Larval Competency of Red Abalone (Haliotis rufescens): A New Timeframe for Larval Distribution.

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1 Larval Competency of Red Abalone (Haliotis rufescens): A New Timeframe for Larval Distribution. Author(s): Thomas B. Mccormick, Lorraine M. Buckley, Gabriela Navas, Gloria Barber, Brianne Billups, Vanessa Gill, Brianna Jones, Nicholas Peterson, Brianna Saylor and James Sayre Source: Journal of Shellfish Research, 31(4): Published By: National Shellfisheries Association DOI: URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Journal of Shellfish Research, Vol. 31, No. 4, , LARVAL COMPETENCY OF RED ABALONE (HALIOTIS RUFESCENS): A NEW TIMEFRAME FOR LARVAL DISTRIBUTION. THOMAS B. MCCORMICK, 1 LORRAINE M. BUCKLEY, 1 GABRIELA NAVAS, 2 * GLORIA BARBER, 1 BRIANNE BILLUPS, 1 VANESSA GILL, 1 BRIANNA JONES, 1 NICHOLAS PETERSON, 1 BRIANNA SAYLOR 1 AND JAMES SAYRE 3 1 Oxnard College, One University Drive, Camarillo, CA 93012; 2 Moss Landing Marine Laboratories, 8272 Moss Landing Road, Moss Landing, CA 95039; 3 California State University Channel Islands, One University Drive, Camarillo, CA ABSTRACT Settling cues in the form of coralline algae were presented to groups of hatchery-reared red abalone (Haliotis rufescens, Swainson 1822) larvae daily from day 4 to day 32 posthatch (temperature, 14 C). Survival of postlarval abalone after settlement was monitored for 30 days to quantify the effect of delayed metamorphosis on the subsequent survival of benthic juvenile abalone. After exposure to live coralline algae, an average of 85% of the larvae metamorphosed and settled the following day. The number of settled postlarvae increased gradually the longer the settling cue was withheld. Postsettlement juveniles were raised in individual containers on mixed diatoms for up to 34 days. Red abalone larvae remained competent to settle 32 days after fertilization. Larvae that metamorphosed from day 4 through day 19 had longer survival during the next 30 days (average, 19%) than those presented with a settling cue from day 20 to day 32 (average, 8%). Statistical analysis using a threshold cut point indicated that the 20-day threshold marked a significant change in subsequent postlarval survival. A receiver operating characteristic curve analysis indicated that the 20-day cut point predicted high or low future survival of postmetamorphic abalone 73.4% of the time. Larvae that swam for less than 20 days had postmetamorphic survival of 8 days or more, whereas survival was 7 days or less for postmetamorphic abalone that had swam previously for days. A larval competency period of 20 days is significantly longer than the 5 7-day larval stage often used to estimate transport times for this species. KEY WORDS: Haliotis rufescens, larval competence, metamorphosis, post-larval survival, red abalone, larvae, distribution INTRODUCTION Dispersal of marine invertebrates is often associated with the early, often planktonic, stage of development. The duration of the larval stage varies greatly depending on the type of larvae. Planktotrophic larvae feed actively and may remain in the plankton for weeks or months; nonfeeding lecithotrophic larvae may spend only a few days in the plankton. For both types of plankton, metamorphosis to benthic forms may be delayed until suitable substrate is encountered (Morse et al. 1979, Hadfield & Strathmann 1996, Wilson & Harrison 1998). Although delayed metamorphosis may extend the larval period, and hence dispersal potential, it may come at a cost of lower postmetamorphic survival and fitness (Pechenik et al. 1998, Roberts & Lapworth 2001). The effects of delayed metamorphosis on subsequent survival have been demonstrated for a range of invertebrate species, including echinoderms (Highsmith & Emlet 1986), bryozoans (Woollacott et al. 1989), gastropods (Pechenik & Eyster 1989), and bivalves (Maldonado & Young 1999). Roberts and Lapworth (2001) and Takami et al. (2002) examined the effect of delayed metamorphosis on growth of postmetamorphic abalone. Both studies found that growth was reduced after longer delays in metamorphosis, suggesting nutritional stress resulting from the limited energy content of the yolk of these lecithotrophic larvae. Roberts and Lapworth (2001) point out that nutritional stress coupled with precocious development of juvenile attributes (Spalding & Morse 1991, Moss 1999) may affect performance of postmetamorphic abalone. The role of the larval stage for abalone dispersal appears to vary by species (Forster et al. 1982, Tegner & Butler 1985, Prince *Corresponding author. kronborgia@gmail.com DOI: / et al. 1987) and by local oceanographic conditions (McShane et al. 1998, Sasaki & Shepherd 1995). Work by Seki and Lan-no (1977) and Morse (1984) indicated that lecithotrophic abalone larvae have a relatively short planktonic duration of 4 10 days. Searcy-Bernal (1999) estimated a 6-day duration of larval competency in red abalone (Haliotis rufescens Swainson 1822), from day 6 to day 12 after fertilization. Searcy-Bernal (1999) then examined survival of larvae 2 days and 6 days after metamorphosis. The time interval between fertilization and hatch varies by species and temperature. For red abalone, this interval is h, approximately one day (McCormick, pers. obs.). The current work was undertaken to define more completely the point when delayed settlement has a deleterious effect on subsequent postmetamorphic survival in red abalone and to provide a better understanding of larval biology and dispersal capabilities. A 2-part experimental design tested the effect of larval age (4 30 days posthatch) on percent settlement and the effect of settlement age on postmetamorphic survival (up to 30 days). MATERIALS AND METHODS Larvae of red abalone were produced from hatchery-raised and wild broodstock held at the Channel Islands Marine Resource Institute (CIMRI) at the Ormond Beach Mariculture Laboratory, Oxnard, CA. Abalone were spawned using hydrogen peroxide (Morse et al. 1977). Eggs were fertilized and hatched, and larvae were cultivated in a flow-through system (Seki & Kan-no 1977, Mill & McCormick 1992, Tong & Moss 1992) at a temperature of 14 C for 4 days. Samples of 25,000 larvae and UV-irradiated seawater were moved to a laboratory at Oxnard College. Aliquots of 100 ml seawater and larvae were taken from a 10-L holding container. Larvae were transferred into well plates (Falcon no. 3043) in 2 steps. First, using 1183

3 1184 MCCORMICK ET AL. a plastic disposable pipette, they were moved with a small volume of seawater to a Petri dish. A 100-mL micropipette was then used to transfer 25 larvae from the Petri dish into each of 24 wells of a tissue culture plate. The transfer and condition of the larvae were observed with a dissecting microscope. Each 3-mL well had previously been filled with 1.5 ml of a stock solution of filtered, antibiotic-treated seawater at a concentration of 7.5 ml antibiotic solution (penicillin G sodium and streptomycin sulfate BP antibiotic; Fisher E) to 1,000-mL UV-irradiated seawater filtered through a 0.22-mm Millipore ExpressPlusfilterandstoredfor24hat14.0 C prior to use (C. Friedman and C. Jackels, University of Washington, pers. comm., 2006). After larvae were placed into each well, it was filled to 3 ml with more antibiotic stock solution to ensure a consistent volume in each well and to prevent larvae from adhering to the water surface. Temperature in the wells was maintained by filling 1 row (6 wells) with larvae and stock solution at a time. The covered culture plate was then moved to the 14 C incubator and the treatment was repeated on another tissue culture plate. After 9 tissue culture plates were filled with larvae and antibiotic stock solution, the covered plates were returned to the 14 C incubator. Larval settlement and subsequent survival was divided into 2 phases. Phase 1 consisted of the introduction of settlement stimuli to replicate groups of 25 swimming larvae starting on day 4 postfertilization and ending at day 32. Each day, a group of 25 larvae were placed in each of 6 wells for a total of 150 larvae. During phase 2, individual settled larvae were each transferred to a separate well and supplied with diatoms as a food source. Survival of these larvae was then monitored daily for 30 days. Phase 1: Larval Competency Coralline algae is known to induce metamorphosis in abalone larvae (Morse et al. 1979) and was used as a settling stimulus in this experiment. Coralline algae was grown at CIMRIÕs hatchery on fiberglass plates over the course of several years. Plates were removed from seawater and submerged in freshwater for 24 h to remove epizootic species. Plates were cut into 1-cm 2 pieces to serve as settlement cues and were stored in the antibiotic stock solution in the incubator for up to 10 days with daily 3-h treatments of light (40 W incandescent) and aeration. Each day until day 32, 6 wells each received a settlement cue. Prior to a row receiving the cue, the number of dead and settled larvae was counted to establish the initial number of swimming larvae. Wells with a settlement cue were checked each day for swimming, settled, or dead larvae. Metamorphosis of each larva was verified by observing the loss of the velum. The first 6 wells of each plate served as a control and did not receive settlement stimuli. Phase 2: Effect of Delayed Settlement on Survival On the day after settlement, up to 4 postlarvae from each well were transferred into individual wells on a separate well plate to observe subsequent survival. Postlarval survival was monitored for up to 32 days or until death. There were several instances during the first 9 days when less than 4 larvae (out of 25) had settled on the day following the introduction of the coralline algae. In these 18 wells (out of 720 wells), the largest time difference between the first and fourth settled larvae replicates transferred from group wells to individual wells was 3 days. Postlarvae were fed a mix of diatoms raised at CIMRIÕs hatchery on fiberglass plates in shaded outdoor tanks receiving flowing, filtered, UV-irradiated seawater. Diatoms, microalgae, and epibenthic zooplankton were scrubbed from the plates with a Scotch Bright scouring pad (3M Corporation) in seawater. The diatom seawater solution was then poured through a 243-mm sieve to remove large particles and zooplankton. The solution was then poured through a 90-mm sieve to retain appropriate-size microalgae. This solution was placed in a 250-mL beaker with constant 40 W incandescent light and aeration at room temperature. When settled larvae were transferred to individual wells, 1 drop of the algae suspension was added. Additional algae were added as needed for the remainder of the experiment. Statistical Analysis The percentage of larvae settled versus the number of days posthatch that the larvae remained swimming without a settlement cue was analyzed with a robust (median) regression analysis (Stata Statistical Software, release 9.0; StataCorp, College Station, TX). The proportion of larvae settled, the dependent variable, was related to the number of days posthatch. A median regression analysis of the data was conducted. The average number of days postmetamorphic individuals survived versus days posthatch swimming was plotted, and an exponential, smoothed curve with upper and lower 95% confidence estimate curves were fitted to the data (Metz 1978) (SPSS, version 14.0; SPSS Inc., Chicago, IL). Next, a binary logistic regression analysis (Hosmer & Lemeshow 1989, Gould & Rogers 1994) was performed to assess a threshold cut point that might be optimal to predict the number days the postmetamorphic juvenile abalone would survive based on the number of days the larvae remained swimming posthatch. A receiver operating characteristic curve analysis was conducted to assess the performance of the day cut point (SPSS Inc.) RESULTS Phase 1: Initial Settlement Larvae provided with the coralline algae settlement cue at 4 days posthatch did not begin settlement until day 6 posthatch, whereas larvae provided with cues on days 5 and 6 posthatch had 77% settlement on day 6 or subsequently the first day they were provided with the cue. In subsequent days, the settlement increased to an average of 85% by day 32, as shown in Figure 1. Settlement was variable from day to day within a range of %. High numbers of larvae settled even when the settlement cue was withheld for 32 days. A robust (median) regression analysis of the percentage of larvae settled versus the number of days posthatch that the larvae remained swimming without a settlement cue yielded a significant (P ¼ 0.021) linear regression with a pseudo r 2 equal to and the equation Percent larvae settled ¼ Days posthatch. More larvae settled as the number of days posthatch increased. Phase II: Survival of Postmetamorphic Juveniles Relative to Duration of Larval Swimming Stage Postsettlement survival was correlated to the duration of the swimming stage. The number of days individuals survived relative

4 LARVAL COMPETENCY OF HALIOTIS RUFESCENS 1185 under the curve (performance measure) of This indicates that 73.4% of the time, the 20 day cut point will predict those individuals that would survive 7 days or less, or 8 days or more. Larvae that received a settlement cue during the first 20 days survived an average of 17 days (95% CI, ) postsettlement. Larvae that received a settlement cue between days 21 and 32 posthatch survived an average of 8 days (95% CI, ) days half that of larvae that received the cue during the first 20 days. DISCUSSION Figure 1. Settlement of larvae when presented settling stimuli 1 32 days after hatch. to age of settlement with an exponential, smoothed curve with upper and lower 95% confidence limits is shown in Figure 2. A threshold cut point that predicts the number of days the larvae postmetamorphic juveniles survive based on the number of days larvae remained swimming was determined with a binary logistic regression. The analysis indicates that day 20 posthatch marks a threshold between high and low survival of the resulting postmetamorphic juveniles. Larvae that encountered the coralline algae from 4 20 days posthatch survived for an average of 17 days (average minimum, 8 days; average maximum, 25 days), and an individual maximum 32 days after settlement and metamorphosis. Larvae that swam more than 20 days posthatch before encountering the coralline algae could be predicted to survive 7 days or less after settlement. A threshold of 20 days posthatch (from the logistic analysis) coincides with a marked decrease in the 95% confidence interval (CI) curves, from days posthatch. The logistic equation given with binary indicator at 20 days is 1:0 : ð1:0 + exp½0:935 0:129ŠÞ 3 Average no: of days individuals survived The receiver operating characteristic curve analysis used to assess the performance of this 20 day cut point yielded an area Larval Competency Period Larvae of Haliotis rufescens remained competent to settle during the entire 32-day experimental period. This capability to delay metamorphosis is similar to that observed for Haliotis discus hannai (24 days (Takami et al. 2002)) and Haliotis iris (30 days (Roberts & Lapworth 2001). The average percentage of red abalone larvae that settled after encountering coralline algae increased gradually during the 32-day trial. This is contrary to reports by Searcy-Bernal (1999), who tested competency of H. rufescens at 6, 8, 10, and 12 days after fertilization (17 C) and found that induction of metamorphosis decreased from 100% to 41% between day 10 and day 12. Repeating the experiment at 16 and 18 C, he concluded that the competency period was very short from day 4 to day 10 postfertilization. However, the current work demonstrates that at 14 C the competency period extends until at least 32 days. Morse (1985) found that larvae can survive for days at 15 C. Effect of Delayed Settlement Although delaying settlement and metamorphosis may increase the chances of encountering a suitable habitat, it reduces subsequent survival of postmetamorphic juveniles. Previous work on delayed metamorphosis with Haliotis iris (Roberts & Lapworth 2001) revealed that the probability of postmetamorphic survival decreased dramatically between day 20 and day 25 postfertilization (which corresponds to days posthatch). Takami et al. (2002) also observed that survival of Haliotis discus hannai was Figure 2. Average days of survival (solid line) for juvenile red abalone as a function of the number of planktonic days prior to encountering a settlement cue. Dashed lines represent 95% confidence limits.

5 1186 MCCORMICK ET AL. correlated with the length of the larval period; postmetamorphic survival decreased from 80% for 5 to 15-day swimmers to 57% for those that swam for 19 days before settlement. In the current work with red abalone, a 20-day swimming period posthatch delineates the point at which survival of the resulting postmetamorphic abalone changed. Larvae that swam 20 days or less survived as postlarvae for an average of 18 days, whereas those that swam more than 20 days survived an average of only 7 days. Dispersal of Red Abalone The red abalone Haliotis rufescens shares with Haliotis iris and Haliotis discus hannai the ability to delay metamorphosis for up to 20 days posthatch without a significant decline in the survival of postsettlement juveniles. This 20-day time frame is 4 times longer than the previously reported competency period of 4 days posthatch required for red abalone to reach settling competency. Longer duration of the competent larval stage offers the potential for dispersal over long distances. Tegner and Butler (1985) conducted a drift tube study examining connectivity between abalone populations on offshore islands and mainland southern California. Based on the premise that the larval stage of abalone persists for 4 7 days, they concluded that little transport between the islands and mainland was possible. Water velocities within the California Current may exceed 20 cm/sec during spring conditions (Miller et al. 1999). If the larval competency period is lengthened to 20 days for calculations, potential transport distance is increased to 350 km, a distance that encompasses most of the southern California Bight and all the islands within it. Although a longer larval stage may not always result in greater larval dispersal (McShane 1992, Sasaki & Shepherd 1995, Prince et al. 1987), it provides the potential. Further study to substantiate larval competency periods for other species of abalone is especially important in light of the number of species with recent population declines. ACKNOWLEDGMENTS This research was supported by the NOAA Minority Serving Institution Environmental Partnership Program grant no. NA09OAR , the Channels Islands Marine Resource Institute, and GenOn Energy. Forster, G. R., G. W. Potts & R. Swinfen Changes in the Ormer populations of Guernsey and Jersey. J. Mar. Biol. Assoc. UK 62: Gould, W. W. & W. H. Rogers Quantile regression as an alternative to robust regression. In: Proceedings of the Statistical Computing Section. Alexandra, VA: American Statistical Association. Hadfield, M. G. & M. F. Strathmann Variability, flexibility and plasticity in life histories of marine invertebrates. Oceanol. Acta 19: Highsmith, R. C. & R. B. Emlet Delayed metamorphosis: effects on growth and survival of juvenile sand dollars (Echinoidea: Clypeasteroida). Bull. Mar. 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