RESEARCH ARTICLE
Thermal Tolerance of the Invasive Belonesox belizanus, Pike Killifish, Throughout Ontogeny JAMES ROY KERFOOT, JR.* Department of Biological Sciences, Florida Institute of Technology, Melbourne, Florida
ABSTRACT
The goal of this study was to characterize the variability of thermal tolerances between lifehistory stages of the invasive Belonesox belizanus and attempt to describe the most likely stage of dispersal across south Florida. In the laboratory, individuals were acclimated to three temperatures (20, 25, or 30◦ C). Upper and lower lethal thermal limits and temperatures at which feeding ceased were measured for neonates, juveniles, and adults. Thermal tolerance polygons were developed to represent the thermal tolerance range of each life-history stage. Results indicated that across acclimation temperatures upper lethal thermal limits were similar for all three stages (38◦ C). However, minimum lethal thermal limits were significantly different at the 30◦ C acclimation temperature, where juveniles (9◦ C) had an approximately 2.0◦ C and 4.0◦ C lower minimum lethal thermal limit compared with adults and neonates, respectively. According to thermal tolerance polygons, juveniles had an average tolerance polygonal area almost 20◦ C2 larger than adults, indicating the greatest thermal tolerance of the three life-history stages. Variation in cessation of feeding temperatures indicated no significant difference between juveniles and adults. Overall, results of this study imply that juvenile B. belizanus may be equipped with the physiological flexibility to exercise habitat choice and reduce potential intraspecific competition with adults for limited food resources. Given its continued dispersal, the minimum thermal limit of juveniles may aid in continued dispersal of this species, especially during average winter temperatures throughout Florida where juveniles could act to preserve remnant populations until seasonal temperatures increase. J. Exp. Zool. 317:266–274, 2012. © 2012 Wiley Periodicals, Inc.
J. Exp. Zool. 317:266–274, 2012
How to cite this article: Kerfoot JR, JR. 2012. Thermal tolerance of the invasive belonesox belizanus, pike killifish, throughout ontogeny. J. Exp. Zool. 317:266–274.
Fishes, as well as other poikilothermic vertebrates, tend to respond to environmental temperature in a mode similar to their response to a traditional ecological resource such as food, searching for optimal levels in their surroundings (Fry, ’47; Magnuson et al., ’79). Fish are extremely sensitive to temperature and those that can successfully thermoregulate can move through habitats in such a way as to maximize time spent at temperatures favorable for optimal physiological performance (Neill, ’79; Richardson et al., ’94; Alexander, ’96; Kassebaum, 2004). Species found in harsher and more variable environments usually have a greater range of tolerance for those factors that make the environment more variable, namely temperature (Strange et al., 2002; Kassebaum, 2004). Introduction of nonnative species may involve removal of individuals from conditions under which the parent species has evolved, effectively releasing them from the control of the
factors such as climatic regime, species interactions, diseases, and parasites that determined the native distribution of the species (Taylor et al., ’84). However, the introduction of a species into a novel environment may include a suite of new pressures to which the species must adapt in order to survive, and according to Shafland and Pestrak (’82), the most important factor affecting the potential range of fishes introduced from the tropics to Florida is their tolerance to seasonally low temperatures. Shafland and Pestrak (’82) identified the lower lethal and sublethal temperatures of 14 introduced fish species in south Florida *Correspondence to: James Roy Kerfoot, Jr., Department of Biological Sciences, Union University, Jackson, TN 38305. E-mail:
[email protected] Received 26 September 2011; Revised 13 December 2011; Accepted 31 January 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jez.1720
© 2012 WILEY PERIODICALS, INC.
THERMAL TOLERANCE OF THE INVASIVE BELONESOX BELIZANUS under controlled laboratory conditions, including pike killifish, Belonesox belizanus. Belonesox belizanus has a native distribution from Rio Antigua, Mexico to northern Costa Rica and were introduced to Miami-Dade County, Florida in November 1957 (Belshe, ’61). Adult B. belizanus had a lower lethal temperature of 9.7 ± 0.6◦ C from an acclimation temperature of 24◦ C (Shafland and Pestrak, ’82). Based on comparisons of lethal temperature tolerances with mean isotherms of winter stream temperatures they estimated that B. belizanus should have the thermal tolerance to expand its northerly range from south Florida to Gainesville, Florida. Shafland and Pestrak (’82) concluded that there are probably several lower lethal temperatures for any given species, each dependent on the existing internal and external conditions of the fish. This conclusion highlights the importance of acclimatization in the thermal tolerance of a species. Shafland and Pestrak (’82) conducted tolerance tests only on adult B. belizanus at one acclimation temperature, thus were unable to determine variation in thermal tolerance across acclimation temperatures. In addition, Shafland and Pestrak (’82) did not investigate thermal tolerance throughout ontogeny, to assess whether age and size of this invasive species mediates its rate of spread and range of distribution in south Florida. The potential differences in thermal tolerances across ontogeny are especially interesting for B. belizanus because their reproduction in south Florida does not appear to be limited by temperature, and reproduction occurs year-round (Turner and Snelson, ’84). A previous study investigating the environmental correlates of the abundance and distribution of B. belizanus in south Florida indicated that data substantially supported AICc models containing temperature as a variable to explain fluctuation in abundance of the fish (Kerfoot et al., 2011). The fact that the magnitude of change in density of B. belizanus correlates with the change in temperature in south Florida and that stagespecific variation in temperature tolerance exists in natural populations (Davidson and Hutchinson, ’38; Reiser and Bjornn, ’79; Bjornn and Reiser, ’91; Baroudy and Elliott, ’94; Ambrose and Hines, ’91) underscores the need to determine if the variation in the ability of individual B. belizanus to tolerate different levels of temperature regimes throughout ontogeny influences the distribution and abundance of this invasive species in south Florida. This study is designed to address the following question: Do the temperature tolerance limits of B. belizanus from south Florida vary throughout ontogeny? The specific hypotheses to be tested are: (1) there is significant variation in the thermal tolerances between B. belizanus life-history stages and (2) temperatures at which individuals cease to feed vary between B. belizanus life-history stages.
MATERIAL AND METHODS Specimen Collection Belonesox belizanus adults (standard length [SL] > 55 mm) and juveniles (25–55 mm in SL) (Turner, ’81) were collected us-
267 ing a 9.5 mm mesh seine, dip nets, and cast nets from the Everglades National Park along highway 9339 that runs through the park (Everglades research permit no. EVER-2005-SCI-0040). Fish were transported live back to the Florida Institute of Technology’s Fish Ecophysiology lab. Belonesox belizanus neonates (SL < 25 mm; Turner, ’81) were raised from live births of fecund females in the laboratory. Collections of B. belizanus were made during the dry season (January–May) in 2008 and 2009. All specimens were housed in the laboratory in 90.9 L aquaria at 25◦ C for 2 weeks prior to the start of the experiment to acclimate the fish to laboratory conditions. Juvenile and adult individuals were fed ad libitum western mosquitofish, Gambusia affinis. Similarly, neonates were fed ad libitum adult brine shrimp, Artemia spp. Mosquitofish were collected from retention ponds on Florida Institute of Technology property, and live adult brine shrimp were acquired from a local aquarium store. Tanks were maintained and cleaned daily to remove uneaten prey and waste material. Experimental Design Acclimation Temperatures. To calculate upper and lower thermal tolerance ranges for this species, individuals from all three life-history stages were randomly assigned to one of three different acclimation temperatures, 20, 25, or 30◦ C. These acclimation temperatures were chosen based on data of the average seasonal temperature fluctuations in south Florida (Kerfoot et al., 2011, South Florida Water Management District database). Fish were acclimated to these temperatures for a period of 2 weeks, which is considered an acceptable time period for acclimation to a new thermal regime (Hill and Matthews, ’80, Strange et al., 2002, Kassebaum, 2004). During the acclimation period, fish were fed daily ad libitum and their tanks cleaned daily to remove waste material. Lethal Maximum and Minimum Temperatures. To assess the upper and lower limits of B. belizanus thermal tolerance across life-history stages, lethal maximum (Lmax—upper tolerance, high temperature threshold) and lethal minimum (Lmin— lower tolerance, low temperature threshold) are two dynamic approaches that were used to evaluate tolerance limits and are defined as the thermal endpoints where death occurs (Hickman and Dewey, ’73; Shafland and Pestrak, ’82; Beitinger et al., 2000; Schofield et al., 2010). All procedures where strictly followed in accordance with the Florida Institute of Technology’s Institutional Animal Care and Use Committee (IACUC) guidelines, Permit no. 99–01. For trials, three 145 cm × 29 cm × 29 cm acrylic tanks were divided into five equal, smaller compartments, yielding 15 24.4 L tanks. These tanks were set up in a re-circulating flow through system where water was pumped into each tank from a common sump. To control thermal levels in the experimental tanks, two 500 W titanium heaters were housed in the sump and an external Delta Star Chiller J. Exp. Zool.
268 (1/4 hp, 115 V, 5.4 Amp, 3,080 BTU, 8/15 gpm) was plumbed into the re-circulating system. A programmable thermal regime was designed to regulate the chiller and heater systems. The Automatically Regulated Thermal Unit Regime (ARThUR) was developed through a collaboration between A. Kunkle (Florida Institute of Technology’s Department of Computer Science) and J. Kerfoot that allowed maintenance of precise thermal gradients throughout the experiment. The set up was designed by A. Kunkle (modified from Schaefer, 2006) and consisted of a process controller (controlanything.com: XR410 Expansion Relay Controller Board with 4 10-Amp SPDT Relays), an analog to digital converter (controlanything.com: AD1216PROXR RS-232 16Channel 12-Bit A/D), two Omega solid state relays (each specific to a chiller or heaters [omega.com: SSRL240DC25]), Type-T thermocouples used to sense temperatures in the sump (omega.com: 5TCGGT3672), and a data-logging computer. ARThUR was programmed to change the temperature in the experimental tanks 1.0◦ C day−1 starting at 0900 hr each day, and to monitor and data-log conditions in the tanks every 30 min. In the event that ARThUR read a ± 0.5◦ C variation in the programmed thermal set point, the automated system would self-regulate by switching on the chiller or heaters at appropriate intervals until the thermal set point was re-established. Individual fish were randomly assigned to an experimental tank at their appropriate acclimation temperature. To ensure that deaths of individuals were attributed only to temperature and not handling stress, individuals remained in the experimental tank for a 24-hr period and were monitored. After the 24-hr period, the temperature was increased (for Lmax trials) or decreased (for Lmin trials) at a rate of 1.0◦ C day−1 until death occurred (following Shafland and Pestrak, ’82). The response variables measured were temperatures at which death and cessation of feeding occurred. In general, the fish ceased to feed a few degrees prior to death. Once the lethal limit was reached, fish were removed from the experimental tank and their SLs measured to the nearest millimeter to ensure that individuals remained in a particular life-history stage throughout the experiment. To avoid any confounding effects of a pseudo-replicated design, Lmin and Lmax trials were staggered and acclimation temperatures randomly assigned. To investigate thermal tolerance through ontogeny of B. belizanus, 30 individuals from each size class (10 individuals per acclimation temperature, with five individuals being used to assess Lmax and five individuals being used to assess Lmin) were used to calculate tolerance ranges, with a total of 90 specimens used.
KERFOOT, JR. Acclimation temperature and life-history stage were independent factors and Lmax and Lmin were used as the dependent variables. Both dependent variables did not meet the assumptions of parametric tests and were rank-transformed following Quinn and Keough (2002). The rank-transformation method is an acceptable technique and can aid in increasing the robustness of factorial ANOVA designs involving nonparametric data (Quinn and Keough, 2002). If an independent factor explained significant variation in the dependent variables, a set of univariate Tukey’s multiple comparisons tests were run to investigate which group within those independent factors was significantly different from the other groups. In addition, to investigate differences in cessation of feeding temperatures between life-history stages and across acclimation temperatures, a single two-way factorial ANOVA was performed on rank-transformed cessation of feeding temperatures during Lmin trials. Cessation of feeding temperatures were used as the dependent variable and life-history stage and acclimation temperatures as independent variables. This analysis was performed using only data from Lmin trials because individuals had a distinct temperature at which they ceased to feed as conditions cooled. This was not the case for Lmax trials where individuals fed until reaching their upper lethal limit. This analysis was performed only on juvenile and adult data to account for differences in prey types between life-history stages. To control for sexual dimorphism in adult pike killifish, Lmax and Lmin values were analyzed using separate Mann–Whitney nonparametric tests with gender as the independent variable and Lmin and Lmax as the dependent variables. Results indicated that there were no significant differences in Lmin or Lmax measurements between genders and thus they could be grouped together and used in subsequent analyses (Lmin—U = 18.5, N = 15, P = 0.681; Lmax—U = 11.5, N = 15, P = 0.067). Finally, to represent the thermal tolerance range of each lifehistory stage, thermal tolerance polygons were developed and measured using average lethal temperatures (Lmin and Lmax) across acclimation temperatures. These average lethal temperatures formed lower and upper bounds of the thermal tolerance polygons. Estimates of thermal tolerance polygons are formed by the area in which the polygon covers and are limited by the lowest and highest acclimation temperatures. Thermal tolerance polygonal areas are given in units of ◦ C2 . All statistical analyses were performed in SPSS 17.0 (IBM Inc., Armonk, NY) and Sigma Plot 2001 (Systat Software Inc., San Jose, CA) at an α-value of 0.050.
RESULTS Statistical Analyses To test the hypothesis that there is significant variation in B. belizanus, thermal tolerance ranges throughout ontogeny and across acclimation temperatures, separate twoway factorial analysis of variance (ANOVA) tests were used. J. Exp. Zool.
Upper lethal thermal tolerance limits (Lmax) ranged between 37.40 and 38.80◦ C, but were not significantly different between life-history stages or across acclimation temperatures (Fig. 1 and Tables 1, 2). Likewise, comparisons of Lmin measurements showed similarities between life-history stages at lower
THERMAL TOLERANCE OF THE INVASIVE BELONESOX BELIZANUS
269
Table 1. Descriptive statistics for lethal thermal limits (Lmin and Lmax) measured across life-history stages and acclimation temperatures. 20◦ C
Neonate Mean (◦ C) SE (◦ C) Juvenile Mean (◦ C) SE (◦ C) Adult Mean (◦ C) SE (◦ C) Figure 1. Mean upper and lower lethal temperature tolerances (Lmax and Lmin) of Belonesox belizanus across life-history stages and acclimation temperatures. Neonates are represented by closed triangles (), juveniles by open squares (), and adults by open circles (◦). Data are given in mean ± SE (standard error of the mean). Lines outline thermal polygons for each life-history stage. Solid lines represent the thermal tolerance polygon for neonates, stippled lines represent juveniles, and dashed lines represent adults.
acclimation temperatures (20 and 25◦ C); with lethal limits hovering between 9.40 and 10.20◦ C (Fig. 1 and Table 1). Conversely, lower lethal limits were dissimilar between life-history stages at an acclimation temperature of 30◦ C, where juveniles had the lowest lethal limit of 9.20 ± 0.20◦ C (Fig. 1 and Table 1). Neonates had the highest Lmin tolerance level at 13.40 ± 0.40◦ C whereas the adults had intermediate Lmin values of 11.00 ± 0.00◦ C. There was a significant effect of life-history stage, acclima-
n
n
n
25◦ C
30◦ C
Lmin
Lmax
Lmin
Lmax
Lmin
Lmax
5 10.20 0.20 5 9.60 0.24 5 9.60 0.24
5 38.00 0.00 5 38.40 0.24 5 37.40 0.24
5 10.00 0.00 5 9.40 0.24 5 10.00 0.00
5 38.80 0.49 5 38.00 0.32 5 38.40 0.24
5 13.40 0.40 5 9.20 0.20 5 11.00 0.00
5 38.00 0.00 5 38.20 0.20 5 37.60 0.24
tion temperature, and an interaction between life-history stage and acclimation temperature on Lmin values (life-history stage: F = 31.563, df = 2,36, P < 0.001; acclimation temperature: F = 13.608, df = 2,36, P < 0.001; life-history stage X acclimation temperature: F = 8.565, df = 4,36, P < 0.001; Table 2). Subsequent Tukey’s multiple comparison tests confirm that the juvenile life-history stage was the group that was significantly different from the others, and that the significant effect of acclimation temperature was due to differences in Lmin values at the 30◦ C acclimation temperature (Table 3). The significant interaction between acclimation temperature and life-history stage effects can be seen from Figure 1; as the acclimation temperature raises from 25 to 30◦ C, the Lmin measurements also increased for two of the three life-history stages. Using Lmin and Lmax measurements across acclimation temperatures, thermal tolerance polygons were calculated for each
Table 2. Results of two-way ANOVAs comparing rank-transformed Lmin and Lmax limits between life-history stages and across acclimation temperatures. The α-level for analyses was set at a significance value of 0.050. Sum of squares and mean square are represented as SS and MS, respectively. Variable
Source
SS
df
MS
F
Significance
Lmax Life-history stage Acclimation Life-history stage × acclimation
500.700 448.900 916.300
2 2 4
250.350 224.450 229.075
2.809 2.518 2.570
0.074 0.095 0.054
Life-history stage Acclimation Life-history stage × acclimation
2,533.433 1,092.233 1,375.033
2 2 4
1,266.717 546.117 343.758
31.563 13.608 8.565
