The effect of egg size and nutrient content on larval performance: implications to protracted spawning in northern pike (Esox lucius Linnaeus)

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1 Hydrobiologia (2008) 601:71 82 DOI /s y INTERNATIONAL PIKE SYMPOSIUM The effect of egg size and nutrient content on larval performance: implications to protracted spawning in northern pike (Esox lucius Linnaeus) Brent A. Murry Æ John M. Farrell Æ Kimberly L. Schulz Æ Mark A. Teece Ó Springer Science+Business Media B.V Abstract Variation in developmental rate from fertilization to swim-up, and body size at swim-up, may affect the growth and survival of young-of-theyear fish. Fish egg size (diameter) is often positively correlated to adult female size, but whether increased egg size equates to higher egg nutrient content and subsequently improved embryo/larval performance, remains unclear. Artificially fertilized northern pike eggs from individual females (total length mm) were cultured under temperature controlled Guest editors: J. M. Farrell, C. Skov, M. Mingelbier, T. Margenau & J. E. Cooper International Pike Symposium: Merging Knowledge of Ecology, Biology, and Management for a Circumpolar Species B. A. Murry (&) J. M. Farrell K. L. Schulz Department of Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Illick Hall, 1 Forestry Drive, Syracuse, NY 13210, USA brent.murry@gmail.com Present Address: B. A. Murry Department of Biology, Central Michigan University, 302 Brooks Hall, Mount Pleasant, MI 48859, USA M. A. Teece Department of Chemistry, Jahn Laboratory, State University of New York, College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA laboratory conditions to test the hypothesis that female body size positively influences egg size and the amount of specific nutrients (carbon, nitrogen, phosphorus, and fatty acids) allocated to eggs. We further hypothesized that greater egg nutrient content would positively influence egg survival, developmental rate, and the size of swim-up fry. These hypotheses were investigated in the context of two different northern pike spawning strategies (early season tributary vs. late season deep shoal spawning). Nutrients were allocated conservatively in northern pike eggs, showing very little variation in nutrient concentrations, but the total mass of all egg nutrients increased linearly with egg dry mass. Neither egg dry mass nor nutrient content (concentration or mass), were related to either egg diameter or female body size. The mass of individual egg nutrients was, however, strongly correlated with egg dry mass (r 2 range 0.62 to 0.99 for individual nutrients) and positively related to the total length of swim-up fry (r 2 = 0.58). The eggs of late spawning pike had significantly greater dry mass (average = 3.02 mg/ egg) and developed more rapidly to swim-up (average = days) than did those of early spawners (average = 2.28 mg/egg, days). Our results indicate that egg quality may be best assessed by egg dry mass, which was correlated with greater nutrient mass and increased swim-up fry body length, as opposed to egg diameter or female body size which showed no correlation to egg nutrient composition and egg/larval performance.

2 72 Hydrobiologia (2008) 601:71 82 Keywords Early life history Development Stoichiometry Fatty acids Introduction The well-documented protracted spawning of northern pike in the upper St. Lawrence River (Farrell et al., 1996, 2006; Farrell, 2001) provides a unique opportunity to examine how variation in egg size and egg nutrient content affects early life development and larval performance across a temporal spawning gradient. Traditionally, upper St. Lawrence River northern pike spawned in early April just after ice-out in flooded coastal tributaries. The majority of spawning now, however, occurs in late May on main river shoals, potentially related to recent habitat changes (Farrell et al., 1996, 2006; Farrell, 2001). Regardless of time and location northern pike spawn at water temperatures between 6 and 14 C (Frost & Kipling, 1967; Farrell, 2001). The temporal protraction occurs because the inland tributaries warm weeks ahead of the main river. Differences in spawning time might influence egg nutrient content through variation in prey availability and timing of endogenous allocation of nutrients to eggs by individual females. Early spawning fish must rely on over-winter (under ice) foraging to supply the necessary raw ingredients for egg allocation, whereas females that delay spawning might benefit by access to prey aggregated in the nearshore for spawning. Further, differences in egg nutrient content are hypothesized to be related to egg size and have a direct influence on egg and larval development. While there were no data on northern pike egg mass found in the literature, typical egg diameters range from 1.5 to 3.1 mm in wild populations (Frost & Kipling, 1967; Chauveheld & Billard, 1983; Farrell et al., 1996). Several authors have described a positive relationship between egg diameter and maternal length (Wright & Shoesmith, 1988; Billard, 1996), but Billard (1996) found that this relationship is highly variable and may be related to female condition, prey availability, and northern pike population structure. Further, we found no studies that linked egg size to embryo/ larval performance for northern pike. The growth and development of fish eggs and embryos are dependent on maternally derived yolk nutrients and energy stores. The nutrient content (i.e., mass and concentration of carbon, nitrogen, phosphorus, and fatty acids) of eggs likely has a substantial effect on both sizes at swim-up and developmental time, but has received little attention as a potential factor related to survival during the critical period. Ashton et al. (1993) showed that developmental time from fertilization to initial exogenous feeding in Chinook salmon (Oncorhynchus tshawytscha Walbaum) is influenced by ambient water temperature and maternally derived nutrient resources in the egg. The effect of temperature on egg and larval developmental time has been studied for northern pike (e.g., Hokanson et al., 1973; Hassler, 1982; Cooper, 2000; Farrell et al. 2006), however, the variation and overall effect of egg size and nutrient content are less well understood. Elements such as carbon, nitrogen, and phosphorus are important to egg and larvae development as major constituents of protein, lipids, and nucleic acids (Sterner & Elser, 2002). Recent research on the role of lipids and especially polyunsaturated fatty acids (PUFA) has shown them to be major egg yolk constituents that have a profound effect on egg and larval development (Ashton et al., 1993; March, 1993; Sargent et al., 1999a, b). The PUFA of primary importance in fish are the long-chain (C 20 C 22 ) omega (x)-3 fatty acids eicosapentaenoic acid (EPA 20:5x- 3), docosahexaenoic acid (DHA 22:6x-3), and the omega (x)-6 arachidonic acid (ARA 20:4x-6) (Ashton et al., 1993; March 1993; Bell & Sargent, 2003). For instance, Czesny & Dabrowski (1998) found a positive correlation between the DHA content and survival of walleye (Sander vitreum Mitchill) embryos. Collectively, PUFA aid in maintaining proper cell membrane structure and are precursors to a group of important hormones collectively known as eicosanoids (prostaglandins and leukotrienes, Sargent et al., 1999a, b). Egg nutrient content is determined not only by mass, but also the relative concentration of nutrients (i.e., C, N, P, and fatty acids). Sargent et al. (1999a, b) discuss the importance of ratios in analyzing fatty acids due to the competitive nature of individual fatty acids in biochemical reactions. For instance, an overabundance of EPA and DHA relative to ARA (and vice versa) can have serious implications to the health of fish, due to eicosanoid hormone regulation. Similarly, Sterner & Elser (2002) have elaborated extensively on the importance of specific ratios of carbon, nitrogen, and phosphorus in affecting the growth of all types of aquatic life. The molar ratio of nutrients within an organism (ecological

3 Hydrobiologia (2008) 601: stoichiometry, Elser et al., 1996) might be used to determine the limiting nutrient for an organism or system process. Generally, phosphorus is considered the major limiting nutrient in freshwater systems. However, at least two studies (Müller-Navarra, 1995; Ballentyne et al., 2003) extended elemental stoichiomentry to include fatty acids (EPA & DHA) and found that growth of Daphnia and sockeye salmon (Oncorhynchus nerka Walbaum) could be limited by fatty acids, when other factors were sufficient. Likely there is no one specific limiting nutrient in nature; rather the limiting nutrient will depend on changing environmental conditions within a given system (Sterner & Schulz, 1998). This concept can be applied to fish eggs as well, as their nutrient composition reflects the female diet, female physiological condition, and surrounding environmental conditions. While the aforementioned studies have illustrated the importance of nutrients and specifically dietary fatty acids to egg/larval development, the question of how variation in egg nutrient content affects larval developmental rate and size has not been addressed for northern pike. We hypothesized that northern pike would exhibit a positive relationship between female body size and egg size (diameter and mass), that larger eggs would have a greater nutrient content, and that egg and larval developmental rate, survival, and size at swimup are positively related to the mass of egg nutrients (i.e., carbon, nitrogen, phosphorus, and fatty acids). Further, due to potential differences in foraging opportunities, we hypothesized that later spawning fish would have greater overall egg nutrient content than early spawners. Our objectives were to first, determine the relationship between female body size and egg size (diameter and dry weight), second evaluate whether egg nutrient content increases with egg size, and finally, to determine if egg/larval developmental rate, final size at swim-up, and survival increase with egg size and/or egg nutrient content. We examined the effect of maternal size on egg quality and larval performance (i.e., survival, developmental rate, and size at swim-up) at the onset and end of the spawning season to determine if observed patterns in egg size, egg nutrient content, and development differed at opposing ends of the reproductive strategy gradient (early shallow tributaries vs. late deepwater spawning; Farrell, 2001; Farrell et al., 2006). Methods Early spawning female northern pike were captured in trapnets set in Delaney Bay on the northeast end of Grindstone Island in the Thousand Islands Region of the St. Lawrence River (Jefferson County, NY, USA) from April 13th to 15th, Nets were set earlier, but no captures were made; thus this collection represents the earliest spawning fish. Northern pike from this site were used exclusively to avoid potential inter-population genetic differences (Bosworth & Farrell, 2006) that may influence egg quality and larval growth. Thirty-six female and several male northern pike were retained from the field and moved to an indoor flow through raceway at ambient river temperature (6 C in April). Twenty-eight females were identified as ripe. The total length (TL, mm) of all fish was measured. The largest male (654 mm) was retained, while 16 females equally distributed among four size classes ( mm, mm, mm, and mm) were selected. The remaining fish were immediately released. The raceway temperature was slowly increased to 12 C overnight (\1 C/h). Previous studies have indicated that optimal egg/larval survival and development occurs at 12 C (Cooper 2000; Farrell & Toner, 2003). Gillnets were used to collect late deepwater (3 6 m) spawning northern pike from Rusho Bay located on the eastern (leeward) side of Grindstone Island, from May 17th to May 20th Ambient river water temperature was 11.5 C during fish collection and the raceway and experimental tank temperatures were maintained at 12 C. Because we do not completely understand the subpopulation genetic structure of the river s pike, we chose Rusho Bay as the late spawning site because it is the closest known late season deepwater spawning location to Delaney Bay. Since mid-may is the end of the spawning season, most fish were already spent. Fiftyfive northern pike were captured during the late season gillnetting and only four ripe females were captured (TL: 529, 571, 656, and 748 mm) along with 10 ripe males. Aside from differences in spawner collection and timing, all other methodologies were identical between the early and late spawning trials. Of the 16 females collected for the early spawning trial, six broods showed no signs of development and were excluded from the study. Thus, total sample size

4 74 Hydrobiologia (2008) 601:71 82 for this study was 10 broods for the early spawning trial and four broods in the late spawning trial. Female body size and egg size relationship Several hundred eggs were manually stripped from each female. The total length (TL, mm) and towel dry wet weight (g) of each fish were recorded. A sample of unfertilized eggs (40 100) from each female was immediately frozen on dry ice and subsequently freeze-dried for chemical analyses and dry weight determination. Nine to twelve batches of three dried eggs were weighed for each brood to determine mean dry weight per egg. Another eggs were retained in water to measure water hardened egg diameter. Water hardened egg diameters were measured within 2 3 days of collection using a digital caliper (n = 33 71/female, measured to the nearest 0.01 mm). Simple linear regression was used to evaluate the relationship between female total length and egg size (diameter and dry weight). Differences in egg diameter and egg mass between early and late spawners was evaluated using analysis of covariance (ANCOVA), where female TL was the covariate and spawning season (i.e., early and late) was the main effect. Egg size and egg quality To determine if egg nutrient composition was an indicator of quality, nutrients were measured first as concentration (percent composition) and, second, as mass per egg for carbon, nitrogen, phosphorus, total fatty acids (TotFA, includes all fatty acids), saturated fatty acids (SFA, 14:0, 16:0, 18:0), monounsaturated, and short-chain (C 18 ) polyunsaturated fatty acids (collectively termed as short-chain unsaturated fatty acids, SCUSFA, 16:4x-1, 16:3x-4, 16:2x-4, 16:1x- 7, 18:4x-3, 18:1x-9, 18:1x-7, 18:3x-3), total longchain (CC 20 ) polyunsaturated fatty acids (PUFA, 20:4x-6, 20:5x-3, 22:5x-3, 22:6x-3, +2 unknown long-chain fatty acids), docosahexaenoic acid (DHA, 22:6x-3), eicosapentaenoic acid (EPA, 20:5x-3), and arachidonic acid (ARA, 20:4x-6). Freeze-dried eggs collected from individual females (see previous section) were used for all chemical analyses. Carbon and nitrogen content were determined with a Costech elemental analyzer coupled to a Thermo Finnigan Delta XP-Plus isotope ratio mass spectrometer at SUNY-ESF (EaSSIL- Environmental Science Stable Isotope Laboratory). A total of lg of crushed egg were weighed into a tin capsule. The resulting percent carbon/ nitrogen composition was multiplied by mean egg mass to yield mean carbon/nitrogen content per egg for each brood. For phosphorus determination, lg of freeze-dried egg were subjected to a potassium persulfate digestion (Langner & Hendrix, 1982) to convert particulate phosphorus into orthophosphate and an Astoria Pacific auto-analyzer used to run a standard molybdate test for phosphate (APHA, 1998). Fatty acid determination was made by thoroughly extracting all lipids from 5 to 10 mg (three eggs) of freeze-dried eggs using three washes with dichloromethane-methanol (2:1). An internal fatty acid standard (19:0, nonadecanoic acid) was added to each sample. Samples were esterified with methanolic hydrochloric acid and subsequently silylated with bis(trimethylsilyl)- trifluoroacetamide (BSTFA, Sigma, 60 min at 60 C) to protect any alcohol groups. Gas chromatographic analysis was performed on a Shimadzu GC-17A using a DB-5 (J&W Scientific) column (30 m, ID 0.25 mm, film thickness 0.25 lm). The column temperature program started at 60 C for 1 min, increased to 140 C at 15 C min -1 of helium and then increased to 300 Cat 4 C min -1 and remained there for 15 min. The split/ splitless injector and detector temperatures were both 250 C, and the column flow was 2.6 ml min -1 of helium. Fatty acids were identified by comparison with known standard mixtures (Restek), and by GC-MS (Shimadzu QP 5050) operating in the electron-impact (EI) mode. Concentrations of fatty acids were determined using the nonadecanoic acid internal standard added prior to esterification. Fatty acid detection limit was 1 ng of fatty acid injected on-column. Simple linear regression was used to evaluate the relationship between egg size (diameter and dry weight) and egg nutrient content (concentration and mass). Larval development and egg quality Four experimental tanks were set-up 2 weeks before fish were collected. Each tank was equipped with its own chiller unit and 100 Watt heater to stabilize

5 Hydrobiologia (2008) 601: temperatures at 12 C. Each tank also contained a custom-made plexiglass/pvc tray that held 16 PVC cups with 2 mm cloth mesh bottoms. The design encouraged water flow through the cups containing the eggs. Due to the 35 day difference between the two trials, semen from a different male was used to dry-fertilize eggs from each trial. The use of a single male within each trial was intended to control for possible genetic variation effects on development and growth within trials. The fertilized eggs were allowed to water harden for 2 3 h in a 12 C water bath, after which they were sorted into the PVC cups within 3 h after fertilization. Two hundred fertilized eggs per female were distributed among four replicate containers (cups) with 50 eggs per cup and each cup was placed into each of the four experimental tanks where they were left to develop. The date and time of fertilization for each brood were recorded at the onset of trial set-up. Eggs were monitored (1 12 h intervals depending upon stage) through swim-up, and the number of minutes to hatch and swim-up for each cup was recorded. The time to hatch, for example, was calculated as the time (minutes) from fertilization to the median time (minutes) between the last observation of an individual as an egg and the first observation as a larvae. OnSet Ò thermographs were installed in each tank and temperature was recorded every half hour. The mean temperature (14 days) of tanks ranged from C (SD = 0.57) to C (SD = 0.67). In order to alleviate inter-tank bias due to the slight temperature differences, the development time is expressed as the number of days at 12 C from fertilization to hatch (DD12 f h ) and from fertilization to swim-up (DD12 f- sw) using the equation: h DD12 x y ¼ X ð#1=2h i CÞ #min x y ½ð#1=2hÞ60 min 24 h 12 CŠ 1 where the subscript x represents the beginning time period and y represents the end time period, P (1/2 h C) is the sum of the half hour temperature measurements in a given tank, the #1/2 h is the number of temperature measurements made, and # min x-y is the number of minutes of stage duration. Developmental time was calculated for each individual larvae and the mean time of each experimental unit (cup) was used to calculate the overall brood mean development time (n = 4). Average survival per clutch (eggs from an individual female) was calculated as the mean percent of eggs that hatched (% survival to hatch) and the mean percent that hatched and reached swim-up (% survival hatch to swim-up) among replicates (n = 4). Finally, the total lengths of all fry that reached swim-up were measured using a digital caliper (measured to nearest 0.01 mm). Six response variables were generated for egg/larval performance: mean percent survival from fertilization to hatch, mean percent survival from hatch to swim-up, mean percent survival from fertilization to swim-up, mean # days at 12 C from fertilization to hatching, mean # days at 12 C from fertilization to swim-up, and the mean swim-up fry total length. Regression was used to evaluate the relationship among female total length, egg size (diameter and dry weight), and egg nutrient content with each of the response variables. Analysis of variance (ANOVA) or ANCOVA was used (egg dry weight or egg diameter as a covariate) to determine if any of the response variables differed between the early and late spawners (main effect). All statistics were calculated using SAS version 8. The assumption of data normality was evaluated through visual inspection of the mean and median, skewness, kurtosis, and normal probability plots; transformations were deemed unnecessary. Similarly, the variances of all early to late season comparisons were examined and were of similar magnitude in all cases. Results Female body size and egg size relationship Mean egg diameter from individual females ranged from 2.18 to 2.42 mm (mean = 2.32 mm, SD = 0.07) and was highly predictable based on female total length (r 2 = 0.76, F 1,13 = 37.10, P \ ; Fig. 1a). If differences in female length were controlled for, then mean egg diameter did not differ between early and late spawners (ANCOVA covariate: F 1,13 = 34.01, P = ; early vs. late: F 1,13 = 0.00, P = ). Mean egg dry weight was much more variable than egg diameter, ranging between 2.06 and 3.40 mg (mean = 2.49 mg, SD = 0.45) and was not related to female total length in either season (early: r 2 = 0.25,

6 76 Hydrobiologia (2008) 601:71 82 (mm) Diameter Egg Mean Dry Mass (mm) Egg Mean Dry Mass (mm) Egg Mean (a) (b) (c) F 1,9 = 2.64, P = ; late: r 2 = 0.01, F 1,3 = 0.02, P = ; Fig. 1b) or to mean egg diameter in either season (early: r 2 = 0.17, F 1,9 = 1.63, P = ; late: r 2 = 0.35, F 1,3 = 1.10, P = , Fig. 1c). Mean egg dry weight, however, was significantly greater in the late spawners (3.02 mg/egg, SD = 0.49) than early spawners (2.28 mg/egg, SD = 0.20, ANOVA: F 1,13 = 17.65, P = ). Egg size and egg quality Female Total Length (mm) Female Total Length (mm) Mean Egg Diameter (mm) Fig. 1 The relationship between female body size and egg size. (a) mean egg diameter (mm) versus female total length (mm), (b) mean egg dry weight (mg) versus female total length (mm), and (c) mean egg dry weight (mg) versus mean egg diameter (mm). Solid symbols are early spawned fry and open circles are late season fry. Error bars represent one standard error of the mean Egg nutrient concentration (% composition), was highly conservative. The concentration of individual Table 1 Mean values (% coefficient of variation = SD/ mean*100) for each egg nutrient constituent Variable Mean concentration (%CV) Mean amount (%CV) C (1.57) 1.26 (18.15) N (2.06) 0.29 (17.00) P 1.04 (8.48) 0.03 (22.56) TotFA (9.46) (22.88) SFA (11.79) (24.59) SCUSFA (10.02) (21.89) TotPUFA (8.65) (23.21) DHA (10.57) (25.22) EPA 4.02 (7.32) 9.39 (22.33) ARA 5.99 (10.36) (21.08) Variable Mean molar ratios C:N 9.30 (1.36) C:P (8.79) N:P (9.18) DHA:EPA 4.96 (8.88) DHA:ARA 3.31 (9.16) EPA:ARA 0.67 (10.07) The early and late spawners are pooled. The left column is the concentration which for C, N, and P is the percent composition, while for the fatty acids it is lg/mg. The right column is the mean mass per egg expressed as mg/egg C, N, and P and lg/ egg for fatty acids. Variation in the molar ratios is displayed in the bottom portion of the table. C, carbon; N, nitrogen; P, phosphorus; TotFA, total fatty acids; SFA, saturated fatty acids; SCUSFA, short chain unsaturated fatty acids; TotPUFA, total polyunsaturated fatty acids; DHA, docosahexaenoic acid (22:6x-3); EPA, eicosapentaenoic acid (20:5x-3); and ARA, arachidonic acid (20:4x-6) constituents was remarkably invariant compared to the mass of nutrients per egg (Table 1), which significantly increased with egg mass (Table 2). Carbon averaged 50.72% of egg mass with a coefficient of variation (CV) of only 1.57%, yet the mass of egg carbon (mean = 1.26 mg) showed a CV of 18.15% due to its strong correlation to mean egg dry weight (r 2 = 0.99, Fig. 2e). Overall the CV for the nutrient mass per egg was in all cases over twice as high as that of the concentration (Table 1). Carbon and nitrogen showed the lowest overall variation among all the constituents, while SFA, DHA, and ARA showed the highest variation in both concentration and mass. Molar ratios also showed relatively low variation consistent with conservative egg nutrient allocation (Table 1).

7 Hydrobiologia (2008) 601: Table 2 Significant regression relationships between the mean egg dry mass (mg) and mean mass of each egg nutrient or the C:N molar ratio Variable (mg/egg) F 1,9 = P-value R 2 C \ N \ P \ C:N molar ratio Variable (lg/egg) F 1,7 = P-value R 2 TotFA SFA SCUSFA TotPUFA DHA EPA ARA Masses of C, N, and P are expressed in mg/egg whereas fatty acids are lg/egg. C, carbon; N, nitrogen; P, phosphorus; TotFA, total fatty acids; SFA, saturated fatty acids; SCUSFA, short chain unsaturated fatty acids; TotPUFA, total polyunsaturated fatty acids; DHA, docosahexaenoic acid (22:6x-3); EPA, eicosapentaenoic acid (20:5x-3); ARA, arachidonic acid (20:4x-6) Egg diameter, routinely used to describe egg size in fish, showed no correlation to any egg nutrient concentration or nutrient mass/egg (F 1,9 \ 1.48, P [ 0.25, r 2 \ 0.16, in all cases; Fig. 2a c). Similarly egg dry weight showed no correlation to egg nutrient concentration (F 1,9 \ 3.37, P [ 0.10, r 2 \ 0.30, in all cases; Fig. 2d). However, egg dry weight did show a strong positive relationship to the mass/egg of all of the major egg constituents including; carbon, nitrogen, phosphorus, all fatty acid components, and the C:N molar ratio (Table 2, Fig. 2e, f). The remaining molar ratios were not related (F 1,9 \ 2.32, P [ 0.16, r 2 \ 0.23, in all cases). There were no significant differences in the concentration of nutrients between early and late spawners (F 1,13 \ 4.44, P [ in all cases). Though the mass of all individual nutrients was greater in late season eggs, the differences became non-significant when egg mass was used as a covariate (F 1,10 \ 4.44, P [ 0.06, in all cases). The egg EPA:ARA and the DHA:ARA ratios, which were not related to egg mass, were significantly higher in the late spawning broods (EPA:ARA F 1,10 = 7.11, P = ; DHA:ARA F 1,10 = 6.23, P = ). The eggs of early spawners had on average 0.64 (SD = 0.05) molecules of EPA to each ARA molecule and 3.17 (SD = 0.18) DHA molecules to each ARA molecule, while the late spawner egg mean EPA:ARA ratio was 0.73 (SD = 0.06) and the DHA:ARA ratio was 3.56 (SD = 0.34). Larval development and egg quality Post-fertilization survival to hatching was highly variable among broods, ranging from 0.5 to 79.9% (mean = 34.3% ± 7.78% SE), but survival from hatching to swim-up was over 94% for all broods. It appears as though under laboratory conditions posthatching survival is fairly consistent; therefore, the remaining discussion will focus on fertilization to hatching survival. Mean fertilization to hatch survival for the early spawner trial was markedly higher (mean = 41.9% ± 9.05% SE) than the survival of the late spawned broods (mean = 15.3% ± 11.59% SE), however, the difference was not statistically significant (ANOVA: F 1,13 = 2.70, P = , Fig. 3a). Survival from fertilization to hatching showed significant negative power relationships to egg dry weight (r 2 = 0.69, P = , Fig. 4), mean mass of carbon (r 2 = 0.72, P = ), nitrogen (r 2 = 0.66, P = ), and phosphorus (r 2 = 0.65, P = ) per egg. It should be noted that these non-linear relationships are largely due to the influence of three data points from the late spawning trial and a single data point from the early season. No other egg nutrient parameters (including fatty acid lg/egg, concentrations, and molar ratios) showed a significant correlation to survival. Due to extremely poor survival of two broods from each trial (less than five swim-up fry out of 200 eggs), only eight of the ten broods in the first trial and two of the four broods in the second trial were used in analyses of swim-up fry mean total length. Variation in swim-up fry mean total length was low and ranged from to mm (mean = mm ± 0.13 mm SE). Swim-up fry from late spawners were on average larger (mean = mm ± 0.10 mm SE) than the fry from early spawners (mean = mm ± 0.11 mm SE), but the difference was not significant when mean egg dry weight was added as a covariate (ANCOVA covariate: F 1,9 = 10.51, P = , early vs. late:

8 78 Hydrobiologia (2008) 601:71 82 N concentration (%) C, (a) (d) P concentration (%) C, N mass mg / egg (b) (e) P mass mg / egg Fatty Acid mass ug / egg (c) (f) Mean Egg Diameter (mm) Mean Egg Dry Mass (mg) Fig. 2 The concentration and mass of individual egg nutrient constituents relative to mean egg diameter (left column) and mean egg mass (right column). C, N, and P concentrations are displayed in the top row (a, d), followed by C, N, and P mass (b, e,) in the middle row and fatty acid mass (c, f) in the bottom row. Solid symbols are early spawned eggs and open circles are F 1,9 = 0.58, P = , Fig. 3b). Therefore differences in mean total length of fry were due to differences in the mean egg dry weight, which was significantly greater in late spawners. Mean egg dry weight described 58.10% of the variation in mean fry total length (F 1,9 = 11.09, P = , r 2 = 0.58, Fig. 5a), whereas female total length and mean egg diameter were unrelated (female TL: F 1,9 = 2.07, P = , r 2 = 0.21, mean egg diameter: F 1,9 = 1.48, P = , r 2 = 0.16). Because of the strong relationship between egg mass and the mass of nutrient constituents, the masses of all nutrients, with the exception of P late season eggs. Carbon measurements are indicated by circles, N by squares, and P by triangles (a, b, d, e). For the fatty acids (c, f), circles indicate total fatty acids, diamonds total PUFA, squares short chain unsaturated fatty acids, and triangles represent measurements of saturated fatty acids (F 1,9 = 3.98, P = , r 2 = 0.33) and ARA (F 1,7 = 5.16, P = , r 2 = 0.46) were significantly correlated to the mean length total of swimup fry (Table 3). The concentrations of C and N and the DHA:ARA molar ratio also showed a positive significant relationship to mean fry total length (Table 3). It should be noted that all of the fatty acid variables showed at least an 89% correlation to one another (most [97%). Overall, the mean mass per egg of individual nutrients (positively related to egg mass) described more variation in mean fry total length than did the concentration values.

9 Hydrobiologia (2008) 601: Hatch - Fert. Proportion Survival Length of Fry (mm) Mean Total Swim-up # Days at 12 C (a) (b) (c) Early Early FHDD12 Late Late FSWDD12 Fig. 3 Differences in egg and larval performance between early (dark columns) and late (light columns) spawned broods; (a) mean percent survival from fertilization to hatching, (b) mean total length of swim-up fry (mm), and (c) developmental time expressed as the number of days at 12 C from fertilization to hatching (FHDD12) and from fertilization to swim-up (FSWDD12). Error bars indicate one standard error of the mean Mean swim-up fry total length was also negatively related to the number of days at 12 C from fertilization to swim-up (F 1,9 = 6.92, P = , r 2 = 0.46, Fig. 5b), but not related to the number of days at 12 C from fertilization to hatch (F 1,9 = 0.38, P = , r 2 = 0.05). There was an interesting dynamic in developmental rates. As developmental time from fertilization to hatching increased, the time from fertilization to swim-up decreased (F 1,12 = 9.01, P = , r 2 = 0.45), meaning that longer developmental time inside the egg translated into a shorter developmental time from hatching to swim-up and larger swim-up fry total length. Developmental time (days at 12 C) from fertilization to hatching was marginally longer in late spawned broods than early ones (ANOVA: F 1,13 = 4.55, P = ) and significantly shorter from Proportion Survival Fert. - Hatch Mean fertilization to swim-up (F 1,12 = 9.79, P = , Fig. 3c), partially explaining why the mean total lengths of late spawned broods were larger than those of the early spawned broods. Development time was not, however, found to be related to any of the measured nutrient variables (regression P [ 0.05 in all cases). Discussion Mean Egg Dry Mass (mg) Fig. 4 Variation in percent survival from fertilization to hatching as a function of mean egg dry weight. Solid symbols are early spawned fry and open circles are late season fry. Error bars indicate one standard error of the mean (mm) Length Total Fry Swim-up (a) Mean Egg Dry Mass (mg) (b) # 12C days Fert. - Swim-up Fig. 5 Major factors influencing the mean total length of swim-up fry (mm): (a) mean egg dry mass (mg) and (b) the mean developmental time (number of days at 12 C) from fertilization to swim-up. Solid symbols are early spawned fry and open circles are late season fry. Error bars indicate one standard error of the mean While most optimal egg size models are based on egg mass (e.g., Sargent et al., 1987) or similarly caloric

10 80 Hydrobiologia (2008) 601:71 82 Table 3 Significant regression relationships between the mean mass, concentration, and molar ratios of egg nutrients to swim-up fry total length (mm) Variable (mg/egg) F 1,9 = P-value R 2 C N Variable (l/egg) F 1,7 = P-value R 2 TotFA SFA SCUSFA TotPUFA DHA EPA Variable (concentration) F 1,9 = P-value R 2 C N Molar Ratio F 1,9 = P-value R 2 DHA:ARA Masses of C and N are expressed in mg/egg whereas fatty acids are lg/egg. The concentrations of C and N are expressed as % content. C, carbon; N, nitrogen; TotFA, total fatty acids; SFA, saturated fatty acids; SCUSFA, short chain unsaturated fatty acids; TotPUFA, total polyunsaturated fatty acids; DHA, docosahexaenoic acid (22:6x-3); ARA, arachidonic acid (20:4x-6) content (Smith & Fretwell, 1974), many researchers report only the egg diameter, assuming that it is indicative of egg quality (e.g., Einum & Fleming, 2002). We found, however, that neither egg diameter nor female total length was related to the concentration or mass of egg nutrients or egg/larval performance. Instead, the mass of every individual egg nutrient was strongly and positively related to egg dry mass. Further, developmental rate and swimup fry total length were related to egg mass, but not egg diameter or female body size. Therefore, at least for northern pike, egg diameter, which can be influenced by water content and environmental stress (Rasanen et al., 2005), is not an accurate assessment of egg quality. Dry mass not only directly measures the collective nutrient mass contained within the egg, but also links increased nutrient content to increased egg/larval performance, and is therefore is a better measure of egg quality. We also observed significant differences in the dry mass of eggs and subsequently egg and larval performance between early- and latespawned broods, indicating that different physiological/ecological trade-offs may exist among early and late spawning strategies of St. Lawrence River northern pike. Similar to findings for walleye eggs (Wiegand et al., 2004; Czesny et al., 2005), the percent content of individual nutrient constituents in northern pike eggs was extremely conservative. For instance, of the four major nutrients measured in this study (C, N, P, totfa), carbon and total fatty acids showed the greatest range in percent composition, both of which were less than 3%. The range in nitrogen content differed by only 1% and phosphorus showed the least variation ranging from 0.85 to 1.18% of egg mass (see also Table 1). The largely invariant concentration of egg nutrients is consistent with stoichiometric concepts (Sterner & Elser, 2002), which suggest that growth is maximized when specific elements (extendable to essential molecules such as fatty acids) cooccur in the proper ratio. The relatively small degree of variation in nutrient concentration of eggs suggests that egg nutrient allocation is constrained by selective pressures to optimize performance, such as pre-swimup development and survival. While the relative concentration of egg nutrients was extremely conservative and was unrelated to egg size (diameter and mass), female body size, or egg/ larval performance, the absolute mass of all egg constituents was more variable and increased linearly with egg dry mass. The mass of nutrients contained in the eggs was also directly and positively related to swim-up fry total length. Swim-up fry total length was significantly related to an increase in all egg nutrients with the exception of phosphorus and ARA, but showed the strongest linear relationship to DHA. Though northern pike are scatter-spawners and do not provide any direct post-fertilization parental care, wild egg mortality rates almost certainly differ among habitat types (Farrell et al., 2006). In the laboratory we observed differences in both egg mass and survival between early and late spawned broods that were utilizing different habitat types for spawning. Survival from fertilization to hatching declined with egg dry mass, where the heaviest eggs (late spawners) showed the lowest mean survival. It could, however, be conjectured that the deep water shoals may have greater expected egg survival, due to fewer

11 Hydrobiologia (2008) 601: egg predators and lower diurnal temperature fluctuations relative to tributaries. The nutrient concentrations of early and late spawned eggs were similar, but late-spawned eggs were significantly heavier and produced significantly larger swim-up fry than did early season eggs. Though the developmental time from fertilization to hatching did not differ between seasons or due to egg mass, late-spawned broods developed to swim-up significantly more rapidly than did early season broods. The trade-off between reduced developmental time and increased swim-up size versus decreased pre-hatching survival may be an adaptive response to delayed spawning. Field observations and modeling efforts indicate that late season deepwater spawning represents an ecological sink largely due to extremely poor post-swim-up survival (Farrell et al., 2006). Predators, including large schools of yellow perch (Perca flavescens Mitchill), rock bass (Ambloplites rupestris Rafinesque), smallmouth bass (Micropterus dolomieu Lacepede), and the recently invasive round goby (Neogobius melanostomus Pallas) are far more abundant in the deepwater shoals than are fry predators in the tributary marshes. Late-spawned fry in deep littoral shoals are also faced with a shorter growing season and lower prey abundance (Farrell et al., 2006). Ultimately heavier, higher quality eggs may be an important reproductive adaptation to maximize the size of young in a situation where predation risk is high and the growing season is short. In conclusion, the dry mass of eggs was the best indicator of egg quality, whereas egg diameter, the traditional metric was completely uninformative. Northern pike eggs were highly conservative in the relative composition of nutrients, but the mass of nutrients was more variable. Increased egg mass (and therefore the mass of individual nutrient constituents) resulted in larger swim-up fry. We found no correlation between egg nutrient content and developmental time, but did observe a decline in egg survival with increased egg mass. Late spawned eggs were heavier than early spawned eggs resulting in larger fry at swim-up, but poor pre-hatch survival. Acknowledgements We thank Aaron Cushing, Kristin Hawley, Jason Toner, and Jerry Mead for their assistance in setting up experimental trials. We also thank Molly M. Ramsey for reading an early draft and providing feedback that improved the manuscript. We especially wish to thank the participants of the International Northern Pike Symposium, 2006 AFS meeting in Lake Placid for meaningful discussions. This project was funded by a grant from the Federal Aid in Sportfish Restoration Act (# FA-5-R), by The National Science Foundation (# DEB ), and by GSEU Professional Development Funds. 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