Northeastern Naturalist Vol. 23, No. 1 M. Surace and G.R. Smith 2016 1 2016 NORTHEASTERN NATURALIST 23(1):1–10 Female Body Size and Reproduction in Western Mosquitofish (Gambusia affinis) from Two Ponds in Central Ohio Margaret Surace1,2 and Geoffrey R. Smith1,* Abstract - Gambusia affinis (Western Mosquitofish) is one of the most widespread and invasive freshwater fish. Herein we describe and compare the body size and reproduction of female Western Mosquitofish from 2 small ponds in central Ohio: Wood Duck (WD) and Olde Minnow (OM). This population is one of the most northern populations of Western Mosquitofish to be studied to date. The 2 ponds differed in water temperature (OM > WD), dissolved oxygen (OM >> WD), and depth (OM > WD). Body size and body condition of Western Mosquitofish collected was similar in the 2 ponds. Litter size averaged 15.2, increased with female size, and did not differ between ponds. This mean litter size is substantially smaller than that reported for other populations at a similar latitude. Females contained embryos or follicles throughout the study (May–July), but the proportion declined over time, especially in WD. Simultaneous presence of embryos and follicles suggests multiple broods per season, and was more common in OM than in WD. Our results indicate that while females from both ponds were similar in size and had similar litter size, there were potential differences in the frequency of reproduction between the ponds. Introduction Non-native fish are a common threat to freshwater ecosystems (Gozlan et al. 2010). To better understand the potential impacts of these fish, and their potential management, a basic understanding of their biology and ecology in their non-native range is necessary (e.g., Elofsson et al. 2012, Guo et al. 2013, Matthews and Marsh- Matthews 2011). Understanding how reproduction of non-native fish varies among introduced populations in different habitats or parts of their introduced ranges may help to understand where they may be able to invade and where they will be able to persist (e.g., Grabowska and Przybylski 2015, Guo et al. 2013, Russell et al. 2012). Gambusia affinis [Baird and Girard] (Western Mosquitofish) is one of the most widespread and invasive freshwater fish (Pyke 2005, 2008). They are known to have numerous negative impacts on the freshwater ecosystems that they invade (reviewed in Pyke 2008). There is a great deal of variation in life-history traits in the live-bearing Western Mosquitofish among their many populations (Johnson and Bagley 2011). In their review, Johnson and Bagley (2011) found a 19.9-fold range in the number of offspring produced by female Western Mosquitofish (5.5–109.2), more variation found than in any other Poeciliid. There are some indications there may be latitudinal or longitudinal trends in the life-history traits of this species 1Department of Biology, Denison University, Granville, OH 43023. 2Current address - 864 Tollis Parkway, Broadview Heights, OH 44147. *Corresponding author - smithg@denison.edu. Manuscript Editor: David Yozzo Northeastern Naturalist 2 M. Surace and G.R. Smith 2016 Vol. 23, No. 1 (e.g., Haynes and Cashner 1995). In addition, life-history traits in Western Mosquitofish can evolve relatively quickly among populations in different environments (e.g., Martin and Leberg 2011, Stearns 1983b, Stockwell and Weeks 1999). Herein we describe and compare the body size and reproduction of female Western Mosquitofish from 2 small ponds in central Ohio. This population is one of the most northern populations of Western Mosquitofish to be studied to date. For example, to our knowledge litter sizes of only 2 other populations at a latitude of >38° N have been published (see Table 1). Our study therefore contributes to a better understanding of the reproduction of Western Mosquitofish from more northern, temperate parts of its introduced range. Site Description Our study was conducted using Western Mosquitofish collected from Wood Duck Pond (WD) and Olde Minnow Pond (OM) on the Denison University Biological Reserve, Granville, Licking County, OH (40°5'N, 82°31'W). Wood Duck Pond (0.86 ha) is characterized by widespread vegetation comprised mostly of Ceratophyllum sp., Elodea sp., and Myriophyllum sp., and by a Lepomis macrochirus Rafinesque (Bluegill) population (J.J. Arrington and J.E. Rettig, Department of Biology, Denison University, Granville, OH, unpubl. data; Schultz and Mick 1998, Smith et al. 2005), whereas OM (0.60 ha) is characterized by le ss-widespread vegetation comprised mostly of Chara sp. and Elodea sp., and has no other known fish populations (J.J. Arrington and J.E. Rettig, unpubl. data; Schultz and Mick 1998) . Table 1. Mean litter sizes of Gambusia affinis from literature reports from north to south. Latitudes were estimated using GoogleEarth. * = size-adjusted mean. Mean Location Latitude litter size Source Cook County, IL, USA 41°44'N 26.1–63.6 Krumholz 1943 Utah Lake, UT, USA 40°12'N 44.7 Billman and Belk 2014 Granville, OH, USA 40°04'N 15.2 This study Hovey Lake, Posey County, IN, USA 37°49'N 21.3 Hughes 1985 Ortaca, Turkey 36°50'N 28.2 Öztürk and Ikiz 2004 Dalaman, Turkey 36°45'N 27.1 Öztürk and Ikiz 2004 Fethiye-Akgöl, Turkey 36°41'N 21.9 Öztürk and Ikiz 2004 Irvine, CA, USA 33°41'N ~29–30 Reznick et al. 2006 Near Roswell, NM, USA 33°23'N 34.5 Swenton and Kodric-Brown 2012 Baghdad, Iraq 33°18'N 8.6 Na’ama and Al-Hassan 1989 Benghazi, Libya 32°05'N 9.0 Jawad and Busneina 2000 Alexandria, Egypt 31°12'N 31.0 Na’ama and Al-Hassan 1989 Basrah, Iraq 30°30'N 3.1 Na’ama and Al-Hassan 1989 Old Fort Bayou, Biloxi Bay, MS, USA 30°24'N 7.9–9.87* Brown-Peterson and Peterson 1990 Southwestern Louisiana, USA 30°11'N 12.2, 22.1 Daniels and Felley 1992 Hawaii, USA 19°53'N 11.9, 15.2 Scribner et al. 1992 Hawaii, USA 19°53'N 21.5 Stearns 1983a Perth, Australia (experimental ponds) 31°57'S 12.5–27.1 Trendall 1983 Perth, Australia (field ponds) 31°57'S 18.3–31.0 Trendall 1982 Northeastern Naturalist Vol. 23, No. 1 M. Surace and G.R. Smith 2016 3 Methods We dip-netted 15 adult females from each pond every other week beginning 28 May 2010 and ending 23 July 2010 for a total of 6 sampling dates in each pond. Once captured, specimens were humanely euthanized and immediately frozen. For each individual, we measured total length (TL) with a plastic ruler (to nearest mm), and obtained body mass (BM) using an electronic balance (to nearest 0.0001 g). We calculated an index of body condition, Fulton’s K (Cone 1989, Stevenson and Woods 2006) for each female. We dissected each individual and counted the numbers of follicles (i.e., yolked eggs) and embryos, and noted the relative size (small vs. large) and developmental stage (early vs. well-developed) of the follicles and embryos, respectively. Litter size was estimated using the number of embryos. We measured depth (cm), water temperature (°C), and dissolved oxygen levels (mg L-1) at 18 locations in each pond on 4 dates during the summer of 2010 using a YSI 550A Dissolved Oxygen Instrument (YSI Incorporated, Yellow Springs, OH). However, these measurements were taken at only 13 of the 18 locations in OM on 1 of those dates (in late May) as a result of meter failure. Due to the relatively small sample sizes for each biweekly sample, we pooled data for females containing embryos across the season for our analyses of TL, BM, Fulton’s K, and litter size. For analyses of TL and Fulton’s K, we conducted analysis of variance (ANOVA) to compare variables between ponds. We analyzed BM using an analysis of covariance (ANCOVA) with TL as the covariate. For this analysis, we log-transformed both BM and TL to linearize the relationship between these 2 variables. We compared the proportion of females with embryos between the 2 ponds by comparing the numbers of females with and without embryos in each pond on each date using a chi-square test. We employed simple linear regression to examine the relationship between number of embryos and female TL, and an ANCOVA to compare litter size between the 2 ponds. The interaction between TL and litter size was not significant and so was removed from the final model. For abiotic pond characteristics, we conducted a 2-way ANOVA with pond and sampling date as factors. Statistical analyses were performed using JMP Pro 10.0 (SAS Institute, Cary, NC). Means are given ± 1 SE. Results Pond characteristics There were a number of significant differences between the 2 ponds and trends over time (Table 2). In general, OM was warmer than WD (Pond: P = 0.027). OM was warmer later in the season than WD (Pond*date interaction: P = 0.0002). Water temperature tended to increase over the season (Date: P < 0.0001). Dissolved oxygen levels were much higher in OM than in WD (Pond: P < 0.0001) and showed a general decline over the season in both ponds (Date: P < 0.0001); there was no significant interaction between pond and date (P = 0.13). OM was deeper on average than WD (P < 0.0001). Depth did not vary across the season (P = 0.84); there was also no interaction between pond and date ( P = 0.93). Northeastern Naturalist 4 M. Surace and G.R. Smith 2016 Vol. 23, No. 1 Female body size. Females containing embryos from the 2 ponds did not differ in TL (OM: 35.6 ± 0.4 mm, n = 65; WD: 35.5 ± 0.4 mm, n = 57; P = 0.92). Body mass of females containing embryos (log-transformed) did not differ between the ponds (P = 0.60). Female body mass (log-transformed) increased with female TL (log-transformed) (P < 0.0001). The relationship between female body mass (log-transformed) and female TL (logtransformed) differed significantly between the 2 ponds (Pond*logTL interaction: P = 0.031). Females from OM increased body mass with TL faster than females from WD (OM: logBM = -5.04 + 3.10logTL, n = 65, r2 = 0.86, P < 0.0001; WD: logBM = -4.34 + 2.66logTL, n = 57, r2 = 0.88, P < 0.0001). Fulton’s K of females containing embryos did not differ between ponds (OM: 1.34 ± 0.02 g cm-3, n = 65; WD: 1.35 ± 0.02 g cm-3, n = 57; P = 0.66). Reproduction Of the 173 females examined, 122 contained embryos. The proportion of females without embryos did not differ between the 2 ponds (OM: 24 of 90, WD: 32 of 89; χ2 1 = 1.38, P = 0.24). Mean litter size for both ponds combined was 15.2 ± 0.7 (n = 122, range = 2 to 53). Mean litter size for females was 15.7 ± 1.0 embryos (n = 65, range = 2 to 53) from OM and 14.6 ± 1.0 embryos (n = 57, range = 3 to 44) from WD. This difference in litter size between ponds was not significant when controlling for female TL (ANCOVA: P = 0.44). Litter size did increase with female TL (ANCOVA: litter size = -24.28 + 1.11[TL]; n = 122, r2 = 0.198, P < 0.0001; Fig. 1). Table 3 reports the frequency of follicles and embryos in females from OM and WD. In OM, embryos were observed on every sampling date, but the proportion of females with embryos dropped on 23 July. Females containing both embryos and follicles were found on the June sampling dates, with a peak frequency on 4 June. Females lacking embryos and follicles were only found later in the study period, with more than 50% lacking embryos and follicles on the 23 July sampling date. Females in WD had the same basic pattern as in OM; however, sampling dates from 25 June to 23 July had higher frequencies of females without fo llicles or embryos. Table 2. Mean (± 1 S.E.) water temperature, dissolved oxygen levels, and depth for 2 small ponds (Olde Minnow [OM] and Wood Duck [WD]) in central Ohio during the summer of 2010. The number of observations is given in parentheses. Late May Mid-June Late June Early July Water temperature (°C) OM 19.6 ± 0.1 (13) 17.8 ± 0.2 (18) 24.0 ± 0.5 (18) 25.3 ± 0.3 (18) WD 20.4 ± 0.2 (18) 17.9 ± 0.2 (18) 22.5 ± 0.1 (18) 24.1 ± 0.3 (18) Dissolved oxygen (mg L-1) OM 14.6 ± 0.5 (13) 12.2 ± 0.6 (18) 13.6 ± 0.5 (18) 12.6 ± 0.3 (18) WD 3.1 ± 0.6 (18) 1.4 ± 0.4 (18) 0.8 ± 0.2 (18) 0.8 ± 0.1 (18) Depth (cm) OM 108.6 ± 9.0 (13) 103.4 ± 7.9 (18) 104.7 ± 9.5 (18) 104.0 ± 9.4 (18) WD 42.4 ± 3.7 (18) 42.5 ± 3.7 (18) 40.8 ± 3.3 (18) 34.6 ± 3.4 (18) Northeastern Naturalist Vol. 23, No. 1 M. Surace and G.R. Smith 2016 5 Discussion In general, females from the 2 ponds sampled in our study had similar body sizes and similar body condition. Other studies have frequently found differences in female body size among Gambusia populations, even when the populations were relatively close together (e.g., Kristensen et al. 2007). In some cases, these differences are related to the water quality of the ponds in question (e.g., Edwards et al. 2010, Hildebrand 1918, Staub et al. 2004). We had expected that WD might have smaller fish than OM since it experienced a winter kill in 2000 and has been hypoxic since then (Smith et al. 2005; this study). WD also contains a Bluegill Figure 1. Relationship between female total length (TL) and the number of embryos for Gambusia affinis (Western Mosquitofish) from 2 ponds (data pooled) in central Ohio. Table 3. The frequency of females with follicles or embryos throughout the study period in Olde Minnow Pond and Wood Duck Pond in central Ohio. Embryos No follicles/ Small Large Early Large and Date embryos follicles Follicles follicles embryos Embryos embryos follicles Olde Minnow Pond 28 May 0 (0%) 3 (20%) 0 (0%) 0 (0%) 3 (20%) 9 (60%) 0 (0%) 0 (0%) 4 June 0 (0%) 0 (0%) 2 (13%) 3 (20%) 3 (20%) 0 (0%) 0 (0%) 7 (47%) 11 June 0 (0%) 0 (0%) 0 (0%) 1 (7%) 1 (7%) 12 (80%) 0 (0%) 1 (7%) 25 June 0 (0%) 0 (0%) 3 (20%) 0 (0%) 0 (0%) 9 (60%) 0 (0%) 3 (20%) 9 July 4 (27%) 0 (0%) 1 (7%) 0 (0%) 1 (7%) 9 (60%) 0 (0%) 0 (0%) 23 July 8 (53%) 3 (20%) 0 (0%) 0 (0%) 3 (20%) 3 (20%) 0 (0%) 1 (7%) Wood Duck Pond 28 May 0 (0%) 0 (0%) 1 (7%) 0 (0%) 5 (33%) 5 (33%) 0 (0%) 4 (27%) 4 June 0 (0%) 0 (0%) 1 (7%) 0 (0%) 3 (20%) 9 (60%) 0 (0%) 2 (13%) 11 June 1 (7%) 1 (7%) 1 (7%) 1 (7%) 0 (0%) 7 (50%) 1 (7%) 2 (14%) 25 June 7 (47%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 5 (33%) 1 (7%) 2 (13%) 9 July 6 (40%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 9 (60%) 0 (0%) 0 (0%) 23 July 7 (78%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (11%) 0 (0%) 1 (11%) Northeastern Naturalist 6 M. Surace and G.R. Smith 2016 Vol. 23, No. 1 population, which has the potential to affect the mosquitofish via competition or predation (Baylis 1982; Coyner et al. 2001; Nowlin and Drenner 2000; G.R. Smith, J.E. Rettig, A. Burger, and E. Tristano, unpubl. data). The differences in water temperature, dissolved oxygen, and water depth we observed would also suggest we might have expected to see differences in body size or condition between the ponds. The lack of any such body size or condition differences between ponds suggests that these traits, at least in our population, may not be driven by temperature or dissolved oxygen. We also have evidence that the diet composition of females differs between these ponds, with females from OM containing higher numbers of prey, in particular zooplankton, and females from WD consuming more snails and fewer zooplankton (M. Surace, G.R. Smith, and J.E. Rettig, unpubl. data). Thus, similarity in diet composition or quantity is unlikely to explain the similarity of body size and condition in the mosquitofish in these 2 ponds. The overall mean litter size for Western Mosquitofish among both ponds pooled was 15.2, which is smaller than reported in many other populations of Western Mosquitofish, especially when compared to populations located at a similar latitude (northern Illinois: 26.1–63.6 [Krumholz 1943], Utah: 44.7 [Billman and Belk 2014]; Table 1). Indeed, in general there is a significant positive relationship between latitude and litter size in Western Mosquitofish from the northern hemisphere (litter size = -3.92 + 0.82latitude; n = 19, r2 = 0.24, P = 0.034), yet the mean litter size for the central Ohio population is well below the predicted litter size for its latitude based on this regression (15.2 vs. 28.9). The latitudinal trend we found in our review is generally consistent with Haynes and Cashner (1995), who determined that Western Mosquitofish shows an increase in adjusted fecundity (litter size per mm) from south to north. Why our populations of Western Mosquitofish have smaller litter sizes than most other populations is not clear . Litter size in both WD and OM showed a similar increase with female size. In general, litter size in Western Mosquitofish increases with female body size (e.g., Belk and Tuckfield 2010, Billman and Belk 2014, Hughes 1985, Jawad and Busneina 2000). The 2 ponds did not have different litter sizes when pooled across the season. It is interesting that the females in our study ponds did not differ in litter size despite differences in abiotic characteristics (water temperature, dissolved oxygen, and depth) between the ponds (this study), as well as a difference in the quality and composition of prey items in the guts of females from these 2 populations (M. Surace, G.R. Smith, and J.E. Rettig, unpubl. data). The lack of difference in litter size between ponds thus parallels the lack of difference in body size and condition between the ponds. Previous studies have shown that populations of Western Mosquitofish can differ in brood size, including populations located near each other geographically (e.g., Brown-Peterson and Peterson 1990, Daniels and Felley 1992, Franssen 2009, Trendall 1982). Litter size in Gambusia can be related to food level, with smaller litters found in females consuming less food or a poorer quality diet (Cech et al. 1992, Trendall 1983). Thermal environment of ponds also appears to potentially drive life-history variation among populations of Western Mosquitofish Northeastern Naturalist Vol. 23, No. 1 M. Surace and G.R. Smith 2016 7 (e.g., Stockwell and Vinyard 2000). As with body size, these explanatory variables appear to have little effect on litter size in our 2 ponds. We found females with embryos and/or follicles throughout the study period, but the proportion declined at the end of the study period. The presence of embryos and follicles in a female indicates that females in our populations may produce multiple broods in a season, and that OM females may do this more than WD females. Females of Western Mosquitofish are known to produce multiple broods per season (e.g., Koya et al. 1998; Krumholz 1943; Trendall 1982, 1983). We also found that the tendency was for OM females to possess higher frequencies of embryos and follicles late in the season compared to those from WD. It may be that the differences in abiotic characteristics and diet composition noted above may be affecting the frequency of reproduction, rather than female size or litter size in the Western Mosquitofish in our ponds. Other studies have found that female Western Mosquitofish are reproductive from late Spring through late Summer throughout their native and non-native ranges (e.g., Reznick and Braun 1987, Self 1940). Indeed, our observations of the reproductive period in our 2 ponds are generally similar to the pattern observed in Western Mosquitofish from Indiana where the proportion of females carrying embryos was highest in June and July (Hughes 1985). Such similarities may exist because the timing of reproduction in Western Mosquitofish can be triggered by temperature (e.g., Medlen 1951) and photoperiod (e.g., Cech et al. 1992). Our results indicate that while female Western Mosquitofish from both ponds were similar in size and had similar mean litter sizes, there were potential differences in the frequency of reproduction between the ponds. It appears that Olde Minnow Pond females may be able to produce more broods per year than females from Wood Duck Pond. It is possible the difference in brood frequency between ponds is related to the differences in water temperature, dissolved oxygen, depth, and diet composition noted earlier (this study; M. Surace, G.R. Smith, and J.E. Rettig, unpubl. data). The fact that body size and litter size did not differ between ponds suggests the factors controlling body size and litter size may be different from those governing brood frequency, at least in our populations. Acknowledgments We thank A. Burger for his help collecting the fish, and J. Rettig for her advice and help with the fish dissections. We also appreciate the comments of 2 anonymous reviewers, which improved the manuscript. This study was done under permit from the Ohio Department of Conservation and was approved by the Denison University Institutional Animal Care and Use Committee. Literature Cited Baylis, J.R. 1982. Unusual escape response by two cyprinodontiform fishes, and a Bluegill predator’s counter-strategy. Copeia 1982:455–457. 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