Abstract
Comparatively, little data are available detailing the geographic variation that exists in the reproductive endocrinology of adult alligators, especially those living in barrier islands. The Merritt Island National Wildlife Refuge (MI) is a unique barrier island environment and home to the Kennedy Space Center (FL, USA). Seasonal patterns of sex steroids were assessed in adult female American alligators from MI monthly from 2008 to 2009, with additional samples collected at more random intervals in 2006, 2007, and 2010. Plasma 17β-estradiol and vitellogenin concentrations peaked in April, coincident with courtship and mating, and showed patterns similar to those observed in adult female alligators in other regions. Plasma concentrations of progesterone, however, showed patterns distinctly different than those reported for alligator populations in other regions and remained relatively constant throughout the year. Plasma DHEA peaked in July around the time of oviposition, decreased in August, and then remained constant for the remaining months, except for a moderate increase in October. Circulating concentrations of DHEA have not been previously assessed in a female crocodilian, and plasma concentrations coincident with reproductive activity suggest a reproductive and/or behavioral role. Interestingly, plasma testosterone concentrations peaked in May of 2008, as has been shown in female alligator populations in other regions, but showed no peak in 2009, demonstrating dramatic variability from year to year. Surveys showed 2009 to be particularly depauperate of alligator nests in MI, and it is possible that testosterone could serve as a strong indicator of breeding success.
Introduction
A great deal has been learned about the reproductive biology of the American alligator (Alligator mississippiensis) in recent decades, fueled in part by increased research efforts following precipitous population declines in the 1960s (Groombridge 1987, Joanen & McNease 1989, Guillette et al. 1997, Lance et al. 2009). While the majority of American alligators reside in freshwater and have been the focus of much of this research, comparatively little data are available detailing the geographical variation that exists in the reproductive endocrinology of adult alligators, especially of alligator populations living in barrier island locales. The Merritt Island National Wildlife Refuge (MI) is a barrier island environment that provides a variety of habitats, including coastal dunes, saltwater estuaries, open ocean, as well as freshwater impoundments. Established in 1963, MI is also home to the Kennedy Space Center, and the heightened security in this area provides an exceptionally unique opportunity to study a population of alligators that has not experienced hunting pressures and has been fully protected for decades. This barrier island habitat also has a considerable influence on diet, and it is not uncommon to find marine prey such as horseshoe crabs (Limulus polyphemus) and blue crabs (Callinectes sapidus) among the stomach contents in these alligators (Nifong et al. 2012, Nifong & Silliman 2013). Mammals and freshwater fishes make up a considerably smaller percentage of the diet in adult MI alligators than in alligators in exclusively freshwater environments or even alligator populations residing near other marine locales, such as coastal Louisiana (Gabrey 2010).
Freshwater vs marine-based diets vary in nutritional and micronutrient content, and although not yet studied in alligators, diet has been shown to alter reproduction as well as circulating concentrations of sex steroids in a variety of other species, including lizards (Cree et al. 1995, Lovern & Adams 2008). Hormones direct much of reproduction and gametogenesis and have considerable influence on reproductive behaviors as well (McEwen 1981, Whittier & Crews 1987, Woolley et al. 2004). While male alligators from MI have been studied extensively (Hamlin et al. 2011), the seasonal reproductive hormone profiles of adult females from this region have yet to be explored.
Studies on adult female alligators carried out in Florida lakes, as well as those carried out near coastal Louisiana, show that courtship and breeding take place in April and May, coincident with elevated concentrations of plasma 17β-estradiol (E2) and testosterone and to a lessor extent progesterone (P4) (Lance 1989, Guillette et al. 1997, Lance et al. 2009). Eggs are laid in June, which aligns with a rapid decline in plasma E2 and testosterone and an elevation in plasma P4 concentration. Approximately 25–68% of female alligators are reproductively active in a given year, indicating that at least some females do not breed each season, although the reasons and mechanisms for skipping breeding opportunities are not yet clear (Guillette et al. 1997).
The sex steroids E2, P4, and testosterone, often show seasonal cyclicity and are highly studied regulators of reproduction in most vertebrates, playing key roles in the production and maturation of gametes (Jones & Swain 1996, Ott et al. 2000, Edwards & Jones 2001, Brasfield et al. 2008, Wack et al. 2008, Smith et al. 2010). In female alligators, as with nearly all oviparous vertebrates, E2 triggers the hepatic production of vitellogenin (VTG), the primary precursor of egg yolk protein (Ho et al. 1982, Ho 1987, Guillette et al. 1997). VTG serves as the primary energy reserve for developing embryos, and this maternally derived resource relies heavily on proper E2 signaling. Plasma E2 concentration is highest during vitellogenesis but is lower and relatively constant throughout gravidity and oviposition in alligators (Guillette et al. 1997, Jones 2011). P4 is assumed to be the primary progestin in reptiles (Jones 2011) and peaks during gravidity in female alligators. Plasma concentrations of testosterone vary throughout the breeding season and show peaks during non-reproductive, late vitellogenic, and late gravid stages (Guillette et al. 1997).
DHEA is an androgen found in a variety of animals including mammals, birds, reptiles, and mollusks and is the most abundant circulating steroid in humans (Garstka et al. 1991, Ando et al. 1992, Croll & Wang 2007). While research on DHEA's role in human health and reproduction has increased dramatically over the past several years (Maninger et al. 2009, Gleicher & Barad 2011), far less is known regarding its role in other vertebrates, especially wildlife models.
DHEA is secreted primarily by the adrenals and to a lesser extent gonads in humans, some primates and other mammals, and songbirds (Wingfield & Hahn 1994, Conley et al. 2004, Soma 2006), although its origin has yet to be studied in alligators. That a variety of animals continue to produce DHEA following castration supports an extra-gonadal source (Ando et al. 1988, Pinxten et al. 2003, Soma 2006) and it appears to be present in relatively high concentrations (ng/ml range) in both males and females in many species (Balthazart 2010). A specific receptor for DHEA has yet to be discovered, and it is thought to act through a variety of receptors and other signaling mechanisms as well as serve as an intermediate in steroidogenesis (Widstrom & Dillon 2004, Labrie et al. 2005, Webb et al. 2006).
DHEA is thought to play roles in lipid regulation, immuno-competence, anti-inflammatory responses, neuroprotection, improved cognition, sexual differentiation of the brain (Chin et al. 2008), and in facilitating non-breeding aggression, although specific mechanisms have yet to be determined (Boonstra et al. 2008). In wildlife, the role of DHEA in non-breeding aggression has been studied fairly extensively. Although circulating testosterone increases the aggression needed for territory defense and courting during breeding seasons, it has been associated with reduced immune function and inflammation and it is therefore physiologically costly to maintain elevated concentrations of testosterone for prolonged periods (Ahmed & Talal 1990, Wilder 1995, Miller & Hunt 1996, Klein 2000, Moller et al. 2005). Conversely, DHEA has been shown to be immuno-stimulatory and anti-inflammatory. It is also thought to play key roles in maintaining aggression outside of the breeding season and does so without the physiological costs of testosterone (Grimble 2001, Daynes & Jones 2002, Poisbleau et al. 2009, Loria & Ben-Nathan 2011). In male red squirrels (Tamiasciurus hudsonicus), the yearly variation in plasma DHEA concentration is positively correlated with population density and is negatively correlated with testes mass (Boonstra et al. 2008). Male song sparrows are highly territorial even in non-breeding seasons when plasma testosterone concentrations are low, and supplementation with exogenous DHEA increases territorial singing, further supporting its role in facilitating aggression and territorial behaviors (Goodson et al. 2005).
In adult male American alligators, DHEA shows a seasonal cyclicity in which it peaks in May following significant reductions in testosterone and then declines in June and again in September, but remains relatively stable for the remaining months (Hamlin et al. 2011). Although DHEA is present in both males and females in a wide range of taxa, and its seasonal cyclicity indicates a role in reproduction, it has not yet been investigated in a female crocodilian.
The goal of this study was to determine the seasonal reproductive hormone profiles of adult female American alligators residing in MI, a unique barrier island environment. This study enhances our knowledge of the geographical variation in the endocrinology that underlies reproduction in female American alligators and determines whether alligators from this unique locale demonstrate distinctive patterns that are not yet observed in alligator populations from other regions.
Materials and methods
Animals and sampling
Adult female American alligators (A. mississippiensis) were captured under State, Federal and Institutional Animal Care and Use Committee (IACUC) permits and guidelines. Alligators were sampled monthly in 2008 and 2009, with additional samples taken at more random intervals in 2006, 2007, and 2010. Blood samples (≈35 ml) were collected from the postcranial supravertebral sinus and were placed into lithium heparin Vacutainer (BD, Franklin, NJ, USA) tubes. Blood was kept on ice until centrifugation at 810 g for 15 min, and the plasma was stored at −20 °C until assayed. Sex was determined by the presence or absence of a penis, and blood samples were collected generally within 10 min of capture, although no sample took longer than 20 min. Body measurements (total length (TL) and snout vent length (SVL)) were recorded to the nearest centimeter and a numbered monel tag was placed in the webbing of the right hind foot to identify recaptured animals. In addition, a low-frequency internal transponder tag was injected behind the skull, with 100% retention for recapture verification.
Eggs were collected from Lake Orange (FL, USA) and were incubated and maintained as described previously (Milnes et al. 2005). Egg yolk was collected at developmental stage 19 and was stored at −80 °C to use in the development of the VTG assay.
RIAs
A solid-phase RIA was conducted as described previously (Hamlin et al. 2011). A pool (five animals) of serially diluted alligator plasma was used to ensure parallelism with the standard curve for each hormone. All antibodies were purchased from Fitzgerald Industries (Concord, MA, USA) using the same lot number for each hormone. Plates for the testosterone, E2, and P4 assay were incubated for 2 h, and plates for the DHEA assay were incubated for 18 h. Interassay variance samples were prepared from a five animal pool of adult alligator plasma samples. Interassay variances for E2, P4, testosterone, and DHEA averaged 10.7, 9.4, 8.7, and 9.8% respectively. Intraassay variation was not >5%.
VTG assay
Lipovitellin (Lv) was purified from the egg yolk of the American alligator by a combination of water-precipitation and gel filtration according to a method described previously (Hiramatsu & Hara 1996) with the following modifications. Three grams of egg yolk were homogenized in a ground glass homogenizer (Iwaki, Tokyo, Japan) with 30 ml of 0.02 M Tris–HCl (pH 9.0) containing 0.5 M NaCl. The egg yolk homogenates were centrifuged (15 000 r.p.m., 20 min, 4 °C) and the resulting supernatant was collected as alligator egg extracts. The extracts (∼30 ml) were precipitated by drop-wise addition into ten times volume (∼300 ml) of ice-cold distilled water and stored overnight at 4 °C. Following centrifugation as described above, the resulting precipitate was collected and dissolved in 12 ml Tris–HCl buffer. Two consecutive gel filtration steps using Sephadex G-200 (GE Healthcare UK Ltd, Little Chalfont, UK) and Superose 6 (GE Healthcare UK Ltd) were performed following the water-precipitation step. The Sephadex G-200 medium was loaded into a 5×90 cm glass column and equilibrated with Tris–HCl buffer. The water-precipitated yolk fraction (∼7.5 ml) was applied onto the column and eluted with Tris–HCl buffer at a flow rate of 40 ml/h at 4 °C. Eluted fractions were collected at a volume of 13 ml/tube. Fractions, which contained Lv as a dominant yolk protein, were collected, pooled, and subjected to the second gel filtration on a Superose 6 column equilibrated with Tris–HCl buffer. Elution of the Lv sample was performed using an FPLC system (GE Healthcare UK Ltd) under conditions described previously (Amano et al. 2007). A single peak was observed at the position corresponding to ∼410 kDa; this peak fraction was collected as purified Lv.
Polyclonal antiserum was raised in the rabbit against purified Lv (anti-Lv) by intradermal injection of each antigen emulsified with an equal volume of Freund's complete adjuvant (Merck). This emulsified Lv was injected into the rabbit (0.25 mg/injection) four times at weekly intervals. Blood was obtained from the ear vein of the immunized rabbit 1 week after the final injection and used for the preparation of antiserum.
The VTG immunoassay was developed in a format of a competitive ELISA. Wells of 96-well microtiter plates (MaxisorpII, Nunc, Thermo Fisher Scientific (Waltham, MA, USA)) were coated with 50 μl PBS solution containing the purified Lv at a concentration of 1 μg/ml at 4 °C overnight. After washing the plate three times with PBS containing 0.1% Tween 20 (TPBS), wells were blocked with 300 μl of blocking reagent (0.5% BlockAce-TPBS). For the standards, plasma of males injected with 0.27 mg/kg E2 every other day for 12 days was serially diluted in blocking reagent from 1:50 to 1:36 450. In parallel, plasma samples were diluted in blocking reagent. The standards and unknown samples (60 μl/well) were pre-incubated in separate non-coated 96-well microtiter plates (Greiner Japan, Tokyo, Japan) with Lv antiserum (60 μl/well, 0.5 μg/ml in blocking reagent) for 1 h at room temperature. After washing the blocked Lv-coated plate as described above, 50 μl of the sample/antiserum or standard/antibody mixtures were transferred into wells of the coated plate from the separate non-coated plate and incubated for 1 h at room temperature. After the wells were washed as described above, 50 μl of the HRP-labeled secondary antibody (goat anti-rabbit IgG, Zymed, Life Technologies) was added to each well and incubated for 1 h at room temperature. After washing the wells as described above, they were filled with 100 μl of a color reaction mixture (TMBZ and TMBZ buffer, Japan EnviroChemicals Ltd, Osaka, Japan). The reactions were carried out for 1 h at room temperature and stopped by addition of 100 μl of 1 M HCl solution. Absorbance of the reactions was determined at 450 nm with an ELISA plate reader (SpectraRainbow, Tecan Japan, Kawasaki, Japan). Levels of VTG in plasma samples were relatively quantified considering the VTG concentration of standard plasma as 1.
Statistical analyses
Differences in plasma hormone and VTG concentrations among months were analyzed using a one-way ANOVA (Statview Software, version 5.0.1; SAS Institute, Inc. (Cary, NC, USA)). If significant variation (P<0.05) existed, a Fisher protected least significant difference test was performed. Non-homogenous data sets (determined with P<0.05 using Scheffé F test) were log transformed to achieve homogeneity prior to ANOVA analyses, although all figures show untransformed values, with averages given as ±s.e.m.
Results
Sampling, morphometric, and environmental data
A total of 220 adult female alligators were captured from MI from 2006 to 2010. Although data from all animals captured were used in the morphometric regressions shown in Fig. 1, only females with an SVL ≥93 cm (183 cm TL) were classified as adults and included in the hormone evaluations. Monthly averages for temperature (°C) and rainfall (cm) are presented in Fig. 2. There were no significant differences in female size (TL or SVL) between months.
Simple regression of total length (cm) vs snout vent length (cm) in adult female American alligators from the Merritt Island National Wildlife Refuge.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
Average monthly temperature (°C) and rainfall (cm) for 2008 (gray line) and 2009 (black line) from the Merritt Island National Wildlife Refuge.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
E2, VTG, and P4
Plasma concentrations of E2 peaked in April (575±114 pg/ml) and dropped gradually to a nadir in August (119±18 pg/ml) (Fig. 3). Plasma concentrations of VTG also peaked in April (5.4±1.7 relative VTG) and dropped rapidly by July (0.20±0.07 relative VTG); plasma VTG concentrations then remained relatively stable throughout the year until March, when a noticeable but insignificant increase could be seen (Fig. 3). Plasma concentrations of E2 and VTG were positively correlated (Fig. 4). Plasma concentrations of P4 were statistically similar throughout the year, with a noticeable but insignificant increase from March to April (Fig. 5). Patterns for E2, VTG, and P4 were similar between 2008 and 2009 during the breeding season.
Monthly variation in mean (±1 s.e.m.) plasma 17β-estradiol (pg/ml) and relative vitellogenin concentration of adult female (≥93 cm SVL) American alligators from the Merritt Island National Wildlife Refuge collected between 2006 and 2010. **Months with different superscripts are significantly different (P≥0.05); n, numbers of sampled animals used in the analyses.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
Simple regression of relative vitellogenin (VTG) vs plasma 17β-estradiol concentration in adult female American alligators from the Merritt Island National Wildlife Refuge.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
Monthly variation in mean (±1 s.e.m.) plasma progesterone (pg/ml) concentrations of adult female (≥93 cm SVL) American alligators from the Merritt Island National Wildlife Refuge collected between 2006 and 2010. n, numbers of sampled animals used in the analyses.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
DHEA and testosterone
Plasma concentrations of DHEA increased in June and peaked in July (42±4 ng/ml), with a precipitous decline in August (27±3 ng/ml) (Fig. 6). There was a slight increase in plasma DHEA in October (32±4 ng/ml) and a gradual decrease by December where plasma concentrations remained relatively stable throughout winter and spring. Patterns in plasma concentrations of DHEA were similar between 2008 and 2009 during the breeding season.
Monthly variation in mean (±1 s.e.m.) plasma DHEA concentration of adult female (≥93 cm SVL) American alligators from the Merritt Island National Wildlife Refuge collected between 2006 and 2010. Months with the same superscript are not significantly different (P≥0.05) within each size class; n, numbers of sampled animals used in the analyses.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
Plasma testosterone concentrations showed a dramatic increase in May (263±109 pg/ml) and a rapid drop in June (71±7 pg/ml) where concentrations remained relatively constant throughout the remaining months (Fig. 7). Patterns in plasma testosterone concentrations were significantly different during the breeding season between 2008 and 2009, with an expected peak in May of 2008, but no peak in May of 2009 (Fig. 8). In addition, 2008 showed a significant increase in July (277±122 pg/ml), which decreased and was relatively constant for the remaining months. In 2009, plasma concentrations of testosterone were not significantly different between months.
Monthly variation in mean (±1 s.e.m.) plasma testosterone (pg/ml) concentration of adult female (≥93 cm SVL) American alligators from the Merritt Island National Wildlife Refuge collected between 2006 and 2010. Months with the same superscript are not significantly different (P≥0.05) within each size class; n, numbers of sampled animals used in the analyses.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
Monthly variation in mean (±1 s.e.m.) plasma testosterone concentration for 2008 and 2009 of adult female (≥93 cm SVL) alligators from the Merritt Island National Wildlife Refuge. Months with the same superscript are not significantly different within each year; n, numbers of sampled animals used in the analyses.
Citation: REPRODUCTION 147, 6; 10.1530/REP-14-0031
Discussion
Adult female alligators from MI showed seasonal concentrations of plasma E2 and testosterone that paralleled seasonal patterns reported in adult female alligators from other locales. Seasonal patterns in plasma P4 concentration, however, were distinctive from previously reported data in other regions (Guillette et al. 1997). This is the first study to show seasonal variation in plasma DHEA concentrations in a female crocodilian and is the most extensive assessment to date, both in terms of the range of hormones analyzed and the number of animals sampled, in a population of adult female American alligators.
As expected, plasma concentrations of VTG paralleled that of E2, a pattern characteristic of most oviparous vertebrates (Figs 3 and 4; Ho 1987). Elevated plasma VTG concentrations are important for proper egg development, and plasma E2 concentrations were sufficiently elevated to elicit hepatic VTG production. Plasma E2 concentrations peaked in April, coincident with courtship and mating, and showed similar patterns to that observed in adult female alligators in other regions, including Florida's freshwater lakes and coastal Louisiana (Lance 1989, Guillette et al. 1997, Lance et al. 2009).
Unlike previously reported seasonal patterns for P4 in alligators from other regions, in which plasma P4 concentrations are lowest in April and then peak during oviposition in June (Lance 1989, Guillette et al. 1997, Lance et al. 2009), this pattern was not observed in MI females. Females in this study maintained relatively stable plasma P4 concentrations throughout the year, with a noticeable yet insignificant increase occurring between March and April. Concentrations of plasma P4 are considerably lower in this study than previous studies, which could highlight a unique difference in MI adult female alligator populations or could reflect differences in assay performance. Antibody specificity has improved greatly in recent years, and it is possible that in older studies, P4 antibodies with lower specificity could have bound with a wide range of progestins, resulting in seemingly higher P4 concentrations. Regardless, it is interesting that distinct seasonal patterns in plasma P4 concentrations were not observed in this study given that robust seasonal patterns have been shown not only in American alligators (Lance 1989, Guillette et al. 1997, Lance et al. 2009) but also in other reptiles as well as birds, which are closely related to archosaurs (Donham 1979, Amey & Whittier 2000). Nile crocodiles (Crocodylus niloticus) do not show distinct seasonal patterns in plasma P4 concentrations, however, and show highly variable concentrations throughout the year in both reproductive and non-reproductive individuals (Kofron 1990).
Although there could be many explanations for the lack of seasonal variability in plasma P4 concentrations in MI alligator populations vs those in other regions, there are three explanations that share scientific support and are particularly plausible.
First, alligators in MI consume a decidedly different diet than those in other locales, as indicated by gut content analyses showing greater percentages of marine prey, especially crustaceans, vs that of alligators from freshwater lakes, or even those of coastal Louisiana (Gabrey 2010, Nifong et al. 2012, Nifong & Silliman 2013). Diet has been shown to alter hormone concentrations in a number of vertebrate models, including alligators. The production of thyroid hormones is reliant upon the availability of iodide, and it has been suggested that the abundance of dietary iodide in the marine prey of MI alligators could alter thyroid hormone concentrations and reduce the seasonal variation seen in alligators from other locales (Boggs et al. 2011). Diet has also been shown to alter concentrations of sex steroids in a variety of species, including reptiles (Cree et al. 1995, Lovern & Adams 2008). Female green anole lizards (Anolis carolinensis) fed higher quality diets showed greater plasma testosterone concentrations than do females on standard or low-quality diets (Lovern & Adams 2008) and produce eggs with elevated yolk testosterone concentrations as well (Warner et al. 2007). It is unclear what effect diet could have on plasma P4 concentrations in American alligators.
Secondly, MI is home to the Kennedy Space Center, and although heightened security in this area removes hunting, and even poaching pressures, stress-related effects associated with shuttle launches and other activities are unclear. Launches and other activities have led to concerns regarding elevated environmental concentrations of aluminum and other heavy metals (Bowden et al. 2012), as well chemicals such as polybrominated diphenyl ethers (PBDEs) used in flame retardants (Guillette & Lowers, unpublished results). Although the influence of aluminum and PBDEs on plasma P4 concentrations in alligators is unknown, evidence in other vertebrates suggests possible effects. Subchronic exposures to aluminum resulted in reduced serum progestogen concentrations in Wistar rats (Alonso et al. 2010). Certain PBDEs have been shown to decrease P4 synthesis in mouse Leydig tumor cells (Han et al. 2012) while other PBDEs have been shown to increase P4 synthesis in the ovarian follicles of pigs (Karpeta & Gregoraszczuk 2010). Proposed mechanisms of action in the latter study include modulation of key steroidogenic genes, many of which are highly conserved among vertebrates (Karpeta et al. 2011).
Thirdly, it is difficult to determine which of the females that were sampled in this study were reproductively active. It is possible that in June, the time period during which other populations of alligators have shown peaks in plasma P4 concentrations, very few reproductively active alligators were captured in this study, reducing the ability to observe expected patterns.
Plasma DHEA peaked in June and July, around the time of oviposition, and then decreased in August and remained relatively constant for the rest of the year, except for a slight increase in October. Concentrations of plasma DHEA are an order of magnitude greater than plasma testosterone concentrations and are similar to those reported for adult male alligators (Hamlin et al. 2011). This is the first study to show the presence of DHEA in a female crocodilian, and seasonal changes in plasma concentrations coincident with reproductive activity suggest a reproductive and/or behavioral role. Although DHEA has been shown to play a variety of beneficial roles in humans and laboratory animals, its role in maintaining non-breeding aggression has been the focus of most wildlife studies (Poisbleau et al. 2009). In adult male alligators, plasma DHEA concentrations peak during periods of intense breeding and courtship, but in females, plasma DHEA concentration was shown to peak during oviposition and early nest guarding. Given that plasma DHEA concentrations in this study were among the highest levels of sex steroids reported in a female crocodilian to date, more work should be conducted to determine DHEA's role in the biology of American alligators (Guillette et al. 1997, Lance et al. 2009).
Previous studies in adult female alligators have shown plasma testosterone concentrations to peak in May, coincident with courtship and mating. Alligators of both sexes display complex behaviors including bellowing (Garrick & Lang 1977, Vliet 1989), and although the role of testosterone in alligator behavior has not yet been studied, testosterone has been shown to affect behavior in a variety of vertebrates, including reptiles and birds (Lindzey & Crews 1986, Lynn 2008). Interestingly, this study showed an expected peak in plasma testosterone concentration in May of 2008, but no peak in 2009. Surveys of alligator nests in MI show 2009 to be an exceptionally poor breeding year, with few nests located despite increased efforts (Lowers, unpublished data). If plasma testosterone concentration correlates with breeding success, this could support the possibility that only non-reproductively active females were captured in May of 2009. In addition, the lack of females captured in 2009 showing the expected peak in plasma testosterone concentration in summer as well would indicate that very few females were reproductively active that year.
Although plasma testosterone concentration in female alligators was markedly different between years that showed normal vs poor nest abundance, and presumably mating success, the patterns of other hormones analyzed in this study were not significantly different during the breeding season between years. Therefore, it is possible that a spike in plasma testosterone concentration is necessary for successful breeding or perhaps increases as a result of successful breeding, while plasma E2 and DHEA concentrations could show seasonal patterns regardless of whether successful breeding takes place.
The reasons for the variation in nest abundance and altered seasonal patterns of plasma testosterone concentration in May between 2008 and 2009 in MI are unclear. Although water temperatures were similar for the month of May in 2008 and 2009 (Fig. 3), rainfall varied considerably, with 1.4 cm in 2008 vs 32.1 cm in 2009. Joanen & McNease (1989) found increased nest abundance during periods when water levels were above average but reduced nest abundance if water levels increased to the point of extreme flooding. Conversely, they found nest abundance was lowest during periods of drought. Although rainfall was markedly different for the month of May, cumulative rainfall for January–July was nearly the same for 2008 and 2009. It is unclear what effect rainfall has on alligator courtship or breeding in MI, however, increased rainfall in May could presumably lead to a reduction in viable nest sites, which could alter the female's decisions to mate. Although highly speculative until this phenomenon is confirmed over multiple years in MI, it is nonetheless an interesting observation. In male alligators, size favors social order, and although smaller adult males are capable of breeding, they show a different pattern of plasma testosterone concentration than larger males. In smaller adult males, instead of peaking mid-season as it does in larger males, plasma testosterone concentrations drop off precipitously to basal levels. Given that these smaller males are physiologically capable of breeding, it is possible that this drop in testosterone could be driven by social cues. It is then conceivable that a female could refrain from breeding due to environmental cues as well.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported in part by grants-in-aid for scientific research (C) (grant number 21510068) (Y Ohta) and scientific research (B) (grant number 24370029) (T Iguchi and S Miyagawa) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Acknowledgements
Appreciation is gratefully extended to the following individuals for assistance in field and/or data collection; Doug Scheidt, Carlton Hall, Stephanie Weiss, Matthew Guillette, Shannon Gann, Carly Garreau, Eric Reyier, Karen Holloway-Adkins, Jim Lyons, Mike Legare, Joe Benjamin, Rebecca Bolt, Mark Mercadante, John Shaffer, and Lynne Phillips.
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(H J Hamlin and R H Lowers contributed equally to this work)
