1
Prey Swimming Behavior and Culture Techniques for Copper,
Sebastes caurinus, and Quillback Rockfish Larvae, Sebastes malinger.
General Introduction:
Live food has been an essential part of marine larval fish culture for almost four
decades (Lavens and Sorgeloos, 1996; Stottrup and McEvoy, 2003). The two most
common live food species for larval fish culture are rotifers, (Brachionus species) and
brine shrimp (Artemia species; Lubezens, 1987; Lubzens, et al., 1989; Sorgeloos, et al.,
2001; Stottrup and McEvoy, 2003). Although much progress has been made with
microparticulate diets in the past 25 years, they can not completely replace live food
(Jones et al., 1993; Liao et al., 2001; Langdon, 2003) however, microparticulate diets
may support larval growth when fed in combination with live foods (Kolkovski et al.,
1997; Cañavate and Fernández-Díaz, 1999; Lazo et al., 2000).
Brachionus rotundiformis (SS-type), B. plicitilis (L-type), and Artemia (Salt
Lake species) were the live food items investigated in this study. Optimal culture
conditions differ among rotifer strains. SS-type shows optimal growth at 28-35C and
30‰, while L-type prefers 18-25C and 20‰ salinity (Stottrup and McEvoy, 2003),
but each species may adapt to non-optimal conditions (Lubzens, 1987; Stottrup and
McEvoy, 2003). Rotifer cultures can be obtained from established colonies or hatched
from resting cysts; however, rotifers must be cultured to obtain high densities, since
high numbers of rotifers cysts are not readily available. In contrast, Artemia are usually
purchased as cysts that are typically hatched over a 24 h period under high light
intensities and with aeration.
Live feed can be enriched by either short or long-term enrichment periods.
Short term enrichment is used to fill the guts of live prey over a period of 6-24 h, while
long-term enrichment refers to daily feeding during the culture process which allows
the live food to not only fill their guts but also to assimilate food for growth (Stottrup
and McEvoy, 2003). Rotifers may empty their guts within 20-30 minutes after harvest
and subsequent feeding to larvae (Stottrup and McEvoy, 2003); consequently, the
2
nutritional value of short-term, enriched rotifers may be reduced, adversely affecting
larval growth and survival. Longer-term enrichment of live food is recommended with
a nutritionally sufficient diet, such as live microalgae (Kreeger et al, 1991; Aragão et
al., 2004). The algal species used for live prey culture can affect the fatty acid content
and composition of prey (Reitan et al 1997). Isochrysis galbana (Tahitian strain, T-Iso)
is often recommended for short and long-term enrichment of live prey due to its high
lipid content and high levels of potentially essential fatty acids.
Importance of live prey items, green-water, and enrichments
There are two developmental types of finfish larvae. First, there are precocial
larvae that appear as mini-adults after the yolk sac is exhausted, exhibiting fully
developed fins and a mature digestive track including a functional stomach (Stottrup
and McEvoy, 2003). These larvae may ingest and digest formulated diets due to the
maturity of their digestive track, allowing them to be reared without the need of a live
prey diet. Salmon and trout are examples of species that have precocial larvae (Stottrup
and McEvoy, 2003). Secondly, there are altricial larvae that remain in a relatively
undeveloped state after the yolk sac is absorbed. Their digestive system seems to be
incapable of processing formulated diets in a manner that allows survival and growth of
the larvae comparable to that of larvae fed on live food (Stottrup and McEvoy, 2003).
A live prey diet is essential for altricial larvae because artificial diets have not yet been
developed for initial growth, proper development, and survival. Laboratory cultures of
altricial larvae are often characterized by massive die offs at the termination of the yolk
sac stage (the “critical period”), when larvae must begin feeding or starve (Fuiman and
Werner, 2002).
Microalgae are often used as a live diet, either fed directly to fish larvae or
indirectly as food for other live prey, such as rotifers or Artemia. Ingested microalgae
may also trigger digestion processes or contribute to the establishment of early larval
gut flora (Reitan et al., 1997). The importance of polyunsaturated fatty acids (PUFA) in
larval fish nutrition has been extensively investigated during the past 20 years
(Wantanabe, 1993; Wantanabe and Kiron, 1994; Sargent et al., 1999).
3
Docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA 20:5n-3), and
arachidonic acid (ARA, 20:4n-6) are thought to be essential fatty acids (EFA) for many
marine species. The specific content of EPA and DHA in some microalgae (e.g. EPA
in Nannochloropsis occulata; (Watanabe, 1979; Watanabe et al., 1983; Koven et al.,
1990; Seto et al., 1992; Sukenik et al., 1993) and DHA in Isochrysis galbana (Lubzens
et al., 1985; Ben-Amotz et al., 1987; Whyte and Nagata, 1990; Sukenik and Wahnon,
1991; Mourente et al., 1993) and their easy mass culture make them attractive
candidates as enrichments and for green-water.
Green-water is the technique of adding microalgae during larval rearing that
acts as a water conditioner by decreasing light intensity and increasing contrast and
larval UV acuity during prey capture (Britt et al., 2001). Green-water also acts as an
initial larval food source and as a means of maintaining the nutritional quality of live
prey before they are ingested by larvae (Reitan et al., 1993, 1997). Given that DHA is
naturally found at very high levels in neural tissue, it is thought to play a specialized
role in neural membrane structure and function (Bell and Dick, 1991; Horrocks and
Yeo, 1999). DHA levels should be higher than those of EPA, by a ratio of 2:1, for
optimal marine larval growth and survival (Su et al., 1997; Reitan et al., 1997;
Copeman et al., 2002). Elevated dietary levels of EPA relative to DHA are postulated
to have a negative impact on larval neural function and thus growth and survival
(Rodriguez et al., 1997). Larvae may also develop high levels of malpigmentation and
poor development when fed elevated ARA relative to EPA (McEvoy et al., 1998;
Estevez et al., 1999). Some live foods, such as rotifers and Artemia, are naturally low
in PUFA; therefore, enrichment of live prey with lipid emulsions, dried or live
microalgae is commonly used to increase their PUFA content and nutritional value.
Rockfish species, biology, and fisheries:
Within the United States, studies on eggs and larvae of most marine finfish
species, such as rockfish (Sebastes sp.), have often stemmed from consideration of
fisheries management issues rather than aquaculture (Berkley et al., 2004; Stahl-
4
Johnson, 1984; Moser and Butler, 1987; Moreno, 1993; Wold, 1991 Watson and
Robertson, 2004; Kusakari, 1991). There are 65 Sebastes species on the Pacific Coast
of North America (Love et al., 2002). Stock assessments prepared from 1999 to 2001
indicated that the biomass of at least seven of the major commercial rockfish species
(bocaccio, canary, yelloweye, dark-blotched, Pacific ocean perch, widow, and cowcod
rockfish) are at or below 25% of that estimated in the 1970’s (Love et al., 2002).
Successful larval and juvenile production will determine the scale of future marine
finfish enhancement efforts in the U.S.(Sheng-Lee, 1997; 2003).
The majority of bony marine fishes are characterized by external fertilization
and development of their eggs. Unlike most marine fishes, rockfish mate with internal
fertilization of eggs, and bear live young (Love et al., 2002). The release of live young
by rockfish species is called parturition. In the Eastern Pacific, rockfish species seem
to have two major seasons of larval production in winter and spring or summer
(Phillips, 1964). Individual fecundity may reach 2,300,000 eggs per female in bocaccio
rockfish (Phillips, 1964) and 2,700,000 in yelloweye (Methot et al., 2003) with weightspecific fecundity reaching 500 eggs per gram body weight (MacGregor, 1970;
Boehlert et al, 1982).
It has only been in the past 25 years that some Northeastern Pacific rockfish
species have been raised past yolk absorption and in some cases to caudal fin formation
(Moreno, 1993; Moser and Butler 1981, 1987; Stahl-Johnson, 1984; Watson and
Robertson, 1999; Wold, 1991; Rust et al.; personal communication). Success in
rockfish larval culture in the Northeastern Pacific is difficult due to a number of factors,
including the relatively small size of larvae at birth, food availability, cool ambient
water temperatures that slow growth rates relative to those of Western Pacific species
(Kendall and Lenarz, 1987). Laboratory cultures of rockfish larvae are often
characterized by high mortalities at the termination of the yolk sac stage or “critical
period” when larvae must begin feeding or starve, (Fuiman and Werner, 2002).
However, NOAA/NWFSC researchers in Washington over the past two years have
reared yelloweye, brown, china, and copper rockfish through caudal fin formation to
the juvenile stage (Rust et al.; personal communication).
5
Effects of temperature transfer on availability of rotifers Brachionus plicitilis and
Brachionus rotundiformis and brine shrimp Artemia metanauplii: application to
rearing larval rockfish, Sebastes species.
Thom Gilbert
Department of Fisheries and Wildlife, Coastal Oregon Marine Experimental Station
Hatfield Marine Science Center, Oregon State University, Newport, Oregon 97365,
USA
Abstract
Rotifers and brine shrimp (Artemia) are important prey items for rearing
marine fish larvae. Their availability in the water column may be reduced when they
are transferred to larval rearing tanks at lower temperature. In this study, Brachionus
rotundiformis (SS-type) and Brachionus plicatilis (L-type) were semi-continuously
cultured and fed on live microalgae (Isochrysis galbana) at 20C. Upon hatching,
Artemia nauplii were fed I. galbana for 24 h at 20C before temperature shock and
handling experiments took place. Temperature shock and handling stress experiments
with prey were conducted to test prey availability for larval culture of Eastern Pacific
rockfish (Sebastes) species. Prey was sampled in 1 mL aliquots in the water column
from 10 L buckets at 10, 14, 18, and 20C after 0, 2, 4, 6, 12, 24, and 48 h suspension
and from four water levels (top, middle, 5 cm above bottom, and bottom). Prey
sampled from the bottom were not swimming while prey in the other three sampled
levels were defined as swimmers. The concentration of initial suspended prey (10 prey
per ml) was reduced after handling and temperature transfer occurred and any recovery
was correlated with temperature. After 6 h at 10ºC, SS-rotifers were almost completely
lost from the water column with bottom samples containing mostly dead rotifers.
While Artemia suffered initial transfer shock, after 24 h 98% of Artemia were
swimming and available in the water column. Rotifers and Artemia should be cultured
6
within a range of target temperatures ± 5 ºC or at similar temperatures compared with
those of larval cultures.
1. Introduction
Live food has been an essential part of marine larval fish culture for almost four
decades (Lavens and Sorgeloos, 1996; Stottrup and McEvoy, 2003). The two most
common live food species for larval fish culture are rotifers, (Brachionus species) and
brine shrimp (Artemia species; Lubezens, 1987; Lubzens, et al., 1989; Sorgeloos, et al.,
2001; Stottrup and McEvoy, 2003). Although much progress has been made with
microparticulate diets in the past 25 years, they can not completely replace live food
(Jones et al., 1993; Liao et al., 2001; Langdon, 2003), however, microparticulate diets
may support larval growth when fed in combination with live foods (Kolkovski et al.,
1997; Cañavate and Fernández-Díaz, 1999; Lazo et al., 2000).
Brachionus rotundiformis (SS-type), B. plicitilis (L-type), and Artemia (Salt
Lake species) were the live food items investigated in this study. Optimal culture
conditions vary among rotifer strains. SS-type shows optimal growth at 28-35C and
30‰ salinity, while L-type prefers 18-25C and 20‰ (Stottrup and McEvoy, 2003),
but each species may adapt to non-optimal conditions (Lubzens, 1987; Lubzens et al.,
1995; Stottrup and McEvoy, 2003). Rotifer cultures can be obtained from established
colonies or hatched from resting cysts; however, rotifers must be cultured to obtain
high densities, since high numbers of rotifers cysts are not readily obtained. In contrast,
Artemia are usually purchased as cysts, which are typically hatched over 24 h period
under high light intensity and with aeration.
It is well known that temperature has an effect on rotifer swimming behavior
(Gatescoupe and Luquet 1981, Korunuma and Fukusho 1987, Snell et al., 1987; Oie
and Olsen 1993; Fielder et al., 2000). Fielder et al. (2000) showed that rotifers suffered
transfer and handling shock upon harvest and concentrations in the water column were
reduced by 50-60%.
7
Live feed can be enriched by either short or long-term enrichment periods.
Short term enrichment is used to fill the guts of live prey over a period of 6-24 h, while
long-term enrichment refers to daily feeding during the culture process which allows
the live food to not only fill their guts but also to assimilate the food for growth
(Stottrup and McEvoy, 2003). Under short-term enrichment, rotifers may empty their
guts within 20-30 minutes after harvest (Lavens and Sorgloos, 1996; Stottrup and
McEvoy, 2003); consequently, the nutritional value of rotifers may be reduced,
adversely affecting larval growth and survival. Longer-term enrichment of live food is
recommended with a nutritionally sufficient diet, such as live microalgae (Kreeger et al,
1991; Aragão et al., 2004). The algal species used for live prey culture can affect the
fatty acid content and composition of prey (Reitan et al 1997). Isochrysis galbana
(Tahitian strain, T-Iso) is often recommended for short and long-term enrichment of
live feeds due to its high lipid content and high levels of potentially essential fatty
acids.
The objectives of this study were (1) to determine if rotifer and Artemia
availability changes following a temperature shock, (2) to assess the difference in live
food availability among species of rotifers and Artemia. Temperatures chosen for the
temperature shock were similar to those used for larval rockfish culture (Stahl-Johnson,
1984; Boehlert and Yoklavich, 1982; Moreno, 1993; Love et al., 2002; Berkeley, et al.,
2004; Watson and Robertson, 2004).
2. Materials and Methods
2.1. Zooplankton culture
Brachionus plicitilis (source: Reed Mariculture San Jose, CA) and Brachionus
rotundiformis (source: Oceanic Institute Hawaii, Oahu, HI) were maintained in an
aerated semi-continuous culture in 100 L tanks at 32‰ salinity, 20C, 7.4-8.4 pH, with
oxygen levels above 5.0mg L-1. Cultures were fed live microalgae, T-Iso ad libitum.
Artemia cysts (source: Salt Creek Inc.) were decapsulated (Stottrup and McEvoy, 2003)
and hatched over a 24 h period in 2 L plastic conical bottles at 20ºC and 32‰ salinity
8
with high aeration and illumination. Upon hatching, nauplii were harvested and placed
into an aerated container at 20C, and enriched with T-Iso microalgae for 24 h before
being used in the temperature experiment as metanauplii.
2.2. Prey culture and harvest
The rotifers used in all experiments were harvested from 100 L cultures, and
concentrated on a 37 m sieve, rinsed with seawater at a similar temperature and
salinity to those of their culture medium, then poured into a 1 L container. Enriched
Artemia were harvested using a 37μm sieve, as described for rotifers. Harvest prey
densities were determined by counting prey in four replicate 0.25 mL aliquots, and the
volume of prey suspension required to provide 10 prey per mL was calculated for each
10 L experimental container.
2.3. Experiments 1-3: Effect of temperature transfer on prey availability
L and SS-rotifers (Experiments 1 and 2) or Artemia (Experiment 3) were
harvested, counted, and added to provide densities of 10 prey per mL in each of the 10
L containers filled with seawater at 10,14, or 18C. As a control for handling stress,
rotifers and Artemia were transferred from cultures at 20˚C to 10 L containers filled
with seawater at the same temperature (20˚C) and salinity as the prey cultures. Five
non-aerated replicate containers were set up per temperature at 32‰ salinity. Prey
availabilities in larval cultures were measured by taking 1 mL samples at four levels in
the water column, top (5 cm below surface), middle, above bottom (5 cm above
bottom), and bottom at 0, 2, 4, 6, 12, 24, and 48 h. Prey sampled from the bottom were
defined as non-swimmers or unavailable, while top, middle, and above bottom samples
were combined and defined as swimmers or available prey. Experiments 1 and 2 were
run simultaneously but Experiment 3 was carried out separately.
2.4. Statistical analyses
The means from each data set were calculated and repeated measures
ANOVA (Ramsey and Schafer 2002 and SAS, 2nd edition 1998) carried out to test for
9
significance differences (p<0.05) among factors. The effect of specific treatment
combinations (i.e the effect of temp within time and time within temp) were tested
using one-way ANOVA’s (Fielder et al. 2000) followed by Student-Newmans-Keuls
(SNK) multiple range test at a significance level of p<0.05. Prey densities were used
for this analysis. (Figures 1-3).
3. Results
For all rotifer experiments, the number of sampled rotifers per mL was always
less than the estimated initial stocking density, indicating that rotifers suffered
temperature and transfer shock (Figures 1 and 2), the loss increasing with the degree of
temperature shock. In experiments with Artemia, there was an initial transfer and
temperature shock but over time Artemia recovered to almost 100% of estimated added
stocking densities (Figure 3).
.
3.1. Experiment 1: Effect of temperature transfer on availability of SS-rotifers
There was a significant effect of sampling time, temperature, and interaction
between temperatures and time on suspended rotifer density (ANOVA; p< 0.001, Table
1a). Mean densities of available rotifers were significantly different among
temperature treatments and some sampling times, depending on the experimental
temperature (SNK p<0.05; Table 1b). There was no significant effect of sampling time
at 18˚C, while rotifers transferred to 14ºC showed a significant increase in densities
after 24 h compared with initial values. In contrast, rotifers transferred to 10C showed
a significant decrease in densities (to zero) at 6 h (Figure 2) and visual inspection at 12
h indicated mortalities.
10
3.2 Experiment 2: Effect of temperature transfer on availability of L-rotifers
There was a significant effect of sampling time and temperature on rotifer
density but there was no temperature by time interaction effect (Table 2; ANOVA;
p<0.05). At most sample times, rotifer densities were highest at 20ºC (Table2b).
Rotifers at all temperatures were initially shocked before slowly recovering over the
experimental period (Figure 3 and Table 2b); however, densities never fully recovered
to 100% of estimated added densities.
3.3. Experiments 3: Effect of temperature transfer on availability of Artemia
Temperature, time, and interaction between temperature and time had
significant effects on densities of Artemia (ANOVA; p< 0.005, Table 3a). Artemia
recovered from temperature transfer (Figure 3) and there were no significant
temperature effects on Artemia initial density after 24 h (Tables 3b and c).
4. Discussion
Live food can survive in a wide range of temperatures and salinities (Oie and
Olsen, 1993; Fielder et al., 2000; Stottrup and McEvoy, 2003) but rapid changes in
salinity or temperature can affect their swimming behavior and metabolism (Epp and
Winston, 1978; Snell et al., 1987; Oie and Olsen, 1993, Fielder et al., 2000).
This study showed that rotifer species were relatively affected by temperature
shock and transfer to lower temperatures but that transfer had greater affects. Rotifers
settled out of the water column and possibly adhered to surfaces (Oie and Olsen, 1993;
Lubzens et al., 1989) when they were subjected to a decrease of 2 to 10ºC from 20ºC.
In contrast to the findings of this study, Oie and Olsen (1993) reported no handling
effects for B. plicitilis and a rapid recovery after 4 min of transfer from 20ºC to 8ºC.
However, these researchers carried out experiments in Petri dishes where movement of
individuals was noted but not swimming activity.
Artemia suffered initial transfer and temperature shock but recovered more
rapidly than rotifers. Since Artemia are positively phototactic (Stottrup and McEvoy,
2003), they migrated towards the surface during the experimental period. Artemia
11
recovery improved with time and temperature in the first 24 h after transfer and
temperature shock. After 24 h, Artemia had recovered in all treatments, with 98% of
initially added Artemia present in the water column.
In conclusion, this study indicates the importance of live prey harvest,
handling, and transfer methods and also the importance of ensuring similar culture
temperatures, when possible, for live prey and fish larvae. When this is not possible,
temperature shock effects on prey swimming activity may be overcome with gentle
aeration or by adding higher densities of prey to account for the loss. An alternate
strategy would be to acclimate prey to larval culture temperatures before being added as
food items (Assavaaree et al., 2001)
Acknowledgements
This research was partly funded by the Hatfield Marine Science Center (HMSC)
Mamie Markham Scholarship Award, National Oceanic and Atmospheric
Administration (NOAA), U.S. Department of Commerce, under grant no.
NA16RG1609 (project no. R/SAQ-04-NSI-NMAI), and NOAA’s National Sea Grant
College program of the U.S. Department of Commerce under grant no. NA16RG1039
(project no. R/SAQ-07). The views expressed herein do not necessarily reflect views of
these organizations.
12
Percent SS-rotifers swimming
100
90
80
70
60
50
40
Temp°C
30
10
14
18
20
20
10
0
0
5
10
15
20
25
30
35
40
45
50
Time (h)
Figure 1: Mean perecent swimming SS-rotifers, B rotundiformis after rapid transfer
from 20 ºC to 10, 14, 18, or 20 ºC. Data are means ± SD (n=5 replicates per treatment).
Experiment 1.
Percent L-rotifers swimming
13
100
90
80
70
60
50
40
30
20
10
0
Temp°C
10
14
18
20
0
5
10
15
20
25
30
35
40
45
50
Time (h)
Figure 2: Mean percent swimming L-rotifers, B. plicitilis after rapid transfer from
20C to 10, 14, 18, or 20C. Data are means ± SD (n=5). Experiment 2.
Percent Artemia swimming
14
100
90
80
70
60
50
40
30
20
10
0
Temp°C
10
14
18
20
0
5
10
15
20
25
30
35
40
45
50
Time (h)
Figure 3. Mean percent swimming Artemia after rapid transfer from 20C to 10, 14,
18, or 20C. Data are means ± SD (n=5 replicates per treatment). Experiment 3.
15
Table 1: Repeated ANOVA (a) and SNK (b and c) analysis of SS-rotifer densities in the
water column at 0-48 h after temperature shock from 20C to 10, 14, or 18C (n = 5
replicates per treatment). Experiment 1.
(a)ANOVA
Source of
Variation
Temp
Time
Time*Temp
DF
Sum of
squares
9.672
0.188
0.414
3
6
18
(b) SNK for time*
Time (h)
Temperature (C)
12
10
14
18
20
0
10
14
18
20
2
10
14
18
20
4
10
14
18
20
6
10
14
18
20
24
10
14
18
20
48
10
14
18
20
Mean
Square
3.224
0.031
0.023
F-value
p-value
921.354
11.007
8.073
<0.0001
<0.0001
<0.0001
(c) SNK for temperature*
Temperature (C)
Time (h)
10
0
2
4
6
12
24
48
14
0
4
2
6
12
24
48
18
0
2
4
6
12
24
48
20
0
2
4
6
24
12
48
*Overall SNK post-hoc tests run separately for each time and temperature. Means
ranked from smallest to largest with those underlined not significantly different at
p>0.05.
16
Table 2: Repeated ANOVA (a) and SNK (b and c) analysis of L-rotifers densities in the
water column at 0-48 h after temperature shock from 20C to 10, 14, or 18C (n = 5
replicates per treatment). Experiment 2.
ANOVA (a)
Source of
Variation
Temp.
Time
Time*Temp
DF
Sum of
squares
3.681
0.597
0.138
3
6
18
(b) SNK for time
Time (h)
Temperature (C)
0
10
14
18
20
2
10
14
18
20
4
10
14
18
20
6
10
14
18
20
12
10
14
18
20
24
10
14
18
20
48
10
14
18
20
Mean Square F-value
p-value
1.227
0.100
0.008
<0.0001
<0.0001
0.0974
204.271
19.812
1.527
(c) SNK for temperature
Temperature (C)
Time (h)
10
0
2 4
6
12
24
48
14
6
0 4
2
12
24
48
18
2
0 4
12
6
24
48
20
0
2 4
6
24
12
48
*Overall SNK post-hoc tests run separately for each time and temperature. Means
ranked from smallest to largest with those underlined not significantly different at
p>0.05.
17
Table 3: Repeated ANOVA (a) and SNK (b and c) analysis of densities of Artemia in
the water column 0-48 h after temperature shock from 20ºC to 10, 14, or 18ºC (n = 5
replicates per treatment). Experiment 3.
ANOVA (a)
Source of
Variation
DF
Sum of
squares
Mean
square
F-value
P-value
Temp
Time
Time*Temp
3
6
18
0.727
2.991
0.389
0.242
0.498
0.022
87.963
444.430
19.264
<0.0001
<0.0001
<0.0001
(b) SNK for time
Time (h)
Temperature (C)
0
10
14
18
20
2
10
14
18
20
4
10
14
18
20
6
10
14
18
20
12
10
14
18
20
24
10
14
18
20
48
10
14
18
20
(c) SNK for Temperature
Temperature (C)
Time (h)
10
0
2
4
6
12
24
48
14
0
2
4
6
12
24
48
18
0
2
4
6
12
24
48
20
0
2
4
6
12
24
48
*Overall SNK post-hoc tests run separately for each time and temperature. Means
ranked from smallest to largest with those underlined not significantly different at
p>0.05
18
Larval culture of Quillback, Sebastes malinger and Copper Rockfish, Sebastes
caurinus.
Thom Gilbert1, Chris Langdon1, Michael Davis2, and Kevin Clifford3
1 Department of Fisheries and Wildlife, Coastal Oregon Marine Experimental Station
Hatfield Marine Science Center, Oregon State University, Newport, Oregon 97365,
USA
2 NOAA/NMFS/AFSC Hatfield Marine Science Center, Newport, OR-97365, USA
3 Oregon Coast Aquarium, Newport, OR-97365, USA
Abstract
Rockfish culture remains at a preliminary stage due to difficulties in obtaining
larvae and establishing optimal culture conditions. Attempts to obtain larvae from
hook-and-line captured wild rockfish have failed due to high larval mortalities;
however maintaining mature reproducing rockfish in tanks is an alternative to obtaining
viable larvae. Visibly gravid females were collected monthly from the Oregon Coast
Aquarium in Newport and held in isolation until they naturally released live larvae. In
this study, we evaluated optimal temperatures, green-water techniques, and grow-out in
static culture conditions for Sebastes caurinus and S. malinger larvae. Survival of
Sebastes caurinus larvae at 18 ºC was <15%, while survival was >40% at 10 and 14 ºC.
Nannochloropsis occulata (Nanno) and Isochrysis galbana, Tahitian strain (T-Iso) were
tested in a green-water and enrichment study with S. malinger larvae. T-Iso was better
than Nanno as a green-water conditioner as well as a food for rotifers, newly hatched,
and enriched Artemia. An average larval growth rate of 0.13 mm per day was observed
for three broods from females of different sizes, 2.95, 0.86, and 0.77 kg. Survival and
19
initial size of larvae were greater for larvae from the largest female. Results from these
experiments will be helpful in optimizing rockfish larval culture conditions for possible
restoration of threatened rockfish species of the Eastern Pacific and for aquaculture.
1. Introduction
1.1. General
Rockfish (Sebastes spp.) populations along the Eastern Pacific have been
commercially fished for decades and recently have been reported as over-fished (Love
et al., 2002; Berkeley et al. 2004a). One approach towards restoration of over-fished
stocks would be to culture and release juveniles. Studies in the U.S. on early life stages
of most marine finfish species, such as rockfish (Sebastes sp.), have stemmed from
consideration of fisheries management issues rather than aquaculture (Berkley et al.,
2004b; Stahl-Johnson, 1984; Moser and Butler,1987; Moreno, 1993; Wold, 1991
Watson and Robertson, 2004; Kusakari, 1991); however, Hubbs-Sea World Research
Institute (HSWRI) in collaboration with NOAA Northwest Fisheries Science Center
(NWFSC) has recently begun preliminary experiments to culture rockfish for possible
stock enhancement (HSWRI, 2006). In Newport, the Oregon Coast Aquarium (OCA)
currently houses yelloweye and bocaccio rockfish which become gravid seasonally in
the display tanks. Captive fish such as these can be a valuable source of larvae for
culture.
There are 65 rockfish species on the Pacific coast of North America (Love et at.,
2002); however, stock assessments from 1999 to 2001 indicated that biomass of at least
seven of the major commercial rockfish species (bocaccio, canary, cowcod,
darkblotched, Pacific ocean perch, widow, and yelloweye rockfishes) are at or below
25% of that estimated in the 1970’s (Berkeley, 2004a; Love et al., 2002). Federal
regulations designed to prevent target harvest and incidental harvest (bycatch) of these
species has resulted in significant cutbacks for commercial and recreational groundfish
harvest. Rockfish landings have decreased from 31,656 metric ton in 1994 to 3,668
metric tons in 2004 (PacFIN, 1994, 2004). Despite the interest in rockfish within the
20
commercial fishery and a need for rebuilding depleted stocks, very little larval culture
research has been carried out.
1.2. Sebastes reproduction
The majority of bony marine fishes are characterized by external fertilization
and development of their eggs. Unlike most marine fishes, rockfish are characterized
by internal fertilization of eggs, and they bear live young (Love et al., 2002). In the
Eastern Pacific, Sebastes seem to have two major seasons of larval production in winter
and spring or summer (Phillips, 1964). Female Sebastes are matrotrophically
viviparous i.e., some energy is transferred directly from the mother to the embryos in
addition to energy stored in the yolk (Boehlert and Yoklavich, 1984). This nourishment
for black rockfish embryos occurs after 27 days of development to about 10 days before
they are released from the mother (Boehlert and Yoklavich, 1984). The total incubation
period for S. caurinus is approximately 41 to 43 days, but may vary with species
(Delacy et al, 1964). Individual fecundity may be as high as 2,300,000 eggs per female
in bocaccio rockfish (Phillips, 1964) and 2,700,000 in yelloweye (Methot et al., 2003)
and weight specific fecundity reaching 500 eggs per gram of body weight (MacGregor,
1970; Boehlert et al, 1982).
1.3. Sebastes larval culture
In the past 25 years, some rockfish species of the Eastern Pacific have been
raised past yolk absorption and in some cases to caudal fin formation (Moreno, 1993;
Moser and Butler, 1987; Stahl-Johnson, 1984; Watson and Robertson, 1999; Wold,
1991, and Rust et al., personal communication). Success in rockfish larval culture is
difficult due to the relatively small size of larvae at birth, availability of high quality
food, and cool ambient water temperatures that slow growth rates relative to Western
Pacific species (Kendall and Lenarz, 1987; Kusakari, 1991). Laboratory cultures of
rockfish larvae are often characterized by high mortalities at the termination of the yolk
sac stage (the “critical period”), when larvae must begin feeding or starve (Fuiman and
Werner, 2002).
21
1.4. Importance of live prey items, enrichments, and green-water
Green-water is the technique of adding microalgae during larval rearing that
acts as a water conditioner by decreasing light intensity and increasing contrast and
larva acuity during prey capture (Britt et al., 2001). Reitan et al. (1993,1997) suggests
that the microalgae decrease light intensity and increase contrast, facilitating prey
capture. Microalgae are commonly used as a live food diet and can be consumed,
directly by larvae or indirectly by boosting the fatty acid content in other live prey, such
as rotifers. Rotifers and Artemia are naturally low in highly unsaturated fatty acids
(HUFA), therefore, enrichment of these prey items with commercially-produced dried
or live phytoplankton is commonly used to increase HUFA levels prior to feeding to
promote larval growth and development.
The importance of HUFA in larval fish nutrition has been extensively
investigated during the past 20 years (Wantanabe, 1993; Wantanabe and Kiron, 1994;
Sargent et al., 1999; Faulk et al., 2005). Docosahexaenoic acid (DHA, 22:6n-3),
eicosapentaenoic acid (EPA 20:5n-3), and arachidonic acid (ARA, 20:4n-6) are
essential fatty acids (EFA) for many marine species. The EPA content of
Nannochloropsis occulata (Watanabe, 1979; Watanabe et al., 1983; Koven et al., 1990;
Seto et al., 1992; Sukenik et al., 1993) and DHA in Isochrysis galbana (Lubzens et al.,
1985; Ben-Amotz et al., 1987; Whyte and Nagata, 1990; Sukenik and Wahnon, 1991;
Mourente et al., 1993) together with their easy mass culture make these algal species
attractive for enrichments of prey and for green-water conditions in larvae cultures.
The objectives of this study were (1) to determine optimal culture temperatures
for rockfish larval growth and survival, (2) to compare Nannochloropsis oculata
(Nanno) and Isochrysis galbana, Tahitian strain (T-Iso) for green-water conditions and
as live food enrichments for larval rockfish culture, (3) to compare larval growth and
survival among different broods of S. caurinus.
22
2. Materials and Methods
2.1. General
Larvae were cultured in either 20 L (Experiment 1 and 2) or 100 L
(Experiment 3) tanks at temperatures ranging from 10-18ºC (Berkley et al., 2004; StahlJohnson, 1984; Moser and Butler,1987; Moreno, 1993; Watson and Robertson, 2004)
Lighting was continuous over 24 hours (Moreno, 1993) at an intensity of 5.34 µ E s-1
m-2. All experiments were performed in a static system with gentle aeration (3-4
bubbles a second; Gilbert, 2006). Every other day a fifty percent water change occurred
and rotifers, Artemia, and T-Iso were added to maintain concentrations of 10, 2, and
100,000 per mL respectively. Seawater was filtered through four canister filters, 50,
10, 0.5, 0.1 µm and then UV sterilized. Water quality parameters were as follows:
salinity for all experiments was 32.5 ±1‰, pH ranged from 7.7-8.2, ammonia levels
<0.2 ppm, and oxygen concentration ranged from 5.5-8.3 mg L-1.
In all experiments, five larvae per liter were stocked on day 1. On day 2, all
dead larvae were removed by siphoning and replaced with live larvae of the same age
before initiating feeding with rotifers. Newly hatched or enriched Artemia were added
on day 8 and fed in combination with rotifers until day 14 when rotifer feedings ceased.
Larval measurements included standard length (SL; Experiments 1 and 2 only), total
length (TL; Experiment 3 only), notochord depth (ND), anus depth (AD), and eye
diameter (ED). All larvae were photographed live using a Spot insight camera attached
to a dissecting microscope (Leica S6D) at 1-2x magnification within 4-6 h of sampling.
Larval size measurements were obtained using Image Pro Plus version (4.5.1) computer
software.
2.2. Broodstock and larval collection
Visibly gravid S. caurinus and S malinger were collected from OCA’s reef
ecosystem exhibit (9 m deep; 1,120,000 L volume). Broodstock collection was from
February to September 2005 using scuba diving and hand nets. Gravid females were
transferred to four 600 L tanks at 12ºC, 32‰ salinity, and held without feeding until
23
natural parturition occurred. All parturitions occurred during the night and newly
released larvae were collected from a soft-mesh liner in the out-flow and from within
the holding tank (see Figure 1) the following morning and immediately transferred to
experimental tanks in order to reduce stress.
2.3. Prey culture and enrichment
Brachionus plicitilis (source: Reed Mariculture San Jose, CA) were
maintained in aerated semi-continuous culture (Stottrup and McEvoy, 2003) 100 L
tanks at 32‰ salinity, 17-18C, pH 7.4-8.4, with oxygen levels above 5.0 mg L-1.
Cultures were fed Nanno and T-Iso from cultures harvested in exponential growth
phase.
Artemia cysts (source: Salt Creek Inc. Salt Lake City, UT) were decapsulated
(Stottrup and McEvoy, 2003) and hatched over 24 hours in 2 L plastic conical bottles at
20ºC, salinity 32‰ with high aeration and continuous illumination. Upon hatching,
Artemia were harvested and either fed to larvae or enriched with microalgae for 24-36
h in an aerated container at 14C. Prey densities were determined by counting prey in
four replicate 0.25 ml aliquots.
2.4. Experiment 1: Optimal temperature for growth and survival of S. caurinus larvae
Larvae released on March 1, 2005 from a female S. caurinus (2.95 kg, 50 cm
total length) were used to determine optimal culture temperature (10, 14, and 18˚C;
Experiment 1) and growth and survival over a 30 day period (Experiment 3). Five
replicate cultures were set up for each temperature treatment together with three starved
control cultures. Cultures were stocked with 100 larvae per 20 L and fed on rotifers,
Artemia, and T-Iso at densities of 10, 2, and 100,000 per mL respectively. The
experiment was terminated when all starved control larvae died (day 16).
2.5. Experiment 2: Green-water/enrichment
Brachionus plicitilis was cultured on T-Iso and Nanno for 2 weeks. Larvae
released on August 12, 2005 from a female S. malinger (0.86 kg, 34 cm total length)
24
were used to compare Nanno and T-Iso for green-water conditions and as live food
enrichments for rearing larval rockfish at 14ºC. Larvae were divided among five
treatments with four replicates per treatment: 1) both rotifers and Artemia cultured and
enriched with Nanno alone and no green-water conditions (Nanno) 2) both rotifers and
Artemia cultured and enriched with Nanno and Nanno added for green-water conditions
(NannoGW) 3) both rotifers and Artemia cultured and enriched with T-Iso alone and
no green-water conditions (T-Iso) 4) both rotifers and Artemia cultured and enriched
with T-Iso and T-Iso added for green-water conditions (T-IsoGW) 5) rotifers cultured
and enriched with T-Iso and T-Iso added for green-water but newly hatched Artemia
nauplii were not enriched before feeding to larvae (T-IsoGWArt). Rotifers were
enriched in culture tanks for 7-9 h and Artemia were enriched at 14ºC for 24-36 h. To
provide equal total cell volumes for green-water conditions, Nanno and T-Iso were
added at 1,000,000 and 100,000 cells per ml, respectively, based on estimated cell
volumes of 4.2 µm3 and 40-50 µm3. Light intensity with green-water conditions ranged
from 4.1-6.9, 1.3-4.9, and 0.4-4.0 µ E s-1 m-2 from the top, middle, and bottom of the
water column respectively. The experiment was terminated and data collected after 20
days of culture.
2.6. Experiment 3: S. caurinus growout
Sebastes caurinus larvae were collected from broods released on March 1 (2.95
kg female), May 20 (0.86 kg female), and May 25 (0.77 kg female), 2005. Larvae were
grown out in duplicate 100 L circular tanks. Ten larval samples from each tank were
collected on days 1, 9, 15, 20, and 30 for growth measurements. Survival was
measured at day 30. Due to reports of uneven larval size distribution in the water
column (Stahl-Johnson, 1984), survival was measured within well-mixed tanks. Larval
samples from day 30 were subtracted from the initial number of stocked larvae in order
to obtain survival estimates.
25
2.7. Statistical analysis
Experimental treatments were compared using standard one-way ANOVA.
Arcsin transformation of percent data was carried out to meet assumptions of ANOVA
and Tukey’s Honest Significant Difference (HSD) multiple range test was used to test
significance and to compare differences among treatments (at a significance level of
p=0.05).
3. Results
3.1. Experiment 1: Optimal temperature for growth and survival of S. caurinus larvae.
There were significant differences in survival between larvae cultured at 18ºC
(13.4%) and both 10 (46.8%) and 14ºC (41.4%) (Figure 2). Larval size measurements
of TL and ND at 14ºC were significantly greater than at 10 and 18ºC (Figures 3 and 4).
3.2. Experiment 2: Live microalgae enrichment and green-water experiment.
Highest survival for S. malinger larvae occurred with T-Iso as a green-water
conditioner with either newly hatched, (T-IsoGwArt; 43%), or T-Iso enriched, (TIsoGw 39.8%), Artemia nauplii (Figure 5). Percent survival of larvae under T-IsoGW
and T-IsoGwArt conditions were significantly higher (Tukey’s HSD; p>0.05) than
those of larvae cultured without T-Iso added to create green-water conditions. Nanno
added as a green-water alga resulted in significantly lower (Tukey’s HSD; p<0.05)
larval survival (5.5%) compared with additions of T-Iso (Figure 5). Friedman’s twoway non-parametric test indicated no significant treatment differences (p>0.05)
between larval size measurements and will not be discussed.
3.3. Experiment 3: Variation in survival among broods of S. caurinus.
Larval survival from the larger sized female (2.95 kg; 50 cm; 33.2%) was
significantly greater at (p<0.05) compared to larval survival from smaller sized females
(0.86 kg; 34 cm; 16.4% and 0.77 kg; 32 cm; 14.3%; Figure 6). Larval growth rates
26
were similar and averaged 0.13 mm d-1 (Table 1); however, initial size of larvae was
greater with larger sized female resulting in significant difference among size
measurements through out culture period (Figure 7; Table 1). Unfortunately, brood
effect was confounded with culture time and conditions.
4. Discussion
Microalgae are widely recognized as important for green-water conditions and
for enrichment of prey species (Reitan et al., 1993, 1997; Su et al., 1997; Faulk et al.,
2005). Results indicate that T-Iso was better than Nanno for green-water and live prey
enrichment for culture of S. malinger larvae. Similarly, Faulk et al., (2005) show Nanno
to be a poor prey enrichment and green-water conditioner, resulting in poor survival of
Ocyurus chrysurus larvae. The superior quality of T-Iso may be due to its higher DHA
content. DHA is naturally found at high levels in neural tissue where it is thought to
play a specialized role in neural membrane structure and function (Bell and Dick, 1991;
Horrocks and Yeo, 1999). Ratios of DHA to ARA or EPA of 2:1 have been suggested
to be optimal for marine larval growth and survival (Reitan et al., 1997; Copeman et al.,
2002). Elevated dietary EPA relative to DHA is postulated to have a negative impact
on larval neural function, growth and survival (Bell et al., 1995; Rodriguez et al.,
1997), while other larvae have developed high levels of malpigmentation and poor
development when fed elevated ARA relative to EPA (McEvoy et al., 1998; Estevez et
al., 1999).
This study suggests 14 ºC as the optimal temperature for growth and survival of
S. caurinus larvae. Rockfish larvae have been cultured at different temperatures: StahlJohnson (1984) at 12-15ºC, Boehlert (1981) at 13-16ºC, Watson and Robertson (2004)
at 11ºC, and Berkeley et al. (2004b) at 10ºC. In the Eastern Pacific, off the Oregon
coast, ambient water temperature ranges from 9-15ºC seasonally. Temperatures for
larval rockfish culture should not exceed 18ºC in the Pacific Northwest. Temperature
transfer can affect the swimming behavior of rotifers and Artemia causing them to settle
out of the water column for prolonged periods of time (Fielder et al., 2000; Gilbert,
27
2006). During larval culture, care should be taken to assure that prey of high nutritional
quality remains available for maximum larval growth and survival.
Berkeley et al. (2004b) found that Sebastes larvae from older females had
growth rates more than three times as fast and survived starvation more than twice as
long as larvae from the youngest females. In support of these findings, results from this
study indicate higher survival of larvae from a larger female; however, growth rate did
not differ among female fish sizes. The initial size of larvae from the bigger female
was greater than that of larvae from smaller females, accounting for significant
differences among size measurements (Table 1). S. caurinus larval average growth rate
was 0.13 mm d-1 a rate that was similar to the findings of Stahl-Johnson (1984) who
reported a rate 0.13-0.14 mm d-1 for S. caurinus larvae, using wild zooplankton and
flow-through conditions.
The Oregon Coast Aquarium (OCA) currently houses hundreds of mature
reproducing rockfish, representing seven different species. Three of the seven species
(yelloweye, canary, and bocaccio rockfish) are listed as over-fished. These rockfish
produce viable larvae at OCA and provide an opportunity for researchers to work with
larval rockfish. Larval collection methods from female isolation systems are being
improved to eventually send larval rockfish to other research facilities for culture.
Efforts to obtain larvae from wild, hook-and-line caught rockfish from the live
commercial fishery of Port Orford, Oregon, failed due to high larval mortality upon
natural release. Five of six gravid china rockfish, S. nebulosus, released only dead
larvae, perhaps due to stress. Stahl-Johnson (1984) also reported high mortality of
naturally released larvae from wild caught S. caurinus and S. auriculatus, as well as
female death from forced larval extrusion.
The results of this project will benefit culture of rockfish species for potential
enhancement of wild populations. There are 65 rockfish species on the Pacific Coast of
North America (Love et al., 2002), however, stock assessments from 1999 to 2000
indicated that biomass of at least seven of the major commercial rockfish species
(bocaccio, canary, cowcod, drakblotched, pacific ocean perch, widow, and yelloweye
rockfish) are at or below 25% of levels estimated in the 1970’s (Berkeley 2004a; Love
28
et al., 2002). Federal regulations designed to prevent targeted harvest and incidental
harvest (by-catch) of these species has resulted in significant cutbacks for commercial
and recreational groundfish harvests of other, non-rockfish species. Rockfish landings
have decreased from 31,656 metric tons in 1994 to 3,668 metric tons in 2004 (PacFIN,
1994, 2004)
Despite an interest in rockfish by the commercial fishery and a need for
rebuilding depleted stocks, very little culture research has been carried out. Further
investigation of optimal culture temperatures, green-water techniques and diets will
improve culture success.
Acknowledgements
This research was partly funded by the Hatfield Marine Science Center (HMSC)
Mamie Markham Scolarship Award, National Oceanic and Atmospheric
Administration (NOAA), U.S. Department of Commerce, under grant no.
NA16RG1609 (project no. R/SAQ-04-NSI-NMAI), and NOAA’s National Sea Grant
College program of the U.S. Department of Commerce under grant no. NA16RG1039
(project no. R/SAQ-07). The views expressed herein do not necessarily reflect views of
these organizations.
29
Chapter 2 Figures
Effluent Flow
Influent Flow
Isolation Tank
Isolation Tank Over
Flow
Water Supply From Filtration System
½” Ball Valve
Fine Mesh Filter Bag
Water Level In Separator
2” True Union Ball Valve
2” System Return Line
Isolation Tank Water Level
Larval Separator
Gravid Female
Figure 1: Diagram of gravid rockfish isolation and larval collection system located at
the Oregon Coast Aquarium.
30
70
Percent Survival
60
a
a
50
40
45.8
41.4
30
b
20
10
13.4
0
10
14
18
Temperature (°C)
Figure 2: Mean percent survival of S. caurinus larvae cultured at different
temperatures. Treatments with similar letters are not significantly different (TukeyKramer’s HSD; p>0.05). Larvae were cultured in 20 L aerated tanks for 16 days with
rotifer, T-Iso, and newly hatched Artemia (added day 8) live prey items stocked at 10,
100,000, and 2 per mL respectively. Data are means ± S.D. (n=5 replicates).
Experiment 1.
31
c
1200
1100
b
Length (µm)
1000
900
800
700
b
Temp °C
b
a
a
a
a
10
b
600
14
18
500
400
300
200
100
Eye Diameter
Notochord Depth
Anus Depth
Larval size measurements
Figure 3: Box plot of S. caurinus larval size measurements cultured at different
temperatures. Treatments with similar letters are not significantly different at each size
measurement (Tukey-Kramer’s HSD; p>0.05). Larvae were cultured in 20 L aerated
tanks for 16 d with rotifer, T-Iso, and newly hatched Artemia (added day 8) live prey
items stocked at 10, 100,000, and 2 per mL respectively. Experiment 1.
32
a
8.5
Length (mm)
8
b
b
7.5
7
7.0
6.6
6.6
6.5
6
5.5
5
10
14
18
Temperature °C
Figure 4: Box plot of S. caurinus larval standard length cultured at different
temperatures. Treatments with similar letters are not significantly different (TukeyKramer’s HSD; p>0.05). Larvae were cultured in 20 L aerated tanks for 16 d with
rotifer, T-Iso, and newly hatched Artemia (added day 8) live prey items stocked at 10,
100,000, and 2 per mL respectively. Experiment 1.
33
b
Percent Survival
60
b
50
40
30
a
20
10
a
a
5.7
6.3
NannoGw
T-Iso
39.8
43.0
2.5
0
Nanno
T-IsoGw
T-IsoGwArt
Enrichment/Green-water Conditions
Figure 5: Mean percent survival of S. malinger larvae cultured for 20 days under
different enrichment and green-water conditions. Nanno: both rotifers and Artemia
cultured cultured and enriched with with Nanno alone and no green-water conditions;
NannoGw: both rotifers and Artemia cultured and enriched with Nanno and Nanno
added for green-water conditions; T-Iso: both rotifers and Artemia cultured and enrich
with T-Iso and no green-water conditions; T-IsoGw: both rotifers and Artemia cultured
and enriched with T-iso and T-Iso added for green-water conditions; T-IsoGwArt:
rotifers cultured and enrich with T-Iso and T-Iso added for green-water but newly
hatched Artemia nauplii were not enriched before feeding to larvae. Treatments with
similar letters were not significantly different (Arcsign transformation; Tukey-Kramer’s
HSD; p>0.05). Larvae were cultured in 20 L aerated tanks at 14 ± 0.3ºC for a period of
20 days with microalgae, Nanno and T-Iso, ration maintained at equal volumes of
100,000 T-Iso cells per mL. Data are means ± S.D. (n=4 replicates). Experiment 2.
34
40
a
Percent Larval Survival
35
30
33.2
25
b
20
15
16.4
b
14.3
10
5
0
2.95kg
50cm
0.86kg
34cm
0.77kg
32cm
Female Sebastes caurinus size
Figure 6: Mean percent survival of S. caurinus larvae culture for 30 d from three
different sized females. Percent survival values with similar letters are not significantly
different (Arcsign transformation; Tukey-Kramer’s HSD; p>0.05). Larvae were
cultured in 100 L aerated tanks at 14 ± 0.3ºC with rotifers, T-Iso, and newly hatched
Artemia (day 8) live prey items stocked at 10, 100,000, and 2 per mL respectively.
Also note that larvae were cultured at different times and with 2 replicates per brood
confounding the results with time and culture conditions. Data are means ± S.D. (n=2
replicates). Experiment 3.
35
11
a
Length (mm)
10
a
9
Female size
2.95 Kg, 50 cm
a
8
b
a
0.86 Kg, 34 cm
7
a
6
b
5
1
b
9
0.77 Kg, 32 cm
b
15
b
20
30
Time (d)
Figure 7: S. caurinus larvae culture for 30 d from three different sized females. Mean
total length values with similar letters are not significantly different (Tukey-Kramer’s
HSD; p>0.05). Larvae were cultured in 100 L aerated tanks at 14 ± 0.3ºC with rotifers,
T-Iso, and newly hatched Artemia (day 8) live prey items stocked at 10, 100,000, and 2
per mL respectively. Also note that larvae were cultured at different times and with 2
replicates per brood confounding the results with time and culture conditions. Data are
means ± S.D. (n=2 replicates).
36
Chapter 2 Tables
S. caurinus
female size
Total
Length,
TL (mm)
Notochord
Depth, ND
(µm)
Eye
Diameter, ED
(µm)
Oil
Globule,
OG (µm)
Growth
Rate
(mm/d)
2.95 kg,
50cm
5.8 ± 0.15
385.3 ± 10.3
375.6 ± 16.3
194.5 ±
14.5
0.129
0.86 kg,
34 cm
5.2 ± 0.10
356.6 ± 16.4
326.5 ± 10.8
142.1 ±
12.4
0.129
0.77 kg,
32 cm
5.2 ± 0.12
342.7 ± 15.1
316.1 ± 13.1
92.1 ± 9.8
0.132
Table 1: Mean initial size measurements ± standard deviations and growth rates of S.
caurinus larvae from three different sized females. Larvae were cultured for a period of
30 d in 100 L aerated tanks at 14 ± 0.3ºC with rotifers, T-Iso, and newly hatched
Artemia (day 8) live prey items stocked at 10, 100,000, and 2 per mL respectively.
Data are means ± S.D. (n=2 replicates).
37
General Conclusions
Live food can survive in a wide range of temperatures and salinities (Oie and
Olsen, 1993; Fielder et al., 2000; Stottrup and McEvoy, 2003) but rapid changes in
salinity or temperature can affect their oxygen consumption rates and swimming
behavior (Epp and Winston, 1978; Snell et al., 1987; Oie and Olsen, 1993, Fielder et
al., 2000).
This study shows that Artemia, L, and SS-rotifers were affected by
temperature shock and transfer to lower temperatures. Rotifers settled out of the water
column and possibly adhered to surfaces (Oie and Olsen, 1993; Lubzens et al., 1989)
when they were subjected to a temperature decrease ranging from 2 to10ºC from a 20ºC
culture temperature. Artemia suffered initial transfer and temperature shock but
recovered more rapidly than rotifers. Since Artemia are positively phototactic (Stottrup
and McEvoy, 2003), they migrated towards the surface during the experimental period.
Artemia recovery improved with time and temperature in the first 24 h after transfer
and temperature shock. After 24 h, Artemia had recovered in all treatments, with 98%
of initially added Artemia present in the water column.
It is important to attempt to obtain similar culture temperatures for prey and
larvae, especially SS-rotifers. When this is not possible, temperature transfer effects on
prey swimming activity may be overcome with gentle turbulence. An alternate strategy
could be to acclimate prey to larval culture temperatures before they are added as prey
(Assavaaree et al., 2001).
This study suggests 14 ºC as the optimal temperature for growth and survival of
S. caurinus larvae. Rockfish larvae have been cultured at different temperatures: StahlJohnson (1984) at 12-15ºC, Boehlert (1981) at 13-16ºC, Watson and Robertson (2004)
at 11ºC, and Berkeley et al. (2004) at 10ºC. In the Eastern Pacific, off the Oregon
Coast, ambient water temperature ranges from 9-15ºC seasonally. Temperatures
recommended for larval rockfish culture should not exceed 18ºC.
Microalgae is widely recognized as important for green-water conditions and
for enrichment of prey species (Reitan et al., 1993, 1997; Su et al., 1997; Faulk et al.,
2005).
Results from this study indicated that T-Iso was better than Nanno as a green-
38
water and live prey enrichment for S. malinger larval culture. Sebastes larval culture in
static tanks is not recommended without T-Iso green-water conditions.
The Oregon Coast Aquarium (OCA) currently houses hundreds of mature
reproducing rockfish, representing seven different species, including yelloweye, canary,
and bocaccio rockfish three of the seven species listed as over-fished. These rockfish
produce viable larvae at OCA and provide an opportunity for researchers to work with
larval rockfish. Larval collection methods from female isolation systems are being
improved to eventually send larval rockfish to other research facilities for culture.
Efforts to obtain larvae from wild hook-and-line caught rockfish from the live
commercial fishery of Port Orford, Oregon, failed due to high larval mortality upon
natural release. Five of six gravid, china rockfish, S. nebulosus, released only dead
larvae, perhaps due to stress. Stahl-Johnson (1984) also reported high mortality of
naturally released larvae from wild caught S. caurinus and S. auriculatus, as well as
female death from forced larval extrusion.
The results of this project will benefit culture of rockfish species for
enhancement of wild populations. There are 65 rockfish species on the Pacific Coast of
North America (Love et al., 2002), however, stock assessments from 1999 to 2000
indicated that biomass of at least seven of the major commercial rockfish species
(bocaccio, canary, cowcod, drakblotched, pacific ocean perch, widow, and yelloweye
rockfish) are at or below 25% of levels estimated in the 1970’s (Love et al., 2002).
Federal regulations designed to prevent targeted harvest and incidental harvests (bycatch) of these species have resulted in significant cutbacks for commercial and
recreational groundfish harvests of other, non-rockfish species. Rockfish landings have
decreased from 31,656 metric tons in 1994 to 3,668 metric tons in 2004 (PacFIN, 1994,
2004)
Despite an interest in rockfish by the commercial fishery and a need for
rebuilding depleted stocks, very little culture research has been carried out. Further
investigation of optimal culture temperatures, green-water techniques and diets will
improve culture success.
39
Bibliography
Aragão, C., Conceicã, L.E., Dinis, M.T., Fuhn, H.J., 2004. Amino acid pools of rotifers
and Artemia under different conditions: nutritional applications for fish larvae.
Aquaculture 234, 429-445.
Assavaaree, M., Hagiwara, A., Ide, K., Maruyama, K., Lubzens, E., 2001. Low
temperature preservation (at 4C) of marine rotifer Brachionus. Aquaculture Research
32: 29-39.
Bell, M.V. and Dick, J.R. 1991. Molecular species composition of the major
diacylglycerophospholipids from muscle, liver, retina, and brain of cod (Gadus
morhua). Lipids 26:565-573.
Ben-Amotz, A., Fishler, R. and Schneller, A. 1987. Chemical composition of dietary
species of marine unicellular algae and rotifers with emphasis on fatty acids. Mar. Biol.
95: 31-36.
Berkeley, S.A, M. Hixon, R. Larson, and M. Love. 2004a. Fisheries sustainability via
population of age structure and spatial distribution of fish populations. Fisheries 29:8
pp23-32.
Berkeley, S.A., Chapman, C., Sogard, S.M., 2004b. Maternal age as a determinant of
larval growth and survival in a marine fish, Sebastes melanops. Ecology 85(5), 12581264.
Boehlert, G.W. 1981. The effects of photoperiod and temperature on laboratory growth
of juvenile Sebastes diploproa and a comparison with growth in the field. Fishery
Bulletin. 79: 789-794.
Boehlert, G.W., W.H. Barss, and P.B. Lamberson. 1982. Fecundity of the widow
rockfish, Sebastes entomelas, off the coast of Oregon. Fish. Bull. 80: 881-884.
Boehlert, G.W. and M.W. Yoklavich. 1984. Reproduction, embryonic energetics, and
the maternal-fetal relationship in the viviparous genus Sebastes (Pisces: Scorpaenidae).
Biol. Bull. 167:354-370.
Britt, L.L., E.R Loew, and W.N. McFarland. 2001. Visual Pigments in the Early Life
Stages of Pacific Northwest Marine Fishes. The Journal of Experimental Biology 204:
2581-2587.
Cañavate, J.P, Fernández-Díaz, C., 1999. Influence of co-feeding larvae with live and
inert diets on weaning the sole Solea senegalensis onto commercial dry feeds.
Aquaculture 174: 255-263.
40
Copeman, L.A., C.C Parrrish, J.A. Brown, and M. Harel. 2002. Effects of
docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth,
survival, lipid composition and pigmentation of yellowtail flounder (Limanda
ferruginea): a live food enrichment experiment. Aquaculture 210:285-304.
Delacy, A.C., Cr Hitz, and R.L. Dryfoos. 1964. Maturation, gestation, and birth of
rockfish (Sebastodes) from Washington and adjacent waters. Washington Dept. Fish.
Res. Paper 2:51-67.
Epp, R.W., Winston, P.W., 1978. The effects of salinity and pH on the activity and
oxygen consumption of Brachionus plicitilis (Rotatoria). Comp. Biochem. Physiol.
59A, 9-12.
Estevez, A., McEvoy, L.A., Bell, J.G., Sargent, J.R. 1999. Growth, survival, lipid
composition and pigmentation of turbot (Scophthalmus maximus) larvae fed live prey
enriched in arachidonic and eicosapentaenoic acids. Aquaculture 180, 321-343.
Faulk, C.K, G.J. Holt, D.A. Davis. 2005. Evaluation of fatty acid enrichment of live
food for yellowtail snapper Ocyurus chrysurus Larvae. Journal of the World
Aquaculture Society 36: 271-281.
Fielder, D.S., Purser, G., Battaglene S., 2000. Effect of rapid changes in temperature
and salinity on availability of the rotifers Brachionus rotundiformis and Brachionus
plicitilis. Aquaculture 189, 85-99.
Fuiman, L.A and R.G. Werner. 2002. Fishery Science. The unique contributions of
early life stages. Blackwell Publishing Co. Malden, MA. Pp 70-71.
Gatesoupe, F., Luquet, P., 1981. Practical diet for mass culture of the rotifer
Brachionus plicitilis: application to larval rearing of sea bass Dicentrarchus labrax.
Aquaculture 27, 149-163.
Gilbert, T.H. 2006. Prey Swimming Behavior and Culture Techniques for Copper, S.
caurinus, and Quillback Rockfish, S. malinger. Oregon State University-Hatfield
Marine Science Center. Newport, OR. Masters Thesis. pp 1-40.
Horrocks, L.A and Yeo, Y.K. 1999. Health benefits of docosahexaenoic acid (DHA).
Pharmacological Research 40:211-225.
HSWRI, 2006. Hubbs-Sea World Research Institute.
http://www.hswri.org/research/researchArea.cfm?areID=5
Jones, D.A., Kamarudin, M.S., Le Vay, L., 1993. The potential for replacement of live
feeds in larval culture. Journal World Aquaculture Society 24, 199-210.
41
Kendal, A.W. and W.H. Lenarz. 1987. Status of early life history studies of northeast
Pacific rockfishes. In Proceedings of the International Rockfish Symposium, 99-128.
Alaska Sea Grant Rep. 87-92. Fairbanks, Alaska.
Kolkovski, S., Koven, W., Tandler, A., 1997. The mode of action of Artemia in
enhancing utilization of microdiet by gilheaded seabream Sparus aurata larvae.
Aquaculture 155, 193-205.
Korunuma, K., Kukusho, K., 1987. Rearing of Marine Fish Larvae in Japan. IDRC,
Ottawa, p 109.
Koven, W.M., Tandler, A., Kissil, G.W., Sklan, D., Friezlander, O., Harel, M. 1990.
The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and
swimbladder development in Sparus aurata larvae. Aquaculture 91, 131-141.
Kreeger, K.E., Kreeger, D.A., Langdon, C.J., and Lowry, R.R, 1991. The nutritional
value of Artemia and Tigropus californicus (Baker) for two pacific mysid species,
Metamysidopsis elongate (Holems) and Mysidopsis intii (Holmquist). J. Exp. Marine
Biol. Ecology 148, 147-158.
Kusakari, M. 1991. Mariculture of kurosoi, Sebastes schlegeli. Environmental
Biology of Fishes 30: 245-251.
Langdon, C. 2003. Microparticle types for delivering nutrients to marine fish larvae.
Aquaculture 227: 259-275.
Lavens, P., Sorgloos, P., 1996. Manual on the production and use of live food for
aquaculture. FAO Fisheries technical paper No. 361 Rome, FAO.
Lazo, J.P., Dinis, M.T., Holt, G.J., Faulk, C., Arnold, C.R., 2000. Co-feeding
microparticulate diets with algae: towards eliminating the need of zooplankton at first
feeding in larval red drum (Sciaenops ocellatus). Aquaculture 188, 339-351.
Liao, I.C., Su, H.M., Chang, E.Y., 2001. Techniques in finfish larviculture in Taiwan.
Aquaculture 200, 1-31.
Love, M.S., M. Yoklavich, and L. Thorsteinson. 2002. The Rockfishes of the
Northeast Pacific. University of California Press. Berkeley, CA. pp. 31-41.
Lubzens, E., Marko, A., Tietz, A. 1985. De novo synthesis of fatty acids in the rotifer
Brachionus plicatilis. Aquaculture 47, 27-37.
Lubzens. E., 1987. Raising rotifers for use in aquaculture. Hydrobiologia 147, 245255.
42
Lubzens, E., Tandler, A., Minkoff, G., 1989. Rotifers as food in aquaculture.
Hydrobiologia 186/187, 387-400.
Lubzens, E., Rankevich, D., Kolodny, G., Gibson, O., Cohen, A., and Khayat, M.,
1995. Physiological adaptations in the survival of rotifers (Brachionus plicitilis, O. F.
Müller) at low temperatures. Hydrobiologia 313/314, 175-183.
MacGregor, J.S. 1970. Fecundity, multiple spawning, and description of the gonads in
Sebastodes. U.S. Fish Wildl. Serv. Spec. Sci. Rep. Fish. 0.596. Washington D.C.
McEvoy, L.A., Estevez, A., Bell, J.G., Shields, R.J., Gara, B., Sargent, J.R. 1998.
Influence of dietary levels of eicosapentaenoic and arachidonic acids on the
pigmentation success of turbot (Scophthalmus maximus L.) and halibut (Hippoglossus
hippoglossus L.). Bull. Aquacult. Assoc. Can. 98-4, 17-20.
Methot, Richard, F. Wallace, and K. Piner. 2003. Status of the yelloweye rockfish off
the West Coast in 2002. Status of the Pacific Coast groundfish fishery through 2003
Stock assessment and fishery evaluation. Portland, OR. pp1-73.
Moreno, G. 1993. Description of early larvae of four northern California species of
rockfishes (Scorpaenidae: Sebastes) from rearing studies. U.S. Dep. Commer., NOAA
Tech. Rep. NMFS 116, 18.
Mourente, G., Rodriguez, A., Tocher, D.R. Sargent, J.R. 1993. Effects of dietary
docosahexaenoic acid (DHA 22:6n-3) on lipid and fatty acid compositions and growth
in gilthead sea bream (Sparus aurata L.) larvae during first feeding. Aquaculture 112,
79-98.
Moser, H.G. and J.L. Butler. 1981. Description of reared larvae and juveniles of the
calico rockfish (Sebastes dallii). Calif. Coop. Oceanic Fish. Invest. Rep. 22:88-95.
Moser, H.G. and J.L. Butler. 1987. Descriptions of reared larvae of six species of
Sebastes. In Widow rockfish, ed. W.H. Lenarz and D.R. Gunderson, NOAA. Tech.
Rep. NMFS 48. Seattle. pp19-29.
Oie G. and Y Olsen. 1993. Influence of rapid changes in salinity and temperature on
the mobility of the rotifer Brachionus plicitilis. Hydrobiologia 255/256: 81-86.
PacFin, 1994, 2004. Pacific Coast Fisheries Information Network.
www.psmfc.org/pacfin/. Pacific Fishery Management Council Groundfish
Management Team Reports. Pacific States Marine Fisheries Commission. Portland,
Oregon. Pp 1-18
Phillips, J.B. 1964. Life history studies on ten species of rockfish (genus: Sebastodes).
Calif. Dept. Fish and Game, Fish Bull. 126. Sacramento.
43
Ramsey, F.L, Schafer, D.W., 2002. The Statistical Sleuth. 2nd edition pp 466-471, 485.
Reitan, K.I., J.R. Rainuzzo, G. Oie, and Y. Olsen. 1993. Nutritional effects of algal
addition in first feeding of turbot (Scophthalmus maximus L.) larvae. Aquaculture 118:
257-275.
Reitan, K.I., Rainuzzo, J.R., Oie, G., Olsen, Y. 1997. A review of the nutritional
effects of algae in marine fish larvae. Aquaculture 155, 207-221.
Rodriguez, C., Perez, J.A., Diaz, M., Izquierdo, M.S., Fernandez-Palacios, H., Lorenzo,
A. 1997. Influence of EPA/DHA ratio in rotifers on gilthead sea bream (Sparus
aurata) larval development. Aquaculture 150, 77-89.
Sargent, J., McEvoy, L., Estevez, A., Bell, J.G., Bell, M., Henderson, J., Tocher, D.R.
1999. Lipid nutrition of marine fish during early development: current status and future
directions. Aquaculture, 179, 217-229.
SAS Institute Inc., 1998. Statview reference. ISBN: 1-58025-162-5. Pp 82-93.
Seto, A., Kumasaka, K., Hosaka, M., Kojima, E., Kashiwakura, M., Kato, T. 1992.
Production of eicosapentaenoic acid by a marine microalgae and its commercial
utilization for aquaculture. In: Kyle, D.J., Raledge, C. (Eds), Industrial Applications of
Single Cell Oils. American Oil Chemists’ Society, Champaign, IL, pp 219-234.
Sheng-Lee, C. 1997. Marine finfish technology in the USA - status and future.
Hydrobiologia 358: 45-54.
Sheng-Lee, C. 2003. Biotechnological advances in finfish hatchery production: a
review. Aquaculture 227: 439-458.
Snell, T.W., Childress, M., Boyer, E.M., Hoff, F.H., 1987. Assessing the status of
rotifer mass cultures. J. World Aquaculture Soc. 18, 270-277.
Sorgeloos, P., Dhert, P., Candreva, P., 2001. Use of brine shrimp, Artemia spp., in
marine fish larviculture. Aquaculture 200, 147-159.
Stahl-Johnson, K.L. 1984. Rearing and development of larval Sebastes caurinus
(copper rockfish) and S. auriclatus (brown rockfish) from the northeast Pacific. M.S.
thesis, Univ. Washington, Seattle. Pp 1-217.
Stottrup, J.G. and L.A. McEvoy. 2003. Live Feeds in Marine Aquaculture. Blackwell
publishing. Oxford, UK. Pp. 11-111.
44
Su, H.M, M.S. Su, and I.C. Liao. 1997. Preliminary results of providing various
combinations of live foods to grouper (Epinephelus coioides) larvae. Hydrobiologia
358: 301-304.
Sukenik, A. and Wahnon, R. 1991. Biochemical quality of marine unicellular algae
with special emphasis on lipid composition: I. galbana. Aquaculture 97: 61-72.
Sukenik, A., Zmora, O., Carmeli, Y. 1993. Biochemical quality of marine unicellular
algae with special emphasis on lipid composistion: II. Nannochloropsis sp. Journal
Aquaculture 117: 313-326
Watanabe, T. 1979. Nutritional quality of living feeds used in seed production of fish.
Proc. 7th Japan-Soviet Joint Symp. Aquaculture, Sept. 1978, Tokyo, pp. 49-66.
Watanabe, T., Kitajima, C., Fujita, S. 1983. Nutritional values of live food organisms
used in Japan for mass propagation of fish: a review. Aquaculture 34: 115-143.
Watanabe, T. 1993. Importance of docosahexaenoic acid in marine larval fish. J.
World Aquaculture Society 24: 152-161.
Watanabe, T. and Kiron, V. 1994. Prospects in larval fish dietetics. Aquaculture 124,
223-251.
Watson, W., Robertson, L.L., 2004. Development of Kelp rockfish, Sebastes
atrovirens (Jordan and Gilbert 1880), and Brown Rockfish, S. auriculatus (Girard
1854), from Birth to Pelagic Juvenile stage, with Notes on Early Development of
Black-and-yellow Rockfish, S. chrysomelas (Jordan and Gilbert 1880), Reared in the
laboratory (Pisces: Sebastidae). NOAA Professional Paper NMFS 3. U.S. Department
of Commerce. Seattle, WA. Pp 1-30.
Whyte, J.N.C., Nagata, W.D. 1990. Carbohydrate and fatty acid composition of the
rotifer, Brachionus plicatilis, fed monospecific diets of yeast or phytoplankton.
Aquaculture 89: 263-272.
Wold, L. 1991. A practical approach to the decription and identification of Sebastes
larvae. MS Thesis: California State University, Hayward. Pp 1-88.
Appendix
Survival Percentage %
45
90
80
70
60
50
40
30
20
10
0
82.0
80.7
69.3
26.7
8.67
0
ir
lA
rt o
C
on
50
o
ro
nt
C
l
A
50
ir
10
ir
A
10
Prey Density and Aeration Treatments
Figure 1: Percent survival of S. malinger larvae cultured with different rotifer densities
(10 and 50 rotifers per mL) and with or without aeration. Larvae were cultured with 10
or 50 rotifers per mL with or without aeration and 100,000 T-Iso cells per mL (starve
controls) in 20 L duplicated tanks for 9 days at 14ºC.
46
90%
Percentage of Larvae
80%
70%
60%
50%
Feeding
40%
Mortality
30%
20%
10%
0%
1
2
3
4
5
6
Time (d)
Figure 2: Percent mortality and percent of S. caurinus larvae initiating feeding on days
1-6 post hatch. Triplicate 10 L tanks with 30 larvae each were fed rotifers each day for
24 hours at 14ºC before sampling for larval mortality and feeding percentages.
Number of Artemia
(In 100,000)
47
800
700
600
500
400
300
200
100
0
782
534
344
299
Hatched
Unhatched
202
75
26
1
73
5
10
15
Artemia cyst wet weight (g)
Figure 3: Hatched and unhatched Artemia from different wet weights of cysts.
Triplicate 1 L conical bottles were used for each treatment of cyst wet weight (1, 5, 10,
and 15g) to determine optimal hatching rate and Artemia hatching totals at 25ºC.
48