North American Journal of Aquaculture 66:42–47, 2004 q Copyright by the American Fisheries Society 2004
Habitat and Substrate Use in Reproduction of Captive Devils River Minnows J. R. GIBSON
AND
J. N. FRIES*
San Marcos National Fish Hatchery and Technology Center, 500 East McCarty Lane, San Marcos, Texas 78666, USA
G. P. GARRETT Heart of the Hills Fisheries Science Center, Texas Parks and Wildlife Department, HC 7, Box 62, Ingram, Texas 78025, USA Abstract.—The Devils River minnow Dionda diaboli is a threatened species once endemic to a portion of the Rio Grande drainage in western Texas and northern Mexico but now found only in the Devils River and San Felipe and Pinto creeks. Because little is known about the biology of this species, it is difficult to establish protocols for maintaining captive populations. We monitored the production of Devils River minnows in two laboratory culture systems containing four substrate types (rocks, gravel, sand, and Spawntex) within each of two habitat types (riffle and pool) that had two subhabitat types each (upper and lower riffle, covered and uncovered pool). A total of 38 adult fish (mean weight 5 1.38 g; mean total length 5 54 mm) were introduced into the two culture systems (19 fish each, approximately equal sex ratio) on 5 September 2001. From 18 September to 6 December, 2,269 young were removed. A significantly greater number (1,922; three-factor nested analysis of variance [ANOVA], Fisher’s least-significant-difference tests, P # 0.05) were in gravel substrate (2–5 cm diameter). However, no differences were found in the counts of young with respect to habitat or subhabitat type. The indoor culture of Devils River minnows is possible using gravel as a spawning substrate and, since parental stock can be manipulated, the technique can be used for establishing refugium populations and for augmenting wild populations.
cies in Mexico was not evaluated. In 2001, a population had been discovered in Pinto Creek, Kinney County, Texas (G. P. Garrett, unpublished data). While the preferred habitat of Devils River minnows is fast moving, spring-fed water over gravel (Harrell 1980; Garrett et al. 1992), they tend to be found most often at confluences of spring runs and streams rather than in the springs themselves (Hubbs and Garrett 1990). In 1998, a conservation agreement for the Devils River minnow was established to protect its remaining habitat and populations (USFWS 1999). The conservation agreement required the maintenance (in captivity) of genetically representative populations of the Devils River minnow as insurance against catastrophic losses, necessitating the development of culture techniques for this minnow. Since 1999, Devils River minnows have been maintained at the Heart of the Hills Fisheries Science Center, Texas Parks and Wildlife Department, where the fish have been successfully reproduced in 11,500-L outdoor systems with pools and riffles. In 2000, attempts to maintain live Devils River minnows at the San Marcos National Fish Hatchery and Technology Center (NFHTC), U.S. Fish and Wildlife Service, in 830-L indoor systems were unsuccessful until live plant material was included. Subsequently, to determine the acceptable spawning habitat, captive fish were provided with several spawning habitat types (combinations of substrate, flow and depth, and cover), and combined counts of eggs and larvae (young) were evaluated across habitat types. Once an acceptable spawning substrate is identified, the development of culture techniques that ultimately will incorporate genetics management can proceed.
The Devils River minnow Dionda diaboli is a threatened fish (USFWS 1999) endemic to a small portion of the Rio Grande drainage basin in Texas and Mexico (Hubbs and Brown 1956; Garrett et al. 1992). Historically, Devils River minnows occurred in the Devils River drainage, in three creeks in Texas, and in two small streams in Mexico (Garrett et al. 1992). In 1989, Garrett et al. (1992) found Devils River minnows only in the Devils River and in two creeks in Texas and speculated that reduced water flow had diminished the abundance and range of the fish. The status of the spe-
Methods * Corresponding author:
[email protected]
Culture systems.—Two culture systems located in a temperature-controlled room without windows
Received March 12, 2003; accepted July 11, 2003
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FIGURE 1.—Diagram of the recirculation system used to test habitat and substrate preferences for egg laying by Devils River minnows, September–December 2001. Shaded areas represent water, arrows the direction of water movement. Tanks were opaque but are shown as transparent. Four habitat types—(1) covered pool, (2) uncovered pool, (3) upper riffle, and (4) lower riffle—were tested, along with the substrate types shown. Two small aquaria for fry, the complete plumbing, and support structures are not shown; figure not to scale.
at the NFHTC were used in this study. Each system (Figure 1) consisted of an 830-L fiberglass tank (Living Stream model LS-900; Frigid Unit, Toledo, Ohio) and two 9.5-L glass aquaria placed over each tank for newly hatched larvae. Temperature was maintained by a heater–chiller unit (2,000 W– 0.5 hp [1 hp 5 746 W]; Model UTCH-3, Universal Marine Industries, Inc., San Leandro, California). Water was circulated through each system by a 0.5hp pump (Model SP125J, Hayward Pool Products, Inc., Elizabeth, New Jersey). An artificial riffle was constructed on one side of each system by suspending a 122-cm 3 31-cm fiberglass tank at a slight angle over the larger tank. Water flowed out of the suspended tank through a 31-cm 3 15cm section removed from the lower end. A set of four substrates was placed in each of the upper and lower portions of the riffle, and in the covered and uncovered pool habitats (Figure 1). Substrates were very coarse sand (0.3–0.5 cm), gravel (2–5 cm), rock (4–8 cm), and dark spawning mats (Spawntex; Blocksom and Co., Michigan City, Indiana). Sections of 10-cm polyvinyl chloride
(PVC) pipe cut in half longitudinally were placed in all areas of the tank to serve as small shelters. Potted plants (Vallisnaria americana and Justicia americana) were placed in the deep, slow-flowing, pool areas of the larger tank (the addition of plants was found previously to be beneficial). Untreated water pumped directly from the Edwards Aquifer was added continuously (15.1 L/h) at the top of the riffle system in each tank, resulting in a turnover of the working volume (467 L) every 1.3 d. This combination of well and recirculation water exiting the riffle was designed to resemble a spring flowing into open water, habitat in which Hubbs (1951) observed the breeding of Nueces roundnose minnow D. serena (5 D. episcopa serena), a closely related species to the Devils River minnow. The water leaving the system drained into a septic field to prevent escapement of fish. During the construction of the culture systems, all Devils River minnows were kept in a 217-L community tank located in the same room. This tank was supplied with well water ranging from
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22–248C in temperature, at 15.1 L/h (14.4-h turnover of working volume). Maintenance.—The fish were fed equal amounts (about 2 g of each) of Spirulina flakes and worm flakes (F01S and F01E, respectively; Aquatic EcoSystems, Inc., Apopka, Florida) twice weekly. Similar amounts of water with debris were siphoned off the bottom of each system during weekly cleaning. Dissolved oxygen (DO), conductivity, water temperature, and pH were measured twice weekly using a Hydrolab multiprobe and DataSonde (Model 2; Hydrolab Corporation, Austin, Texas). Total gas saturation was recorded twice weekly using a gas saturometer (Model DS-1B, Sweeney Aquametrics, Stony Creek, Connecticut). Ammonia levels were measured once weekly using an Accumet ammonia ion selective electrode (Fisher Scientific, Pittsburgh, Pennsylvania) attached to a Denver Instruments Model 250 pH-ISE conductivity meter (Denver Instruments Company, Denver, Colorado). Spawning.—The fish collected from the Devils River and a tributary, Phillips Creek, during August 2000 were introduced into culture systems on 5 September 2001. Small groups (four to five individuals) were netted at random and anesthetized with 75 mg/L MS-222 (tricaine methanesulfonate; Argent Chemical Laboratories, Redmond, Washington). A single fish was removed and measured for total length (TL) and weighed in water on a tared balance. Nuptial tubercles had not developed yet, so gender was determined by coloration (some fish had a blue sheen on the head and were presumed to be males), and the fish was placed into a test tank. This was repeated until all fish were placed, alternating by presumed gender, into both culture systems (19 in each, approximately equal sex ratios). The fish ranged in weight from 0.82 to 2.39 g (mean 5 1.38 g) and in total lengths from 45 to 64 mm (mean 5 54 mm). These fish were exposed to one annual cycle of photoperiod and temperature change (temperature ranged from 18– 248C; photoperiod ranged from 10 to 14 h daylight) compressed within a 4-month duration based, in part, on data collected in the area by Valdes-Cantu and Winemiller (1997). This increased the potential number of seasonal studies on the same small group of presumably short-lived fish. Water temperature was changed by setting the heater–chiller unit which maintained temperature to 618C of set point. Fluorescent lights totaling 420 W were suspended 1.8 m above the bottom of the tank. Lights containing four 55-W bulbs (Hamilton Super Sun Lite, Hamilton Technology Cor-
poration, Gardena, California) were placed directly over each tank above the pool area containing plants. The photoperiod for all light fixtures was controlled using 24-h timers. Weekly, all substrates, PVC shelters, and plants were individually removed from the systems and examined for young. The larvae removed would be no more than a few days old at most. We assumed that they would not have yet developed swim-up behavior and were likely in the substrate in which they were originally deposited. The substrates and plants were thoroughly washed with water into a glass tray. If present, eggs or larvae were removed from the water or the surface of substrate with a plastic pipette, counted, and placed in fry aquaria. Since substrates could contain multiple broods with eggs and different-sized larvae, all were combined as ‘‘young.’’ Time-lapse video of both tanks was used to observe fish behavior. Statistical analysis.—The count data for young (eggs and larvae combined), which were summed for all 11 collection periods since temporal effects were not tested, were not normally distributed (probability plot analysis and D’Agostino-Pearson K2 test; Wilkinson 1998; Zar 1999) but became so after log transformation. Additionally, data transformation substantially reduced heteroscedasticity (residual plot analysis and Bartlett’s test for homogeneity; Wilkinson 1998; Zar 1999). To examine the differences in egg laying in the several habitats and substrate types, the transformed data were analyzed using three-factor nested ANOVA (Ott 1993; Wilkinson 1998). Independent variables were substrate (rock, gravel, sand, or Spawntex), nested within each of two subhabitats (upper or lower riffle, covered or uncovered pool), each nested within two habitat types (riffle or pool). There were two replications (two culture systems). To separate means, Fisher’s least-significant-difference tests were conducted for analyses (which indicated significant differences at P , 0.05). Results and Discussion Water quality was similar in both tanks. Temperature (mean 5 20.58C; range 5 17.5–23.58C) and DO (mean 5 4.3 mg/L; range 5 3.2–5.3 mg/ L) were within acceptable ranges for warmwater fishes as given in Parker and Davis (1981). Generally, pH (mean 5 7.9; range 5 7.2–8.2) was higher and DO was lower than those recorded for the Devils River by Valdes-Cantu and Winemiller (1997) or measured during the collection of minnows used in this study (pH 5 7.35–7.87; DO 5 7.23–8.06 mg/L). Conductivity (mean 5 0.618
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TABLE 1.—Mean number of Devils River minnow young (eggs and larvae) collected from several substrates and habitat types in two artificial streams at the San Marcos National Fish Hatchery and Technology Center, San Marcos, Texas, from 18 September to 6 December 2001. Different letters indicate significant differences (three-factor nested analysis of variance on log-transformed total counts followed by Fisher’s least-significant-difference tests; P # 0.05; two replicates). Substrate Habitat
Subhabitat
Rocks
Gravel
Sand
Riffle
Upper Lower
130.5 10.5
65.0 29.0
2.5 1.0
12.0 3.0
Pool
Covered Uncovered
9.5 1.0
535.5 331.5
0.0 0.0
1.0 2.5
961.0 z
3.5 x
Total
151.5 y
mS/cm; range 5 0.505–0.697 mS/cm) was slightly higher than the levels measured during collection (0.466–0.528 mS/cm). Total gas saturation (mean 5 93.8%; range 5 72.5–104.6%) was similar to the levels measured in the wild at the time of collection (92.0–107.6%) and was below the potentially harmful levels noted by Bouck (1980). Ammonia always was below detectible levels ( ,0.05 mg/L). After introduction to the experimental systems, the minnows were observed (using time-lapse video) swimming throughout all areas of the culture system. Most of the daytime was spent schooling in the pool area under the overhanging riffle section, generally near the downstream side of the riffle where the water flowed into the pool section of the tank. Some of the more noticeably chalky blue-colored fish hovered directly over the gravel and rock trays, chasing off other fish. This might have been territorial behavior exhibited by males in breeding coloration. Larvae were first discovered in the gravel trays in one tank on 19 September 2001, 2 weeks after introduction. Larvae and eggs were found in the gravel of the other tank the next day. Production continued to 15 November. Young were removed almost every week. The longest hiatus in production was 2 weeks. A total of 2,306 young was produced from both tanks: 1,302 in one tank and 1,004 in the other. One group of 37 larvae was removed from the analysis due to ambiguity about their substrate association (experimental error), leaving 2,269 young. The largest weekly production, 358 eggs, was on 1 November. Reproduction occurred during a temperature and photoperiod change coinciding with the fall and winter seasons. Reproduction started when the temperature and photoperiod were reduced to 228C and 12.5 h/d, and continued during further reductions to the min-
Spawntex
Total 210.0 43.5 253.5 546.0 335.0 881.0
18.5 yx
imum of 188C and 10 h/d. As these settings were increased, production ceased in the one tank at 198C and 10.5 h/d, and in the other tank at 20 8C and 11 h/d. No young were noticed during the remaining seasonal cycle. The eggs (mean diameter 5 1.59 mm; range 5 1.40–1.78; N 5 24) were transparent with a faint yellow hue and were slightly adhesive. They seemed to be concentrated in one area of the tray, with most attached to the inside bottom of the tray itself or deep in the substrate. Few eggs or larvae were visible when looking at the top surface of the substrate trays. The larvae tended to swim downward and underneath structure. At 5–7 d posthatch (when the yolk was greatly diminished) most occasionally swam up into the water column to feed. By 10–14 d, most fry maintained buoyancy and schooled at middepth in the water. The fry were observed readily eating live brine shrimp or flaked or fry food (150–250mm feed; Gold Fry-3, Aurum Aquaculture, Ltd., Kirkland, Washington). Table 1 summarizes the results of the statistical analysis. There were significant differences among the means of young collected from the four substrates (F3,25 5 7.79, P 5 0.001). The mean number of young collected from gravel (961.0) was significantly higher (least significant difference; P 5 0.035) than the means for rocks (151.5), Spawntex (18.5), and sand (3.5). The mean number of young collected from rocks was significantly higher than the mean for sand (least significant difference; P 5 0.027). However, there were no significant differences (F1,25 5 1.38, P 5 0.251) between the mean number of young collected from riffles (253.5) and pools (881.0). There also were no significant differences (F2,25 5 1.57, P 5 0.228) among means for upper riffles (210.0), lower riffles (43.5), covered pools (546.0), and uncovered pools
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(335.0). No eggs or larvae were observed on or around the plants or PVC shelters. Comparable laboratory studies on habitat and substrate use by spawning minnows were not found. Jacobsen (1979) showed that the minnow (also known as the Eurasian minnow) Phoxinus phoxinus prefers stony over sandy substrates despite depth of water or age of fish outside of spawning season, and Burkhead and Jelks (2001) showed a reduction of reproductive success in tricolor shiner Cyprinella trichroistia with increased levels of suspended sediment. All species of Dionda are likely to be broadcast spawners (releasing eggs and sperm over unprepared substrate, the most common and primitive mode of cyprinid spawning; Johnston 1999). Other modes of spawning include (1) crevice spawning (laying eggs within substrate or submerged structure), as is found with tricolor shiners (Burkhead and Jelks 2001); (2) egg clustering (laying eggs on a cavity ceiling and guarding them), as is found with fathead minnow Pimephales promelas (Denny 1987; Johnston 1999); and (3) pit building (the male excavates a pit in the substrate with his mouth), as is found with central stoneroller Campostoma anomalum (Miller 1962) and redspot chub Nocomis asper (Maurakis and Roston 1998). Neither nest construction nor egg clustering were observed during our study of Devils River minnows. Hubbs (1951) observed schools of Nueces roundnose minnows spawning during April in the Nueces River, Texas, in about 25 mm of cool (17– 188C) water. Roundnose minnow D. episcopa spawned in the Guadalupe River, Texas, from January to August, with major peaks in April and May and minor peaks in July and August (Wayne and Whiteside 1985). Devils River minnows did not show definitive seasonality in our study since it encompassed only one simulated annual cycle. Additionally, we did not observe seasonality during subsequent work with these fish following the same compressed cycle. Perhaps they might not exhibit strong seasonality in captivity with only light and temperature being altered since other factors (such as prey availability, flow variation, and interactions with other species) were not incorporated into the compressed seasonal cycle. Alternatively, Devils River minnows might not exhibit seasonality even in the wild where they can inhabit relatively constant temperature, spring-fed waters. Nueces roundnose minnows have heavy, nonadhesive eggs that lodge in gravel (Hubbs 1951). The Devils River minnow eggs in our study were
slightly adhesive, but one group of eggs was not adhesive at all. Typically, cyprinids eggs are temporarily adhesive immediately after expulsion (Braum 1978), but in Devils River minnows, the eggs may be nonadhesive when laid and later become adhesive after they have descended down into the spaces between the gravel. Dionda spp. generally are found in clear, springfed waters of streams in the arid and semitropical regions of southwestern North America (Mayden et al. 1992). Manantial roundnose minnows D. argentosa are limited to the spring-fed headwaters and spring runs of the Devils River and San Felipe and Sycamore creeks (Hubbs et al. 1991), where they are sympatric with Devils River minnows (Mayden et al. 1992). At least three other pairs of sympatric species of Dionda occur in Mexico where they appear to select different habitats in which to live and spawn within their common range (Hubbs and Miller 1974, 1977; ContrerasBalderas and Verduzco-Martinez 1977). After a flooding event on the Devils River, Harrell (1978) reported a shift in habitat location for Devils River minnows from channels towards or into riffles and the reverse situation for manantial roundnose minnows. How the Devils River minnow and the more abundant manantial roundnose minnow segregate within their shared habitat is not completely understood. Determining the specific habitat requirements of Devils River minnows will facilitate the recovery goals of refugium establishment, habitat management, resource protection, and reintroduction of the species. We conducted only a single test run and confounding factors (such as proximity to inflow) weakened the results. Nonetheless, for purposes of culture, gravel had the highest number of young. Subsequent work with this species and gravel for spawning has yielded over 1000 young (.20 mm TL). Culture in captivity for establishing refugium populations and for augmenting wild Devils River minnow populations is possible, although genetics have not yet been considered. Additionally, more work on habitat use, especially in the wild, is needed to determine other ecological requirements for this species. Acknowledgments We thank Lynn Lindsay for pioneering the project, Steve Hamby for his help with water quality measurements; the laboratory at the A. E. Wood State Fish Hatchery, Texas Parks and Wildlife Department, for the use of their facility; and Tim Bonner, Tom Brandt, Bob Edwards, Paula Power, and Kirk Winemiller for their reviews and com-
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ments. This research was funded by the Biological Resource Division of the U.S. Geological Service. References Bouck, G. R. 1980. Etiology of gas bubble disease. Transactions of the American Fisheries Society 109: 703–707. Braum, E. 1978. Ecological aspects of the survival of fish eggs, embryos, and larvae. Pages 102–131 in S. D. Gerking, editor. Ecology of freshwater fish production. Wiley, New York. Burkhead, N. M., and H. L. Jelks. 2001. Effects of suspended sediment on the reproductive success of the tricolor shiner, a crevice-spawning minnow. Transactions of the American Fisheries Society 130:959– 968. Contreras-Balderas, S., and J. Verduzco-Martinez. 1977. Dionda mandibularis, a new cyprinid fish endemic to the upper Rio Verde, San Luis Potosi, Mexico, with comments on related species. Transactions of the San Diego Society of Natural History 18:259– 266. Denny, J. S. 1987. Guidelines for the culture of fathead minnows Pimephales promelas for use in toxicity tests. U.S. Environmental Protection Agency, Environmental Research Laboratory, 600/3-87/001, Duluth, Minnesota. Garrett, G. P., R. J. Edwards, and A. H. Price. 1992. Distribution and status of the Devils River minnow, Dionda diaboli. The Southwestern Naturalist 37: 259–267. Harrell, H. L. 1978. Response of the Devil’s River (Texas) fish community to flooding. Copeia 1978:60– 68. Harrell, H. L. 1980. Dionda diaboli Hubbs and Brown, Devils River minnow. Page 153 in D. S. Lee, C. R. Gilbert, C. H. Hocutt, R. E. Jenkins, D. E. McAllister, and J. R. Stauffer, Jr., editors. Atlas of North American freshwater fishes. North Carolina State Museum of Natural History, Raleigh, North Carolina. Hubbs, C. 1951. Observations on the breeding of Dionda episcopa serena in the Nueces River, Texas. The Texas Journal of Science 3:490–492. Hubbs, C., and W. H. Brown. 1956. Dionda diaboli (Cyprinidae), a new minnow from Texas. Southwestern Naturalist 1:69–77. Hubbs, C., R. E. Edwards, and G. P. Garrett. 1991. An annotated checklist of the freshwater fishes of Texas, with keys to identification of species. Texas Journal of Science, Supplement 43:1–56. Hubbs, C., and G. P. Garrett. 1990. Reestablishment of Cyprinodon eximius (Cyprinodontidae) and status of Dionda diaboli (Cyprinidae) in the vicinity of Dolan
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Creek, Val Verde Co., Texas. The Southwestern Naturalist 35:446–478. Hubbs, C. L., and R. R. Miller. 1974. Dionda erimyzonops, a new, dwarf cyprinid fish inhabiting the Gulf coastal plain of Mexico. Occasional Papers of the Museum of Zoology, University of Michigan 671:1–17. Hubbs, C. L., and R. R. Miller. 1977. Six distinctive cyprinid fish species referred to Dionda inhabiting segments of the Tampico Embayment drainage of Mexico. Transactions of the San Diego Society of Natural History 18:267–336. Jacobsen, O. J. 1979. Substrate preference in the minnow (Phoxinus phoxinus L.). Polskie Archiwum Hydrobiologii 26:371–378. Johnston, C. E. 1999. The relationship of spawning mode to conservation of North American minnows (Cyprinidae). Environmental Biology of Fishes 55: 21–30. Maurakis, E. G., and W. Roston. 1998. Spawning behavior in Nocomis asper (Actinopterygii: Cyprinidae). Virginia Journal of Science 49:199–202. Mayden, R. L., R. H. Matson, and D. M. Hillis. 1992. Speciation in the North American genus Dionda (Teleostei: Cypriniformes). Pages 710–746 in R. L. Mayden, editor. Systematics, historical ecology, and North American freshwater fishes. Stanford University Press, Palo Alto, California. Miller, R. J. 1962. Reproductive behavior of the stoneroller minnow, Campostoma anomalum pullum. Copeia 1962:407–417. Ott, R. L. 1993. An introduction to statistical methods and data analysis. Duxbury Press, Belmont, California. Parker, N. C., and K. B. Davis. 1981. Requirements of warmwater fish. Pages 21–28 in L. J. Allen and E. C. Kinney, editors. Proceedings of the Bioengineering Symposium for Fish Culture. American Fisheries Society, Fish Culture Section, Publication 1, Bethesda, Maryland. USFWS (U.S. Fish and Wildlife Service). 1999. Endangered and threatened wildlife and plants; final rule to list the Devils River minnow as threatened. Federal Register 64:202(20 October 1999):56596– 56609. Valdes-Cantu, N. E., and K. O. Winemiller. 1997. Structure and habitat associations of Devils River fish assemblages. The Southwestern Naturalist 42:265– 278. Wayne, L. M., and B. G. Whiteside. 1985. Reproduction data on Dionda episcopa from Fessenden Spring, Texas. Texas Journal of Science 37:321–328. Wilkinson, L. 1998. SYSTAT 8.0 for Windows. SPSS, Inc., Chicago. Zar, J. H. 1999. Biostatistical analysis. Prentice Hall, Inc., Upper Saddle River, New Jersey.
