National Science Foundation Research Coordination Network:
Integrating Ecology and Endocrinology in Avian Reproduction
(E-BIRD-USA)
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National Science Foundation Research Coordination Network: |
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Current position: Post-doc with Dr. Alexander Kitaysky, University of Alaska Fairbanks
Goals and collaborator. The goal of this project is to better understand mechanisms of avian endocrine maternal effects by quantifying the effects of steroid exposure in ovo on the expression of steroid receptors in the brain. I am currently working in an endocrinology lab, and sought to learn some molecular neurobiology techniques. To this end, I traveled to the University of Edinburgh, Centre for Integrative Physiology to work with Dr. Simone Meddle. She is a neurobiologist with extensive experience using in situ techniques. Our goal was to assess the effects of androgens and corticosterone on the expression of steroid receptors in the brains of quail hatchlings.
Research accomplished. During my visit, I sectioned 28 quail brains and conducted in situ riboprobe/oligoprobe hybridization for steroid receptors and enzymes. Unfortunately, it takes time for the radioactivity from the probes to generate silver grains on the photographic emulsion used to visualize the mRNA, so the results of this aspect of the study are pending. I plan to return to University of Edinburgh later this summer to quantify receptor expression in several regions of the brain. I assayed hatchling plasma for androgens, which were mostly undetectable. However, based on the small subset of chicks with detectable testosterone, chicks from eggs injected with testosterone had higher plasma testosterone at hatching, while chicks from corticosterone-treated eggs appeared to have lower testosterone. I am currently validating the quantification of steroids in egg waste from these birds, and hope to assess whether exposure to elevated yolk steroids induced positive or negative feedback on endogenous steroid production in ovo.
Products & broader impacts. We hope to produce at least 2 publications as a result of this collaboration. My collaboration with Dr. Meddle will continue in the future, and I am using what I learned in her lab to establish some in situ assays in the Kitaysky lab here at University of Alaska Fairbanks. It was extremely valuable to have the opportunity to expand the scope of my research using techniques that I do not have local access to, and I believe that this mechanistic approach will provide much needed information for those of us interested in maternal effects.
My dissertation research has focused on life history trade-offs between reproduction and survival in Florida Scrub-Jays at Archbold Biological Station, under the guidance of Dr Stephen Schoech, University of Memphis. A potential proximate parameter driving variation in yearly reproductive effort and consequent survival is a birds’ immunocompetence across seasonal stages. I have primarily focused on the interaction between an individual’s reproductive effort and immune function by comparing immune measures when energetic requirements were relatively low (pre-breeding) and when they would be at their maximum - when feeding dependent young and concurrently initiating their annual molt. By tracking reproductive effort and survivorship of individuals for which I had immune measures, I hope to be able to determine the ultimate effects of these proximate factors. Many factors could contribute to variation in or the magnitude of a bird’s immune capability, including energetic constraints during reproduction and molt, as well as exposure to disease. I have been able to infer a disease state from various blood measures, such as the ratio of immunoglobulins to other plasma proteins and from leukocyte counts. However, what I lacked was an actual measure of disease state to test the assumptions that elevated immunoglobulins and the presence of specific leukocyte types were due to exposure to a pathogen or parasite. To remedy this shortfall, I collaborated with Dr. Robert Ricklefs and visited his laboratory at the University of Missouri - St. Louis to investigate the prevalence of avian malaria in the research population of Florida Scrub-Jays. Additionally, a recent graduate of Dr Ricklef’s had developed a natural antibody assay, which I wanted to learn and add to my repertoire of immune measures to apply to my studies of Florida Scrub-Jays.
Methods: During February of 2006 I spent almost two weeks delving into the voodoo world of DNA extraction and PCR techniques in the Ricklef’s lab. With the help of Ms. Julia Gray, I was first taught extraction techniques and methods to apply to unknown “problem” samples. This included learning proper decanting and drying techniques and adding specific enzymes, such as proteinase K, to break down samples that were overloaded with protein (as were most of my samples). After a few introductory lessons I was left to my own to extract the DNA from approximately 300 Florida Scrub-Jay blood samples that had been collected during the two different life history stages referenced above over a two year period. Recall that the first of these periods was before breeding and the second was near the end of the breeding season during June, July and August when FSJ’s are caring for dependent young. This later period is the wet season with high vector concentration and is the period I would expect to find the highest prevalence of malaria in the FSJ population. Following DNA extraction, the Ricklef’s laboratory uses their own pair of primers (343F and 496R, Fallon et al. 2003) to initially screen for a 286 bp fragment of rRNA coding sequence from the mDNA of the malaria genome. This fragment is conserved across Plasmodium spp., Haemoproteus spp., and Leukozytozoan spp. If an initial screen is positive, a second set of primers is used to capture a larger fragment that is subsequently used for sequencing and identification of the specific parasite. I am currently waiting for sequencing data to identify which particular species and strains of parasites infect Florida Scrub-Jays.
Results: This first trip only allowed me to conduct the initial screening and, to my great surprise, we had a 41% positive rate for at least one of the above mentioned Apicomplexa genera. It was gratifying to find that the ‘positives’ from the PCR DNA screening process included those samples for which we had previously identified Plasmodium, Haemoproteus, and Leucozytozoan spp. from blood smears. However, it is important to note that our blood smear screening yielded only a 9-11% positive rate per year. Clearly, the molecular method of identifying infected birds is a powerful and valuable tool to add to my arsenal and will provide a far more accurate measure of disease state. My initial analyses of the data suggest that more birds are infected during the post-breeding period than during the pre-breeding period. I am currently teasing apart if this is related to reproductive effort or actual time of year by investigating if breeders are more likely to be infected than non-breeding helpers. Given that the investment in reproduction by non-breeding helpers is far less than that of breeders, if immune capability is down-regulated during reproduction one might expect greater infection rates in breeders than in non-breeders. However, my initial analysis found that non-breeders are more likely to be infected than breeders. Less surprising is the finding that wildland birds have greater infection rates than suburban birds. I am currently analyzing correlations between the malarial data and a suite of other physiological measures including, stress (hetrophil/lymphocyte ratios, corticosterone), reproductive hormone levels (estradiol and testosterone), and a number of measures of immune response (Total white blood cell, IgG levels, bacterial killing capacity, agglutination and lysis activity). I am also assessing whether these data explain reproductive effort and, ultimately, survival.
I also learned and conducted the agglutination and lysis assay derived from avian plasma. An assay, which measures the ability of the plasma components (IgG and IgM) to agglutinate rabbit red blood cells, following which the complement immune system is activated and lysis of the blood cells can be scored. I was able to then use this test on samples stored from previous seasons and examine these two immune factors and their relationship to multiple variables. The only difference I found was breeders have a slightly greater agglutination response than non-breeders. This greater immune response may suggest that breeders are in slightly better condition than non-breeders and is supported by the lower malarial prevalence in breeders. As the breeding population has a mean age greater then the non-breeding population age may also play a role. I therefore tested for a relationship with age and could find no relationship between age and agglutination or lysis score.
Broader Impacts: I am currently in the process of disseminating this information via two papers. The first paper describes the disease states of Florida Scrub-Jays and the second relates the effect of malaria on reproductive onset and activity. This project has been a successful collaboration and both Dr. Ricklefs and I wish to extend the work across another year. I plan to make another visit to University of Missouri - St. Louis in the coming year for further analyses and lab work. Furthermore, during my visit I presented my work to new graduate students in the biology department and encouraged them to work in cross-disciplinary fields.
For my research project at Boise State University, I examined how exposure to elevated levels of corticosterone (using corticosterone-filled silastic tubing implants) during the nestling period influenced various morphological and physiological aspects of development, including feather formation and immunocompetence. To fully explore these developmental parameters, I needed to learn two techniques, both of which were readily accomplished the University of Memphis with Dr. Steve Schoech and Ms. Lynda Leppert.
First, by using an Ocean Optics USB2000 Spectrometer, I analyzed the reflectance of feathers collected from American kestrels (Falco sparverius) subjected to different hormonal regimes during the developmental period.
Secondly, I learned how to perform leucocyte counts on blood smears made from those same birds. For that technique, I was shown how to stain slides, make differential counts, interpret the relative abundance of different white blood cells, and search for and identify blood parasites.
My goals were to gain insight into stress-induced effects on immunocompetence and variability in plumage characteristics. By integrating growth, immune response, and quality of feather production in response to one week of elevated levels of corticosterone, I was able to elucidate some of the short-term effects of physiological stress during the nestling stage. By examining feathers grown during the period of exposure to elevated corticosterone, I saw that plumage coloration, known to affect mate choice, was not significantly affected by hormone treatment. Although there was a slight trend for bluer secondaries in the control birds, the trend was not very strong. Additionally, when examining the appropriate leukocyte ratios (specifically, heterophil to lymphocyte, or H/L), I saw that the increase of exogenous corticosterone did not significantly affect H/L either during exposure, or after a washout period.
At the completion of this trip, I had learned several new techniques. In addition to knowing how to prepare feathers for color analysis, I also learned how to operate the software, the spectrometer, and how to statistically analyze the reflectance data. Additionally, I learned how to stain blood smear slides, what the various leukocytes looked like under an oil immersion objective, the best way to count these various cells, and what the different cell counts meant.
From a broader perspective, I also made several advances in my graduate career. In addition to increasing the number of laboratory techniques in which I am experienced, I also made several new contacts at the University of Memphis. In the field of biology, where collaboration can lead to some of the most exciting work, it is very important to have these networking opportunities. Additionally, I am currently a Masters student who is applying to several PhD programs. Opportunities such as the NSF RCN Exchange Visit allow me to increase my funding history as well as demonstrate my commitment to producing the best science possible. My graduate career has benefited not only with an expanded repertoire of laboratory techniques and a more diverse and solid resume, but also with the benefit of having made several new contacts in the biological academic community.
Purpose of exchange visit: Measure changes in PRL levels in response to a stressor in known-aged common terns (Sterna hirundo).
In many organisms reproductive performance increases with age, however we currently have little information about the physiological mechanisms that underlie this commonly observed pattern. Prolactin (PRL) is a hormone that is involved in regulating parental behaviors in vertebrates and has recently been shown to decrease in response to stressors in birds. When the value of current reproduction is high relative to the value of survival and future reproduction, as it is expected to be in older parents, PRL levels may be predicted to decline less rapidly in response to stressors. I tested this expectation in a long-lived seabird, the common tern (Sterna hirundo) in collaboration with Drs. Olivier Chastel, Ian Nisbet, and Ellen Ketterson. This exchange visit made it possible for me to travel to Dr. Chastel’s lab at the Centre d’Etudes Biologiques de Chizé (CEBC) in France to learn a new hormone assay necessary to test this hypothesis.
Technique learned: PRL assay
To measure changes in PRL levels in response to handling and restraint stress, known-age terns were captured on the nest during incubation from May-July 2005 on Bird Island, MA. An initial blood sample was collected within the 0-3 min of capture. Terns were then placed in individual holding tubes so subsequent samples could be drawn after 30 and 50 min. In Dr. Olivier Chastel’s lab, André Lacroix taught me how to measure PRL levels in these samples using a heterologous radioimmunoassay (RIA). The assay employs purified chicken PRL standard and a rabbit-derived antibody. The dose response curve for tern plasma was found to parallel that for chicken plasma, indicating that the assay could be used to measure relative plasma PRL levels in common terns. All samples were run in duplicate in a single assay and the intra-assay coefficient of variation was 8.9% (n=4).
Results and broader impacts: PRL decreases less rapidly in response to stressors in older parents.
We found that PRL levels declined significantly less rapidly in response to handling stress with age (F1,51=4.12, p=0.048, linear regression, Figure 1). Our results are consistent with other avian studies that have shown that PRL levels decrease in response to stressors; however, this is the first study to demonstrate that this occurs more slowly in older parents. PRL is involved in regulating incubation behavior in birds and baseline PRL levels and PRL level dynamics have been shown to be correlated with avian parental behaviors. Therefore, given that PRL levels decreased less rapidly in response to stressors with age, older parents may be expected to abandon their offspring less quickly in the face of stressors than younger parents. We suggest that modulation of the PRL response to stressors may be an important mechanism that facilitates an increase in reproductive performance with age. This exchange visit greatly aided my academic development by allowing me an opportunity to learn a new assay technique that was necessary for the completion of my dissertation. In addition, it provided me with an invaluable opportunity to collaborate with other scientists that are interested in similar research questions.
Purpose of visit: The purpose of this exchange visit was to gain experience in the techniques used in staining and sectioning neural tissue, techniques to determine the volume of brain regions, and to observe immunocytochemistry techniques in the lab of Dr. Scott MacDougall-Shackleton (Dept. of Psychology, University of Western Ontario). Most temperate songbirds exhibit strong seasonal and sexual differences in both song production and the area of the song control system (SCS). Males sing, females typically do not, and the SCS in males is larger during the breeding season than during the non-breeding season. The Northern cardinal (Cardinalis cardinalis) is unique from many other species of temperate songbird in that both sexes sing during the breeding season and song production is nearly monomorphic. Also, cardinals occasionally sing outside of the breeding season. This research helped initiate collaboration between Dr. MacDougall-Shackleton and myself and began the analysis of seasonal and sexual differences of the area of the SCS in cardinals.
Techniques performed during visit: Perfused brains (10 male and 10 female northern cardinals collected at two time points [December 2004 and March 2005], from Kent Farm Bird Observatory, Bloomington, IN) were sectioned in the coronal plane at 40μm in a cryostat (-18oC, 12o blade angle). Sections of tissue containing the areas of interest (high vocal center or HVC, Area X, and the robust nucleus of the archistriatum or RA) were collected into tissue wells containing 0.1M phosphate buffered saline (PBS; pH 7.5). Alternate sections were kept for future GnRH immunocytochemistry (cryoprotected and stored at -20oC). Sections collected for SCS area measurements were mounted on microscope slides and Nissl stained. Stained sections were assessed for, first, area of the HVC (reported below). Additional analyses will assess the areas of Area X and RA. Last, I observed the steps for GnRH immunocytochemistry (part of an ongoing experiment in a different species).
Results: There was a significant effect of sex on the area of HVC in cardinals with males having a larger HVC than females (2-way ANOVA, F=46.199, P<0.0001; Figure 1). There was a significant effect of season with HVC larger during the spring than the winter in both sexes (F=5.540, P=0.033; Figure 1). However, there was no sex by season effect (P=0.59). (Hold mouse pointer over image to read caption.)
Perspective: Many of the studies investigating the neural controls of song production in birds have focused on species with either complete dimorphism in song production, or in species that duet but with females possessing smaller, less-complex repertoires than males. In northern cardinals female song is equally as complex as male song, and females sing almost as frequently. Female cardinals have significantly smaller HVC area than males, although other SCS nuclei have yet to be measured. This size difference is similar to what is observed in those species where females have a lesser song repertoire compared to males. Our findings also show that in both sexes there is a difference in the size of the HVC from breeding to non-breeding. However, the difference in area observed in cardinals is not as pronounced as that observed in other temperate breeding species. Future research will investigate other parts of the SCS for seasonal and sexual differences, and GnRH immunocytochemistry will be performed on preserved sections. These findings will continue to expand the growing body of knowledge addressing the neural controls of sexually dimorphic and monomorphic behavior.
In a broader context, this research exchange visit gave me the opportunity to obtain new techniques that I can now apply to future research projects. I will use these techniques in my new position at the University of Southern Mississippi in collaborative research with other faculty and with students. Last, this visit helped promote a collaborative research effort between Dr. MacDougall-Shackleton and myself investigating female song production, a topic of growing interest.
Please see Buddy's report (PDF).