Integrative and Comparative Biology Advance Access originally published online on March 29, 2006
Integrative and Comparative Biology 2006 46(3):334-346; doi:10.1093/icb/icj023
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Linking stages of life history: How larval quality translates into juvenile performance for an intertidal barnacle (Balanus glandula)
Oregon Institute of Marine Biology and Department of Biology, University of Oregon PO Box 5389 Charleston, OR 97420, USA
Correspondence: 1E-mail: remlet{at}uoregon.edu
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Many marine invertebrates with complex life cycles produce planktonic larvae that experience environmental conditions different from those encountered by adults. Factors such as temperature and food, known to impact the larval period, can also affect larval size and consequently the size of newly settled juveniles. After documenting natural variation in the size of cyprids (the final larval stage) of the barnacle Balanus glandula, we experimentally manipulated temperature and food given to larvae to produce cyprids of differing sizes but within the size range of cyprids found in the field. In a set of trials in which larvae of B. glandula were raised on full or reduced rations in the laboratory and subsequently outplanted into the field as newly metamorphosed juveniles, we explored the effects of larval nutrition and size on juvenile performance. Larvae that received full rations throughout their feeding period produced larger cyprids (with more lipid and protein). These larger cyprids grew faster as juveniles and sometimes survived better in the field than juveniles from larvae that had their food ration reduced in the last feeding instar. For naturally settling barnacles brought into the laboratory within 2 days of settlement and fed, we found that initial juvenile size was a good predictor of juvenile size even after 2 weeks of growth. By manipulating food given to juveniles that were derived from larvae fed either full or reduced rations, we found that larval nutritional effects persisted in juveniles for 2–3 times the period that larvae experienced altered food rations.
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Variation in offspring size has been a focal point for the study of life history evolution since Lack (1947)
examined regional, seasonal, and taxon-specific patterns of reproduction in birds (for example, Vance 1973a, 1973b; Smith and Fretwell 1974; Stearns 1992). Greater parental investment and the production of fewer and larger offspring must increase the fitness of offspring enough to offset the drop in fecundity associated with larger offspring. In other words, with everything else being equal, larger offspring should outperform smaller offspring in most environments. Numerous laboratory studies show the benefits of increased offspring size while others show costs or at least no benefit (reviewed in Moran and Emlet 2001; Fox and Czesak 2000; Pechenik 2006). Experimental field evidence in support of this prediction can be found in studies on terrestrial plants (for example, Stanton 1984); fishes (for example, Einum and Fleming 2000); and several marine organisms including snails (Moran and Emlet 2001), mussels (Phillips 2002), bryozoans (Marshall and others 2003; Marshall and Keough 2004), and colonial ascidians (Marshall and others 2006). While theoretical considerations of size-fecundity trade-offs continue to be developed, our understanding of the causes of selection for increased or decreased offspring size remains limited due, in part, to a strong need for experimental studies executed in the field.
For organisms with open populations and widely dispersing offspring, the experience offspring have before reaching the local adult habitat may impact the quality of arriving offspring and their ability to recruit into the local population (for example, Pechenik and others 1998). For feeding larvae, the nutrition obtained during the pelagic period may determine initial juvenile size or post-settlement performance, and for competent larvae, delays in metamorphosis may reduce juvenile performance. With considerable attention focused on larval availability as a determinant of recruitment (for example, Gaines and Roughgarden 1985; Shanks 1995; Morgan 2001; Underwood and Keough 2001) variation in larval quality may modulate the effects of larval supply.
In consideration of both evolutionary and ecological consequences of offspring size, we present results from sampling natural populations and from combined laboratory and field experiments on barnacles that demonstrate variation in cyprid size and show that offspring size effects early juvenile performance. First, we show that samples of cyprids of the intertidal barnacle Balanus glandula Darwin vary in cyprid carapace area within and across samples. Then we examine how the factors of temperature and food availability affect cyprid size. Next, in a series of experiments where we raise larvae on different rations, let them metamorphose, and outplant them in the field, we examine the link between larval nutrition and juvenile performance measured as growth and survivorship. We document variation in size and growth of naturally settling barnacles. Last, we manipulate food rations given to juveniles to determine whether juveniles can recover from reduced larval rations.
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Studies on natural populations of cyprids and juveniles
On many occasions since 1994 we have collected plankton using a 135 µm mesh net in the mouth of Coos Bay (18 m depth). When samples contained large numbers of cyprids that could be identified to a particular species (with the cyprid key by Standing 1981
), we collected and preserved (in 4% formalin in SW) individuals by species. Each sample of cyprids was placed in a Syracuse dish with a small amount of water, and cyprid areas were drawn in the lateral view at a magnification of ca. x65 with the aid of a camera lucida attached to a dissecting microscope. As an additional measure of size, we estimated cyprid volume from the carapace area, assuming that cyprids were prolate spheroids with a 2:1:1 ratio of length, height, and breadth. We validated volume estimates for cyprids by showing that dry organic mass (DOM) corresponded to estimated volumes for laboratory-reared cyprids. Newly settled barnacles of B. glandula were obtained by deploying slate plates (8 cm x 8 cm x 1 cm and containing rectilinear arrays of small, conical pits that are 1–2 mm deep, spaced at intervals of 0.5–1 cm) along a breakwater at an intertidal height of +1 m. Plates were checked every other day and cleared with a brush if little or no settlement had occurred. On two occasions (July 1995 and June 1996) settlement occurred in numbers high enough to warrant the survival and growth of settlers. All plates were brought into the laboratory on the second day after settlement and the locations of individuals were mapped. Up to 30 juveniles on each plate were mapped and drawn at a magnification of ca. x65 (decreasing to x33 or less as barnacles grew) with the aid of a camera lucida attached to a dissecting microscope. The quasi-circular attachment area of each barnacle, whose actual shape approximated the frustum of a cone, was drawn. One-third of the plates were then redeployed in the field, and surviving individuals were drawn on all subsequent sample days. Maps and our experience with changes in coloration and size due to calcification and age allowed us to distinguish experimental barnacles from other natural settlers that colonized the plates during intervals in the field. Natural settlers were removed using a dental pick, so growth and survival were not influenced by competitors.
The remaining two third of the plates were kept in the laboratory and maintained in containers with seawater. Barnacles on half of the laboratory plates were fed the diatom Skeletonema costatum at final densities of 1 x 105 cells/ml, and the barnacles on the other half of the plates were maintained in 0.45 µm filtered seawater (FSW) without food. Water was changed every other day. For each treatment (field, fed, or starved) naturally settled barnacles were assigned to one of the three groups (smallest 1/3, middle 1/3, or largest 1/3) based on their initial sizes on day 2 after settlement. Mean size of individuals in the smallest 1/3 and largest 1/3 were plotted and sizes were analyzed at the end of the study (14 or 16 days after settlement).
Studies on larvae raised in the laboratory
Adult barnacles of B. glandula Darwin with egg lamellae that were ready to hatch were collected from intertidal locations on the exposed coast or within the Coos Bay estuary near Charleston, Oregon, USA (43.3450°N, 124.3217°W). Lamellae from a single adult or a single adult with its lamellae were placed in separate 50 ml plastic tubes with seawater and transported back to the Oregon Institute of Marine Biology. In the laboratory, lamellae from each adult were placed in separate 1 liter beakers filled with 0.45 µm FSW and exposed to light (color temperature: 3250 K) from fiber-optic illuminators to induce hatching of stage I nauplii. When more than 6000 larvae were obtained from a single adult they were used in an experiment. Larvae hatched as stage I nauplii, soon molted to stage II, and were set up in experiments within a few hours of hatching.
Two sequential trials of an experiment were carried out to examine the influence of temperature and food ration on larval size. In each trial 4000 stage II nauplii from each of the four adults were placed into sixteen 3.8 l glass jars. One thousand larvae from a single parent were placed in each jar with 2 l of FSW for an initial density of 1 larva/2 ml FSW, and there were four jars for each parent. In temperature trial 1, two jars of larvae from each parent were haphazardly chosen and placed in an incubator at 9°C and the remaining two jars for each parent were placed in a sea table with water maintained at 16–17.5°C. In temperature trial 2, the jars of larvae were placed in an incubator set at 16°C or in a sea table with water held at 9–10°C. Culture water in jars was stirred with reciprocating paddles at 10–12 r.p.m. (for example, Strathmann 1987). From the start of each trial, larvae in one jar at the higher temperature and one jar at the lower temperature from each of the four parents were fed the diatom Skeletonema costatum at a final density of 1 x 105 cells/ml and larvae in the other jar from each of the parents were fed one-fourth of this ration, 2.5 x 104 cells/ml. Larval culture water was changed every 2 days by pouring the jar contents into a cup with a 100 µm mesh bottom partially submerged in FSW, removing larvae from the cup with a baster pipette, and placing them into a clean jar containing FSW at the appropriate temperature (9 or 16°C). Larvae were fed their assigned food ration immediately following water changes. Larvae progressed through development at different rates as a function of food and temperature. When the cyprid stage was reached, cyprids were harvested and preserved in 4% buffered formalin for the later measurement of carapace area (n = 30 per jar).
Outplant experiments consisted of raising larvae on two distinct food rations until they metamorphosed, settling cyprids on plates that were later placed in the field, and monitoring survival and growth of juveniles for 12–50 days after settlement. Four outplant experiments were conducted. For each experiment 6000 stage II nauplii from each of two adults were placed into twelve 3.8 l glass jars. Jars were set up as described above, but with six jars for each parent. All larvae were cultured at ambient temperatures (12–14°C) in a table with running seawater, and each jar was stirred as described above. Initially, all jars of larvae were fed the diatom S. costatum at a final density of 1 x 105 cells/ml. As described above, larval culture water was changed every 2 days, and larvae were subsequently fed. When a majority of larvae reached the transition between naupliar stages V and VI, three jars from each parental group were haphazardly assigned to a full ration treatment and the three remaining jars were assigned to a reduced ration treatment (one-fourth of the full ration or 2.5 x 104 cells/ml). The two food ration treatments of larvae passed though the final feeding instar, reached the cyprid stage in approximately the same amount of time, and yielded equivalent numbers of cyprids.
One or two days after the cultures were dominated by cyprids, competent cyprid larvae from each jar were introduced to separate containers filled with FSW and field-seasoned slate plates (with arrays of pits as described above). Plates were seasoned by placing them in the field on the vertical wall of a concrete jetty at an intertidal height of 1.3 m above the chart datum for a period of 14–28 days, and naturally settled barnacles were removed prior to use. At the same time cyprids were allowed to settle, and a sample of 30 cyprids from each culture jar was preserved in buffered formalin for the later measurement of cyprid size. Cultured larvae were allowed to settle on the plates, attach, and metamorphose for 2 (occasionally 3) days. One or two plates of juveniles were obtained from each jar of cultured larvae. After the metamorphosis juveniles were mapped and outplanted in the field on the same concrete jetty at +1.3 m tidal height for each experiment. Plates were retrieved at 2–10 day intervals to measure juvenile basal area and to record mortality. Natural barnacles that settled near juveniles from cultured larvae were removed using a dental pick. Monitoring took place in the laboratory during one low tide, after which the plates were re-assigned a random position in the linear sequence along the jetty wall.
Prior to outplanting in the field, up to 30 juveniles on each plate were drawn as described earlier. Survivors of these 30 individuals were drawn on all subsequent sample days and all other survivors were counted.
Constituent analysis
In three of the four outplant trials (1, 3, and 4), the number of cyprids was sufficient for additional samples of cyprids to be taken for measurement of DOM and for the analysis of protein and lipid content. For these samples, aliquots of known number of cyprids (usually 75) were concentrated in tubes using a microcentrifuge (momentary spin button was depressed for <1 s). Larvae were resuspended and shaken in distilled water for <30 s and concentrated with another pulse spin, and water was removed and the tubes with pellets of larvae were stored at –80°C for later analysis.
To determine DOM, frozen larval samples were transferred to small aluminum dishes (pre-ashed at 450°C for 6 h) and were dried at 60–80°C to constant mass. Samples were then weighed (total dry mass) to the nearest microgram value on a mass-calibrated balance (Mettler ME30) and subsequently ashed at 450°C for 6 h. Ashed samples were weighed (ash mass) on the same balance, and DOM was calculated as the difference between the total dry mass and ash mass and expressed as DOM per larva. One to three samples per jar of larvae were obtained in the three experiments.
For protein and lipid analysis, frozen larval samples were lyophilized for 12–24 h and stored at –80°C until ready for use. Lyophilized larvae were homogenized in 1000 µl distilled water using a sonicator (Sonics & Materials, Inc.). Samples were sonicated in an ice water bath for 1.5–2 min until sample was thoroughly homoginized.
Protein content was measured using a Protein Assay Kit (Bio-Rad Cat. # 500-0002), based on the method of Bradford (1976) with bovine serum albumin as the standard. Protein composition of cyprids was determined using the procedure devised by Holland and Gabbot (1971), as modified by Mann and Gallager (1985) and Jaeckle and Manahan (1989), with the following modifications: (1) The TCA-insoluble pellet was dissolved in 500 µl of 0.5 M NaOH and (2) the alkaline protein solution was consequently acidified using 300 µl of 0.835 M HCl. Total lipid content was measured using a tripalmitin standard and the methods of Holland and Gabbott (1971), Holland and Hannant (1973), and Mann and Gallager (1985), with the following modifications: (1) no second extraction was conducted; (2) no purification of the lipid containing chloroform layer was conducted; and (3) sulfuric acid charring step was performed at 150°C.
Juvenile feeding experiments
To determine how long the larval food ration affected juvenile size, we exposed juveniles from larvae that received the full ration to either a full ration or a reduced ration as juveniles. We did the same for juveniles of larvae that had received the reduced ration in their last feeding instar. Larvae for these experiments were raised as described above and allowed to settle on slate plates. Two days after the settlement, juvenile attachment area was recorded and equal numbers of plates of juveniles from each larval food treatment were haphazardly assigned to either full ration (1 x 105 cells/ml) or reduced ration (2.5 x 104 cells/ml) of S. costatum. We placed plates of barnacles into containers with FSW and stirred them with paddles to keep the diatoms suspended. Containers were maintained at ambient temperatures of 12–14°C; water was changed every other day, followed by feeding of juveniles.
Statistical analyses
The effects of temperature and food ration on cyprid size were analyzed separately for the two trials with a two-factor analysis of variance. Mean carapace area of cyprids from each jar was the response variable; temperature and food were considered fixed factors.
For the outplant trials, the size of cyprids reared in the laboratory was analyzed for the effects of two factors: food ration given to nauplii was considered a fixed factor and parent was considered a random factor with replicate jars (random) nested within both the primary factors. To give added power to tests for parent, the trials were tested together with trial considered as a random factor and parent nested within trial. Mean sizes from jars were used in the analysis. The relationship of cyprid size (mean carapace area from jars) and juvenile size at 2 days post-metamorphosis (mean attachment area for plates) was evaluated using the Pearson correlation.
Survival of juveniles was examined separately for the four trials. Percent survival on plates on the last day of each trial was analyzed, and the data were arcsin transformed prior to the analysis. Parent (a random factor) and treatment (food ration given to nauplii, a fixed factor) were first analyzed in a two-factor ANOVA. If the interaction term was not significant, it was dropped from the model and the test was rerun to examine the main effects.
In the two trials that manipulated both larval and juvenile food rations to determine how long the effect of the larval ration persisted in the juvenile stage, initial size of juveniles were compared with a nested ANOVA where treatment (four levels: full larval–full juvenile, full larval–reduced juvenile, reduced larval–full juvenile, and reduced larval–reduced juvenile) was the main factor and replicate plates were the nested factor. Separate ANOVAs were run for each trial. Where significant main effects were found pairwise comparisons were made with a Bonferroni adjustment (Systat 9.0).
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Field samples of cyprids
Cyprids of B. glandula occur in coastal waters of Coos Bay, Oregon, from March to November, but their abundance during this period can vary daily. Seven samples taken in different months in 1995 and 1996 showed modal carapace areas that fluctuated around the seasonal mean of 2.0 x 105 µm2 (Fig. 1). Samples of carapace areas were not different from normal distributions (one sample Kolmogorov–Smirnov tests, P-values ranged from 0.33 to 0.99). Each sample showed considerable variation (coefficients of variation ranged from 7.4 to 9.5%). The estimated volume of the smallest individual in each sample was 63 to 75% of the estimated volume of the modal size, depending on the sample. The estimated volume of the largest individual in each sample was 123 to 142% of the estimated volume of the modal size, depending on the sample.
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A sample of cyprids of B. glandula taken in Friday Harbor, Washington, had notably larger individuals than those found off Oregon (Fig. 1). The sample of carapace areas was also not different from a normal distribution (P = 0.45) and had a coefficient of variation that was 10.1%. The estimated volume of the modal size of this sample was 140% of that estimated for Oregon cyprids with a carapace area of 2.0 x 105 µm2.
Effects of rearing-temperature and food on cyprid size
Cyprid size increased when nauplii experienced high food rations or low temperatures, a response consistent between the two trials (Fig. 2). Separate two-way ANOVAs on the trials indicated that both food and temperature influenced cyprid size significantly but did not interact significantly (Table 1). The largest cyprids occurred in cultures held at low temperatures and fed high food rations, whereas the smallest cyprids occurred in cultures held at high temperatures and fed low food rations. Mean areas of cyprids that received the full ration as larvae, especially those reared at the lower temperature (9°C), approached the sizes of cyprids collected near Coos Bay Oregon (Fig. 1). Areas of cyprids that received reduced rations were generally smaller than those sampled in the field. In particular, the mean area of cyprids from the low food, high temperature cultures from trial 1 were much smaller that any larvae observed in the field samples. Estimates of cyprid volumes made from means of carapace areas ranged from 98 to 34% of the volume of cyprids with the seasonal mean carapace area of 2.0 x 105 µm2. We did not determine whether the smallest cyprids could metamorphose and survive as juveniles.
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Outplant trials
Cyprid sizes
In four trials, with offspring of two parents in each trial, larvae fed a full ration consistently metamorphosed into larger cyprids than larvae whose ration was reduced in the final feeding instar (Fig. 3A, Table 2). A combined statistical analysis of all the four trials showed no significant interaction of food and parent (P = 0.652). Even though larvae receiving the full ration always produced larger cyprids than those receiving a reduced ration, a significant interaction of food and trial (P = 0.025) indicated that the magnitude of the food effect was unequal across trials. Parent also significantly affected cyprid size (P < 0.001). Cyprids of trial 3, especially those that received the full ration as larvae, were larger than those in trial 4 (Fig. 3A). Comparison of cyprid sizes in these trials with sizes of those naturally occurring in similar times showed that trial 3 cyprids that received the full ration were at the modal size of natural larvae, but the other groups of laboratory raised cyprids were below the modal size (Fig. 1 graphs for May and June 1996).
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Cyprids from the two different larval feeding treatments also differed in DOM and in their amounts of protein and lipid (Table 3). Larvae receiving the full ration produced cyprids with DOMs of 9–12.5 µg/larva while those receiving the reduced ration produced cyprids with 7–8.8 µg/larva, which was 60–80% of the mass of their full ration counterparts. In trials 3 and 4, the relative estimated volume (determined from carapace area) of cyprids from full and reduced rations were in close agreement with the relative masses determined from DOM (Table 3).
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This agreement supports the use of carapace area to estimate volume and to compare within and across samples. Cyprids that received the full ration as nauplii had more protein, more lipid, and higher lipid to protein ratios than their reduced ration counterparts. However, the recovery of organics in the form of lipid and protein from the constituent analysis was low, ranging from 40 to 60% of the DOM (Table 3). Carbohydrate and chitin (not analyzed) would account for some of the missing fraction, but a sizable portion of the total was lost.
Juvenile size at metamorphosis
Larger cyprids metamorphosed into larger juvenile barnacles than did smaller cyprids (Fig. 3B). Each of the four trials showed similar patterns, with results from trials 1, 3, and 4 having statistically significant positive correlations. Results from trial 2 were not statistically significant; limited settlement (none by parent C) and consequently limited sample size reduced power in this trial. In all trials, the cyprids from full ration treatments typically produced larger juveniles than those from reduced ration treatments. In each of the trials 1 and 3, when one jar receiving the full ration produced cyprids smaller than the other jars receiving full rations, the resulting juveniles were also small compared with juveniles from other full ration jars (Fig. 3A and B).
Survival
The effect of larval food ration on survival in the field of newly metamorphosed juveniles varied with trials (Fig. 3C). All trials showed the highest mortality in the first 2–6 days after introduction to the field. During this period, juveniles derived from larvae that received the full food ration had higher survival than their reduced ration counterparts. In trial 1, survival appeared to vary between parents and treatments. However, a two-factor ANOVA on survival at day 12 showed (no interaction and) no effect of parent (F = 4.15; df = 1,9; P = 0.072) or food treatment (F = 1.56; df = 1,9; P = 0.243). Trial 2 ran the longest and had the highest survival of all the trials, with 43 and 49% of the juveniles from full and reduced ration treatments, respectively, surviving for 52 days. In this trial, the overlap between treatments in juvenile survival suggested no differences between treatments, and this was supported by an ANOVA run on survival at day 52 (F = 0.43; df = 1,7; P = 0.539). Trials 3 and 4, which had the largest and most balanced samples of outplanted plates, both showed similar patterns across parent. Separate two-factor ANOVA of survival at day 16 in trial 3 and day 12 in trial 4 showed no interaction between parent and treatments. Reruns of tests with treatment and parent only revealed nonsignificant effects of parent (Trial 3, F = 0.11; df = 1,17; P = 0.743; Trial 4, F = 0.19; df = 1,18; P = 0.669) and significant effects of treatment (Trial 3, F = 46.01; df = 1,17; P < 0.001; Trial 4, F = 10.76; df = 1,18; P = 0.004).
Growth
When each of the four trials ended (due to low numbers of survivors, see Fig. 3C), most juveniles that received the full larval ration were larger than those that received the reduced larval ration. For parent A in trial 1 and for the five parents evaluated in trials 2–4, the rations fed to their larvae established differences in sizes of newly metamorphosed juveniles, which were consistently maintained or increased slightly in the 12–52 days that juveniles were followed in the field. Juveniles of larvae that received the reduced ration took 4, 8, or ca. 12 days to reach equivalent basal areas of related juveniles that received the full larval rations (Fig. 3D). The time differences to reach equivalent size correspond well with the magnitude of the differences in organic content of the cyprids obtained using specific trials (compare Fig. 3D and Table 3 for trials 1, 3, and 4).
For parent B in trial 1 juveniles from the different food treatments followed similar growth patterns until day 10 after settlement when juveniles derived from larvae that received a reduced ration failed to grow as quickly as their full ration counterparts.
Growth of naturally settling barnacles
On two occasions when they were brought into the laboratory, initial juvenile size of naturally settling B. glandula continued to affect juvenile size for up to 2 weeks after settlement (Fig. 4). Naturally settled barnacles assigned to the large group based on their initial size (largest 1/3 on day 2 after settlement) remained larger than the small group (smallest 1/3 on day 2 after settlement) regardless of whether they were fed or not. When fed the diatom S. costatum, juvenile barnacles increased their attachment areas by three- to 4-fold over 2 weeks. When they were starved, the barnacles initially increased their attachment areas by a factor of 0.5 but size became fixed by day 6–8 after settlement. Mortality in the laboratory for both fed and unfed barnacles was low, ranging from 0 to 16%, with no pattern related to food or initial juvenile size.
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The original design of these studies included groups of newly settled barnacles that were mapped and returned to the field. In the 1995 study, mortality was very high, with no juveniles remaining alive on day 6 after settlement. In the 1996 study also mortality was high (75–90% by day 4 and 85–96% by day 8 after settlement). Plots of the smallest 1/3 and largest 1/3 groups followed in the field show essentially no growth during this period (Fig. 4).
Persistence of larval nutritional effects
In typical experiments where the food ration was reduced, sixth stage nauplii received this reduced (1/4) ration for 5 or 6 days prior to metamorphosis. This interval represented 33–43% of the total feeding larval period, which ranged from 12 to 18 days in different experiments. In both trials where larval and juvenile rations were manipulated, the initial sizes of juveniles that received the full ration as larvae were significantly larger than juveniles that received the reduced ration as larvae. (Trial A: treatment, P = 0.001; pairwise comparisons: initial juvenile size differed according to larval ration P < 0.01, but not within ration P = 1.0. Trial B: treatment, P < 0.001; pairwise comparisons: initial juvenile size differed according to larval ration P < 0.001, but not within ration P = 1.0.) When juveniles from each larval ration treatment received the opposite ration after metamorphosis, their growth rates were affected. Juveniles that received a full ration as larvae grew more slowly when they received a reduced ration than when they received a full ration. Juveniles that received reduced ration as larvae grew more rapidly when they received a full ration than when they received a reduced ration (Fig. 5). In two experiments, it took 9 and 12 days, respectively, for the juvenile food ration to negate the effects on attachment area that were created by the larval food ration (where treatment of full larval ration–reduced juvenile ration crossed that of reduced larval ration–full juvenile ration). Continued exposure of juveniles to full or reduced rations resulted in growth that aligned them according to their juvenile ration by 23 and 20 days after metamorphosis in the respective experiments (Fig. 5). These studies show that the larval nutritional effects on juvenile size persisted for longer periods (up to 2- and 3-fold) than the period of the larval treatment despite efforts in the laboratory to reverse the effect.
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At the time of settlement, marine invertebrate larvae may vary in size and quality due to condition of the parents or their experiences in the larval environment. Larval size or quality may in turn affect post-settlement success though impacts on juvenile growth and survival (for example, Emlet 1986
; Emlet and Hoegh-Guldberg 1997; Marshall and others 2003; Marshall and Keough 2004). The present study shows that the size of the cyprid (the final larval stage) of the intertidal barnacle B. glandula varies in nature, that this variation can be reproduced in the laboratory, and that the size of the cyprid may have consequences for juvenile growth and survival in the field. The later results are based on experimental manipulations of larval nutrition that alter cyprid size and energetic content, juvenile size at metamorphosis, and subsequent juvenile performance. The effects of larval nutrition and juvenile size varied across field trials, with larger larvae and juveniles growing more and surviving better than smaller larvae and juveniles or both groups performing similarly.
Variation in cyprid size
For cyprids of B. glandula collected in the coastal waters of Oregon, the estimated volume of the smallest individual in each sample was 63–75% of estimated volume of the modal size depending on the sample. The estimated volume of the largest individual in each sample was 123–142% of the estimated volume of the modal size depending on the sample. In laboratory studies that manipulated both temperature and food during the larval period, estimates of cyprid volumes, calculated from means of carapace areas, ranged from 98 to 34% of the volume of cyprids with the seasonal mean carapace area of 2.0 x 105 µm2. Lower temperature and abundant food during the larval period both lead to larger cyprids than those of larvae raised at higher temperatures or lower food rations.
For larvae raised at a constant temperature, the estimated volume of cyprids (determined from carapace area) that as nauplii were fed full or reduced rations corresponded to their measured masses for two of the three trials of larvae for which both measurements were made (trials 1, 3, and 4 in Table 3). Cyprids from larvae that received the reduced ration had estimated volumes that were 86, 65, and 72% of those of cyprids from full ration larvae in trials 1, 3, and 4, respectively.
Variation in size of cyprids is known for other barnacles. Barnes (1953) reported size variation in cyprids of the intertidal Semibalanus balanoides and subtidal Balanus crenatus, finding several modal sizes in single samples and changes in modal sizes across samples through time. For both of these species, he attributed variation in cyprid sizes of single samples to mixing of larvae from adult populations from different environments: different intertidal heights for S. balanoides, and shallow or deeper populations of the subtidal B. crenatus. He attributed seasonal differences in modal sizes of cyprids of B. crenatus and Verruca stroemia to temperatures during egg and embryonic development, with larger cyprids occurring when adults brood at lower temperatures and smaller cyprids when adults brood at higher temperatures (Barnes 1953). Patel and Crisp (1960) demonstrated experimentally for four balanomorph species that adults held and fed at lower temperatures (10–15°C) produced larger eggs and embryos than conspecifics held and fed at higher temperatures (25–30°C). Crisp (1962) reported that nauplii and cyprids of S. balanoides from (cooler) artic waters (74°N) were substantially larger than those from (warmer) British waters (53°N), and he noted a general latitudinal trend within species of increasing cyprid size with latitude. We also found substantially larger cyprids in the sample from Friday Harbor Washington than in samples from off Oregon, but we are uncertain about the causation.
The variation in cyprid size that we produced by manipulations of temperature and food ration in the larval environment was on the scale of the observed variation in nature. We do not know how differences in temperature or food ration while adults are growing eggs of brooding embryos would add to the variation we produced. Finally, we have noted changes across time in modal sizes of samples from off Oregon, but have been unable to find significant physical correlates of these differences (Emlet, unpublished data).
Relationship between the larval and juvenile stages
Numerous laboratory-based studies have shown that larval diet affects rates of development and size of cyprids (for example, West and Costlow 1987; Anil and Kurian 1996; Qiu and Qian 1997; Hentschel and Emlet 2000). Other laboratory studies have shown that larval diet, energy content, or delayed metamorphosis can impact settlement or post-metamorphic growth (for example, Lucas and others 1979; Pechenik and others 1993; Thiyagarajan and others 2002, 2003).
By following natural populations, Connell (1961) noted that cyprid size varied across the day of settlement for S. balanoides and that larger cyprids produced larger juvenile barnacles. Jarrett and Pechenik (1997), also working with S. balanoides, reported significant relationships between cyprid length and organic content and between cyprid length and juvenile growth rates of barnacles settling in the field but grown in the laboratory. Organic content and growth rates differed among barnacles grouped by day of settlement. Jarrett (2003) extended these observations on natural populations, showing that cyprid organic content decreased through the season. Jarrett (2003) also examined cyprid metamorphic success and juvenile growth rate, and found that both of these differed across days of settlement and were related to cyprid organic content for groups brought into the laboratory but not for those remaining in the field.
The present study demonstrated experimentally a direct link between larval diet, cyprid size, cyprid organic content, and juvenile size in the laboratory and early juvenile survival and growth in the field. Reduced food rations always resulted in smaller cyprids and juveniles compared with those from larvae that received full rations. Outplanted juveniles from larvae that received full rations performed the same or better than juveniles from larvae that received reduced rations in the final feeding instar. Two of four trials (3 and 4, Fig. 3C) had significantly higher survival of juveniles from full ration larvae compared with those from reduced ration larvae.
The size differences produced by different larval diets were maintained or slightly increased over the time that juveniles were followed in the field. Growth rates were highest for trial 1, where most juveniles had exponential growth trajectories (Fig. 3D). The slowest growth rates occurred in trial 4, which suffered very high mortality, and in trial 2, which was in the field during fall and winter, when air temperatures are low and planktonic food is less abundant compared when spring and summer.
Consistent with our findings that cyprids of B. glandula are in the plankton from spring through fall Menge (2000) and Connelly and colleagues (2001) documented recruitment of B. glandula during this same interval. Trials 3 and 4, outplanted in late spring and early summer, showed significant effects of larval diet on survival, but all barnacles in these trials experienced high mortality. Trial 1, conducted in August, had high mortality rates but no effect of larval diet on survival. In contrast, Trial 2, outplanted in fall, had the lowest mortality rates of barnacles (Fig. 3C). The nonsignificant effect of larval diet on this group of barnacles may indicate that larval nutritional effects vary with season, but further replication of these studies within and across seasons will be necessary to determine whether this is so.
High post-settlement mortality rates are well known to occur for many benthic invertebrates (for example, reviews by Hunt and Scheibling 1997; Gosselin and Qian 1997) and for barnacles in particular (for example, Connell 1961; Wethey 1985; Gosselin and Qian 1996; Jarrett 2000). High mortality (85–100% in 6–8 days after settlement) also occurred on the two occasions where naturally settling cyprids were tracked for growth and survival. High juvenile mortality in the field prevented growth comparisons with juveniles brought into the laboratory, where almost no mortality occurred. When juveniles were grouped by size on day 2 after settlement the larger juveniles retained their size advantage for 2 weeks whether they were fed or starved (Fig. 4).
Larval experiences not only impact juvenile performance, but they may persist even when food regimes given to juveniles change. In laboratory studies, the effects of the larval food ration on juvenile size persisted longer than the time that time larvae received the different food rations. It took ca. two times the period that larvae received unequal rations (full or reduced) for changes in the juvenile food ration to undo the affects of the larval food ration. It took more than three times this same larval period for juvenile size, measured as basal area, to reflect the effects of juvenile food ration, regardless of the larval food ration (Fig. 5).
It is clear from the present study and from several others (for example, Phillips 2002; Pechenik and others 1998; Marshall and Keough 2004; Pechenik 2006) that larval experiences have the potential to impact juvenile success and performance. However, demonstration of an effect of larval experiences on juvenile performance for natural populations is less certain, probably due to increased environmental variability that natural populations experience. The present study showed effects of larval size on juvenile survival in two out of four outplant experiments. We also documented two events of naturally settling barnacle larvae that did not survive long enough to measure effects of larva size on juvenile growth or survival in the field. These findings indicate that further and numerous experimental outplants together with specific monitoring of physical and biological factors that may impact performance will be necessary to confirm the suggestion of laboratory studies that larval nutrition effects "carry over" and play an important role in natural population dynamics.
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Acknowledgements |
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Many people have contributed to this research effort. Undergraduate students Christie Heidel, Eric Crandall, Sonora Rasmussen, Kelly Glenn, and Lila Elliot collected and analyzed data associated with natural populations of cyprids and juveniles. Former graduate students Amy Moran and Bruce Miller contributed suggestions and help with both field and laboratory experiments. We thank all the people mentioned above and the Director and staff at OIMB for facilitating this work. We also thank the symposium organizers, Diana Padilla and Ben Miner for their hard work. We are sure that "Larval Marvel" would have been proud to attend this memorial symposium in his honor. The research was supported by NSF grants OCE 9416590 and OCE-9911682.
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2Present address: Department of Evolution, Ecology, and Marine Biology, University of California, Santa Barbara, CA 93106–9610, USA
From the Symposium "Complex Life-Histories in Marine Benthic Invertebrates: A Symposium in Memory of Larry McEdward" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2005, at San Diego, USA.
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