THE SOUTHWESTERN NATURALIST 49(3):311–315
SEPTEMBER 2004
SEASONAL CHANGES IN 17-ß ESTRADIOL OF THE RIO GRANDE CHUB (GILA PANDORA) IN SOUTH-CENTRAL NEW MEXICO COLLEEN A. CALDWELL,* S. ADAM FULLER, WILLIAM R. GOULD, PAUL R. TURNER, DENNIS M. HALLFORD
AND
U.S. Geological Survey, New Mexico Cooperative Fish and Wildlife Research Unit, Box 30003 MSC 4901, Las Cruces, NM 88003 (CAC) U.S. Fish and Wildlife Service, Mora National Fish Hatchery and Technology Center, Highway 434, Mile Marker 2, Mora, NM 87732 (SAF) University Statistics Center, New Mexico State University, MSC 3CQ, Las Cruces, NM 88003 (WRG) Department of Fishery and Wildlife Sciences, New Mexico State University, Box 30003 MSC 4901, Las Cruces, NM 88003 (PRT) Department of Animal and Range Sciences, New Mexico State University, Box 30003 MSC 3-I, Las Cruces, NM 88003 (DMH) *Correspondent:
[email protected]
ABSTRACT Timing of gametogensis and thus spawning can be inferred through changes in plasma concentrations of gonadal hormones. In preparation for ovulation and spawning, mean concentrations of 17ß-estradiol in a population of Rio Grande chub (Gila pandora) occupying the Rio Bonito, New Mexico, peaked at 37.6 ng/mL on 16 June and declined to 1.50 ng/mL by 11 August. Similarly, the gonadal somatic index (GSI) increased from 9.02 on 21 May (n 5 9) to 11.85 on 16 June (n 5 2) and declined to 6.10 on 11 August (n 5 2). Peak concentrations of 17ßestradiol and elevated GSI in June coincided with peak daylength for the year (14 h and 12 min) and average water temperature of 15.18C. Concentrations of 17ß-estradiol remained low through 3 October indicating no additional spawning events in the Rio Grande chub population. We demonstrated 17ß-estradiol is a nondestructive and thus useful tool in estimating timing of spawning in a wild fish population. RESUMEN El periodo de gametoge´nesis y desove se puede inferir a trave´s de cambios en las concentraciones de hormonas gona´dicas en la sangre. En preparacio´n para la ovulacio´n y desove, la concentracio´n promedio de 17ß-estradiol en una poblacio´n de Gila pandora del rı´o Bonito, Nuevo Me´xico, alcanzo´ su punto ma´ximo de 37.6 ng/mL el 16 de junio y disminuyo´ a 1.50 ng/ mL el 11 de agosto. Del mismo modo, el ´ındice gonadal soma´tico (IGS) se incremento´ de 9.02 el 21 de mayo (n 5 9) a 11.85 el 16 de junio (n 5 2) y disminuyo´ a 6.10 el 11 de agosto (n 5 2). El punto ma´ximo de 17ß-estradiol y el elevado IGS en junio coincidieron con el ma´ximo fotoperiodo del an ˜ o (14 h y 12 minutos) y una temperatura promedio del agua de 15.18C. Las concentraciones de 17ß-estradiol permanecieron bajas hasta el 3 de octubre indicando que no hubo eventos adicionales de desove en la poblacio´n de Gila pandora. Demostramos que el 17ßestradiol es una herramienta uÚ til y no destructiva para estimar el periodo de desove en poblaciones silvestres de peces.
Reproductive cycles in fishes can be characterized using gonadal-somatic indices, which require sacrificing fish, or by nondestructive methods, such as blood profiles of gonadal steroids. Fish ovaries synthesize a series of steroid molecules that regulate gametogenesis of the gonads, resulting in ovulation. Gonadal development and ovulation in fishes are under the
control of the hypothalamo-pituitary-gonadal axis, which coordinates rapid responses to environmental cues (Redding and Patin ˜ o, 1993). The teleost pituitary secretes gonadotropins to stimulate biosynthesis of gonadal steroid hormones, which, in turn, mediate gametogenesis (Nagahama, 1994). We can infer the timing of gametogenesis through changes in blood con-
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centrations of 17ß-estradiol. Release of 17ß-estradiol by the ovary, and thus increased plasma concentrations, stimulates hepatic synthesis of vitellogenin (yolk protein) for incorporating into developing oocytes (Nagahama, 1994). Developing oocytes then undergo a period of growth prior to maturation and ovulation as a prerequisite for fertilization. In response to environmental cues, inhibition in the negative feedback of progesterone and maturation-inducing factors at the pituitary results in the decline of 17ß-estradiol, thereby promoting final maturation of oocytes and ovulation (Scott and Sumpter, 1983a). Monitoring seasonal profiles of reproductive hormones in blood of teleosts provides information on the cycle of reproductive readiness and spawning. In addition, using hormones as a monitoring tool has the added advantage of requiring small amounts of blood without sacrificing the test specimen. Rising levels of 17ßestradiol in the blood have been used as indicators of gonadal development and spawning in a variety of fishes, including goldfish, Carassius auratus (Kobayashi et al., 1988), common carp, Cyprinus carpio (Barry et al., 1990), sea bass, Dicentrarchus labrax (Man ˜ano´s et al., 1997; Prat et al., 1999), and rainbow trout, Oncorhynchus mykiss (Scott et al., 1980; Scott and Sumpter, 1983b). The reproductive biology of Rio Grande chub (Gila pandora) is not well documented. Koster (1957) reported that spawning occurred within riffles in late spring. Based on gonadal changes in a northern New Mexico population, spawning presumably occurred from late spring to early summer (March to June) (Rinne, 1995). Thus, our objectives were to characterize seasonal profiles of 17ß-estradiol and environmental variables (i.e., photoperiod, water temperature, stream discharge) as determinants for spawning in a population of Rio Grande chub from a small perennial stream in south-central New Mexico. METHODS The study area was on the Rio Bonito, a headwater tributary of the Pecos River in southcentral New Mexico (Fig. 1). Fish were sampled (March to October 1999) from 200-m of the Rio Bonito using a Smith Root backpack electrofisher. Fish $125 mm total length were anaesthetized using 20 mg/L of MS-222 (Tricaine MethaneSulfonate, buffered with 20 mg/L calcium bicarbonate). Total length (61 mm) and mass (60.1 g) were recorded,
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FIG. 1 Map of Rio Bonito, New Mexico, study area.
and a 0.4-mL blood sample was drawn from the haemal sinus using a 1-cc syringe and 25-gauge needle. Sexual dimorphism (coloration and genital papilla) was not apparent throughout the entire study. Thus, blood was collected from both males and females ranging in length from 125 to 229 mm (n 5 88 on 6 March; n 5 14 on 10 April; n 5 70 on 21 May; n 5 99 on 26 June; n 5 40 on 23 July; n 5 6 on 11 August; n 5 23 on 29 August; n 5 59 on 3 October). Fish were resuscitated and returned to the stream at a central site within the study area. Blood samples were placed on ice, allowed to clot, and centrifuged within 6 h. Serum was removed and frozen (2108C) until analysis. Concentrations of 17ß-estradiol were determined from serum from both sexes using a commercially available solidphase enzyme immunoassay (Diagnostic Product Corporation, Los Angeles, California), as modified by Richards et al. (1999). In general, both male and female fish produce 17ß-estradiol, and both display similar hormonal profiles (albeit reduced in males) (Kadmon et al., 1985; Mayer et al., 1990) throughout the reproductive season. Despite the lower concentrations of 17ß-estradiol in males, when these concentrations were averaged with hormone concentrations in females throughout the study, we anticipated seasonal profiles would clearly indicate gametogenesis. Accuracy of the 17ß-estradiol assay, calculated as the percent exogenous 17ß-estradiol recovered from pooled charcoal-stripped channel catfish serum, averaged 97% (n 5 6). Estimates of within and be-
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tween variability of the assay obtained from separate samples of pooled channel catfish sera were 3.6% and 2.7%, respectively. The sensitivity of the assay was 2.0 picograms/mL. The stress of capture and confinement has been shown to alter gonadal steroid concentrations in many fish species (Pottinger et al., 1999). To assess handling and recapture effects on individual hormone levels through time, 158 fish were implanted with a 15-mm long Passive Integrated Transponder (peritoneal cavity posterior to left pectoral) on 6 March (n 5 72), 10 April (n 5 15), and 21 May (n 5 71). Steroid concentrations in fish recaptured at later sample collections (n 5 16) compared to initial captures, using Wilcoxon Rank Sum test (Hollander and Wolfe, 1999), indicated no detectable difference (17ß-estradiol: Z 520.10, P 5 0.92). Thus, repeated capture had no effect on 17ß-estradiol concentrations. Small samples of female fish were sacrificed on 4 sample collections (21 May, n 5 9; 16 June, n 5 2; 23 July, n 5 15; 11 August, n 5 2) and preserved in 10% formalin to determine gonadal somatic index (GSI). Gonadal somatic index was determined by dividing ovary weight (g) by total fish weight (g) prior to gonad removal and multiplying by 100 to obtain percent GSI. The GSI estimate for the July sample was lost because of poor preservation. Stream discharge data were obtained from a United States Geological Survey gauge (number 08389055, installed 3 April 1999) located 4 km upstream from the study site. A Stowaway TidBit data logger (Onset Computer Corporation, Pocasett, Massachusetts) was used to measure water temperature at 6-h intervals (0000, 0600, 1200, and 1800), from which mean daily water temperature was obtained. The data logger was lost during a storm event just prior to the May collection. From 16 June through 23 July, water temperatures were determined with a hand-held chemical thermometer at 0800 and 1500 h and a mean daily temperature was obtained. A datalogger was installed 29 August and recorded temperature for the remainder of the study. Photoperiod was obtained for the study site on each sample date (Lammi, 2001). Daylength (h) was obtained from official sunrise (0.83 degrees or 50 arc-min) below the horizon and official sunset (0.83 degrees or 50 arc-min) below the horizon.
RESULTS Mean concentrations of 17ß-estradiol in Rio Grande chub peaked at 37.6 nanograms/mL (SE 5 4.38) on 16 June and declined to 1.5 ng/mL (SE 5 0.39) on 11 August (Fig. 2a). Mean GSI followed a similar pattern in the sample collections, increasing from 9.02 (SE 5 1.57) on 21 May to 11.85 (SE 5 5.75) on 16 June and subsequently decreasing to
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FIG. 2 2a) Mean plasma concentrations of 17ßestradiol (ng/mL) in Rio Grande chub from March through October 1999. Error bars represent SE on either side of the mean. 2b) Mean daily discharge (m3/s) and temperature (8C) in the Rio Bonito, New Mexico, throughout the 1999 study. United States Geological Survey gauge (number 08389055) was installed April 1999.
6.10 (SE 5 1.84) on 11 August. In addition, while processing fish in both June sample collections, males released milt with light pressure. Thus, gametogenesis and presumably spawning occurred sometime between the steroidal and GSI peaks on 16 June and samples collected 11 August. The rise in concentrations of 17ß-estradiol coincided with an increase in photoperiod; the pre-ovulatory peak of 17ß-estradiol occurred during maximum daylength (14 h and 12 min) on 16 June (Fig. 2a). Although water temperature increased from 11.08C on 10 April to 15.18C on 16 June, water temperature continued to increase and peaked at 18.08C on 29 August, when hormonal concentrations were below pre-ovulatory levels (1.47 ng/mL) (Fig. 2b). The rise in concentrations of 17ß-estradiol
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coincided with a series of small episodic discharge events (peak events ranged from 0.02 to 0.16 m3/s) separated by baseline flow that averaged 0.006 m3/s (60.0004, range 0.003 to 0.008 m3/s) (Fig. 2b). An increase in discharge occurred from 25 July through 11 August (range 0.45 to 1.59 m3/s), while 17ß-estradiol concentrations continued to decline until the end of the study. DISCUSSION The seasonal profile of 17ß-estradiol for Rio Grande chub indicated gametogenesis was completed in the Rio Bonito during a 2-month window, and concentrations of 17ß-estradiol remained at or below baseline concentrations, reflecting no additional gonadal changes in the population. Thus, the concomitant pre-ovulatory peak of 17ß-estradiol and GSI in June indicated spawning occurred during mid summer (mid June to mid August) in the Rio Bonito, when photoperiod was at a maximum. Similar responses to photoperiod have been observed in other species of fish in which gametogenesis, as indicated by fluctuations in 17ß-estradiol, was accompanied by changes in GSI (MacKenzie et al., 1989). Duston and Bromage (1987) demonstrated that photoperiod worked to synchronize the maturation cycle of teleosts with respect to seasonal changes in the environment rather than directly influencing reproduction. Increasing daylength is an important cue from which fish can reliably extract information from their environment indicating the arrival of suitable spawning conditions (Munro, 1990). In addition, water temperature has been identified as an important environmental influence and predictor in the timing of spawning of Gila species. Captive populations of bonytail (Gila elegans), roundtail chub (Gila robusta), and humpback chub (Gila cypha) spawned at temperatures that ranged from 18 to 218C (Hamman, 1981, 1982), which was slightly higher than the temperature range in the Rio Bonito when fish were presumed to have spawned (15 to 188C). Spawning was also observed in wild populations of roundtail chub and bonytail in response to increasing water temperature (18.58C) (Vanicek and Kramer, 1969). Predictable and reliable factors such as daylength and temperature promote gonadal development in teleosts (Sumpter, 1990) until synchronizing cues (i.e., stream flow) signal
suitable spawning conditions (Hontela and Stacey, 1990). Stream discharge might also represent an environmental cue by influencing spawning in stream fishes to accelerate, delay, or inhibit oocyte maturation and ovulation. Additional work, however, is needed to determine how specific environmental factors (singly or in combination) might regulate the seasonal pattern in reproductive condition and spawning of this native fish. This study was funded by the United States Bureau of Land Management, Roswell Office, and T&E, Inc., a private research foundation supporting the protection of native fauna and flora in the Southwest. Additional financial assistance was provided by the United States Geological Survey New Mexico Cooperative Fish and Wildlife Research Unit and the Agricultural Experiment Station, New Mexico State University. We thank E. Gonzales, C. Courtright, S. Niemela, J. Bak, M. Ahlm, C. Gatton, and M. Kritter who helped with the fieldwork, and G. Parker for upkeep of the field station and his knowledge of the study area. We thank R. DuBey for his assistance with the figure of the study area and our internal and external reviewers for guidance during manuscript preparation.
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