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Elements and innovations in the cultivation of red abalone Haliotis rufescens
Aquaculture, 39 (1984) 375-392 Elsevier Science Publishers B.V., Amsterdam
375 - Printed in The Netherlands
ELEMENTS AND INNOVATIONS IN THE CULTIVATION ABALONE HALIOTIS RUFESCENS
OF RED
EARL E. EBERT and JAMES L. HOUK Marine Culture Laboratory, Marine Game, Monterey, CA 93940 (U.S.A.)
Resources
Branch,
California
Department
of Fish and
ABSTRACT Ebert. E.E. and Houk, J.L., 1984. Elements and innovations Haliatis r&rem. Aquaculture, 39: 375-392.
in the cultivation
of red abalone
Seven elements are described for cultivation of the red abalone, ~~l;#r~s rz&scem broodstock management; spawning induction, fe~ili~ation, and early development; larval ~ult~vat~on~ forage culture; settlement and early-juvenile-stage cultivation; weaning; and grow-out. Cultivation methods and equipment are described. Spawnable abalones are maintained on a year-round basis. Larvae are cultured in a flow-through apparatus that yields over 90% to the settling stage. A diatom slurry method is used to provide forage to the early benthonic and juvenile stages. Cultivation problems are described. Included are those with bacteria, Vibrio spp., and the maintenance of suitable diatom substrates for juvenile stages.
INTRODUCTION
Cultivation of abalone, Huliotis spp., has spanned nearly a 50-year period. However, there was a gap of 17 years between the first major contribution by Murayama (1935) and that of Ino (1952). During the 1970s there was a surge in valuable contributions on various aspects of abalone cultivation (e.g., Leighton, 1972, 1974, 1977; Shibui, 1972; Kikuchi and Uki, 1974a,b,c; Kanno, 1975; Morse et al, 1977, 1979; Seki and Kan-no, 1977, 1981). As an outgrowth of these investigations interest in cultivating abalone, an economically valuable shellfish, has spread from Japan to various parts of the world. Research and development projects, or production hatcheries, are in operation in Australia, the British Isles, Canada, Chile, France, Mexico, and the United States (Californian. Pioneering efforts to cultivate North American haliotids began in 1940 (Carlisle, 1962). However, more than 20 years elapsed before renewed interest surfaced to cultivate North American haliotids, and more specifically the red abalone, H. rufeseens. Spurred by a report on Japanese abaione-hatchery methods by Cox (1963) an initial small laboratory was constructed in 1964 at Morro Bay, on the central California coast, to test red abalone-cultivation methods (F.L. Clogston, unpublished observations). Clogston demonstrated 0044~8486/84/$03.00
0 1984 Elsevier Science Publishers B.V.
376
that red abalones could be mass cultivated, and his efforts created a surge of interest from the private sector, as welt as university and government scientists. In 1965 Pacific Mariculture (now Pigeon Point Aquaculture Center), a commercial shellfish hatchery situated along the central California coast about 40 miles south of San Francisco at Pigeon Point, began to mass cultivate the red abalone. Pacific Maricuiture became the first private company to mass produce and sell relatively large quantities of red abalone seed. However, abalone cultivation was unpro~table for Pacific Mariculture for various reasons and they opted to discontinue their red abalone-hatchery operations. The first commercial abalone hatchery in California, designed specifically for that purpose, was California Marine Associates (now Ester0 Bay Mariculture). This hatchery, located along the central California coast near Cayucos, has been in operation since 1968. The 1970s witnessed a continued interest in red abalone cultivation in California. In 1970 the California Department of Fish and Game commenced red abalone-cultivation research at their Granite Canyon Laboratory, located on the central California coast near Monterey (Ebert et at, 1974). Following this, two more commercial abalone hatcheries were established. These were Monterey Abalone Farms, which began in late 1971, and the Ab Lab, located along the coast south of Santa Barbara at Port Hueneme, which commenced operations in 1974. Additionally, an Abalone Seeding Association was formed by a group of commercial abalone fishermen and processors in late 1974 to hatch and seed abalone larvae as a means of augmenting the fishery. Southern California Edison, a major California electricity producer has also sponsored an abalone cultivation project. The broad objectives of the red abalone research and development project at the California Department of Fish and Game’s Granite Canyon Laboratory are (i) to develop further the technological and biological methods for mass cultivation and to disseminate this information, and (ii> to provide seed abalone for rebuilding impacted abalone-fishing grounds. METHODS AND MATERIALS
Cultivation methods for the red abalone, Huliotis rufescens, are divided into seven major elements, presented serially in the following pages. Some methods within specific elements are continually being subjected to experimentation to seek refinements. Where this is the case, the specific experiments and findings are presented with those elements. Broodstock is maintained in 15liter polyethylene plastic containers. Each container offers about 0.34 m* surface area, including the lid. The container lids are perforated and have a central hole to accommodate a water-inlet pipe that extends to the container bottom. Ambient temperature seawater (9 lS”C), sand-filtered to about 20 pm, and ultraviolet WV) treated, is supplied to each container at a 3-4 liter/min flow rate. Containers are cleaned weekly,
371
and fresh giant kelp, Mucrocystis spp., is supplied, to excess, at the same time. Broodstock is maintained separately according to geographic origin and sex. Variation of the above “standard” broodstock methods were tested. These included: (i) using raw seawater (no filtration or UV treatment), (ii) maintaining two containers in total darkness, and (iii) different abalone densities (30, 60, or 90/container). Also we measured and weighed abalones monthly, and recorded the amount of kelp, in wet weight, consumed by abalones weekly in each container. This afforded a calculation of the feeding rate. This is expressed as the feed consumption per abalone at its mean weight for the period and was derived using the formula: Fr-F2 x 100 t x w
where F, = feed given, F, = feed remaining, t = time in days, and W = mean abalone weight. Food-conversion efficiency was calculated and defined as the wet weight of animal gain per wet weight of food ingested. A gonad-indexing method was developed in order to assess the abalone’s maturation stage through gross examination of the gonad bulk and color. The abalone gonad envelops the digestive gland and together they form a rather large cone-shaped appendage positioned on the right side of the abalone’s foot just beneath the shell. The gonad is easily viewed by turning the abalone upside-down and pushing the foot aside. The testis is cream colored and the ovary is dark green. The gonad index is:
o-
Immature, sex indeterminate, greyish-brown mass.
1 - Gamete development color differentiated, determination is easy color; however, female 2 - Gametes fully envelop gonad not bulky.
has on for sex
the digestive
gland is easily viewed as a
initiated; gametes appear in a patchy pattern, the surface of the digestive gland. Sex males at this stage because of the creamish determination is difficult.
the conical appendage,
sex easily determined
but
3 - Same condition as for index stage 2 except that the gonad is quite bulky. This bulk extends to the gonad tip. Abalones to be used for spawning induction are preferably in index category 3; however, when these are not available, we use abalones that are between index categories 2 and 3. Gonad indexing was done monthly at the same time that the abalones were weighed and measured. However, indexing was done only in three containers that each held 30 abalones. Two of these containers were maintained in continuous darkness; one received raw seawater, the other filtered. The third container had a natural photoperiod and was supplied with filtered seawater. Gonad indices for all abalones in a container were totaled and weighed according to the number of abalones at each index (e.g., 3 at category 3 = 9; 8 at category 2 = 16). Comparison of the weighted gonad index, by broodstock container, afforded a measure of gonad conditioning
378
success according to the conditions Spawning induction, fertihation,
that were imposed.
and ear& development
Spawning induction is accomplished by exposing sexually mature abalones to heavily UV-irradiated seawater (Kikuchi and Uki, 1974a). In our system we use a REFCO’ water purifier, model RL-IO-1P (Refco International, Inc., San Leandro, California), and adjust the pumping rate of the l-pm filtered, ambient-temperature seawater to about 150 ml/min. Male and female abalones are held in separate containers (the same kind that are used for broodstock), one or two abalones per container depending upon their size. The water purifier is activated by a timer set to come on at 0600 h on a “spawning day.” A 3- to 4-h exposure to the UV-treated seawater is generally sufficient to trigger spawning. After spawning the ova are collected by siphon and distributed into 15liter plastic containers. They are fertilized within 30 min postspawning. The ova are slightly more dense than seawater and settle to the bottom of the container. Excess sperm are removed by decantation beginning about 15 min postfertilization. This is repeated two or three times or until the water is clarified. It is necessary to wait 5-10 min between decantations in order for the ova to settle. Ova that do not settle after this time are discarded. Following the last decantation the culture container is refilled. One-Nrn filtered, UVtreated seawater at 15°C is used for all culture-water exchanges. It is desirable to end up with a single layer of ova on the culture container bottom. Using a file, the shells of spawned abalones are marked with a v-shaped notch just to the right of the most anterior respiratory pore; these animals then are returned to their broodstock container: The shellnotch serves as a spawning-history reference mark. Lurval cultivation
Fertilized ova develop to the veliger stage in the hatching container. They are positively phototactic and rise to the culture surface. The veligers are dipped (by beaker) or siphoned into l-liter beakers to obtain a density estimate. This is done by aliquot sample. First, a uniform larval distribution is obtained with a swizzle stick. Aliquot samples are taken with a l-ml pipette. The pipette is passed diagonally through the larvae. Three to five aliquots are taken. The number taken is dependent upon the variation in larval counts between aliquot samples. If this variation exceeds 50% then five aliquots are taken. The veligers are distributed into 8-liter larval rearing apparatuses at a concentration of 5/ml (40,000 per culture). The larval rearing apparatus basically consists of a container within a container. The inner container (larval chamber) consists of a 20-cm diameter PVC pipe section, 36 cm tall, and screened 2.5 cm above the bottom (90 pm NITEX). The outer container is about 2’7 cm in diameter and 30 cm tall. In operation water flows downward
‘Mention
of manufacturer’s
name does not imply endorsement
319
through the inner container, through the NITEX screening, and exits from the outer container via a standpipe (Fig. 1). Larvae are cultured at 15°C using 3pm filtered, UV-treated seawater at a 200-300 ml/min flow rate.
Fig. 1. The larval-culture rearing apparatus; a, water inlet; b, outer culture container; c, top of water outlet; d, inner (larval rearing) container; e, bottom of water outlet; f, water-exit ports from larval-rearing container; g, base support; h, 90-pm NITEX screening.
Forage culture Abalone larvae do not require supplemental food. They subsist upon and nutrients from their relatively large yolky egg. Upon settlement metamorphosis, the abalone subsists on diatoms. These diatoms must be less than 10 pm in greatest dimension so that the young abalones can ingest them. To meet this requirement we developed a diatom-slurry system. The diatom-slurry system entails the collection of diatoms that have passed through selective filtration, adventitiously settled on a substrate, and grown. These diatoms are removed by wiping, refiltered, and concentrated in a slurry. We use white polyethylene plastic containers for diatom settlement and culture substrates. Each container offers about 2,900 cm* of surface area. Ambient temperature seawater, filtered to 1 pm, is supplied to each container. Water flow rates are not closely monitored, but are less than 0.5 liter/min. Illumination is provided by overhead fluorescent lamps (Westinghouse, Chroma SO>. The lamps are positioned 30 cm above the diatom cultures. Normally, after a 7-day growth period, the diatoms are harvested. This is done by draining each container and wiping down the diatom film with a latex glove. The diatoms are rinsed with l-pm filtered seawater and poured first through a j-pm and then a l-pm filter bag. Diatoms that pass through the lpm bag filtration are retained for the larvae that are ready to be settled. The diatoms that do not pass through the filter bags are retained for feeding older juvenile abalones. One diatom container will generally yield 1 liter of diatom
380
slurry (l-pm bag filtered) at a concentration of 5.0 x 105/ml. Part of this yield (about 6.8 x 10’ diatom cells) is used to reinoculate the original diatom-culture container. The reinoculated container is refilled with l-pm filtered seawater and held static for 24-48 h to allow the diatoms to settle and attach. After this time a gentle water flow rate is reestablished. Numbers of diatom cells from the concentrated slurry used to inoculate various sizes of abalone settling tanks are: 3.5 x 107/12-liter tank; 4.0 x 107/14-liter tank; 1.3 x 108/73-liter tank; 3.3 x 108/176-liter tank; and 3.5 x 108/302-liter tank. Abalones 5 mm and larger can readily forage on macroalgae. We generally use giant kelp, Mucrocystis spp., for these abalones. Occasionally, bull kelp, Nereocystis luetkeana, and feather boa kelp, Egregia menziesii, are used. Settlement and ear~~uve~ile-stage cuit~vation
The abalone settling tanks are constructed from fiberglass and have a white gel coat finish. They are circular with a dished bottom. The tanks are about 107 cm wide and have a sidewall depth of about 25 cm. The bottom slopes downward to about a 33-cm depth at the center bottom drain opening. The drain opening accommodates about a 3.8-cm diameter standpipe. These tanks hold about 260 liters and offer about 17,000 cm2 of wetted surface area. The settling tanks are located in a greenhouse that incorporates translucent plastic siding in order to maximize solar illumination. Preparation of settling tanks for abalone larvae generally commences 24 h before larval seeding. Clean tanks are used. They are filled with l-hrn filtered, UV-treated seawater, at 15°C. The tanks are inoculated with a diatom slurry (described in forage culture section). The following day, when the abalone larvae are introduced, the diatom inoculum has formed a very faint tannishcolored film. Abalones are seeded at densities ranging from 1.2 to 2.0/cm2 (20,000 to 35,00O/tank). Aeration is provided to these tanks. The l-pm filtered, 15°C seawater is supplied at a reduced rate (about 0.2 - 0.3 liter/min) for the first 24 h, but increased to 5 to 6 literlmin thereafter. About 2 weeks after the abalones are seeded the tanks are drained and rinsed. A 90-E;Lrn screen is positioned beneath the drain to collect dead abalones, and those live individuals that were dislodged. The tanks are drained and rinsed weekly thereafter. About 1 month following settlement, the filtration level is reduced to about 50-75 Wm. The tanks are covered and uncovered periodically to regulate diatom growth. Upon reaching about 5 mm in length the abalones are transferred to weaning tanks. Weaning
Weaning tanks are used for abalones that can forage on giant kelp, but have a strong preference for diatoms (abalones in the 5-10 mm shell lengths). The weaning tanks are both round and trough shaped. The round tanks have a 95cm diameter by 25-cm depth with about 15,000 cm2 of wetted surface area. The trough-shaped tanks are 2.4 m long by 0.57 m wide by 0.28 m deep and offer just over 30,000 cm2 of wetted surface area. The weaning tanks are allowed to develop a rich diatom film that is occasionally supplemented with a diatom slurry. Giant kelp and abalone habitats are also added. The habitats
38
consisted of various lengths of PVC pipe sections, generally about 8-10 cm in diameter, that are cut in half lengthwise. These pipe sections secure the kelp and provide shelter for the increasingly light-sensitive young abalones. Abalones in the weaning tanks receive raw seawater at ambient temperature at a 4-8 liter/min flow rate. These tanks are drained and rinsed weekly, and a fresh supply of giant kelp is also added at this time. Abalone densities in the weaning tanks are not closely regulated, but their numbers are judged and maintained in response to their grazing rate. Grow-Out
The tanks used for abalone grow-out are constructed from fiberglass and have two configurations: round and trough shaped. There are two sizes of round tanks: 302 liters that offer about 2.3 m2 of wetted surface area and 181 liters that offer about 1.5 m2 of wetted surface area. The trough-shaped tanks are about 2.08 m by 0.56 m by 0.32 m deep (about 606-liter capacity) and offer about 3.8 m2 of wetted surface area. All of the aforementioned wetted surface areas do not include habitats. All of the grow-out tanks have habitats that cover most of the tank bottoms. These habitats are made from 10.2-cm diameter PVC pipe sections that are cut in half lengthwise. They have 2.5-cm diameter holes drilled in the top, spaced at about 15-cm intervals, and very similar to those illustrated by Inoue (1976). The habitats shelter the abalones (they tend to become progressively more light sensitive), and secure the giant kelp proximal to them. Grow-out tanks are stocked with 2000 to 3000 abalone (12 mm and larger). Raw, ambient temperature seawater at a 6-8 liter/min flow rate is supplied to each tank. These tanks are also aerated. RESULTS
Broodstock
management
The methods we used promoted gamete development and maturation, and ripe abalones were available year-round. The experiments designed to examine possible broodstock-management improvements revealed that abalones maintained in continuous darkness had the highest feeding rates, growth rates (length and weight), and food conversion efficiencies (Tables I-III; Figs. 2,3). Abalone gonad indices were recorded in three containers, monthly, for 1 year. However, after 2 months, it became impossible to weigh and compare the indices because abalones were withdrawn and spawned for various Therefore, experiments. gonad-maturation success, by container, was measured according to the number of mature abalones that were withdrawn and induced to spawn. The abalones that were maintained in continuous darkness on filtered seawater yielded the most spawnings (171, including one male that spawned twice and a female that matured and was induced to spawn on three separate The abalone container maintained on a natural photoperiod on occasions. filtered seawater produced 15 spawners, including five that spawned twice. The third abalone container (continuous darkness and raw seawater) yielded only
(“Cl
13.6 13.8
13.8 14.2
10.7 10.7 10.6 11.2 12.4 13.1 13.2 12.9
1979-1980
11/14-12/14 12/14-l/14
1114-2113 2/13-3114
3/14-4114 4/14-5/14 S/14-6/11 6/11-7115 7115-8115 8/15-9112 9/12-10114 10/14-11114
30 30 30 30 27 27 27 27
30 30
30 30
N
“Conditions: natural photoperiod,
Mean temp.
Time period
45.1 45.5 45.8 45.9 46.6 46.9 47.4 47.9
43.6 44.5
42.7 43.3
End
filtered seawater.
44.5 45.1 45.5 45.8 45.9 46.6 46.9 47.4
43.3 43.6
42.6 42.7
Start
0.6 0.4 0.3 0.1 0.7 0.3 0.5 0.3
0.3 0.9
0.1 0.6
Shell length increase (mm)
growth, and conversion
Mean shell length (mm)
Juvenile red abalone food consumption,
TABLE 1
14.7 15.2 15.5 15.8 16.8 16.0 16.0 17.9
12.7 14.0
II.5 11.9
Start
15.2 15.5 15.8 16.8 16.0 16.0 17.9 18.2
14.0 14.7
Il.9 12.7
End
Mean weight (gl
0.5 0.3 0.3 1.0 -0.8 0.0 1.9 0.3
1.3 0.7
0.4 0.8
(g)
Weight increase
446 382 456 485 609 582 699 562
712 708
405 568
(gl
Kelp consumed
3.2 2.7 3.5 3.2 4.4 4.8 4.8 3.7
5.9 5.5
3.8 4.8
(%l
Feeding rate
efficiency on a diet of giant kelp, Mucrocysfvstis spp.”
3.4 2.4 2.0 6.2 0.0 0.0 7.3 1.4
5.5 3.0
3.0 4.2
P/o)
Conversion efficiency
(“G)
13.6 13.8 13.3 14.2 10.7 10.7 10.6 11.2 12.4 13.1 13.2 12.9
1979-1980
11/14-121’14 12/14-L/14 l/14-2/13 2/13-3114 3/14-4/14 4/14-s/14 5/14-6/11 6/11-7/15 7/‘15-8/15 8/15-9/‘12 9/12-10114 10/14-11/14
30 30 30 30 30 30 30 25 25 25 25 25
N End 42.3 44.3 46.6 49.5 51.1 52.6 54.0 54.0 55.7 57.0 58.8 60.2
Start
41.2 42.3 44.3 46.6 49.5 51.1 52.6 54.0 54.0 55.7 57.0 58.8
Mean shell length (mm)
1.1 2.0 2.3 2.9 1.6 1.5 1.4 0.0 1.7 1.3 1.8 1.4
Shell length increase (mm)
“Conditions: photaperiod is 24 h dark, filtered seawater.
Mean temp.
Time period
10.1 11.9 13.2 15.0 18.5 20.0 22.9 23.4 24.8 24.8 27.9 31.1
Start 11.9 13.2 15.0 18.5 20.0 22.9 23.4 24.8 24.8 27.9 31.1 32.2
End
Mean weight (g)
1.8 1.3 1.8 3.5 1.5 2.9 0.5 1.4 0.0 3.1 3.2 1.1
(8)
Weight increase
578 654 1138 1278 905 954 740 878 952 1050 1202 1138
(g)
Kelp consumed
5.8 5.4 9.0 8.5 4.9 4.9 4.1 4.3 5.0 5.7 5.1 4.6
(+%I
Feeding rate
Juvenile red abalone food consumption, growth, and conversion efficiency on a diet of giant kelp, Mucrocysris spp?
TABLE II
9.3 6.0 4.1 8,2 5.0 9.1 2.0 4.0 0.0 7.4 6.6 2.4
(%I
Conversion efficiency
(“Cl
13.5
1979-1980
11/14-12/14 12/14-l/14 l/14-2/13 2/13-3114 3/14-4J14 4114-V 14 5/14-6/11 6/11-7115 7/15-8/1S 8/15-9112 9/12-10114 10/14-11114
aConditions: photoperiod
10.7 10.7 10.6 11.4 12.5 13.1 13.4 13.0
13.8 13.3 14.2
Mean temp.
Time period
47.8 49.7 51.8 53.8 54.8 56.9 59.3 61.6 63.5
44.5 47.8 49.7 51.8 53.8 54.8 56.9 59.3 61.6
30 30 30 30 27 23 23 23 17
is 24 h dark, raw seawater.
39.4 41.6 44.5
End
38.3 39.4 41.6
Start
Mean shell length (mm)
1.9
2.9 3.3 1.9 2.1 2.0 1.0 2.1 2.4 2.3
1.1 2.2
Shell length increase (mm)
growth, and conversion
30 30 30
N
Juvenile red abalone food consumption,
TABLE III
13.4 16.2 19.4 22.5 23.1 26.4 30.2 31.1 38.1
8.3 9.6 10.8
Start
38.1 37.6
9.6 10.8 13.4 16.2 19.4 22.5 23.1 26.4 30.2 31.1
End
Mean weight (g)
-0.5
2.6 2.8 3.2 3.1 0.6 3.3 3.8 0.9 7.0
1.3 1.2
(gl
Weight increase
4.9 5.3 6.4 5.8 4.6 4.0
1420 1111 1033 946 1200 1298 1151 1183 790
(%)
Feeding rate
8.4 5.0 11.4 10.7 6.7 5.5
681 478 1243
(g)
Kelp consumed
efficiency on a diet of giant kelp, ~~c~oc~~~is spp?
8.6 9.0 1.9 7.4 6.7 1.8 13.6 0.0
6.3 5.9
5.7 7.5
(%>
Conversion efficiency
385
’ I
351
’
2
’
3
’
4
’
5 TIME
’
6
’
7
8
’
9
’
IO
II
12
(months)
Fig. 2. Comparative growth rates (shell length) of red abalone broodstock subjected to different variables: curve 1, natural photoperiod, n = 30; curve 2, 24 h dark, n = 30; curve 3, natural photoperiod, n = 90; curve 4, natural photoperiod, n = 60; curve 5, 24 h dark, n = 30; curves 1-4, filtered seawater; curve 5, raw seawater. I
r-4
I
/ / /
,L------------J 2 3 4 5 I TIME
6
7
8
9
IO
II
I2
(months)
Fig. 3. Comparative weight increases of red abalone broodstock See Fig. 2 caption for explanation of variables.
subjected
to different variables.
spawners. The elapsed time between spawnings for the multiple spawners ranged from 3 to 6 months. two
Spawning induction, fertilization, and earb development Abalones were induced to spawn on demand throughout the year, using the heavily UV-irradiated seawater technique (Kikuchi and Uki, 1974a). Males generally respond to the UV treatment in about 3.5 h; females take approximately 15-30 min longer. Their ova are about 225 pm in diameter (Fig. 4a). We find that excess sperm are not detrimental to the success of provided that the excess is removed within 15-30 min fertilization,
386
postfertilization. Fertilization success is routinely in excess of 95O/n. Abalone development at 15°C proceeds to the two-cell stage in 2 h. The next few divisions occur at hourly intervals, and at about 15 h postfertilization trochophore development is complete; they measure about 160 wrn by 195 pm, and are rotating within the egg membrane (Fig. 4b). Shortly after this (20 h postfertilization), the trochophores commence to rupture the egg membranes, and “risers” appear on the culture surface. Veliger-stage sheil development is complete at about 30 h postfertilization, at which point they measure about 210 pm by 270 km (Fig. 4c) and are ready for transfer to the larval-rearing culture containers. This developmental sequence is similar to that previously described by Shibui (1972) and Leighton (1974). Larval cultivation
The larval rearing period is 6 days at 15°C. Survivorship during this period averages close to 90%. Occasionally, a pathogenic bacteria, yibrio spp., proliferates in a culture and results in minor to extensive larval mortality. This bacterial infection, when it appears, generally beomes obvious about day 4 or 5. Grossly characterized, the Vibrio spp. infection appears as agglutinated clumps of larvae on the culture bottom screen that have a yellowish coloration, rather than the normal dark green. Vibrio spp. are effectively treated with Neomycin sulfate (50 mg/liter). However, when the infection is severe, we discard the culture rather than attempt this treatment. After 6 days, the veliger larvae have developed to a stage close to metamorphosis. This stage is recognized when the veligers have four branches on the cephalic tentacles, and the foot is sufficiently developed whereby the veliger can pull itself upright (on its foot) and propel itself about by ciliary action. We term this stage the “gliding stage.” These veligers are ready for transfer to the settling tanks. Forage culture
The diatom-slurry method provides sufficient diatoms for initial tank inoculations for just-metamorphosed abalones, which exhibit a low rate of foraging. At this stage, and at the stocking density specified, the diatom-cell growth rate is considerably greater than the rate of consumption by the abalone. As a result, we generally cover the abalone settling tanks 4 or 5 days after larval settlement to retard diatom growth. Tanks are uncovered and covered periodically in response to the abalone grazing rate and the diatom cell density. These subjectively governed adjustments are used to balance the abalone grazing rate with diatom celf production. Within 2 months, the abalone grazing rate sharply increases and the tanks are left uncovered. When the abalone grazing rate exceeds diatom cell production, additional diatom inocula, using the larger coarse bag-filtered diatoms, are added. Settkment and early-juvenile-stage cultivation
Under the conditions described, most larvae (ca. 90%) cease swimming within 24 h, and the remainder settle within 3 days after distribution into the settling tanks. When the juvenile abalones are 2 weeks old, they average about
387
Fig. 4. Red abalone d~vei~pmental stages at 15°C: a, ova at fertil~ation (225 pm), note sperm on ova membranes; b, tro~hophore stage (160 pm x 195 pm), 15 h post fertilization, just prior to rupturing the membrane; c, veliger stage (210 Frn x 270 pm), 30 h postf~rtilization~ d, early juvenile stage (490 pm), 2 weeks old; e, early juvenile stage (0.8 mm), 6 weeks old; f, juvenile, first respiratory pore formation (2.1 mm). about 8 weeks old.
388
490 pm in length, by 6 weeks about 0.8 Frn long, and the first respiratory pore notch forms at about 8 weeks, when they measure 2.1 mm long. Most abalone mortality (close to 90°~) occurs during the first month postsettlement, and most of this during the initial 2 weeks in the settlement tanks. Approximately one-half of the initial mortality consists of veligers that fail to metamorphose. Culture success is quite variable. Five representative culture trials revealed an average 7.5% survivorship (ranging from 1.9 to 13.5%) during an approximate 3-month period (Table IV). TABLE IV Red abalone settlement tank cultivation results Trial
1 2 3 4 5
No. seeded
35,000 35,000 26,000 24,000 20,000
Yield
Shelt length (mm)
Density
Trial
. (#/cm 21
period (days)
(#)
F%~
Mean
Range
2.0 2.0 1.5 1.4 1.2
85 85 90 78 133
4,200 650 3,500 1,800 522
12.0 1.9 13.5 7.5 2.6
2.9 2.2 3.2 3.0 12.5
1.8- 5.5 1.8- 5.0 1.2- 5.5 l.O- 5.0 6.6-16.7
Weming Abalone mortality in the weaning tanks is low (less than 2%) and is generally confined to the transfer period, from handling and mechanical damage. Abalones at these sizes (5-10 mm) have a very high grazing rate and, even with frequent additions of diatom-slurry inocula, manage to keep the film “well-grazed.” This forces the abalones to graze on macroalgae (giant kelp blades). Grow-our
The abalone growth rate in all grow-out tanks was quite similar. During the first year in these tanks, the abalones averaged about 2 mm/month (12-36 mm). At larger sizes (5-8 cm), the growth rate declined to about 1 mm/month. Abalone mortality in the grow-out tanks was low, generally less than lo/o,and most of this mortality was confined to the initial transfer period, from mechanical damage or handling stress. RISCUSSrON
Conditioning of II. rufescensfor spawning is relatively simple when compared to that of certain other abalone species. Temperature or photoperiod or both are frequently cited as critically important to the maturation process (Kikuchi and Uki, 1974a). Neither of these two factors appear to be limiting for red
389
abalone-gonadal maturation, under the conditions we have thus far explored. The poor maturation success observed with abalones held in continuous darkness, on raw seawater, may be attributed to (natural) spawnings that went undetected. Typically, two or more uninduced spawnings occur in the laboratory annually, with abalones that are receiving raw seawater. We speculate that something in the raw seawater triggers the spawning, but is not present in the filtered seawater. Our filtered seawater supply does receive a low-dosage IJV treatment following passage at high flow rate through the laboratory sand filters. Spawning induction, jkrtilization, arrd early development
We consider the UV spawning-induction method that was developed by Kikuchi and Uki (1974a) to be one of the most significant contributions to abalone cuItivation. Prior to this breakthrough, it was di~cult to obtain synchronous red abalone spawners. Red abalones respond to both UV and the hydrogen peroxide (Morse et al., 1977) spawning-induction techniques. We prefer the former technique because it requires no chemical additions, and we can program the UV unit timer clock to activate at an early hour. Therefore, the abalones are close to spawning when the normal workday begins. This allows a full day for setting up cultures, and most importantly, the abalones hatch early on the following day. Probably the two most critical procedures in cultivating abalones are the collection of ova from the spawning container and the transfer of veliger-stage larvae to the larval-rearing apparatus. These procedures are critical because they represent possible contamination sources. Scrupulous sanitary procedures should be applied to maintain a clean spawning container, and the transfer of any contaminants along with the ova must be avoided. Likewise, the bottom of the larval-hatching container contains shed egg membranes and undeveloped, or aberrantly developed, ova that can serve as contaminants, Therefore, only “high-rising” veliger larvae should be transferred. Application of these procedures should eliminate the need for antibiotics during the larvalrearing period. Larval cultivation
The development of a flow-through larval-rearing apparatus represents a distinct advancement in abalone-cultivation procedures (cf. Morse et al., 1979). Larvae can be cultivated at higher densities (up to 201ml) with good survivorship, and the culture seawater does not require periodic exchanges. Only two culture conditions must be monitored and maintained. These are temperature and water-flow rate. The water flow must be maintained at a low rate whereby the larvae are uniformly distributed and are not forced against the culture-bottom screen. Although we use only an 8-liter capacity larval-rearing apparatus, this apparatus appears amenable to scaling up for large commercial operations. Forage culture
Maintaining an adequate diatom film in abalone-settling tanks is a major
390
problem in the overall cultivation process. Problems arise from variable abalone grazing rates according to size, grazing intensity relative to abalone density settlement success, seasonal diatom-growth rates, and selective grazing by abalones. Alternate covering/uncovering of the settlement tank in response to the early (low grazing rate) abalone-settlement period is not inefficient or labor intensive. Frequently, however, the diatom film is insufficient on the settling-tank walls where abalones prefer to settle, and too dense on the tank bottom. It is undesirable to allow a thick, often filamentous, diatom film to develop on the tank bottom; this obstructs the young abalone’s mobility and harbors micropredators. The diatom film on the tank bottom is controlled through vigorous tank rinsing or by brushing. If abalone-settlement success is reasonably good after 2 months in the settling tanks (10% or more) then it becomes increasingly difficult to maintain an adequate diatom film, and forage can become limiting. The occasional diatom inoculations from the slurry system help, but this has proved insufficient for maintaining an adequate diatom film when natural diatom production is low (November to April). One possible solution is to increase the vertical surface area in the settling tanks. This is now being studied. We do not completely understand the etiology of the high abalone mortality that we have observed during the initial 2 weeks in our settling tanks. Known pathogenic bacteria, V&o spp., are present and implicated. Copepods, Tigriopus caiifornicus, unidentified nematodes, and ciliate protozoans are often associated with these abalone mortalities. These mortalities also appear unrelated to our initial abalone-stocking densities. Abalone cultivators frequently use a settlement and metamorphosis inducer for fully developed veliger-stage larvae (F.L. Clogston, unpublished observations; Morse et al., 1979; Seki and Kan-no, 1981). We consider this unnecessary with our cultivation methods; although our initial abalonemortality rates are high in the settling tanks, the limiting factors are vertical substrates and insu~cient diatoms. We estimate that maximum abalone production in one of our settling tanks, for 3 months, is about 5000 abalone (about 14% success or 1114 cm*); typically, however, a lower success rate is observed. We cannot provide adequate diatoms for this number of abalones after 3 months without incurring serious stunting; therefore, the abalones must be transferred to weaning tanks. Weaning
The abalone-weaning-tank period represents the most inefficient element in our cultivation methods. It represents an “extra step,” but we have not been able to circumvent it. It was not planned for in our original abalone-cultivation scheme. Abalone at 5-10 mm require considerably more diatoms than we can provide in the grow-out tanks. Early attempts to force these young abalones to feed on macroalgae (giant kelp) resulted in a temporary cessation in growth and increased mortality.
391
Grow-Out
Abalones grow equally well in both the circular and trough-shaped tanks. Initially we thought that water-flow characteristics in the circular tanks would enhance abalone-growing conditions. A negative aspect of the circular tanks is the constraint imposed on abalone habitats. Such habitats require more complex construction in order to fit uniformly in a round configuration. We intend to layer the habitats and thus increase available surface area and abalone numbers. This can be more easily accomplished using trough-shaped tanks. It is anticipated that such tanks will support 5000 abalones (l/7 cm2, excluding the habitat surface area), up to 3 cm in length, with a 6-8 liter/min water-flow rate (about a 90 min tank-turnover period). ACKNOWLEDGMENTS
This work was supported in part by the Bartlett Commercial Fisheries Research and Development Act (P.L. 88-309), Project #743, “Shell~sh Culture Investigations.” REFERENCES Carlisle, J.G.. Jr., 1962. Spawning and early life history of Huliotis ru&cens Swainson. Nautilus, 76(2):44-48. Cox, K.W., 1963. Report of visit to abalone hatcheries in Japan, March, 1963. State of California, the Resources Agency, Department of Fish and Game, Marine Resources Operations, MRO Reference No. 63- 1, Ebert, E.E., Haseltine, A.W. and Kelly, R.O., 1974. Seawater system design and operations of the Marine Culture Laboratory, Granite Canyon. Calif. Fish and Game, 60(1):4-14. Ino, T., 1952. Biological studies on the propagation of Japanese abalone (genus Ha~i~~;s). Bull. Tokai Reg. Fish. Res. Lab., 5 (in Japanese with English summary~~ 102 pp. Inoue, M.. 1976. Awabi. in: Suisan Zoyoshoku Deeta Bukku. (Abalone. In: Fisheries Propagation Data Book). Published by Suisan Suppan (translated by M. Mottet). Kan-no, H. 1975. Recent advances in abalone culture in Japan. Proceedings of the First International Conference on Aquaculture Nutrition, October, 1975:195-21 I. Kikuchi, S. and Uki, N., 1974a. Technical study on artificial spawning of abalone, genus Hulioris. II. Effect of ultraviolet irradiated seawater to induce spawning. Bull. Tohoku Reg. Fish. Research Lab., No. 33:80-92. (In Japanese with English abstract.) Kikuchi, S. and Uki, N., 1974b. Technical study on artificial spawning of abalone genus Huliotis. 111. Reasonable sperm density far fertilization. Bull. Tohoku Reg. Fish Res. Lab., 3467-71. (In Japanese with English abstract.) Kikuchi, S. and Uki, N., 1974~. Technical study on artificial spawning of abalone. genus Hai~~~is. V. Relation between water temperature and advancing sexual maturity of Hul~otj~ discus Reeve. Bull. Tohoku Reg. Fish. Res. Lab., 34:77-85. (In Japanese with English abstract.) Leighton. D.L., 1972. Laboratory observations on the early growth of the abalone, Haiiotis .sormsm/. and the effect of temperature on larval development and settling success. Fish. Bull., 70(2):373-381. Leighton, D.L., 1974. The influence of temperature on larval and juvenile growth in three species of southern Califonia abalones. Fish. Bull., 72(4):1137-l 145. Leighton, D.L., 1977. Some problems and advances in culture of North American abalones (t;laliotrs). Paper presented to the First Symposium in the Latin American Aquaculture Association, Maracay, Venezuela, Nov. 5-12. 1977. Morse, D.E., Duncan, H., Hooker, N. and Morse, A., 1977. Hydrogen peroxide induces spawning in molluscs, with activation of prostagiand~n endoperoxide synthetase. Science, 196298-300.
392
Morse, D.E., Hooker, N., Jensen, L., and Duncan, H., 1979. Induction of larval abalone settling and metamorphosis by y-aminobutyric acid and its congeners from crustose red algae: II: Applications to cultivation, seed-production and bioassays; principal causes of mortality and interference. Proc. World Maricul. Sot. IO:8l-91. Murayama, S., 193.5. On the development of the Japanese abalone, ~Q~~o?~s g~g~~fe~. Jour. Coil. Agric., X111(3):227-233. Seki, T. and Kan-no, H., 1977. Synchronized control of early life in the abalone, Haiioris discus hannai Ino, (Haliotidae, Gastrpoda). Bull. Tohoku Reg. Fish. Res. Lab. No. 38. October, pp 143-153, (in Japanese with English summary.) Seki, T. and Kan-no, H., 19Rl. Induced settlement of the Japanese abalone, H&Otis discus hannai, veliger by the mucous trails of the juvenile and adult abalones. Bull. Tohoku Reg. Fish. Res. Lab., No. 43:29-36. (In Japanese with English summary.) Shibui, T., I972. On the normal development of the eggs of Japanese abalone, Haliotis disrus hannai Ino. and ecological and physiological studies of its larvae and young. Bull. Iwate Pref. Fish. Exp. Stat. (2):1-69.
375 - Printed in The Netherlands
ELEMENTS AND INNOVATIONS IN THE CULTIVATION ABALONE HALIOTIS RUFESCENS
OF RED
EARL E. EBERT and JAMES L. HOUK Marine Culture Laboratory, Marine Game, Monterey, CA 93940 (U.S.A.)
Resources
Branch,
California
Department
of Fish and
ABSTRACT Ebert. E.E. and Houk, J.L., 1984. Elements and innovations Haliatis r&rem. Aquaculture, 39: 375-392.
in the cultivation
of red abalone
Seven elements are described for cultivation of the red abalone, ~~l;#r~s rz&scem broodstock management; spawning induction, fe~ili~ation, and early development; larval ~ult~vat~on~ forage culture; settlement and early-juvenile-stage cultivation; weaning; and grow-out. Cultivation methods and equipment are described. Spawnable abalones are maintained on a year-round basis. Larvae are cultured in a flow-through apparatus that yields over 90% to the settling stage. A diatom slurry method is used to provide forage to the early benthonic and juvenile stages. Cultivation problems are described. Included are those with bacteria, Vibrio spp., and the maintenance of suitable diatom substrates for juvenile stages.
INTRODUCTION
Cultivation of abalone, Huliotis spp., has spanned nearly a 50-year period. However, there was a gap of 17 years between the first major contribution by Murayama (1935) and that of Ino (1952). During the 1970s there was a surge in valuable contributions on various aspects of abalone cultivation (e.g., Leighton, 1972, 1974, 1977; Shibui, 1972; Kikuchi and Uki, 1974a,b,c; Kanno, 1975; Morse et al, 1977, 1979; Seki and Kan-no, 1977, 1981). As an outgrowth of these investigations interest in cultivating abalone, an economically valuable shellfish, has spread from Japan to various parts of the world. Research and development projects, or production hatcheries, are in operation in Australia, the British Isles, Canada, Chile, France, Mexico, and the United States (Californian. Pioneering efforts to cultivate North American haliotids began in 1940 (Carlisle, 1962). However, more than 20 years elapsed before renewed interest surfaced to cultivate North American haliotids, and more specifically the red abalone, H. rufeseens. Spurred by a report on Japanese abaione-hatchery methods by Cox (1963) an initial small laboratory was constructed in 1964 at Morro Bay, on the central California coast, to test red abalone-cultivation methods (F.L. Clogston, unpublished observations). Clogston demonstrated 0044~8486/84/$03.00
0 1984 Elsevier Science Publishers B.V.
376
that red abalones could be mass cultivated, and his efforts created a surge of interest from the private sector, as welt as university and government scientists. In 1965 Pacific Mariculture (now Pigeon Point Aquaculture Center), a commercial shellfish hatchery situated along the central California coast about 40 miles south of San Francisco at Pigeon Point, began to mass cultivate the red abalone. Pacific Maricuiture became the first private company to mass produce and sell relatively large quantities of red abalone seed. However, abalone cultivation was unpro~table for Pacific Mariculture for various reasons and they opted to discontinue their red abalone-hatchery operations. The first commercial abalone hatchery in California, designed specifically for that purpose, was California Marine Associates (now Ester0 Bay Mariculture). This hatchery, located along the central California coast near Cayucos, has been in operation since 1968. The 1970s witnessed a continued interest in red abalone cultivation in California. In 1970 the California Department of Fish and Game commenced red abalone-cultivation research at their Granite Canyon Laboratory, located on the central California coast near Monterey (Ebert et at, 1974). Following this, two more commercial abalone hatcheries were established. These were Monterey Abalone Farms, which began in late 1971, and the Ab Lab, located along the coast south of Santa Barbara at Port Hueneme, which commenced operations in 1974. Additionally, an Abalone Seeding Association was formed by a group of commercial abalone fishermen and processors in late 1974 to hatch and seed abalone larvae as a means of augmenting the fishery. Southern California Edison, a major California electricity producer has also sponsored an abalone cultivation project. The broad objectives of the red abalone research and development project at the California Department of Fish and Game’s Granite Canyon Laboratory are (i) to develop further the technological and biological methods for mass cultivation and to disseminate this information, and (ii> to provide seed abalone for rebuilding impacted abalone-fishing grounds. METHODS AND MATERIALS
Cultivation methods for the red abalone, Huliotis rufescens, are divided into seven major elements, presented serially in the following pages. Some methods within specific elements are continually being subjected to experimentation to seek refinements. Where this is the case, the specific experiments and findings are presented with those elements. Broodstock is maintained in 15liter polyethylene plastic containers. Each container offers about 0.34 m* surface area, including the lid. The container lids are perforated and have a central hole to accommodate a water-inlet pipe that extends to the container bottom. Ambient temperature seawater (9 lS”C), sand-filtered to about 20 pm, and ultraviolet WV) treated, is supplied to each container at a 3-4 liter/min flow rate. Containers are cleaned weekly,
371
and fresh giant kelp, Mucrocystis spp., is supplied, to excess, at the same time. Broodstock is maintained separately according to geographic origin and sex. Variation of the above “standard” broodstock methods were tested. These included: (i) using raw seawater (no filtration or UV treatment), (ii) maintaining two containers in total darkness, and (iii) different abalone densities (30, 60, or 90/container). Also we measured and weighed abalones monthly, and recorded the amount of kelp, in wet weight, consumed by abalones weekly in each container. This afforded a calculation of the feeding rate. This is expressed as the feed consumption per abalone at its mean weight for the period and was derived using the formula: Fr-F2 x 100 t x w
where F, = feed given, F, = feed remaining, t = time in days, and W = mean abalone weight. Food-conversion efficiency was calculated and defined as the wet weight of animal gain per wet weight of food ingested. A gonad-indexing method was developed in order to assess the abalone’s maturation stage through gross examination of the gonad bulk and color. The abalone gonad envelops the digestive gland and together they form a rather large cone-shaped appendage positioned on the right side of the abalone’s foot just beneath the shell. The gonad is easily viewed by turning the abalone upside-down and pushing the foot aside. The testis is cream colored and the ovary is dark green. The gonad index is:
o-
Immature, sex indeterminate, greyish-brown mass.
1 - Gamete development color differentiated, determination is easy color; however, female 2 - Gametes fully envelop gonad not bulky.
has on for sex
the digestive
gland is easily viewed as a
initiated; gametes appear in a patchy pattern, the surface of the digestive gland. Sex males at this stage because of the creamish determination is difficult.
the conical appendage,
sex easily determined
but
3 - Same condition as for index stage 2 except that the gonad is quite bulky. This bulk extends to the gonad tip. Abalones to be used for spawning induction are preferably in index category 3; however, when these are not available, we use abalones that are between index categories 2 and 3. Gonad indexing was done monthly at the same time that the abalones were weighed and measured. However, indexing was done only in three containers that each held 30 abalones. Two of these containers were maintained in continuous darkness; one received raw seawater, the other filtered. The third container had a natural photoperiod and was supplied with filtered seawater. Gonad indices for all abalones in a container were totaled and weighed according to the number of abalones at each index (e.g., 3 at category 3 = 9; 8 at category 2 = 16). Comparison of the weighted gonad index, by broodstock container, afforded a measure of gonad conditioning
378
success according to the conditions Spawning induction, fertihation,
that were imposed.
and ear& development
Spawning induction is accomplished by exposing sexually mature abalones to heavily UV-irradiated seawater (Kikuchi and Uki, 1974a). In our system we use a REFCO’ water purifier, model RL-IO-1P (Refco International, Inc., San Leandro, California), and adjust the pumping rate of the l-pm filtered, ambient-temperature seawater to about 150 ml/min. Male and female abalones are held in separate containers (the same kind that are used for broodstock), one or two abalones per container depending upon their size. The water purifier is activated by a timer set to come on at 0600 h on a “spawning day.” A 3- to 4-h exposure to the UV-treated seawater is generally sufficient to trigger spawning. After spawning the ova are collected by siphon and distributed into 15liter plastic containers. They are fertilized within 30 min postspawning. The ova are slightly more dense than seawater and settle to the bottom of the container. Excess sperm are removed by decantation beginning about 15 min postfertilization. This is repeated two or three times or until the water is clarified. It is necessary to wait 5-10 min between decantations in order for the ova to settle. Ova that do not settle after this time are discarded. Following the last decantation the culture container is refilled. One-Nrn filtered, UVtreated seawater at 15°C is used for all culture-water exchanges. It is desirable to end up with a single layer of ova on the culture container bottom. Using a file, the shells of spawned abalones are marked with a v-shaped notch just to the right of the most anterior respiratory pore; these animals then are returned to their broodstock container: The shellnotch serves as a spawning-history reference mark. Lurval cultivation
Fertilized ova develop to the veliger stage in the hatching container. They are positively phototactic and rise to the culture surface. The veligers are dipped (by beaker) or siphoned into l-liter beakers to obtain a density estimate. This is done by aliquot sample. First, a uniform larval distribution is obtained with a swizzle stick. Aliquot samples are taken with a l-ml pipette. The pipette is passed diagonally through the larvae. Three to five aliquots are taken. The number taken is dependent upon the variation in larval counts between aliquot samples. If this variation exceeds 50% then five aliquots are taken. The veligers are distributed into 8-liter larval rearing apparatuses at a concentration of 5/ml (40,000 per culture). The larval rearing apparatus basically consists of a container within a container. The inner container (larval chamber) consists of a 20-cm diameter PVC pipe section, 36 cm tall, and screened 2.5 cm above the bottom (90 pm NITEX). The outer container is about 2’7 cm in diameter and 30 cm tall. In operation water flows downward
‘Mention
of manufacturer’s
name does not imply endorsement
319
through the inner container, through the NITEX screening, and exits from the outer container via a standpipe (Fig. 1). Larvae are cultured at 15°C using 3pm filtered, UV-treated seawater at a 200-300 ml/min flow rate.
Fig. 1. The larval-culture rearing apparatus; a, water inlet; b, outer culture container; c, top of water outlet; d, inner (larval rearing) container; e, bottom of water outlet; f, water-exit ports from larval-rearing container; g, base support; h, 90-pm NITEX screening.
Forage culture Abalone larvae do not require supplemental food. They subsist upon and nutrients from their relatively large yolky egg. Upon settlement metamorphosis, the abalone subsists on diatoms. These diatoms must be less than 10 pm in greatest dimension so that the young abalones can ingest them. To meet this requirement we developed a diatom-slurry system. The diatom-slurry system entails the collection of diatoms that have passed through selective filtration, adventitiously settled on a substrate, and grown. These diatoms are removed by wiping, refiltered, and concentrated in a slurry. We use white polyethylene plastic containers for diatom settlement and culture substrates. Each container offers about 2,900 cm* of surface area. Ambient temperature seawater, filtered to 1 pm, is supplied to each container. Water flow rates are not closely monitored, but are less than 0.5 liter/min. Illumination is provided by overhead fluorescent lamps (Westinghouse, Chroma SO>. The lamps are positioned 30 cm above the diatom cultures. Normally, after a 7-day growth period, the diatoms are harvested. This is done by draining each container and wiping down the diatom film with a latex glove. The diatoms are rinsed with l-pm filtered seawater and poured first through a j-pm and then a l-pm filter bag. Diatoms that pass through the lpm bag filtration are retained for the larvae that are ready to be settled. The diatoms that do not pass through the filter bags are retained for feeding older juvenile abalones. One diatom container will generally yield 1 liter of diatom
380
slurry (l-pm bag filtered) at a concentration of 5.0 x 105/ml. Part of this yield (about 6.8 x 10’ diatom cells) is used to reinoculate the original diatom-culture container. The reinoculated container is refilled with l-pm filtered seawater and held static for 24-48 h to allow the diatoms to settle and attach. After this time a gentle water flow rate is reestablished. Numbers of diatom cells from the concentrated slurry used to inoculate various sizes of abalone settling tanks are: 3.5 x 107/12-liter tank; 4.0 x 107/14-liter tank; 1.3 x 108/73-liter tank; 3.3 x 108/176-liter tank; and 3.5 x 108/302-liter tank. Abalones 5 mm and larger can readily forage on macroalgae. We generally use giant kelp, Mucrocystis spp., for these abalones. Occasionally, bull kelp, Nereocystis luetkeana, and feather boa kelp, Egregia menziesii, are used. Settlement and ear~~uve~ile-stage cuit~vation
The abalone settling tanks are constructed from fiberglass and have a white gel coat finish. They are circular with a dished bottom. The tanks are about 107 cm wide and have a sidewall depth of about 25 cm. The bottom slopes downward to about a 33-cm depth at the center bottom drain opening. The drain opening accommodates about a 3.8-cm diameter standpipe. These tanks hold about 260 liters and offer about 17,000 cm2 of wetted surface area. The settling tanks are located in a greenhouse that incorporates translucent plastic siding in order to maximize solar illumination. Preparation of settling tanks for abalone larvae generally commences 24 h before larval seeding. Clean tanks are used. They are filled with l-hrn filtered, UV-treated seawater, at 15°C. The tanks are inoculated with a diatom slurry (described in forage culture section). The following day, when the abalone larvae are introduced, the diatom inoculum has formed a very faint tannishcolored film. Abalones are seeded at densities ranging from 1.2 to 2.0/cm2 (20,000 to 35,00O/tank). Aeration is provided to these tanks. The l-pm filtered, 15°C seawater is supplied at a reduced rate (about 0.2 - 0.3 liter/min) for the first 24 h, but increased to 5 to 6 literlmin thereafter. About 2 weeks after the abalones are seeded the tanks are drained and rinsed. A 90-E;Lrn screen is positioned beneath the drain to collect dead abalones, and those live individuals that were dislodged. The tanks are drained and rinsed weekly thereafter. About 1 month following settlement, the filtration level is reduced to about 50-75 Wm. The tanks are covered and uncovered periodically to regulate diatom growth. Upon reaching about 5 mm in length the abalones are transferred to weaning tanks. Weaning
Weaning tanks are used for abalones that can forage on giant kelp, but have a strong preference for diatoms (abalones in the 5-10 mm shell lengths). The weaning tanks are both round and trough shaped. The round tanks have a 95cm diameter by 25-cm depth with about 15,000 cm2 of wetted surface area. The trough-shaped tanks are 2.4 m long by 0.57 m wide by 0.28 m deep and offer just over 30,000 cm2 of wetted surface area. The weaning tanks are allowed to develop a rich diatom film that is occasionally supplemented with a diatom slurry. Giant kelp and abalone habitats are also added. The habitats
38
consisted of various lengths of PVC pipe sections, generally about 8-10 cm in diameter, that are cut in half lengthwise. These pipe sections secure the kelp and provide shelter for the increasingly light-sensitive young abalones. Abalones in the weaning tanks receive raw seawater at ambient temperature at a 4-8 liter/min flow rate. These tanks are drained and rinsed weekly, and a fresh supply of giant kelp is also added at this time. Abalone densities in the weaning tanks are not closely regulated, but their numbers are judged and maintained in response to their grazing rate. Grow-Out
The tanks used for abalone grow-out are constructed from fiberglass and have two configurations: round and trough shaped. There are two sizes of round tanks: 302 liters that offer about 2.3 m2 of wetted surface area and 181 liters that offer about 1.5 m2 of wetted surface area. The trough-shaped tanks are about 2.08 m by 0.56 m by 0.32 m deep (about 606-liter capacity) and offer about 3.8 m2 of wetted surface area. All of the aforementioned wetted surface areas do not include habitats. All of the grow-out tanks have habitats that cover most of the tank bottoms. These habitats are made from 10.2-cm diameter PVC pipe sections that are cut in half lengthwise. They have 2.5-cm diameter holes drilled in the top, spaced at about 15-cm intervals, and very similar to those illustrated by Inoue (1976). The habitats shelter the abalones (they tend to become progressively more light sensitive), and secure the giant kelp proximal to them. Grow-out tanks are stocked with 2000 to 3000 abalone (12 mm and larger). Raw, ambient temperature seawater at a 6-8 liter/min flow rate is supplied to each tank. These tanks are also aerated. RESULTS
Broodstock
management
The methods we used promoted gamete development and maturation, and ripe abalones were available year-round. The experiments designed to examine possible broodstock-management improvements revealed that abalones maintained in continuous darkness had the highest feeding rates, growth rates (length and weight), and food conversion efficiencies (Tables I-III; Figs. 2,3). Abalone gonad indices were recorded in three containers, monthly, for 1 year. However, after 2 months, it became impossible to weigh and compare the indices because abalones were withdrawn and spawned for various Therefore, experiments. gonad-maturation success, by container, was measured according to the number of mature abalones that were withdrawn and induced to spawn. The abalones that were maintained in continuous darkness on filtered seawater yielded the most spawnings (171, including one male that spawned twice and a female that matured and was induced to spawn on three separate The abalone container maintained on a natural photoperiod on occasions. filtered seawater produced 15 spawners, including five that spawned twice. The third abalone container (continuous darkness and raw seawater) yielded only
(“Cl
13.6 13.8
13.8 14.2
10.7 10.7 10.6 11.2 12.4 13.1 13.2 12.9
1979-1980
11/14-12/14 12/14-l/14
1114-2113 2/13-3114
3/14-4114 4/14-5/14 S/14-6/11 6/11-7115 7115-8115 8/15-9112 9/12-10114 10/14-11114
30 30 30 30 27 27 27 27
30 30
30 30
N
“Conditions: natural photoperiod,
Mean temp.
Time period
45.1 45.5 45.8 45.9 46.6 46.9 47.4 47.9
43.6 44.5
42.7 43.3
End
filtered seawater.
44.5 45.1 45.5 45.8 45.9 46.6 46.9 47.4
43.3 43.6
42.6 42.7
Start
0.6 0.4 0.3 0.1 0.7 0.3 0.5 0.3
0.3 0.9
0.1 0.6
Shell length increase (mm)
growth, and conversion
Mean shell length (mm)
Juvenile red abalone food consumption,
TABLE 1
14.7 15.2 15.5 15.8 16.8 16.0 16.0 17.9
12.7 14.0
II.5 11.9
Start
15.2 15.5 15.8 16.8 16.0 16.0 17.9 18.2
14.0 14.7
Il.9 12.7
End
Mean weight (gl
0.5 0.3 0.3 1.0 -0.8 0.0 1.9 0.3
1.3 0.7
0.4 0.8
(g)
Weight increase
446 382 456 485 609 582 699 562
712 708
405 568
(gl
Kelp consumed
3.2 2.7 3.5 3.2 4.4 4.8 4.8 3.7
5.9 5.5
3.8 4.8
(%l
Feeding rate
efficiency on a diet of giant kelp, Mucrocysfvstis spp.”
3.4 2.4 2.0 6.2 0.0 0.0 7.3 1.4
5.5 3.0
3.0 4.2
P/o)
Conversion efficiency
(“G)
13.6 13.8 13.3 14.2 10.7 10.7 10.6 11.2 12.4 13.1 13.2 12.9
1979-1980
11/14-121’14 12/14-L/14 l/14-2/13 2/13-3114 3/14-4/14 4/14-s/14 5/14-6/11 6/11-7/15 7/‘15-8/15 8/15-9/‘12 9/12-10114 10/14-11/14
30 30 30 30 30 30 30 25 25 25 25 25
N End 42.3 44.3 46.6 49.5 51.1 52.6 54.0 54.0 55.7 57.0 58.8 60.2
Start
41.2 42.3 44.3 46.6 49.5 51.1 52.6 54.0 54.0 55.7 57.0 58.8
Mean shell length (mm)
1.1 2.0 2.3 2.9 1.6 1.5 1.4 0.0 1.7 1.3 1.8 1.4
Shell length increase (mm)
“Conditions: photaperiod is 24 h dark, filtered seawater.
Mean temp.
Time period
10.1 11.9 13.2 15.0 18.5 20.0 22.9 23.4 24.8 24.8 27.9 31.1
Start 11.9 13.2 15.0 18.5 20.0 22.9 23.4 24.8 24.8 27.9 31.1 32.2
End
Mean weight (g)
1.8 1.3 1.8 3.5 1.5 2.9 0.5 1.4 0.0 3.1 3.2 1.1
(8)
Weight increase
578 654 1138 1278 905 954 740 878 952 1050 1202 1138
(g)
Kelp consumed
5.8 5.4 9.0 8.5 4.9 4.9 4.1 4.3 5.0 5.7 5.1 4.6
(+%I
Feeding rate
Juvenile red abalone food consumption, growth, and conversion efficiency on a diet of giant kelp, Mucrocysris spp?
TABLE II
9.3 6.0 4.1 8,2 5.0 9.1 2.0 4.0 0.0 7.4 6.6 2.4
(%I
Conversion efficiency
(“Cl
13.5
1979-1980
11/14-12/14 12/14-l/14 l/14-2/13 2/13-3114 3/14-4J14 4114-V 14 5/14-6/11 6/11-7115 7/15-8/1S 8/15-9112 9/12-10114 10/14-11114
aConditions: photoperiod
10.7 10.7 10.6 11.4 12.5 13.1 13.4 13.0
13.8 13.3 14.2
Mean temp.
Time period
47.8 49.7 51.8 53.8 54.8 56.9 59.3 61.6 63.5
44.5 47.8 49.7 51.8 53.8 54.8 56.9 59.3 61.6
30 30 30 30 27 23 23 23 17
is 24 h dark, raw seawater.
39.4 41.6 44.5
End
38.3 39.4 41.6
Start
Mean shell length (mm)
1.9
2.9 3.3 1.9 2.1 2.0 1.0 2.1 2.4 2.3
1.1 2.2
Shell length increase (mm)
growth, and conversion
30 30 30
N
Juvenile red abalone food consumption,
TABLE III
13.4 16.2 19.4 22.5 23.1 26.4 30.2 31.1 38.1
8.3 9.6 10.8
Start
38.1 37.6
9.6 10.8 13.4 16.2 19.4 22.5 23.1 26.4 30.2 31.1
End
Mean weight (g)
-0.5
2.6 2.8 3.2 3.1 0.6 3.3 3.8 0.9 7.0
1.3 1.2
(gl
Weight increase
4.9 5.3 6.4 5.8 4.6 4.0
1420 1111 1033 946 1200 1298 1151 1183 790
(%)
Feeding rate
8.4 5.0 11.4 10.7 6.7 5.5
681 478 1243
(g)
Kelp consumed
efficiency on a diet of giant kelp, ~~c~oc~~~is spp?
8.6 9.0 1.9 7.4 6.7 1.8 13.6 0.0
6.3 5.9
5.7 7.5
(%>
Conversion efficiency
385
’ I
351
’
2
’
3
’
4
’
5 TIME
’
6
’
7
8
’
9
’
IO
II
12
(months)
Fig. 2. Comparative growth rates (shell length) of red abalone broodstock subjected to different variables: curve 1, natural photoperiod, n = 30; curve 2, 24 h dark, n = 30; curve 3, natural photoperiod, n = 90; curve 4, natural photoperiod, n = 60; curve 5, 24 h dark, n = 30; curves 1-4, filtered seawater; curve 5, raw seawater. I
r-4
I
/ / /
,L------------J 2 3 4 5 I TIME
6
7
8
9
IO
II
I2
(months)
Fig. 3. Comparative weight increases of red abalone broodstock See Fig. 2 caption for explanation of variables.
subjected
to different variables.
spawners. The elapsed time between spawnings for the multiple spawners ranged from 3 to 6 months. two
Spawning induction, fertilization, and earb development Abalones were induced to spawn on demand throughout the year, using the heavily UV-irradiated seawater technique (Kikuchi and Uki, 1974a). Males generally respond to the UV treatment in about 3.5 h; females take approximately 15-30 min longer. Their ova are about 225 pm in diameter (Fig. 4a). We find that excess sperm are not detrimental to the success of provided that the excess is removed within 15-30 min fertilization,
386
postfertilization. Fertilization success is routinely in excess of 95O/n. Abalone development at 15°C proceeds to the two-cell stage in 2 h. The next few divisions occur at hourly intervals, and at about 15 h postfertilization trochophore development is complete; they measure about 160 wrn by 195 pm, and are rotating within the egg membrane (Fig. 4b). Shortly after this (20 h postfertilization), the trochophores commence to rupture the egg membranes, and “risers” appear on the culture surface. Veliger-stage sheil development is complete at about 30 h postfertilization, at which point they measure about 210 pm by 270 km (Fig. 4c) and are ready for transfer to the larval-rearing culture containers. This developmental sequence is similar to that previously described by Shibui (1972) and Leighton (1974). Larval cultivation
The larval rearing period is 6 days at 15°C. Survivorship during this period averages close to 90%. Occasionally, a pathogenic bacteria, yibrio spp., proliferates in a culture and results in minor to extensive larval mortality. This bacterial infection, when it appears, generally beomes obvious about day 4 or 5. Grossly characterized, the Vibrio spp. infection appears as agglutinated clumps of larvae on the culture bottom screen that have a yellowish coloration, rather than the normal dark green. Vibrio spp. are effectively treated with Neomycin sulfate (50 mg/liter). However, when the infection is severe, we discard the culture rather than attempt this treatment. After 6 days, the veliger larvae have developed to a stage close to metamorphosis. This stage is recognized when the veligers have four branches on the cephalic tentacles, and the foot is sufficiently developed whereby the veliger can pull itself upright (on its foot) and propel itself about by ciliary action. We term this stage the “gliding stage.” These veligers are ready for transfer to the settling tanks. Forage culture
The diatom-slurry method provides sufficient diatoms for initial tank inoculations for just-metamorphosed abalones, which exhibit a low rate of foraging. At this stage, and at the stocking density specified, the diatom-cell growth rate is considerably greater than the rate of consumption by the abalone. As a result, we generally cover the abalone settling tanks 4 or 5 days after larval settlement to retard diatom growth. Tanks are uncovered and covered periodically in response to the abalone grazing rate and the diatom cell density. These subjectively governed adjustments are used to balance the abalone grazing rate with diatom celf production. Within 2 months, the abalone grazing rate sharply increases and the tanks are left uncovered. When the abalone grazing rate exceeds diatom cell production, additional diatom inocula, using the larger coarse bag-filtered diatoms, are added. Settkment and early-juvenile-stage cultivation
Under the conditions described, most larvae (ca. 90%) cease swimming within 24 h, and the remainder settle within 3 days after distribution into the settling tanks. When the juvenile abalones are 2 weeks old, they average about
387
Fig. 4. Red abalone d~vei~pmental stages at 15°C: a, ova at fertil~ation (225 pm), note sperm on ova membranes; b, tro~hophore stage (160 pm x 195 pm), 15 h post fertilization, just prior to rupturing the membrane; c, veliger stage (210 Frn x 270 pm), 30 h postf~rtilization~ d, early juvenile stage (490 pm), 2 weeks old; e, early juvenile stage (0.8 mm), 6 weeks old; f, juvenile, first respiratory pore formation (2.1 mm). about 8 weeks old.
388
490 pm in length, by 6 weeks about 0.8 Frn long, and the first respiratory pore notch forms at about 8 weeks, when they measure 2.1 mm long. Most abalone mortality (close to 90°~) occurs during the first month postsettlement, and most of this during the initial 2 weeks in the settlement tanks. Approximately one-half of the initial mortality consists of veligers that fail to metamorphose. Culture success is quite variable. Five representative culture trials revealed an average 7.5% survivorship (ranging from 1.9 to 13.5%) during an approximate 3-month period (Table IV). TABLE IV Red abalone settlement tank cultivation results Trial
1 2 3 4 5
No. seeded
35,000 35,000 26,000 24,000 20,000
Yield
Shelt length (mm)
Density
Trial
. (#/cm 21
period (days)
(#)
F%~
Mean
Range
2.0 2.0 1.5 1.4 1.2
85 85 90 78 133
4,200 650 3,500 1,800 522
12.0 1.9 13.5 7.5 2.6
2.9 2.2 3.2 3.0 12.5
1.8- 5.5 1.8- 5.0 1.2- 5.5 l.O- 5.0 6.6-16.7
Weming Abalone mortality in the weaning tanks is low (less than 2%) and is generally confined to the transfer period, from handling and mechanical damage. Abalones at these sizes (5-10 mm) have a very high grazing rate and, even with frequent additions of diatom-slurry inocula, manage to keep the film “well-grazed.” This forces the abalones to graze on macroalgae (giant kelp blades). Grow-our
The abalone growth rate in all grow-out tanks was quite similar. During the first year in these tanks, the abalones averaged about 2 mm/month (12-36 mm). At larger sizes (5-8 cm), the growth rate declined to about 1 mm/month. Abalone mortality in the grow-out tanks was low, generally less than lo/o,and most of this mortality was confined to the initial transfer period, from mechanical damage or handling stress. RISCUSSrON
Conditioning of II. rufescensfor spawning is relatively simple when compared to that of certain other abalone species. Temperature or photoperiod or both are frequently cited as critically important to the maturation process (Kikuchi and Uki, 1974a). Neither of these two factors appear to be limiting for red
389
abalone-gonadal maturation, under the conditions we have thus far explored. The poor maturation success observed with abalones held in continuous darkness, on raw seawater, may be attributed to (natural) spawnings that went undetected. Typically, two or more uninduced spawnings occur in the laboratory annually, with abalones that are receiving raw seawater. We speculate that something in the raw seawater triggers the spawning, but is not present in the filtered seawater. Our filtered seawater supply does receive a low-dosage IJV treatment following passage at high flow rate through the laboratory sand filters. Spawning induction, jkrtilization, arrd early development
We consider the UV spawning-induction method that was developed by Kikuchi and Uki (1974a) to be one of the most significant contributions to abalone cuItivation. Prior to this breakthrough, it was di~cult to obtain synchronous red abalone spawners. Red abalones respond to both UV and the hydrogen peroxide (Morse et al., 1977) spawning-induction techniques. We prefer the former technique because it requires no chemical additions, and we can program the UV unit timer clock to activate at an early hour. Therefore, the abalones are close to spawning when the normal workday begins. This allows a full day for setting up cultures, and most importantly, the abalones hatch early on the following day. Probably the two most critical procedures in cultivating abalones are the collection of ova from the spawning container and the transfer of veliger-stage larvae to the larval-rearing apparatus. These procedures are critical because they represent possible contamination sources. Scrupulous sanitary procedures should be applied to maintain a clean spawning container, and the transfer of any contaminants along with the ova must be avoided. Likewise, the bottom of the larval-hatching container contains shed egg membranes and undeveloped, or aberrantly developed, ova that can serve as contaminants, Therefore, only “high-rising” veliger larvae should be transferred. Application of these procedures should eliminate the need for antibiotics during the larvalrearing period. Larval cultivation
The development of a flow-through larval-rearing apparatus represents a distinct advancement in abalone-cultivation procedures (cf. Morse et al., 1979). Larvae can be cultivated at higher densities (up to 201ml) with good survivorship, and the culture seawater does not require periodic exchanges. Only two culture conditions must be monitored and maintained. These are temperature and water-flow rate. The water flow must be maintained at a low rate whereby the larvae are uniformly distributed and are not forced against the culture-bottom screen. Although we use only an 8-liter capacity larval-rearing apparatus, this apparatus appears amenable to scaling up for large commercial operations. Forage culture
Maintaining an adequate diatom film in abalone-settling tanks is a major
390
problem in the overall cultivation process. Problems arise from variable abalone grazing rates according to size, grazing intensity relative to abalone density settlement success, seasonal diatom-growth rates, and selective grazing by abalones. Alternate covering/uncovering of the settlement tank in response to the early (low grazing rate) abalone-settlement period is not inefficient or labor intensive. Frequently, however, the diatom film is insufficient on the settling-tank walls where abalones prefer to settle, and too dense on the tank bottom. It is undesirable to allow a thick, often filamentous, diatom film to develop on the tank bottom; this obstructs the young abalone’s mobility and harbors micropredators. The diatom film on the tank bottom is controlled through vigorous tank rinsing or by brushing. If abalone-settlement success is reasonably good after 2 months in the settling tanks (10% or more) then it becomes increasingly difficult to maintain an adequate diatom film, and forage can become limiting. The occasional diatom inoculations from the slurry system help, but this has proved insufficient for maintaining an adequate diatom film when natural diatom production is low (November to April). One possible solution is to increase the vertical surface area in the settling tanks. This is now being studied. We do not completely understand the etiology of the high abalone mortality that we have observed during the initial 2 weeks in our settling tanks. Known pathogenic bacteria, V&o spp., are present and implicated. Copepods, Tigriopus caiifornicus, unidentified nematodes, and ciliate protozoans are often associated with these abalone mortalities. These mortalities also appear unrelated to our initial abalone-stocking densities. Abalone cultivators frequently use a settlement and metamorphosis inducer for fully developed veliger-stage larvae (F.L. Clogston, unpublished observations; Morse et al., 1979; Seki and Kan-no, 1981). We consider this unnecessary with our cultivation methods; although our initial abalonemortality rates are high in the settling tanks, the limiting factors are vertical substrates and insu~cient diatoms. We estimate that maximum abalone production in one of our settling tanks, for 3 months, is about 5000 abalone (about 14% success or 1114 cm*); typically, however, a lower success rate is observed. We cannot provide adequate diatoms for this number of abalones after 3 months without incurring serious stunting; therefore, the abalones must be transferred to weaning tanks. Weaning
The abalone-weaning-tank period represents the most inefficient element in our cultivation methods. It represents an “extra step,” but we have not been able to circumvent it. It was not planned for in our original abalone-cultivation scheme. Abalone at 5-10 mm require considerably more diatoms than we can provide in the grow-out tanks. Early attempts to force these young abalones to feed on macroalgae (giant kelp) resulted in a temporary cessation in growth and increased mortality.
391
Grow-Out
Abalones grow equally well in both the circular and trough-shaped tanks. Initially we thought that water-flow characteristics in the circular tanks would enhance abalone-growing conditions. A negative aspect of the circular tanks is the constraint imposed on abalone habitats. Such habitats require more complex construction in order to fit uniformly in a round configuration. We intend to layer the habitats and thus increase available surface area and abalone numbers. This can be more easily accomplished using trough-shaped tanks. It is anticipated that such tanks will support 5000 abalones (l/7 cm2, excluding the habitat surface area), up to 3 cm in length, with a 6-8 liter/min water-flow rate (about a 90 min tank-turnover period). ACKNOWLEDGMENTS
This work was supported in part by the Bartlett Commercial Fisheries Research and Development Act (P.L. 88-309), Project #743, “Shell~sh Culture Investigations.” REFERENCES Carlisle, J.G.. Jr., 1962. Spawning and early life history of Huliotis ru&cens Swainson. Nautilus, 76(2):44-48. Cox, K.W., 1963. Report of visit to abalone hatcheries in Japan, March, 1963. State of California, the Resources Agency, Department of Fish and Game, Marine Resources Operations, MRO Reference No. 63- 1, Ebert, E.E., Haseltine, A.W. and Kelly, R.O., 1974. Seawater system design and operations of the Marine Culture Laboratory, Granite Canyon. Calif. Fish and Game, 60(1):4-14. Ino, T., 1952. Biological studies on the propagation of Japanese abalone (genus Ha~i~~;s). Bull. Tokai Reg. Fish. Res. Lab., 5 (in Japanese with English summary~~ 102 pp. Inoue, M.. 1976. Awabi. in: Suisan Zoyoshoku Deeta Bukku. (Abalone. In: Fisheries Propagation Data Book). Published by Suisan Suppan (translated by M. Mottet). Kan-no, H. 1975. Recent advances in abalone culture in Japan. Proceedings of the First International Conference on Aquaculture Nutrition, October, 1975:195-21 I. Kikuchi, S. and Uki, N., 1974a. Technical study on artificial spawning of abalone, genus Hulioris. II. Effect of ultraviolet irradiated seawater to induce spawning. Bull. Tohoku Reg. Fish. Research Lab., No. 33:80-92. (In Japanese with English abstract.) Kikuchi, S. and Uki, N., 1974b. Technical study on artificial spawning of abalone genus Huliotis. 111. Reasonable sperm density far fertilization. Bull. Tohoku Reg. Fish Res. Lab., 3467-71. (In Japanese with English abstract.) Kikuchi, S. and Uki, N., 1974~. Technical study on artificial spawning of abalone. genus Hai~~~is. V. Relation between water temperature and advancing sexual maturity of Hul~otj~ discus Reeve. Bull. Tohoku Reg. Fish. Res. Lab., 34:77-85. (In Japanese with English abstract.) Leighton. D.L., 1972. Laboratory observations on the early growth of the abalone, Haiiotis .sormsm/. and the effect of temperature on larval development and settling success. Fish. Bull., 70(2):373-381. Leighton, D.L., 1974. The influence of temperature on larval and juvenile growth in three species of southern Califonia abalones. Fish. Bull., 72(4):1137-l 145. Leighton, D.L., 1977. Some problems and advances in culture of North American abalones (t;laliotrs). Paper presented to the First Symposium in the Latin American Aquaculture Association, Maracay, Venezuela, Nov. 5-12. 1977. Morse, D.E., Duncan, H., Hooker, N. and Morse, A., 1977. Hydrogen peroxide induces spawning in molluscs, with activation of prostagiand~n endoperoxide synthetase. Science, 196298-300.
392
Morse, D.E., Hooker, N., Jensen, L., and Duncan, H., 1979. Induction of larval abalone settling and metamorphosis by y-aminobutyric acid and its congeners from crustose red algae: II: Applications to cultivation, seed-production and bioassays; principal causes of mortality and interference. Proc. World Maricul. Sot. IO:8l-91. Murayama, S., 193.5. On the development of the Japanese abalone, ~Q~~o?~s g~g~~fe~. Jour. Coil. Agric., X111(3):227-233. Seki, T. and Kan-no, H., 1977. Synchronized control of early life in the abalone, Haiioris discus hannai Ino, (Haliotidae, Gastrpoda). Bull. Tohoku Reg. Fish. Res. Lab. No. 38. October, pp 143-153, (in Japanese with English summary.) Seki, T. and Kan-no, H., 19Rl. Induced settlement of the Japanese abalone, H&Otis discus hannai, veliger by the mucous trails of the juvenile and adult abalones. Bull. Tohoku Reg. Fish. Res. Lab., No. 43:29-36. (In Japanese with English summary.) Shibui, T., I972. On the normal development of the eggs of Japanese abalone, Haliotis disrus hannai Ino. and ecological and physiological studies of its larvae and young. Bull. Iwate Pref. Fish. Exp. Stat. (2):1-69.