Zoology 127 (2018) 20–26
Contents lists available at ScienceDirect
Zoology journal homepage: www.elsevier.com/locate/zool
Standard metabolic rates of early life stages of the diamondback terrapin (Malaclemys terrapin), an estuarine turtle, suggest correlates between life history changes and the metabolic economy of hatchlings
T
Christopher L. Rowe University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, P.O. Box 38, Solomons 20659, MD, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: activity costs dispersal energy allocation parental investment residual yolk respiration
I estimated standard metabolic rates (SMR) using measurements of oxygen consumption rates of embryos and unfed, resting hatchlings of the diamondback terrapin (Malaclemys terrapin) three times during embryonic development and twice during the early post-hatching period. The highest observed SMRs occurred during mid to late embryonic development and the early post-hatching period when hatchlings were still reliant on yolk reserves provided by the mother. Hatchlings that were reliant on yolk displayed per capita SMR 135 % higher than when measured 25 calendar days later after they became reliant on exogenous resources. The magnitude of the difference in hatchling SMR between yolk-reliant and exogenously feeding stages was much greater than that attributed to costs of digestion (specific dynamic action) observed in another emydid turtle, suggesting that processing of the yolk was not solely responsible for the observed difference. The pre-feeding period of yolk reliance of hatchlings corresponds with the period of dispersal from the nesting site, suggesting that elevated SMR during this period could facilitate dispersal activities. Thus, I hypothesize that the reduction in SMR after the development of feeding behaviors may reflect an energy optimization strategy in which a high metabolic expenditure in support of development and growth of the embryo and dispersal of the hatchling is followed by a substantial reduction in metabolic expenditure coincident with the individual becoming reliant on exogenous resources following yolk depletion.
1. Introduction Patterns of standard metabolic rate (“SMR,” the metabolic rate of a resting, post-absorptive individual; e.g. Bennett and Dawson, 1976) during development in reptiles are quite variable among species. For most turtles and crocodilians, SMR peaks shortly prior to hatching, corresponding with maximum embryonic size and rapid developmental rates. However, in snakes SMR increases exponentially throughout development and in lizards and some turtles, the pattern of SMR is sigmoidal, reaching a maximum prior to hatching and then remaining elevated (a “plateau” pattern; discussed by Peterson and Kruegl, 2005). Previous research on emydid turtles suggests that species of this family display a peaked pattern or perhaps a plateau (Peterson and Kruegl, 2005). The emydid turtle, Malaclemys terrapin (the diamondback terrapin), is an estuarine endemic distributed throughout much of the Gulf of Mexico and Atlantic coasts of North America. The diamondback terrapin is unique among the emydids in being an obligate estuarine organism. In Maryland, USA, diamondback terrapins nest up to three
times per season from June through August and on average produce a clutch of 13 eggs (Roosenburg and Dunham, 1997). Hatchlings possess abdominal yolk stores at hatching which are depleted over the ensuing several weeks (see Rowe et al., 2017). Thus, during emergence and initial dispersal from the nest (when hatchlings are not yet feeding exogenously), the hatchlings are fueled by maternally-derived resources which are eventually depleted, requiring that the individuals develop feeding behaviors and become energetically independent from the mother. Dispersal of hatchling diamondback terrapins is not well-documented. Recent evidence suggests that, in a population in Jamaica Bay, New York, USA, hatchlings disperse a short distance from the nest prior to burrowing into the terrestrial substrate until the following spring or summer (Duncan and Burke, 2016). However, such a strategy has not been reported in other populations of this species. Diamondback terrapins and some other turtles in the northern portions of their range are known to overwinter in the nest itself (e.g. Costanzo et al., 2006, 2008; Pfau and Roosenburg, 2010), but leaving the nest and establishing a new, subterranean overwintering site has only been observed or
E-mail address:
[email protected]. https://doi.org/10.1016/j.zool.2018.03.001 Received 13 October 2017; Received in revised form 21 January 2018; Accepted 6 March 2018 Available online 14 March 2018 0944-2006/ © 2018 Elsevier GmbH. All rights reserved.
Zoology 127 (2018) 20–26
C.L. Rowe
Legler, 1978; Tucker et al., 2007) to induce oviposition. After oviposition, eggs were held for 12 – 16 days at 22 °C to retard development until facilities became available to conduct the study at higher temperature. This initial holding temperature (22 °C) is near or slightly below the minimum recorded in local nests (Jeyasuria et al., 1994; pers. obs.). Considering results from higher temperature incubations, 22° C probably supports very limited development (see the results of Jeyasuria et al., 1994, that demonstrated extremely slow development at 26 °C). Following the initial holding period at 22 °C, eggs were incubated through hatching at 31 °C (a female-producing temperature; Jeyasuria et al., 1994) in a single incubator (Fisher Iso-temp; Fisher Scientific, Asheville, NC). The incubation substrate consisted of a 1.1:1 mix of well water:vermiculite which was misted daily with well water to replace evaporative loss. Upon pipping and hatching, animals were placed in moist Spanish moss for 1 – 3 d at 25 °C until the external portion of the residual yolk had been absorbed. I subsequently held the hatchlings individually in 1 L plastic containers with 50 ml estuarine water (salinity = 16 psu; filtered 0.5 μm; replaced every 2 d) in a 25 °C laboratory on a 12:12 h light cycle with full spectrum + UV lights. Mean temperature of the holding room (logged every 6 h) was 24.8 ± 0.02 °C. Hatchlings were offered chopped clams daily, beginning two weeks after hatching began. Initiation of feeding responses by the hatchlings was interpreted to correspond approximately with depletion of yolk stores provisioned by the female (see Rowe et al., 2017). Average time to initiation of feeding was 18.7 ± 0.7 days posthatching (52.4 ± 4.4 degree days as defined below). Because of the temperature shifts during incubation and housing, I report developmental times in degree days above 22 °C (“DD > 22”), calculated as: [DD > 22 = (calendar day after oviposition)*(incubation or holding temperature − 22) + (# of hatchling assays to date * 36)]. Thus the first 16 days of holding the eggs at 22 °C did not contribute to the total degree day count, which reflected only the time eggs were incubated at 31 °C or hatchlings were held at 25 °C. The final expression in the equation applies only to hatchlings and adjusts the DD > 22 calculations for the pre-assay period (three days) and the assay period (one day) during which hatchlings were held at 31 °C rather than 25 °C (the value 36 in this expression reflects these four days during which temperature was 9 °C above the 22 °C baseline). One egg from each clutch, which was incubated along with the assayed eggs but excluded from assays, was dissected at the time of each egg assay to estimate developmental stages of the embryos. Developmental stages of M. terrapin have not been described. Therefore, stages assigned here (see Table 1) are based on stages described for another emydid turtle, Trachemys scripta, by Greenbaum (2002) and should be considered approximate. Data loggers (Onset Hobo Onset Hobo Pro V2; Onset Corporation, Pocasset, MA) recorded temperatures hourly in the incubators used for incubating eggs and in the incubator holding specimens during SMR assays. Average temperature (daily mean) in the egg incubator during the 22 °C holding period was 22.1 ± 0.3 °C. During the 31 °C egg
suggested to occur in the Jamaica Bay, NY population of M. terrapin (Draud et al., 2004; Muldoon and Burke, 2012; Duncan and Burke, 2016). Observations from other populations of M. terrapin have suggested that the hatchlings disperse into areas where living vegetation or flotsam and matted wrack are present to provide protection (Burger, 1976; Pitler, 1985; Lovich et al., 1991). Regardless of the diamondback terrapins’ post-hatching dispersal strategy, the activities involved must be fueled by energy stores (as residual yolk or somatic tissue) since the animals are not yet feeding at this stage (as suggested by laboratory studies; see Rowe et al., 2017). Somatic stores of triacylglycerol (a primary fuel for reptilian embryonic and neonatal metabolism; Rowe et al., 1995; Thompson and Speake, 2002) can be relatively large in hatchling turtles (Nagle et al., 1998, 2003; Thompson et al. 1999) and could serve as an alternate or supplemental fuel source to residual yolk during the non-feeding, posthatching period (Nagle et al., 2003; Van Dyke et al., 2011). Determining the energetic costs of dispersal and the energetic substrates that fuel it remains a difficult challenge due to a general lack of knowledge of dispersal behaviors and hatchling metabolic rates in diamondback terrapins and most other turtles. A recent study evaluating maternal influences on offspring SMR in M. terrapin unexpectedly revealed that elevated SMR during late embryonic development appeared to be sustained through the early posthatching period when hatchlings were still reliant on yolk, but then declined substantially in older hatchlings that had begun exogenous feeding (assumed to concur approximately with depletion of the residual yolk; Rowe et al., 2017). However, that study (Rowe et al., 2017) was not designed for direct comparisons between embryonic and hatchling SMR, since it evaluated embryonic and hatchling SMR at different temperatures. I conducted the current study to better ascertain the pattern in SMR during embryonic and early hatchling development in M. terrapin. Using animals from four clutches tested at the same temperature during both the embryonic and hatchling periods, I estimated SMR at three intervals during the embryonic period and two intervals after hatching. Here I describe the pattern of SMR throughout development in embryonic and hatchling M. terrapin. I evaluate the results with respect to the role that residual, maternal energy subsidies may play in fueling dispersal, thereby affecting the energetic economy of the individual during its early life history. 2. Materials and Methods This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland Center for Environmental Science. Values given in the text, tables, and figure are expressed as means ± 1 SE. I captured female diamondback terrapins over four days as they came ashore to nest at Naval Air Station Patuxent River, Lexington Park, MD, USA. I transported them to the laboratory where I administered oxytocin via intraperotineal injection (30 IU/kg; Ewert and
Table 1 Sizes of specimens used for assays of metabolic rates. DD > 22 = degree days above 22 °C (see text for calculation), ww = wet weight, CL = carapace length. Embryonic developmental stages are approximate, based upon descriptions of another emydid turtle (Greenbaum, 2002). Values are means (1 SE). N = 3 eggs per clutch except Clutch C during Assay 2 where N = 2 eggs. N = 4 hatchlings per clutch except Clutch C where N = 3 hatchlings. Clutch
A B C D
Eggs (whole)
Size at Hatching
Assay 1 (DD > 22 = 117; Stage 18)
Assay 2 (DD > 22 = 234; Stage 22)
Assay 3 (DD > 22 = 297; Stage 25)
ww (g)
ww (g)
ww (g)
10.06 (0.11) 9.29 (0.37) 11.78 (1.38) 11.75 (0.42)
10.25 (0.07) 9.34 (0.77) 12.30 (0.42) 11.59 (0.70)
10.50 (0.10) 9.64 (0.71) 12.31 (0.59) 11.82 (1.02)
(mean DD > 22 = 387)
ww (g) 7.04 7.11 8.94 7.67
21
Hatchlings
(0.06) (0.54) (0.29) (0.65)
Assay 4 (DD > 22 = 405; Yolk-reliant)
CL (mm) 29.1 28.4 30.5 29.6
(0.1) (0.8) (0.4) (0.5)
ww (g) 6.91 7.12 8.83 7.69
(0.02) (0.58) (0.29) (0.64)
Assay 5 (DD > 22 = 480; Exogenously feeding) ww (g) 6.68 7.62 6.79 8.22
(0.16) (0.69) (0.57) (0.39)
CL (mm) 30.6 31.4 30.4 31.7
(0.2) (0.6) (0.9) (0.5)
Zoology 127 (2018) 20–26
C.L. Rowe
identically. Hatchlings were placed in 800 ml chambers containing brackish water (salinity = 16 psu; filtered to 0.5 μm) to a depth of 1 cm (50 ml) to prevent desiccation of the hatchlings and analyzed at 31 °C following a three day holding period at that temperature. For the final assay of hatchlings, conducted after the animals had reached feeding stage, hatchlings were unfed for the final 48 h prior to the assay to avoid or reduce influences of digestion on oxygen consumption. I weighed eggs or hatchlings (wet weight, ww) after each assay and measured hatchling carapace length (CL) at time of hatching and following the final assay. Prior to analysis, data collected during the first three hours of any assay were deleted to remove acute effects of handling. For eggs, the average VO2 from the remaining measurements was used as an estimate of SMR. To estimate SMR in hatchlings, I calculated the mean of the lowest half of the values (after the first three hours of measurements were discarded) for each hatchling (Rowe et al., 2017). This was intended to exclude any periods of elevated oxygen consumption due to unobserved activity during the assays that would cause overestimation of SMR. The truncated data sets were evaluated by ANCOVA (wet weight = covariate) on per capita rates (μl O2/min) and by ANOVA on mass-adjusted rates (μl O2/g min; calculated as per capita rate/wet mass) to determine effects of clutch, DD > 22, and clutch* DD > 22 on SMR. While analysis of per capita rates by ANCOVA is preferred (e.g. Packard and Boardman, 1988) due to the allometric relationship between body weight and SMR (e.g. Schmidt-Nielsen, 1984), I also report results for mass-adjusted SMR so that my results can be directly compared to other values in the literature that are expressed in that fashion. Tukey’s pairwise comparisons followed significant models. Data satisfied the assumptions of normality (normal plots of residuals) and equivalence of variances (Hartley’s test on average degrees of freedom; Neter et al., 1990) required for parametric analysis without transformation. A level of significance = 0.05 was used in all tests.
incubation period, average daily incubator temperature was 30.9 ± 0.1 °C. Average daily temperature in the incubator used for SMR assays was 31.0 ± 0.3 °C. Standard metabolic rates were measured as oxygen consumption rates (VO2) of eggs or hatchlings. Standard metabolic rate reflects those maintenance and activity costs required for short-term survival. In young, growing animals, SMR measurements may also capture metabolic investments in growth which are difficult to distinguish from maintenance investments (see Parry, 1983; Nagy, 2000; Beaupre and Zaidan, 2001). Furthermore, while exogenously feeding animals can be starved prior to measurement to ensure that they are in a post-absorptive state, younger animals reliant on existing yolk stores cannot be starved (unless yolk is surgically removed; see Van Dyke et al., 2011), and their metabolic rates likely include expenditures associated with yolk digestion and associated anabolic investments in protein synthesis (Parry, 1983; Beaupre and Zaidan, 2001). For example, Beaupre and Zaidan (2001) observed that CO2 production rates (VCO2) were elevated in yolk-reliant neonatal rattlesnakes relative to their predicted values based upon measures of older individuals, reflecting the combined yolk processing costs and investments in anabolism. However, Van Dyke et al. (2011) saw no effect of yolkectomy on VCO2 of hatchling Apalone mutica, which suggests a lack of a significant metabolic elevation associated with yolk processing in that species of turtle. In the current study, my measurements of VO2 inherently included these processes since the animals were young and growing and the embryos and newly hatched animals were digesting maternal provisions of yolk. Thus, it is possible that the values reported here for SMR of yolk-reliant individuals might be inflated somewhat above the specific costs of maintenance if yolk processing and growth during embryonic/early hatchling development present significant costs in M. terrapin. I used 12 eggs from four clutches to obtain measurements of embryonic SMR (VO2). Three eggs each were randomly chosen from three clutches (A, B, and D) and all three eggs from a partial clutch (Clutch C) were used in the assays. I attempted to use the same eggs for all SMR assays, however mortality or suspected poor health of three eggs (evidenced by discoloration, softening, or changes to the general appearance of the shell) prior to the second or third assays required that I substitute alternate eggs from the same clutches. These substitute eggs had been incubated identically to those originally chosen for use in the assays. During the second assay of embryonic SMR, a leak developed in one chamber (containing a Clutch C egg) producing nonsensical data which were excluded from the data set, resulting in 11 observations for SMR during this assay. Oxygen consumption rates of individual eggs were measured following slight modifications from the methods of Rowe et al. (2017). Briefly, eggs were placed in individual, 800 ml respirometry chambers containing Styrofoam packaging material (“peanuts”) which cradled the egg in the upper half of the chamber above the level of well water (40 ml) included to prevent desiccation. The chambers were housed in a dark, 31 °C incubator and connected to a computer-controlled, closed circuit, micro-respirometer (Micro Oxymax™, Columbus Instruments, Columbus, Ohio, USA) which measured oxygen consumption at ∼1 h intervals over 24 h. To prevent hypoxic conditions from forming in the sealed respirometry chambers, the respirometer was configured to automatically refresh the chambers with ambient air if O2 concentration declined more than 0.5 % during a sampling interval. A standard (a discharging 8.4 volt medical grade battery that consumed O2 at a constant rate; Duracell Procell™ Zinc-air, # M1342) was included in each assay. The micro-respirometer was calibrated prior to each assay with a verified gas mixture. Oxygen consumption of hatchlings was measured using the same instrument and configuration as was used for eggs. However I used 15 hatchlings in each assay (four each from Clutches A, B, D and three from Clutch C) which consisted of the same pool of individuals used for the egg assays plus an additional hatchling randomly selected from the pool of hatchlings from Clutches A, B, and D that had been incubated
3. Results Sizes and approximate developmental stages at the time of each assay are presented in Table 1. Mean hatching date was ∼387 degree days, corresponding to 55 calendar days of incubation (12 d at 22 °C and 43 d at 31 °C). The pattern in SMR versus time after oviposition was broadly peaked in shape such that maximum per capita SMR occurred from mid to late in the embryonic period to shortly after hatching (Fig. 1). After residual yolk had presumably been depleted and animals began to feed (between SMR Assays 4 and 5) hatchling SMR declined substantially; per capita SMR of yolk-reliant hatchlings (Assay 4, mean = 15.89 μl O2 consumed/min) was 135 % higher relative to SMR of feeding-stage animals (Assay 5, mean = 6.77 μl O2 consumed/min). For mass-adjusted values, this difference was 122 % (2.09 μl/g min vs 0.94 μl/g min). A statistical main effect of DD > 22 on per capita rates resulted from SMR during Assay 1 (stage 18 embryos) and Assay 5 (feeding-stage hatchlings) being significantly lower than SMR during Assays 2 (stage 22 embryos), 3 (stage 25 embryos), and 4 (yolk-reliant hatchlings) and Assay 1 being lower than Assay 5 (Table 2, Fig. 1). Per capita SMR was highest in Assays 3 and 4, but Assay 4 did not differ from Assay 2 (Fig. 1). A main effect of DD > 22 was also present in massadjusted rates, with the pattern in SMR being Assay 1 < Assay 5 < Assay 2 < Assay 3 = Assay 4 (Table 2, Fig. 1). Main effects of clutch were present such that SMR followed the pattern Clutch D = Clutch B < Clutch A = Clutch C for per capita and mass-adjusted rates (Table 2, Fig. 1). Significant interactions between clutch and DD > 22 reflected significant clutch effects during Assays 2–4 that were not present initially (Assay 1) and had mostly converged after feeding began (prior to Assay 5; Table 2, Fig. 1). 4. Discussion There are three described patterns in per capita metabolic rate 22
Zoology 127 (2018) 20–26
C.L. Rowe
during embryonic development in reptiles: 1. an exponential increase (observed in snakes, similar to altricial birds), 2. a sigmoidal pattern (in lizards and some turtles, similar to most precocial birds), and 3. a peaked pattern in which a maximum is reached prior to hatching followed by a decline (in many crocodilians and turtles, similar to precocial ratite birds; see Vleck et al., 1979, 1980; Thompson, 1989; Peterson and Kruegl, 2005; Reid et al., 2009). The vast majority of studies of patterns of SMR during embryonic development of turtles describe the latter, peaked pattern (see Lynn and Von Brand, 1945; Ackerman, 1981; Gettinger et al., 1984; Webb et al., 1986; Thompson, 1989; Whitehead and Seymour, 1990; Leshem et al., 1991; Birchard and Reiber, 1995; Booth, 1998a,b; Booth and Astill, 2001; Peterson and Kruegl, 2005; Reid et al., 2009; Ligon and Lovern, 2012). Based on scant data and interpolation of graphs from published sources, Peterson and Kruegl (2005) hypothesized that, unlike most groups of turtles displaying a peaked pattern in metabolic rate during embryonic development, emydid turtles display a sigmoidal “plateau” pattern in in which a maximum is reached prior to hatching and maintained through hatching. However, this prediction was not borne out by their results, which showed a peaked pattern in which metabolic rate declined prior to hatching in the emydid turtle (Chrysemys picta; Peterson and Kruegl, 2005). The emydid turtle that I studied (M. terrapin) displayed a pattern in per capita SMR that appears as a broad peak; in mid to late embryonic development, per capita SMR reached a maximum which was sustained through the early post-hatching period while the embryos were reliant on residual yolk stores, but then declined substantially after exogenous feeding had begun (Fig. 1). While it appears that there was some decline in SMR between the final egg assay (Assay 3) and the first hatchling assay (yolk-reliant hatchlings, Assay 4; see Fig. 1), the values for these assays did not differ statistically (Fig. 1). When evaluated as mass-adjusted rates, my results display a similar pattern in which SMR in Assays 3 and 4 were the highest and did not differ statistically (Fig. 1). However, as noted below, the use of massadjusted rates can be quite misleading as contributions to total wet mass from metabolically inactive tissues as well as reductions in total mass at hatching will bias the observed pattern in SMR. Early in development (DD > 22 117), when embryos were still relatively small (approximately stage 18; Greenbaum, 2002), SMR was relatively low. As incubation continued, SMR increased, likely reflecting the increased size and complexity of the embryos (see Ackerman, 1981; Birchard and Reiber, 1995). Shortly after hatching (DD > 22 405), when hatchlings were still reliant on stored yolk, SMR remained about as high as it was during the mid to late embryonic period (DD > 22 297). Thus maximum SMR of these early life stages was experienced from mid embryonic development through the early posthatching period, prior to depletion of yolk stores by the hatchlings (Fig. 1). This is roughly similar to the pattern observed in three species of turtles by Lynn and Von Brand (1945) who noted that “young hatched turtles” had similar respiration rates as late-stage embryos. Gettinger et al. (1984) reported that respiration rates plateaued late in embryonic development in two turtle species. However Gettinger et al. (1984) did not assay animals after hatching so it is not possible to determine if or when SMR subsequently declined after hatching. After the diamondback terrapin hatchlings had begun feeding exogenously, their SMR dropped to levels lower than those observed for all but the first measurement of SMR (stage 18 embryos). This is consistent with observations by Rowe et al. (2017) who suggested that the higher SMRs measured in yolk-reliant individuals may have been elevated by costs associated with digesting/metabolizing yolk. A similar decline in SMR after hatching was observed by Van Dyke et al. (2011) for A. mutica. In A. mutica, VCO2 peaked 10 d before hatching and then declined throughout the remaining embryonic period and the posthatching period (measured for 40 d post-hatching; Van Dyke et al., 2011). Yolkectomy did not affect post-hatching growth in A. mutica, suggesting that yolk was not a critical substrate for fueling anabolic processes (Van Dyke et al., 2011). The yolk did not appear to
Fig. 1. Per capita (top panel) and mass-adjusted (bottom panel) oxygen consumption rates by embryonic and hatchling diamondback terrapins from four clutches measured at 31 °C. The vertical dashed line indicates mean hatching date (degree day 387) and the vertical dotted line indicates the mean date of initiation of feeding activity (degree day 450). Calculation of degree days is described in the text. Results of statistical comparisons of assay date versus oxygen consumption are shown; dates that do not contain the same lower case letter differed statistically at α = 0.05. Values are means ± 1 SE. Table 2 Statistical results for the effects of degree days above 22 °C (DD > 22), clutch, and their interaction on per capita (μl O2/min) and mass-adjusted (μl O2/g min) standard metabolic rates in embryos and hatchlings of the diamondback terrapin. Per capita rates were analyzed by ANCOVA (wet mass = covariate) and mass-adjusted rates were analyzed by ANOVA.
Per capita rates Wet mass DD > 22 Clutch DD > 22*Clutch Error Total
df
SS
MS
F
p
1 4 3 12 44 64
14.19 2435.38 115.16 163.94 175.07 3463.00
14.19 608.84 38.39 13.66 3.43
3.57 153.02 9.65 3.43
0.066 < 0.001 < 0.001 0.001
df 4 3 12 45 64
SS 26.68 1.01 1.08 1.98 33.14
MS 6.67 0.34 0.09 0.04
F 151.40 7.61 2.04
p < 0.001 < 0.001 0.042
Mass-adjusted rates DD > 22 Clutch DD > 22*Clutch Error Total
23
Zoology 127 (2018) 20–26
C.L. Rowe
was not measured during the fourth assay when still yolk-reliant. The large decline in SMR after reaching feeding stage might suggest an energy-saving strategy employed by M. terrapin hatchlings when yolk subsidies from the mother have been exhausted. A switch from a lifestyle exclusive of feeding activities during the early post-hatching period when reliant on endogenous resources to a lifestyle in which exogenous resources must be located and harvested prior to assimilation would present additional energetic costs to the individual in the form of respiratory expenditures in support of activity (see Schekkerman and Visser, 2001). Furthermore, yolk-reliant hatchlings are ensured a constant provision of energy, whereas later when actively foraging they are more likely faced with limits to resource availability. Thus there are considerable energetic constraints that develop upon achieving energetic independence from the mother following yolk depletion. From the view of resource allocation-based life history theory, it may be advantageous to reduce metabolic energy expenditures when the environment places limits on the availability of resources or when costs of living are high (e.g. Congdon et al., 2001). The observed reduction in metabolic energy expenditure associated with the switch from endogenous resources (yolk) to live prey that likely require substantial investments in the form of activity (foraging) costs is congruent with such predictions. The pre-feeding period during which hatchling terrapins are reliant upon residual yolk stores is coincident with the period that the hatchlings would be emerging and dispersing from the nests. Thus, elevated SMR of hatchlings while reliant on yolk relative to that after reaching feeding stage could prove advantageous to the animals in fueling dispersal from the nesting site. As noted above, Van Dyke et al. (2011) suggested that emergence from the nest was not fueled by yolk in A. mutica, although the energetically-intensive, post-emergence dispersal period was not evaluated. A high SMR could facilitate activity (Taigen, 1983; Bennett, 1980), and thus dispersal distance or speed, which presumably would reduce risks of predation (e.g. Wilbur and Morin, 1988). Indeed, high SMR facilitating an active lifestyle has been considered a driver of the evolution of endothermy (discussed in Bennett, 1980; Taigen, 1983; Gebczynski and Konarzewski, 2009). It is possible that the elevated SMR that I observed during the first few weeks after hatching may be advantageous in supporting high rates of activity, but after the initial period of dispersal the high SMR would become an energetic liability, favoring a strategy that reduces SMR after the dispersal period. A better understanding of the dispersal dynamics of hatchling terrapins, which remains elusive due to conflicting observations and hypotheses (e.g. Burger, 1976; Pitler, 1985; Lovich et al., 1991; Muldoon and Burke, 2012; Duncan and Burke, 2016) and limits to tracking technology, is needed to more rigorously evaluate the energetic correlates that I propose. For example, observations from a population in New York, USA, indicating that hatchlings disperse from the nest and then excavate subterranean refuges for overwintering (Duncan and Burke, 2016) suggest additional energetically-intensive behaviors during dispersal beyond those associated only with lateral movement through the habitat. Fuel for these activities must derive either from somatic reserves (such as triacylglycerol; Rowe et al., 1995; Nagle et al., 2003) or residual maternal subsidies in the form of yolk, assuming the hatchlings have not reached feeding stage by the time of burial. Mass-adjusted estimates of SMR reported here should be viewed with caution, as they reflect the total wet mass of the egg or hatchling. With respect to the eggs, non-metabolized or metabolically inactive components such as shell, shell membranes, yolk, or water would have contributed to the total mass, having an overall reducing effect on the estimate of mass-adjusted SMR of the embryo itself. Thus the true, mass-adjusted SMR of the embryos was somewhat higher than that shown in Fig. 1. On the other hand, estimates of per capita SMR did not consider animal mass explicitly and should not have been affected by metabolically inactive materials present in the egg. This may explain the slight difference in the observed patterns of SMR versus time
significantly support the initial emergence activity of hatchling A. mutica either, as yolk mass did not correlate with time required for emergence from the nest (Van Dyke et al., 2011). The role of the yolk in fueling post-emergence dispersal activities was not evaluated by Van Dyke et al. (2011). Assuming that the onset of exogenous feeding corresponds approximately with depletion of the residual yolk, all SMR measurements except the final one took place while the terrapins were reliant on maternally-contributed energy in the form of yolk. After beginning to feed and achieving independence from the mother's energetic influence, SMR declined substantially to less than half that of yolk-reliant hatchlings. A similar pattern was observed in an earlier study with this species in which exogenously feeding hatchlings had reduced SMR relative to earlier in development when still reliant on yolk (Rowe et al., 2017). The decline in metabolic expenditure after the onset of exogenous feeding could in part have been an artefact of comparing results from animals that were metabolizing yolk stores (thus influenced by costs of digestion, or Specific Dynamic Action, “SDA”; McCue, 2006) with animals that had achieved feeding stage but were unfed prior to and during the assays (e.g. Klaassen et al., 1987; Beaupre and Zaidan, 2001; Rowe et al., 2017). In another emydid turtle (Trachemys scripta elegans), SDA by hatchlings fed animal flesh (shrimp, mealworms) elevated SMR above the pre-feeding rate by about at 40 − 50 % (Pan et al., 2004). I observed a 135 % elevation in mean, per capita SMR of M. terrapin hatchlings when comparing SMR of unfed individuals that had reached feeding stage (final assay: mean across clutches = 7.74 μl O2 consumed/min) to the higher SMR of younger, yolk-reliant individuals (fourth assay: 18.25 μl O2 consumed/min). The magnitude of the difference in SMR between yolk-reliant and feeding-stage hatchlings observed here was thus considerably larger than that attributed to SDA by Pan et al. (2004) for T. s. elegans. Estimates of SDA Scope (postprandial SMR/preprandial SMR; McCue, 2006) for turtles from several families reviewed in McCue (2006) ranged from 1.4 to 2.7, with that for the emydid T. scripta elegans being 1.5 (Pan et al., 2004; McCue, 2006). The SDA Scope calculated for my data (as yolk-reliant per capita SMR/yolkindependent per capita SMR) is 2.35, considerably greater than that for the emydid T. s. elegans, but within the range of values observed in other turtle families (Pan et al., 2004; McCue, 2006). The dearth of studies of SDA in emydid turtles (see McCue, 2006) precludes generalizing about costs of SDA in this group. Thus, while evaluating my results in the light of limited, previous work on SDA is difficult, the large differences in SMR I observed between yolk-reliant and feeding-stage animals relative to the estimates of SDA provided by Pan et al. (2004) for the emydid T. s. elegans seem to suggest that SDA was not wholly responsible for the high SMR of yolk-reliant hatchling M. terrapin. Furthermore, in the smooth softshell turtle (A. mutica), yolkectomy did not influence SMR of hatchlings, suggesting that processing of residual yolk does not induce an SDA-like response in that species (Van Dyke et al., 2011). As noted above, growth itself requires metabolic investment (e.g. Parry, 1983), and the respiratory signature of this investment may be in part responsible for the SDA response (Parry, 1983; Brown and Cameron, 1991). The growth investment also may extend beyond the period of elevated respiration rates attributed to SDA in exogenously feeding animals, possibly confounding measures of SDA with investments in growth (Parry, 1983). It is possible that elevated growth investments were captured in my measurements of respiration in yolk-reliant individuals that were not observed in the exogenously feeding ones (e.g. Beaupre and Zaidan, 2001; Peterson and Kruegl, 2005). This would suggest elevated growth during the early post-hatching period relative to later, after yolk had been catabolized, which is congruent with the pattern observed in neonate rattlesnakes (Beaupre and Zaidan, 2001), but which is not borne out by hatchling wet weights here (Table 1). Measurements of carapace length (CL) at hatching and after the final assay indicate an increase in average dimensional size in ¾ of the clutches during this period (Table 1), but CL 24
Zoology 127 (2018) 20–26
C.L. Rowe
between per capita and mass-adjusted values (Fig. 1); since total wet weight declined from egg to hatchling, the mass-adjusted values for hatchlings are artificially inflated by the inclusion of the mass term in the denominator. Consistent with results from a prior study (Rowe et al., 2017), there was a significant effect of clutch on SMR of embryos and hatchlings prior to achieving feeding stage, yet this this clutch effect largely disappeared and SMR converged by the final assay of feeding-stage animals (Fig. 1). Rowe et al. (2017) suggested that this clutch effect likely has little significance to individual fitness due to its transience, and may reflect differences among females in the yolk components allocated to the eggs during vitellogenesis. During the late embryonic and early post-hatching period, diamondback terrapins experienced high SMR fueled by maternal energy contributions. When exogenous feeding began and maternally-derived stores were presumably depleted, SMR declined substantially. This pattern suggests an energy conservation strategy in which metabolic expenditures decline when maternally-provisioned energetic resources are no longer available. Furthermore, the high SMR coincident with the period of dispersal from the nest may be advantageous in that it allows for increased activity that could not be accomplished with less metabolic support (e.g. Nagle et al., 2003). It is also possible that the residual yolk is simply a result of females overprovisioning their eggs with fuel and has little significance to the hatchlings (e.g. Van Dyke et al., 2011). On the other hand, in species in which active dispersal follows emergence, the presence of residual yolk could be a valuable asset for fueling activity, especially in resource-poor habitats (Nagle et al., 2003). This line of reasoning suggests that hatchling terrapins that retain relatively high stores of residual yolk might be better equipped for the initial dispersal period and experience a survival advantage during this energetically-intensive period over individuals that retain less yolk after hatching. If so, residual yolk stores may represent a critical component of maternal energy allocation and have important functional significance for hatchling diamondback terrapins. This hypothesis could be definitively evaluated via yolkectomy and characterization of hatchlings’ capacity for, and metabolic expenditures during, activities associated with dispersal from the nesting site.
Brown, C.R., Cameron, J.N., 1991. The relationship between specific dynamic action (SDA) and protein synthesis rates in the channel catfish. Physiol. Zool. 64, 298–309. Burger, J., 1976. Behavior of hatchling diamondback terrapins (Malaclemys terrapin) in the field. Copeia 1976, 742–748. Congdon, J.D., Dunham, A.E., Hopkins, W.A., Rowe, C.L., Hinton, T.G., 2001. Resource allocation based life history trait values: A conceptual basis for studies of environmental toxicology. Environ. Toxicol. Chem. 20, 1698–1703. Costanzo, J.P., Baker, P.J., Lee, R.E., 2006. Physiological responses to freezing in hatchlings of freeze-tolerant and-intolerant turtles. J. Comp. Physiol. B 176, 697–707. Costanzo, J.P., Lee, R.E., Ultsch, G.R., 2008. Physiological ecology of overwintering in hatchling turtles. J. Exper. Zool. A 309A, 297–379. Draud, M., Bossert, M., Zimnavoda, S., 2004. Predation on hatchling and juvenile diamondback terrapins (Malaclemys terrapin) by the Norway rat (Rattus norvegicus). J. Herpetol. 38, 467–470. Duncan, N.P., Burke, R.L., 2016. Dispersal of newly emerged diamond-backed terrapin (Malaclemys terrapin) hatchlings at Jamaica Bay, New York. Chelonian Conserv. Biol. 15, 249–256. Ewert, M.A., Legler, J.M., 1978. Hormonal induction of oviposition in turtles. Herpetologica 34, 314–318. Gebczynski, A.K., Konarzewski, M., 2009. Locomotor activity of mice divergently selected for basal metabolic rate: a test of hypotheses on the evolution of endothermy. J. Evol. Biol. 22, 1212–1220. Gettinger, R.D., Paukstis, G.L., Gutzke, W.H.N., 1984. Influence of hydric environment on oxygen consumption by embryonic turtles Chelydra serpentina and Trionyx spiniferus. Physiol. Zool. 57, 468–473. Greenbaum, E., 2002. A standardized series of embryonic stages for the emydid turtle Trachemys scripta. Can J. Zool. 80, 1350–1370. Klaassen, M., Guri, S., Bech, C., 1987. Metabolic rate and thermostability in relation to availability of yolk in hatchlings of black-legged kittiwake and domestic chicken. Auk 104, 787–789. Leshem, A., Ar, A., Ackerman, R.A., 1991. Growth, water, and energy metabolism of the soft-shelled turtle (Trionyx triunguis) embryo: Effects of temperature. Physiol. Zool. 64, 568–594. Ligon, D.B., Lovern, M.B., 2012. Interspecific variation in temperature effects on embryonic metabolism and development in turtles. ISRN Zoology 2012, 13. http://dx. doi.org/10.5402/2012/846136. Lovich, J.E., Tucker, A.D., Kling, D.E., Gibbons, J.W., 1991. Behavior of hatchling diamond-back terrapins Malaclemys terrapin released in a South Carolina USA salt marsh. Herpetol. Rev. 22, 81–83. Lynn, W.G., Von Brand, T., 1945. Studies on the oxygen consumption and water metabolism of turtle embryos. Biol. Bull. 88, 112–125. McCue, M.D., 2006. Specific dynamic action: A century of investigation. Comp. Biochem. Physiol. A. 144, 381–394. Muldoon, K.A., Burke, R.L., 2012. Movements, overwintering, and mortality of hatchling diamond-backed terrapins (Malaclemys terrapin) at Jamaica Bay, New York. Can. J. Zool. 90, 651–662. Nagle, R.D., Burke, V.J., Congdon, J.D., 1998. Egg components and hatchling lipid reserves: Parental investment in kinosternid turtles from the southeastern United States. Comp. Biochem. Physiol. B 120, 145–152. Nagle, R.D., Plummer, M.V., Congdon, J.D., Fischer, R.U., 2003. Parental investment, embryo growth, and hatchling lipid reserves in softshell turtles (Apalone mutica) from Arkansas. Herpetologica 59, 145–154. Nagy, K.A., 2000. Energy costs of growth in neonate reptiles. Herpetol. Monogr. 14, 378–387. Neter, J., Wasserman, W., Kutner, M.H., 1990. Applied Linear Statistical Models: Regression, Analysis of Variance, and Experimental Designs, Third Edition. Richard D. Irwin, Inc., Boston. Packard, G.C., Boardman, T.J., 1988. The misuse of ratios, indices, and percentages in ecophysiological research. Physiol. Zool. 61, 1–9. Pan, Z.-C., Ji, X., Lu, H.-L., 2004. Influence of specific dynamic action of feeding in hatchling red-eared slider turtles Trachemys scripta elegans. Acta Zool. Sinica 50, 459–463. Parry, G.D., 1983. The influence of the cost of growth on ectotherm metabolism. J. Theor. Biol. 101, 453–477. Peterson, C.C., Kruegl, A., 2005. Peaked temporal pattern of embryonic metabolism in an emydid turtle (Chrysemys picta picta). J. Herpetol. 39, 678–681. Pfau, B., Roosenburg, W.M., 2010. Diamondback terrapins in Maryland: research and conservation. Radiata 19, 2–34. Pitler, R., 1985. Life history notes. Malaclemys terrapin terrapin (northern diamondback terrapin), Behavior. Herpetol. Rev. 16, 82. Reid, K.A., Margaritoulis, D., Speakman, J.R., 2009. Incubation temperature and energy expenditure during development in loggerhead sea turtle embryos. J. Exper. Mar. Biol. Ecol. 378, 62–68. Roosenburg, W.M., Dunham, A.E., 1997. Allocation of reproductive output: Egg- and clutch-size variation in the diamondback terrapin. Copeia 1997, 290–297. Rowe, J.W., Holy, L., Ballinger, R.E., Stanley-Samuelson, D., 1995. Lipid provisioning of turtle eggs and hatchlings − total lipid phospholipid, triacylglycerol and triacylglycerol fatty acids. Comp. Biochem. Physiol. B 112, 323–330. Rowe, C.L., Woodland, R.J., Funck, S.A., 2017. Metabolic rates are elevated and influenced by maternal identity during the early, yolk-dependent, post-hatching period in an estuarine turtle, the diamondback terrapin (Malaclemys terrapin). Comp. Biochem. Physiol. A 204, 137–145. Schekkerman, H., Visser, G.H., 2001. Prefledging energy requirements in shorebirds: Energetic implications of self-feeding precocial development. Auk 118, 944–957. Schmidt-Nielsen, K., 1984. Scaling: Why is Animal Size so Important? University of Cambridge, New York.
Acknowledgements E. Crandall and R. Woodland reviewed drafts of this manuscript and E. Crandall also helped in the field and lab. K. Rambo provided access to the field collection site at Naval Air Station Patuxent River. This project was facilitated by a charitable donation to my laboratory from Mrs. G. Lane and I dedicate this article to her memory with sincere gratitude. This is contribution 5442 from the University of Maryland Center for Environmental Science. References Ackerman, R.A., 1981. Growth and gas exchange of embryonic sea turtles (Chelonia, Caretta). Copeia 1981, 757–765. Beaupre, S.J., Zaidan, F., 2001. Scaling of CO2 production in the timber rattlesnake (Crotalus horridus), with comments on cost of growth in neonates and comparative patterns. Physiol. Biochem. Zool. 74, 757–768. Bennett, A.F., 1980. The metabolic foundations of vertebrate behavior. Bioscience 30, 452–456. Bennett, A.F., Dawson, W.R., 1976. Metbolism. In: In: Gans, C., Dawson, W.R. (Eds.), Biology of the Reptilia, vol. 7. Academic Press, New York, pp. 127–223. Birchard, G.F., Reiber, C.L., 1995. Growth, metabolism, and chorioallantoic vascular density of developing snapping turtles (Chelydra serpentina): Influence of temperature. Physiol. Zool. 68, 799–811. Booth, D.T., 1998a. Effects of incubation temperature on the energetics of embryonic development and hatchling morphology in the Brisbane river turtle Emydura signata. J. Comp. Physiol. B 168, 399–404. Booth, D.T., 1998b. Incubation of turtle eggs at different temperatures: Do embryos compensate for temperature during development? Physiol. Zool. 71, 23–26. Booth, D.T., Astill, K., 2001. Incubation temperature, energy expenditure and hatchling size in the green turtle (Chelonia mydas), a species with temperature-sensitive sex determination. Aus. J. Zool. 49, 389–396.
25
Zoology 127 (2018) 20–26
C.L. Rowe Taigen, T.L., 1983. Activity metabolism of anuran amphibians − implications for the origin of endothermy. Am. Nat. 121, 94–109. Thompson, M.B., 1989. Patterns of metabolism in embryonic reptiles. Respir. Physiol. 76, 243–256. Thompson, M.B., Speake, B.K., Russell, K.J., McCartney, R.J., Surai, P.F., 1999. Changes in fatty acid profiles and in protein ion and energy contents of eggs of the Murray short-necked turtle, Emydura macquarii (Chelonia, Pleurodira) during development. Comp. Biochem. Physiol. A 122, 75–84. Thompson, M.B., Speake, B.K., 2002. Energy and nutrient utilisation by embryonic reptiles. Comp. Biochem. Physiol. A 133, 529–538. Tucker, J.K., Thomas, D.L., Rose, J., 2007. Oxytocin dosage in turtles. Chelonian Conserv. Biol. 6, 321–324. Van Dyke, J.U., Plummer, M.V., Beaupre, S.J., 2011. Residual yolk energetics and
postnatal shell growth in smooth softshell turtles Apalone mutica. Comp. Biochem. Physiol. A 158, 37–46. Vleck, C.M., Hoyt, D.F., Vleck, D., 1979. Metabolism of avian embryos: Patterns in altricial and precocial birds. Physiol. Zool. 52, 363–377. Vleck, C.M., Vleck, D., Hoyt, D.F., 1980. Patterns of metabolism and growth in avian embryos. Am. Zool. 20, 405–416. Webb, G.J.W., Choquenot, D., Whitehead, P.J., 1986. Nests, eggs, and embryonic development of Carettochelys insculpta (Chelonia: Carettochelidae) from Northern Australia. J. Zool. 1, 521–550. Whitehead, P.J., Seymour, R.S., 1990. Patterns of metabolic rate in embryonic crocodilians Crocodylus johnstoni and Crocodylus porosus. Physiol. Zool. 63, 334–352. Wilbur, H.M., Morin, P.J., 1988. Life History Evolution in Turtles. In: In: Gans, C., Huey, R.B. (Eds.), Biology of the Reptilia, vol. 16 Alan R. Liss, New York pp. 387–439.
26
