Settlement and Metamorphosis of Red Abalone (Haliotis Rufescens)
Settlement and Metamorphosis of Red Abalone
(Haliotis rufescens) Larvae: A Critical Examination
of Mucus, Diatoms, and ~-Aminobutyric Acid
(GABA) as Inductive Substrates
A Thesis
Presented to
The Faculty of the Department of Biology
San Jose State University
In Partial Fulfillment
of the Requirements for the Degree
Master of Arts
By
Marc Slattery
December, 1987 iii
Abstract
Settlement and metamorphosis of red abalone,
~~~~~~ larvae in the presence of three inductive cues
(mucus, diatoms, and j'-aminobutyric acid) was tested without the use of antibiotics. Larval settlement differed between substrates. Mucus from juvenile abalones yi significantly higher settlement. Settlement varied during the year and was highest between August and mid
September. Metamorphosis and survival (to the development of the first respiratory pore) was variable among the substrates. At 11 weeks, approximately 50 %, 20 %, and 0 % of the larvae had survived on mucous, diatom, and GABA substrates, respectively. In all treatments an initial high rate of mortality and stunting of some larvae suggested the abalone were feeding inefficiently. iv
Acknowledgements
I would like to express my sincerest gratitude to the many people who provided support and encouragement through out the course of my research and manuscript preparation. I am indebted to the members of my committee: Dr. James w. Nybakken, Dr. Michael S. Foster, and Dr. Gregor M. cailliet, who provided invaluable experience, time, and patience in critical review of my thesis. Dr. Daniel E. Morse's enlightening comments are also appreciated. My deepest gratitude is extended to Mr. Earl E. Ebert, of the
Fish and Game Marine Culture Lab, for sharing his insights and ideas on abalone culture.
Thanks to Dr. Phil Law and Allen Grover for their technical assistance. Special thanks to the staff of the
Marine Culture Lab for their continued cooperation. The help of Lynn McMasters, and the faculty, staff, and students of Moss Landing Marine Labs is gratefully acknowledged.
My family and friends have been a source of constant encouragement for which I am extremely grateful. Thanks to Dr. Roy s. Houston who taught me to look at "la pintura grande." Finally, this thesis is dedicated in loving memory to Kim Peppard. v
Table of contents
Page
Abstract iii
Acknowledgements iv
List of Tables vi
List of Figures vii
Introduction 1
Materials and Methods 7
Results 12
Discussion 14
References 21
Tables 27
Figures 30
Appendix A. 32
Appendix B. 33 vi
List of Tables
1. ANOVA Summary Table showing the effects of 27 water treatment (FSW and FSW + GABA), substrate (clean plastic, diatoms, 24 hour mucus, and 72 hour mucus), period, and interactions (between treatment, substrate, and period) on larval settlement.
2. Tukey's Studentized Range Groupings for 28 mean larval settlement with respect to substrate (a), and period (b).
3 • ANOVA Summary Table showing the effects of 29 substrate (diatoms, diatoms + GABA, and diatoms + mucus), period, and interactions (between substrate and period) on larval survival. vii
List of Figures
1. Mean Settlement of abalone larvae in 30 FSW and FSW + GABA on 72 hour mucous (a), 24 hour mucous (b), clean plast (c), and diatoms (d) substrates during ten periods from 5/28/85 to 12/4/85.
2. Mean Survival of abalone larvae on diatoms 31 + mucous, diatoms, and diatoms + GABA substrates during three trials; 5/28/85, 9/10/85, and 12/4/85. 1
Introduction
A critical stage in the life history of marine inverte brate larvae occurs during the termination of the planktonic or dispersive stage. The transformation from larva to
juvenile involves two distinct processes: settlement and metamorphosis (Chia, 1978; Crisp, 1974; Hadfield, 1984).
Settlement has been described as a behavioral change typically characterized by the active searching for and orientation to certain environmental factors (Crisp, 1974;
Hadfield, 1984). Metamorphosis is a non-reversible state that involves anatomical and physiological changes in the
organism (Bonar, 1976; Scheltema, 1974).
The specific factors involved in larval settlement and metamorphosis are quite variable (see for example Burke,
1983). However, the classic model appears to be a simple
stimulusjresponse system, possibly under some neuronal
control (Chia, 1978; Hadfield, 1978). Larvae grow until
they reach a time of "competence" that enables them to
respond to certain environmental cues (Burke, 1983). This
typically corresponds to development of sensory structures
by the organisms (Bonar, 1976; Crisp, 1974; Morita, 1972;
Morse et al., 1979a). 2
Settlement is affected by several factors including age, diet, and physio-chemical characteristics of the available substrate (Hadfield, 1984). In the presence
of the requisite stimulus the larva responds with species
specific tissue morphogenesis (Bonar, 1976). Metamorphos
can be delayed if the correct environmental cues are not
encountered (Burke, 1983; crisp, 1974). It is the nature of
the cues that triggers settlement and metamorphosis in the
larva (Baloun and Morse, 1984).
The red abalone, Haliotis rufescens, is highly prized
as food for humans, much research has been done on its
commercial culture potential (Ault, 1982; Kan-no, 1975;
Kikuchi and Uki. 1974; Leighton, 1972; Leighton et al.,
1981; Morse et al., 1977 and 1979a; Seki and Kan-no, 1977
and 198la). According to Ault (1982), Horse et (1979a)
and Mottet (1978), red abalone larvae, held in cultures at
15°C, become competent within 6-7 days following the
development of eyespots, a muscular foot, and cephalic
tentacles. Morse et (1979a; 1979b) noted that mari-
culture of this species was not economically feasible
because the processes of settlement and metamorphos were
retarded in the absence of a naturally required specific
biochemical cue. This resulted in high rates of mortality 3
when the larva's yolk supplies were exhausted. However, juvenile abalone {l-20 mm) were most commonly observed in natural "nursery habitats" of crustose red algae including
Lithophyllum, Lithotharnnion, and Hildenbrandia, suggesting the settling cues might be associated with these species
(Morse et al., 1979a; 1980). Many invertebrate species, including tubeworms (Gee, 1965), chitons (Barnes and Gonar,
1973; Rumrill and cameron, 1983), limpets (Steneck, 1982), and asteroids (Barker, 1977) have also been reported to settle preferentially on crustose red algae.
Morse et al. (l979a; 1980) subsequently discovered that a /-aminobutyric acid (GABA) mimetic peptide and phycoerythobilin were the settlement inducing agents sequestered at the surface of the crustose red algae. Foliose reds, greens, browns, and cyanobacteria were also found to contain a GABA mimetic peptide and biliproteins
(Morse et ~~ 1984; Morse and Morse, 1984). However, the larvae required contact with the biochemical cues in order to trigger settlement and metamorphosis (Morse et
1980) . The morphogenetic inductive molecules were freed during mucus sloughing by the epithelial cells of crustose reds and contact occurred when larvae randomly tested the substrate (Morse and Morse, 1984). The requirement of 4
larval contact assured that the abalone would recruit to a desirable habitat which provided inductive molecules, shelter, camouflaging pigments, and nutrition (Morse et al.,
1979a; 1979b; 1980).
Three methods have been most often used to initiate settlement and metamorphosis. The first involved the use of a 1 ~M solution of GABA in filtered seawater. In the presence of antibiotics, Morse et (1979a; l979b) achieved 93% settlement and metamorphosis of abalone larvae at that concentration. They noted that higher concentra tions inhibited metamorphosis and lower concentrations slowed the process. However, in the absence of antibiotics, complete mortality of the larvae was reported (Morse et al.,
1979b). A second method used diatom mats, the predominant diet of newly settled larvae, as the inductive substrate
(Grant, 1981; Ina, 1966; Ebert and Houk, 1984). An average of 7.5% of the larvae settled, by the California Department of Fish and Game's Marine Culture Lab, on diatom mats survived to 3 months age (Ebert and Houk, 1984). Finally, the mucous material secreted by the foot of juvenile and adult abalones has been used to successfully induce settle ment and metamorphosis of larval abalone in Japanese hatcheries with a 12% survival rate (Kan-no, 1975; Seki, 5
1980; Seki and Kan-no, l98lb).
The advantage of GABA for use in rnariculture is the high percentage of metamorphosis, whereas the advantage of diatoms or mucus is that they are simple, inexpensive methods. However, there is little agreement within the scientific community as to which method is best for use in rnariculture. Akashige et al. (1981) reported that GABA actually narcotized the velar cilia of the planktonic larvae causing the veligers to "fall" out of solution and die on the substrate. Morse (pers. corn.) noted the "weakly
inductive" potential of mucus, but suggested that mucus was a breeding ground for a "microbial overgrowth" that could kill the larvae. To further complicate the situation, there has been little consistency in experimental design among the investigators, making comparisons difficult.
Results were obtained from experiments involving several hundred (Morse et al., 1979b) to several million (Grant,
1981) larvae in vessels of variable volumes (Mottet, 1978).
The importance in determining the best substrate for
settlement and metamorphosis of abalone larvae is unequiv
ocal. In addition, the need for standardization in determi
nation of a "best method" is clear. The purpose of this study was to compare settlement, metamorphosis, and 6
survival rates of red abalone larvae on mucous, diatom, and clean substrates in the presence and absence of GABA without the benefit of antibiotics. The experimental design addresses the need for development of low cost mariculture systems through questions of settlement substrate pref erence, inductive cue strength, and survival rates of abalone larvae in standard culture vessels. 7
Materials and Methods
The studies were conducted at the California Department of Fish and Game's Marine Culture Laboratory over an eight month period from June 1985 to February 1986. The lab is located at Granite canyon, an exposed section of coastline, approximately 12 miles south of Monterey. The existing seawater system, assembled by Ebert et al. (1974), was modified with the addition of 5, 3, 1, and 0.5 micron in-line cartridge filters. This effectively reduced culture contamination by copepods, nematodes and bacteria. Lab oratory broodstock were utilized for all larval production
(Ebert and Houk, 1984).
Abalone were spawned with the ultraviolet irradiated seawater technique described by Kikuchi and Uki (1974). The ova were fertilized, washed, and left to develop to the veliger stage for 30 hours in 15°C UV treated seawater
(Ebert and Hamilton, 1983). The "healthiest" larvae, as determined by their swimming behavior, were held in con centrations of 5 per ml in 8 liter culture tubes and maintained for 6-7 days at 15°C (Ebert and Houk, 1984).
The competent larvae, which exhibited the characteristic behavioral (exploration and orientation) and morphological 8
(sense organ development and cephalic tentacle protrusion) changes described by Mottet (1978) and Seki and Kan-no
{l98la), were utilized in the experiments. Subsamples of larvae (McCallum, 1979) from the cultures, were collected to estimate total numbers of individuals in the ten substrate preference trials (5/28/85, 6/17/85, 7/1/85, 7/15/85,
8/5/85, 8/20/85, 9/10/85, 9/23/85, 10/6/85, and 12/4/85) and three survival trials (5/28/85, 9/10/85, and 12/4/85).
Settlement
The substrate preference of settling abalone larvae was tested in two plastic troughs. Each settling trough was
147.32 em long, 30.48 em wide, and 25.40 em deep. Seawater filtered (FSW) through 1 micron filters at 15°C was added to each trough. A solution of GABA was added to one trough to a final concentration of 1 pM, as described by Morse al. (1979b), but without the antibiotics.
Substrates for these troughs were prepared by first placing 64 plastic petri dishes 60.96 em below two Chroma-50 flourescent tubes. Forty eight dishes were innoculated with
100 ml of a diatom slurry solution (Ebert and Houk, 1984).
The remaining 24 dishes were innoculated with 100 ml of 9
filtered seawater. The diatoms settled within three days and coated the petri dishes with a thin tan film.
All 64 petri dishes were transferred to a water table after the first three days. Five juvenile abalone (8 rnrn) were placed in each of 16 dishes containing a diatom film. The dishes were covered with a plastic screen to contain the grazing abalone. Filtered seawater was deliver ed to the water table and all dishes remained immersed for
72 hours. A second batch of juvenile abalone was placed in
16 additional dishes containing the diatom film. These animals were allowed to graze their designated dishes for 24 hours. The remaining 16 petri dishes were left with an ungrazed diatom film.
After 72 hours on the water table the abalones were removed, leaving four substrates available for use in the settlement experiment: 16 petri dishes with a diatom film,
16 with a 24 hour mucous film, 16 with a 72 hour mucous film, and 16 clean dishes. Eight petri dishes, represent ing each substrate, were randomly positioned in each settling trough. Approximately 5000 larvae were added to each trough and a nytex screen was used to cover the cultures. The petri dishes were removed from each settling trough 10
after 72 hours. A Wild dissecting microscope was used to count the number of larvae that settled in each dish.
Metamorphosis
Twelve small tanks were utilized to test the survival of abalone larvae on various substrates. Each tank was constructed by welding a PVC sheet to a section of 15.24 em diameter PVC pipe. A capillary filtration standpipe was fashioned for each tank from 1.27 em diameter PVC pipe and
90 micron mesh screen. The tank volumes were 1.2 liters and provided 540.5 sq ern of wetted surface area. Seawater filtered with 0.5 micron screen was delivered to each tank at a rate of 100 rnl per min.
The twelve tanks were placed on a wet table 91.44 em under two Chroma-50 flourescent tubes. Each tank was innoculated with 1000 rnl of a diatom slurry. Within three days the diatoms had settled and produced a tan film on each tank surface. Ten juvenile abalone (8-15 rnrn) were added to each of four tanks and allowed to graze the substrate for three days. The abalones were removed from the tanks after providing a fine mucous film on the sub strate. Four additional tanks were inoculated with a 11 solution of GABA to a final concentration of 1 jlM. Three treatments were therefore available: 1) diatoms, 2) diatoms + mucus, and 3) diatoms + GABA. Two hundred larvae were added to each tank during the three trials (5/28/85, 9/10/85, and 12/4/85) of the survival experiment. The tanks were covered with a screen to prevent contamination of the cultures. Sampling for mortalities occurred after 3, 6, 9, and 11 weeks (12 weeks in trial 1).
The numbers of abalones exhibiting new shell growth were counted at these times.
A Three-Way factorial mixed effect model ANOVA was utilized to determine the effects of water treatment,
substrates, and time on the settling of larval abalone
(Winer, 1971). A Tukey's Studentized Range test was used to compare the means. A Two-Way ANOVA was used to examine the effect of period and substrate on larval survival (Zar,
1984). A nonparametric Tukey type test was used to deter mine significant differences between the samples. 12
Results
Settlement
Larval settlement was significantly affected by substrate (P< .001) and by period (P< .001) (3 Way ANOVA;
Table 1). However, the effect of water treatment on larval settlement was not significant. All two way inter- action effects (substrate x treatment, substrate x period, and treatment x period) had a significant influence on larval settlement (P< .001). The three way interactions effect was not significant.
There were significant differences between larval settlement on mucous substrates and on diatom and clean plastic substrates (Tukey Test; Table 2a). Mean settling differences were not significant between the 72 hour mucous (X= 63.99; SD = 32.20) and 24 hour mucous (X= 57.99; SD = 31.55) substrates. Similiarly, no difference was noted between the clean plastic (X= 44.23; SD = 31.74) and diatom (X= 43.35; SD = 33.27) substrates. Larval settlement varied with time (3 Way Anova;
Table 1). A significantly high number of larvae (X ~ 76.31; . SD = 25.36) settled during the 8/20/85 trial (Tukey ' 13
Table 2b and Figure 1) . Larval settlement was significantly lower (X= 20.59; SD = 25.66) during the 5/28/85 trial (Tukey Test; Table 2b and Figure 1).
Metamorphosis
Survival of larvae was markedly different (2 Way Anova;
P Approximately 50% of the larvae, in tanks with a mucous treatment, survived through development of the first respiratory pore, at 11 to 12 weeks, {Figure 2). The 0% larval survival in the GABA treatments was significantly lower (0.005 treatments (nonparametric Tukey Test q= 4.30). The survival of larvae in tanks with a mucous substrate was not signifi cantly different from survival on diatom substrates ( 2.36). No signifcant difference was noted between survival in the diatom and GABA treatments ( 1.94). All treatment tanks, in each trial run, exhibited a high initial mortality through week 3 of the experiment. 14 Discussion Settlement The results obtained suggest that red abalone larvae will settle on several substrates including diatoms, mucus, and clean plastic, but settlement on mucus was signifi cantly higher. This agrees with the findings of Seki and Kan-no (1981b) who found the larvae of Haliotis discus hannai settle preferentially on the mucous trails of adult abalones. Based on these findings it seems larval red abalone utilize mucus, or some associated component of the mucus, as an inducer of settlement and metamorphosis. Mucus discrimination has been reported in adult gastropods (Peters, 1964; Lowe and Turner, 1976) that used their cephalic tentacles to "taste" the substrate. Seki and Kan-no (1981a) have observed Haliotis discus hannai veligers testing substrates with their cephalic tentacles prior to settlement. However, the presence of ungrazed diatoms in the mucous film may also provide some inductive cue to the larvae. The amount of mucus present did not significantly affect the number of larvae that settled. This might be 15 due to a decrease in mucous production over time by the grazers held for 72 hours. Culley and Sherman (1985) reported mucous production was related to substrate texture. Production was decreased when the pedal surface was protect ed from abrasion. Another possibility is that the inductive agent within the mucus might degrade or change over time periods longer than 24 hours. For example, Seki and Kan-no (1981b) noted different settling rates of abalone larvae on mucus collected from grazing, crawling, and stimulated adults. This suggested that the secreted mucus was of a different chemical nature. These hypotheses could fee- tively explain the similar inductive potential of 72 hour and 24 hour mucus in my experiments. Larval settlement on each substrate was unaffected by the presence of GABA. Akashige et (1981) noted the velar cilia of the larvae were paralyzed by GABA and they suggested that it caused the larvae to settle un naturally. I observed no difference in settlement of larvae on mucous substrates in FSW and on mucous substrates in FSW + GABA. This seems to suggest that if larvae are narco tized by GABA they are still capable of a certain degree of substrate selectivity or GABA does not incapacitate the larvae as reported. The fact that higher settlement was 16 noted on mucous substrates than diatom or clean substrates suggests the larvae are actively testing the substrate for a preferred inductive agent. Morse et al. (1979a) described the induction of metamorphosis by GABA as a stereochemically specific system. However, I observed larval selection of a preferred substrate in the presence of GABA. Perhaps the inductive cue in mucus utilizes a separate pathway to trigger larval settlement behavior or GABA is not blocking all the receptor sites. The lack of statistically signifi cant differences of larval settlement on clean plastic in the two water treatments is consistent with the findings of Morse et al. (1979b) and indicates the importance of antibiotics when using GABA as an inductive cue. The most surprising result suggested that larval settlement varied with time. This is confusing since all trials were subjected to a controlled set of conditions. Lannan (1980) observed increased larval survival when fertilization occured during an optimal period in the adult's gametogenic cycle and confirmed a genetic component (in addition to environmental factors) in the role of larval settlement success. Abalones exhibit extreme variability in gametogenic cycles (Mottet, 1978). The April to July peak spawning season in red abalones (Ault, 1982) suggests 17 genetic variation may be important in my experiments. It is interesting to note that larvae from the third settlement (6/30/85) group were collected during a natural spawning of abalones held in raw seawater in the lab. These larvae did not exhibit a high degree of settlement suggesting other factors were also affecting the larval set. Metamorphosis Although the results suggest red abalone larvae can settle quite successfully on many substrates, meta morphosis and survival were highly variable on each. The high rate of survival on the mucous substrate seems signifi cant as this was the preferred inductive cue for larval settlement. The mucus of gastropods has been well studied (Calow, 1974; Crisp, 1967; Cook, 1971; Grenon and Walker; 1980; Hughes, 1978; Lowe and Turner, 1976; Peters, 1964) and is probably quite important ecologically. Seki and Kan-no (1981b) noted abalone larvae of Haliotis discus hannai settled on mucus produced by adults of the same species, as well as other abalone species, suggesting a common agent within the mucus. It follows that the mucus is an important inductive agent for larval abalone settlement. However, 18 there is little evidence to support a gregarious settling strategy in natural populations of abalone larvae. It appears this system may have been historically important, perhaps as a precursor to the coraline algae inductive system described by Morse et ~ (1980). Survival noted on the diatom substrate is interesting as it compares quite favorably with the survivorship observed by Ebert and Houk (1984) in their hatchery tanks. They reported yields ranging from 1.9 to 13.5 % dependent on larval stocking densities. Similar survivorship was reported in Japanese hatcheries utilizing mass culture methods (Kan-no, 1975). Most researchers agree the initial diet is most important in determining survival of post settled larvae (Garland et al., 1984; Imai, 1967; Seki, 1980). This might help explain the initial high mortality observed during the first three weeks of the experiments. The evidence suggests the larvae are feeding in- e iciently in the culture containers. Recent studies have shown newly settled larvae were incapable of digesting large pennate diatoms (>10 microns) due to the slow development of the radula and fed almost exclusively on bacteria grazed from the substrate (Garland et al., 1984). Th suggests another food source, absent in my cultures, might 19 be crucial during the early (1-3 weeks) juvenile develop ment. The higher rate of larval survival in mucous cultures compared to diatom cultures indicates the nutritional value of mucous material (Calow, 1974). Larvae may be utilizing mucoproteins, mucopolysaccharides, bacteria, or some as yet undetermined component of the mucus during the initial 3 weeks of settlement. The poor survival of larvae settling in the presence of GABA superficially supports the argument of Akashige et al. (1981) that narcotized larvae are incap able of feeding. However it seems more likely the absence of antibiotics, a requisite component of the GABA inductive system (Morse, pers. com.), had a greater effect on larval survival, The o % survival recorded in tanks containing GABA is consistent with the results reported by Morse et al. (1979b, 1980) in which they compared survival in penicillin and streptomycin treated cultures with untreated cultures. I used no antibiotics in my cultures; however, my experimental design allowed for equal infection of all cultures. It is surprising that larvae in mucous and diatom cultures persisted, with varying degrees of success, while larvae in GABA cultures did not survive. Characteristic grazing of 20 the diatom~ilm (Ebert and Houk, 1984~ Morse et , 1979a; Mottet, 1978) was evident in the mucous and diatom cultures but not in the GABA culture. I observed stunting of larvae and a lack of new shell growth in the GABA cultures. Approximately 10 % of the larvae in the diatom and mucous cultures also exhibited stunting; this probably represent ed the "normal 11 situation or was an artifact of the hypo thesized incomplete diet. These results confirm the importance of antibiotics in culture work utilizing GABA. My results indicate red abalone larvae settle prefer entially on the mucous trails left by grazing juvenile and adult abalone. In addition, the highest rates of meta morphosis and survival were recorded on the mucous sub strate. Many invertebrate species have larvae which settle preferentially in the optimal adult habitats (Burke, 1983; Crisp, 1974; Scheltema, 1974). Morse and his colleagues (1980) proposed an evolutionary bond between abalone larvae and certain species of crustose red algae which appeared to be optimal "nursery habitats" for juvenile abalones. My results suggest this coevolution was secondary in develop ment to the mucus inductive system. Furthermore, my results strongly suggest mucus is the most important inductive agent for the settlement and metamorphosis of larval red abalone. 21 \ References Akashige, s., Seki, T., Kan-no, H. and Nomura, T., 1981. Effects ofl'-aminobutyric acid and certain neurotrans mitters on the settlement and the metamorphosis of the larvae of Ha1iotis discus hannai Ino (Gastropoda). Bull. Tohoku Reg. Fish. Res. Lab., 43:37-45. Ault, J.S., 1982. Aspects of the laboratory reproduction of the red abalone, Haliotis rufescens Swainson. M.S. Thesis, Humboldt State University. 77pp. Baloun, A.J. and Morse, D.E., 1984. Ionic control of settlement and metamorphosis in larval Haliotis rufescens (Gastropoda). Biol. Bull., 167:124-138. Barker, M.F., 1977. Observations on the settlement of the brachiolaria larvae of Stichaster australis (Verrill) and Coscinasterias calamaria (Gray) (Echinodermata: asteroidea) in the laboratory and on the shore. J. Exp. Mar. Biol. Ecol., 30:95-108. Barnes, J.R. and Goner, J.J., 1973. The larval settling response of the lined chiton Tonicella lineata. Marine Biol., 20:259-264. Bonar, D.B., 1976. Molluscan metamorphosis: A study in tissue tranmsformation. Amer. Zool. 16:573-591. Burke, R.D., 1983. The induction of marine invertebrate larvae: Stimulus and response. can. J. Zool., 61:1701-1719. Calow, P., 1974. Some observations on locomotary strategies and their metabolic effects in two species of freshwater gastropods, Ancylus fluviatilis Mull. and Planorbis contortus Linn. Oecologia (Berl.), 16: 149-161. Chia, F.S., 1978. Perspectives: Settlement and metamorph osis of marine invertebrate larvae. in: Settlement and metamorphosis of marine invertebrate larvae. (ed. Chia, F.S. & Rice, M.E.). Elsevier, New York. 290 pp. Crisp, D.J., 1967. Chemical factors inducing settlement in Crassostrea virginica (Gmelin). J. Anim. Eco1., 36:329-335. 22 Crisp, D.J., 1974. 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Prentice-Hall Inc., New Jersey. 718 pp. 27 Source of Variation DF ss MS F p Total 639 733936.34 Cells 79 349270.84 4421.15 6.44 <0.001 Treatment 1 8555.63 8555.63 2.52 <0.5 Substrate 3 50270.53 16756.85 9.48 <0.001 T X S 3 16589.56 5529.85 8.35 <0.001 Period 9 177707.38 19745.26 28.75 <0.001 T X p 9 30551.09 3394.57 4.94 <0.001 s X p 27 47719.81 1767.40 2.57 <0.001 T X s X p 27 17876.84 662.11 0.96 <0.5 Error 560 384665.50 686.90 Table 1. ANOVA summary Table showing the effects of water treatment (FSW and FSW + GABA), substrate (clean plastic, diatoms, 24 hour mucus, and 72 hour mucus), period, and interactions (between treatment, substrate, and period) on larval settlement. 28 a) N Mean SD Substrate Tukey Grouping 160 63.99 32.20 72 hour mucus 160 57.94 31.55 24 hour mucus J 160 44.23 31.75 clean plastic 160 43.35 33.27 diatoms J b) N Mean SD Period/(date) Tukey Grouping 64 76.31 25.36 6 (08/20/85) 64 72.19 13.93 7 (09/10/85) 64 64.67 20.14 5 (08/05/85) J 64 61.56 26.49 10 (12/04/85) J 64 54.67 40.60 9 ( 10/06/85) 64 53.39 39.43 4 (07/15/85) 64 48.48 33.49 8 (09/23/85) 64 38.11 21.97 3 (07/01/85) ] 64 33.92 25.77 1 (05/28/85) 64 20.59 25.66 2 (06/17/85) ] Table 2. Tukey's studentized Range Groupings for mean larval settlement with respect to substrate (a), and period (b);~= 0.05, DF = 560, MS = 686.9027. Means with overlapping bars are not significantly different. 29 Source of Variation DF ss MS F p Total 143 404809.49 2830.84 Cells 11 370001.41 33636.49 127.56 <0.001 Substrate 2 283742.76 141871.38 538.01 <0.001 Period 3 79849.24 26616.41 100.94 <0.001 S X p 6 6409.40 1068.23 4.05 <0.001 Error 132 34808.08 263.70 Table 3. ANOVA Summary Table showing the effects substrate (diatoms, diatoms + GABA, and diatoms+ mucus), period, and the inter actions (between substrate and period) on larval survival. 160 160 b 140 a 140 120 120 100 eo 0~~~~~~~/~~~~~~~~~~' 2 3 4 s s 1 a s 10 2 3 4 5 a 1 a 9 10 160 160 140 c d 140 lili£1 FSW 120 120 ~ FSW+GABA 100 100 2: 3 4 5 s 1 a g 10 2 3 4 s 6 7 ll 9 10 Period Period Figure 1. Mean Settlement of abalone larvae in FSW and FSW + GABA on 72 hour mucous (a), 24 hour mucous (b), clean plastic (c), and diatoms (d) substrates during ten periods from 5/28/85 to 12/4/85. Error bars = + 1 SD. w Histograms =X larvae from 8 petri dishes. 0 31 200 180 -o- Dlatoms+Mucus 160 -+- Diatoms -1!8- DlatomS+GABA CIJ ro 140 t: ro -1 120 CIJ :> :J 100 =1:1: c co CIJ 80 :E 60 40 20 0 0 3 6 9 12 Weeks Figure 2. Mean Survival of abalone larvae on diatoms + mucous, diatoms, and diatoms + GABA sub strates during three trials; 5/28/85, 9/10/85, and 12/4/85. Error bars = ± 1 SD. Symbols = X of 12 replicates for each substrate. 32 Treatment FSW FSW + GABA Substrate Period N Miiii olsan plaliltic 1 a 13.75 4.87 32.38 12.60 2 8 11.00 11. OJ 22.38 9.49 3 a 24.13 18.57 41.38 9.77 4 8 32.38 14.84 49.00 17.36 5 8 84.88 45.63 74.63 73.68 6 8 61.38 28.36 57.38 58.20 7 e 61.13 19.89 66.25 23.18 8 a 49,50 12.85 44.75 25,61 9 a 33.00 10.58 47.13 13.62 10 a u.88 10.43 33.38 10.00 diatoms 1 8 5.25 2.76 16.88 14.52 2 8 13.00 9.15 31.50 19.84 3 8 22.38 19.70 35.00 10.85 4 8 34.38 13.07 46.50 13.75 5 B 59.63 21.96 36,00 33.45 6 8 90.38 28.55 85.13 57.68 7 B 75.00 :n. 79 49.75 32.39 a 8 32.88 19.85 38.25 20.01 9 a 48.38 15.77 44.88 24.29 10 8 53.00 U.62 48.88 17.78 24 hr mucus 1 8 42.50 5.61 43.75 16.48 2 8 13.00 7.!56 21.63 20.56 3 B 46.50 13.14 44.88 14.05 4 8 52.25 23.96 60.88 19.58 5 8 97.75 29.03 49.00 31.43 6 8 83.63 25.29 53.63 40.57 7 B 90.75 23.06 76.50 43.45 8 8 48.50 18.72 55.50 23.18 9 8 74.13 20.50 49.63 27.93 10 B 53.00 19.62 48.88 17.78 7:il hr mucus 1 8 73.13 34.26 44.13 19.23 2 8 21.50 13.94 30.75 13.72 3 8 4 6. B8 31.52 43.75 26.67 4 8 eo. 38 27.45 71.38 38,64 5. 8 74.63 36.21 40.88 25.80 6 B 8·L50 29.52 94.50 36.15 7 8 116.75 51.73 61.38 23.72 8 8 68.50 24.10 50,00 16.63 9 8 91.38 22.35 48.88 27.75 10 8 99.63 16.517 56.88 14.47 Appendix A. Mean Settlement of abalone larvae in water treatments (FSW and FSW + GABA) over four substrates (clean plastic, diatoms, 24 hour mucus, and 72 hour mucus) during ten periods (5/28/85 to 12/4/85). N = number of dishes sampled. 33 Substrate Period N Mean SD Mucus 1 12 160.08 18.39 2 12 122.58 18.09 3 12 105.25 17.53 4 12 93.58 33.42 Diatoms 1 12 112.83 15.25 2 12 65.75 18.25 3 12 45.00 12.93 4 12 32.67 8.93 GABA l 12 34.42 14.76 2 12 8.58 6.56 3 12 0.50 1.17 4 12 0.17 0.39 Appendix B. Mean Survival of abalone larvae on three substrates (diatoms + mucus, diatoms, and diatoms + GABA) during four periods (3, 6, 9, and 12 weeks). N = number of tanks sampled.