into consideration both cell density and areal spread. The technique allows for examination of the effects of both abiotic and biotic factors on the growth of Labyrinthula apart from its seagrass host, separating the effects of environmental condition on the host from their effects on the pathogen.

Biomarkers of Dissolved Oxygen Stress in Oysters: A Tool for Restoration and Management Efforts

The frequency and intensity of anoxic and hypoxic events are increasing worldwide, creating stress on the organisms that inhabit affected waters. To understand the effects of low dissolved oxygen stress on oysters, hatchery-reared oysters were placed in cages and deployed along with continuously recording environmental data sondes at a reef site in Mobile Bay, AL that typically experiences low oxygen conditions. To detect and measure sublethal stress, we measured growth and survival of oysters as well as expression of three biomarkers, heat shock protein 70 (HSP70), hypoxia inducible factor (HIF) and phospho-p38 MAP kinase, in tissues from juvenile and adult oysters. Survival rates were high for both juvenile and adult oysters. Expression levels of each of the 3 isoforms of HSP 70 were negatively correlated to dissolved oxygen (DO) concentrations, suggesting that HSP 70 is useful to quantify sublethal effects of DO stress. Results for HIF and phospho-p38 MAP kinase were inconclusive. Test deployments of oysters to assess expression of HSP 70 relative to environmental conditions will be useful, in addition to measuring abiotic factors, to identify appropriate sites for restoration, particularly to capture negative effects of habitat quality on biota before lethal impacts are incurred.

A SIMPLE TRANSPORT PROCEDURE FOR JUVENILE CALICO SCALLOPS, ARGOPECTEN GIBBUS (LINNAEUS, 1758)

Abstract A simple procedure for transporting live calico scallop, Argopecten gibbus (Linnaeus, 1758), juveniles for grow-out culture is described. The effects of 4 simulated transport periods (9, 16, 20 and 24 h), and subsequent recovery were examined. Success was assessed by survival rate and growth during 2-wk recovery. Survival rate was of 100% for all tested transport periods immediately following transport and following 2-wk recovery. Shell growth averaged 1.76–2.31 mm over the 2-wk period, yielding a growth rate 0.88–1.2 mm.week−1; comparable to average growth rate at same site. There was no significant difference in growth among transport exposure time. Monitored levels of heat shock protein 70 (HSP 70) during manipulation did not indicate any physiologic stress. Successful application of this procedure to a real life 14 h airway transport of another subtropical/tropical scallop species, demonstrates its use for transport of two scallop species juveniles ranging in size from 8–40 mm, without detriment to subsequent survival or growth.

Heat Shock Protein 70 Expression in Juvenile Eastern Oysters, Crassostrea virginica (Gmelin, 1791), Exposed to Anoxic Conditions

ABSTRACT Anoxic water events in conjunction with summer high temperatures are thought to be one of the causes of declines in natural oyster reefs on the eastern shore of Mobile Bay. Work is underway to determine whether tolerance to low oxygen can be selected for in hatchery-produced oysters. As a component of this work, the expression of heat shock protein 70 (HSP 70) was examined in control (normoxia) and anoxia-challenged juvenile oysters. Parental Eastern oysters, Crassostrea virginica were collected from 2 sites, Cedar Point Reef (CP), an area considered to have normoxic conditions, and White House Reef (WH), an area suspected to experience periodic anoxia. F1 generation oysters were produced from CP and WH parents that survived an anoxic exposure of 96 h. Control F1 generation oysters from both parental stocks not exposed to anoxia were also produced. The F1 generation oysters were subsequently exposed to anoxia or control normoxic conditions, and differences in expression of HSP 70 were examined. Nitrogen was used to create the anoxic conditions for both the parental and F1 generations. Three HSP 70 isoforms—2 constitutive forms (HSC 77 and HSC 72) and 1 inducible form (HSP 69)—were expressed in both anoxia- and normoxia-exposed oysters from all groups. Although there were differences among groups of oysters from the 2 sites, there were no differences in the expression of HSC 77 and HSC 72 between the control and anoxia-treated oysters within a group. Interestingly, the expression of HSP 69 was higher in oysters exposed to normoxia than the ones from anoxia treatments. These differences are thought to reflect a combination of responses to nutritional stress in the controls and facultative anaerobiosis and metabolic arrest in the anoxia groups.

Population Structure of the Gulf Pipefish in and around Mobile Bay and the Northern Gulf of Mexico

The genetic structure of inshore aquatic populations can be influenced by a number of factors, including coastal configurations, flow rates, and local adaptation. Properties such as salinity and temperature can differ significantly along the coasts and into the bays and rivers that contribute to these systems. Within these environments, low migratory euryhaline species provide a unique system to examine how these factors influence population structure, even when these populations are continuously distributed. In this study, we utilized microsatellite data to assess the population structure of 7 Gulf pipefish populations located in and around Mobile Bay and the northern Gulf of Mexico. Global F (ST) values (F (ST) = 0.025) suggest moderate levels of genetic structure among the populations. Local genetic structure was present among all coastal pipefish populations. Significant levels of genetic structure were also observed between coastal and estuarine populations (P < 0.05), with bay populations being distinct from their coastal counterparts. Cluster analysis suggests 2 parental populations, with one consisting mainly of estuarine individuals and the other comprising mainly coastal individuals. However, although these populations were genetically distinct, the genetic data also suggested moderate levels of migration between coastal and estuarine areas. We suggest that the differentiation between these populations is likely due to the colonization of individuals from coastal populations followed by limited dispersal out of the bay. It is also possible that different selective pressures between coastal and estuarine habitats may be contributing to the genetic differences between these populations.