EFFECTS OF SALINITY AND TEMPERATURE ON THE GROWTH, SURVIVAL, WHOLE
BODY OSMOLALITY, STRESS RESISTENCE, AND mRNA EXPRESSION OF NA+/K+ ATPASE IN RED PORGY LARVAE, Pagrus pagrus
Andrew D. Ostrowski
A Thesis Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Master of Science
Center for Marine Science
University of North Carolina Wilmington
2009
Approved by
Advisory Committee
Dr. Thomas Shafer
Dr. Frederick Scharf
Dr. John Selden Burke
Dr. Wade O. Watanabe
Chair
Accepted by
DN: cn=Robert D. Roer, o=UNCW,
ou=Dean of the Graduate School &
Research, email=roer@uncw.edu,
c=US
Date: 2010.01.21 14:53:35 -05'00'
_____________________
Dean, Graduate School
This thesis has been prepared in the style and format
consistent with the journal
Aquaculture
ii
TABLE OF CONTENTS
ABSTRACT ....................................................................................................................... vi
ACKNOWLEDGEMENTS ............................................................................................. viii
DEDICATION ................................................................................................................... ix
LIST OF TABLES .............................................................................................................. x
LIST OF FIGURES ............................................................................................................ xi
Introduction
1.1 Red Porgy ...................................................................................................................... 1
1.2 Red Porgy Aquaculture ................................................................................................. 2
1.3 Larval Development
1.3.1 Yolk Sac larvae .............................................................................................. 3
1.3.2 Preflexion larvae ........................................................................................... 4
1.4 Effects of temperature and salinity on marine finfish larvae
1.4.1 Temperature .................................................................................................. 5
1.4.2 Salinity........................................................................................................... 6
1.4.3 Temperature and Salinity Interactions.......................................................... 8
1.5 Objectives ...................................................................................................................... 9
1.5.1 Null hypothesis ......................................................................................................... 10
Methods
2.1 Experimental Animals ................................................................................................. 11
2.2 Experimental System ................................................................................................... 11
2.3 Experimental Design ................................................................................................... 12
iii
2.4 Experimental Conditions ............................................................................................. 13
2.5 Experimental Procedures
2.5.1 Growth and Survival .................................................................................... 13
2.5.2 Whole-body Osmolality................................................................................ 14
2.5.3 Survival Activity Index ................................................................................. 14
2.5.4 mRNA expression of Na+/K+ ATPase......................................................... 15
2.5.5 RNA extraction and Reverse Transcription.................................................. 15
2.5.6 Sequencing of Na+/K+ ATPase................................................................... 16
2.5.7 Quantitative PCR ......................................................................................... 16
2.6 Analytical methods ...................................................................................................... 17
Results
3.1 Growth
3.1.1 Notochord Length......................................................................................... 18
3.1.2 Wet Weight ................................................................................................... 21
3.1.3 Dry Weight ................................................................................................... 24
3.2 Survival........................................................................................................................ 27
3.3 Whole-body osmolality ................................................................................................ 30
3.4 Survival activity index ................................................................................................. 33
3.5 mRNA expression of Na+/K+ ATPase ........................................................................ 35
Discussion
4.1Growth.......................................................................................................................... 39
4.2 Survival........................................................................................................................ 43
iv
4.3 Whole-body osmolality ................................................................................................ 45
4.4 Survival activity index ................................................................................................. 46
4.5 mRNA expression of Na+/K+ ATPase ........................................................................ 47
Conclusions ...................................................................................................................... 49
LITERATURE CITED ..................................................................................................... 51
v
ABSTRACT
Atlantic Red porgy, Pagrus pagrus, is an important demersal finfish species in the
snapper-grouper complex off the southeastern United States. Stocks have declined severely due
to overfishing even with management measures in place during the past decade. In the
Mediterranean, and more recently in the U.S., red porgy has been found to be a viable candidate
for aquaculture with its high market value and its ability to spawn freely in captivity. Optimal
rearing conditions for larviculture are currently being developed at the UNCW. This paper
examines the combined effects of temperature and salinity on eggs, yolk-sac larvae, and early
feeding-stage larvae of red porgy.
To determine the optimal temperature and salinity conditions for culture from hatching to
day 16 post-hatching (d16ph), embryos were stocked into black 15-L tanks at 60/L under four
temperatures (17, 19, 21, and 23ºC) and two salinities (24 and 34 g/L) in a 4 x 2 factorial design.
Light intensity was 500 lx, aeration was 90 mL/min, and photoperiod was 16 L:8 D. Significant
(P < 0.05) effects of temperature and salinity on growth (notochord length, wet and dry weight),
survival, whole-body osmolality, and mRNA expression of Na+/K+ ATPase were observed with
minimal interactive effects. Under both salinities, growth increased with increasing temperature.
On d16ph, wet weight (mg) at 21 and 23ºC (2.03 and 2.91) was significantly (P < 0.05) higher
than at 17 and 19ºC (0.20 and 0.69). Under all temperatures, salinity had no effect on growth but
had a significant (P < 0.05) effect on survival to d16ph, with greater survival at 24 g/L (18.4%)
than at 34 g/L (6.77%). Salinity significantly (P < 0.05) affected whole body osmolality
(mOsm/kg) on d2ph (342 in 24 g/L vs. 522 in 34 g/L) and on d17ph (412 vs. 481), but not on d6
or 11ph. Except for d6ph, when whole body osmolality at 23ºC (401) was significantly lower
than at 21ºC (683), no clear temperature related effects were observed throughout the study. On
vi
d16ph, when all larvae were challenged with a sublethal salinity increase to 40 g/L, there was a
significant (P < 0.01) interaction between temperature and salinity on survival activity index
(SAI) at 48h post-transfer. SAI decreased with increasing temperatures in 24 g/L treatments, but
increased with increasing temperatures in 34 g/L treatments. In a separate sublethal salinity (44
g/L) challenge, larvae from 34 g/L treatments did not show an increase in mRNA expression of
Na+/K+ ATPase after 24 or 48 h post transfer. However, fish in the 24 g/L treatments showed a
significant (P < 0.05) increase in mRNA expression of Na+/K+ ATPase after 24 h followed by a
decrease after 48 h. Within the ranges tested, a temperature of 23 ºC and a salinity of 24 g/L are
optimal for culture of red porgy embryos, yolk-sac, and first-feeding stage larvae to d16ph.
vii
ACKNOWLEDGEMENTS
I would like to first thank my thesis advisor, Dr. Wade Watanabe for his expertise and
knowledge in aquaculture and for generously taking time to teach me and prepare me for future
endeavors, for this, I feel truly prepared for whatever comes my way. Thank you to my
committee members for their time, commitment, and suggestions on my thesis: Dr. Thomas
Shafer for his assistance with the mRNA expression of Na+,K+-ATPase assays, Dr. Frederick
Scharf, and Dr. John Selden Burke. Although he was not on my committee, I would also like to
thank Dr. James Morris Jr. for his help on the data analysis, keeping a continuous supply of red
porgy eggs, and encouragement along the way.
I cannot begin to thank Mr. Frank Montgomery for all his time he has spent on this
project. I am truly fortunate to have had the opportunity to have worked with him for three
spawning seasons. He has had a tremendous impact on the outcome of this project as well as my
life outside the four walls of the cramped larvilab.
I would also like to thank Troy Rezek for his thorough training in larval experiments and
for giving up his weekends and evenings to make sure fish were collected for everything. I
would also like to thank my fellow staff members of the Aquaculture Program for their
contributions and giving up their time in sampling, for that, I cannot purchase enough donut
holes to repay you. These people include Pat Carroll, Chris Bentley, Amanda Myers, Dustin
Allen, Walker Wright-Moore, Tyler Gibson, and Chelsea McDougall. I would also like to thank
my fellow graduate students at UNCW that I have had the opportunity to meet along the way.
This research was funded by grants from the USDA, CSREES, and North Carolina
Fisheries Resource Grant Program.
viii
DEDICATION
To Kendall, who has encouraged me every step of the way and whose love and support
has gotten me through everything. Words cannot express how much you mean to me.
ix
LIST OF TABLES
Table
Page
1. Growth in notochord length (mm) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23ºC, n = 8) and salinities (24 and 34 g/L, n = 16). In
Table 1a, both salinities were combined under each temperature. In Table 1b, all
temperatures were combined under each salinity. At each age, probability values are
shown (ANOVA). Means in the same row followed by the same letter are not
significantly different (Tukey’s, P < 0.05). NS= not significant (P > 0.05) ......... 20
2. Growth in wet weight (mg) (mean ± s.e) of larval red porgy at different temperatures (17,
19, 21, and 23ºC, n =8) and salinities (24 and 34 g/L, n =16). In Table 2a, both salinities
were combined under each temperature. In Table 2b, all temperatures were combined
under each salinity. At each age, probability values are shown (ANOVA). Means in the
same row followed by the same letter are not significantly different (Tukey’s, P < 0.05).
NS= not significant (P > 0.05). Asterisk (*) indicates interaction ....................... 23
3. Growth in dry weight (mg) (mean ± s.e) of larval red porgy at different temperatures (17,
19, 21, and 23 ºC, n =8) and salinities (24 and 34 g/L, n =16). In Table 3a, both salinities
were combined under each temperature. In Table 3b, all temperatures were combined
under each salinity. At each age, probability values are shown (ANOVA). Means in the
same row followed by the same letter are not significantly different (Tukey’s, P < 0.05).
NS= not significant (P > 0.05) .............................................................................. 26
4. Survival (%) (mean ± s.e) of larval red porgy at different temperatures (17, 19, 21, and 23
ºC, n =8) and salinities (24 and 34 g/L, n =16). In Table 4a, both salinities were
combined under each temperature. In Table 4b, all temperatures were combined under
each salinity. At each age, probability values are shown (ANOVA). Means in the same
row followed by the same letter are not significantly different (Tukey’s, P < 0.05). NS=
not significant (P > 0.05) ....................................................................................... 29
5. Whole body osmolality (mOsm/kg) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). In
Table 5a, both salinities were combined under each temperature. In Table 5b, all
temperatures were combined under each salinity. At each age, probability values are
shown (ANOVA). Means in the same row followed by the same letter are not
significantly different (Tukey’s, P < 0.05). NS= not significant (P > 0.05). A dagger (†)
indicates means that were significantly (t-test, P < 0.05) different ....................... 32
6. mRNA expression Na+/K+ ATPase (mean ± s.e) of d16ph larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). In
Table 6a, both salinities were combined under each temperature. In Table 6b, all
temperatures were combined under each salinity. At each time period, probability values
are shown (ANOVA). NS= not significant (P > 0.05). Dagger (†) indicates a significant
(P < 0.05) difference from the time 0 value .......................................................... 38
x
LIST OF FIGURES
Figure
Page
1. Growth in notochord length (mm) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16).
Salinities were combined under each temperature (Fig. 1a), while temperatures were
combined under each salinity (Fig. 1b). Asterisks indicate significant treatment
differences and probability levels (*0.05, **0.01, ***0.001, ANOVA)............... 19
2. Growth in wet weight (mg) (mean ± s.e) of larval red porgy at different temperatures (17,
19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were combined
under each temperature (Fig. 2a), while temperatures were combined under each salinity
(Fig. 2b). Asterisks indicate significant treatment differences and probability levels
(*0.05, **0.01, ***0.001, ANOVA) while i denotes a significant interaction ..... 22
3. Growth in dry weight (mg) (mean ± s.e) of larval red porgy at different temperatures (17,
19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were combined
under each temperature (Fig. 3a), while temperatures were combined under each salinity
(Fig. 3b). Asterisks indicate significant treatment differences and probability levels
(*0.05, **0.01, ***0.001, ANOVA) ..................................................................... 25
4. Survival (%) (mean ± s.e) of larval red porgy at different temperatures (17, 19, 21, and 23
ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were combined under each
temperature (Fig. 4a), while temperatures were combined under each salinity (Fig. 4b).
Asterisks indicate significant treatment differences and probability levels (*0.05, **0.01,
***0.001, ANOVA) .............................................................................................. 28
5. Whole body osmolality (mOsm/kg) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16).
Salinities were combined under each temperature (Fig. 5a), while temperatures were
combined under each salinity (Fig. 5b). Asterisks indicate significant treatment
differences and probability levels (*0.05, **0.01, ***0.001, ANOVA). Dagger (†)
indicates a significant (P < 0.05) difference from the d2ph value......................... 31
6. Survival Activity Index at 48h (mean ± s.e) of larval red porgy at different temperatures
(17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16) following a salinity
challenge to 40 g/L and under zero feeding conditions. Salinities were combined under
each temperature (Fig. 6a), while temperatures were combined under each salinity (Fig.
6b). A significant (P < 0.01, Two-way ANOVA) temperature x salinity interaction was
observed ................................................................................................................ .34
7.
mRNA expression of Na+/K+, ATPase (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16) after a
salinity challenge to 44 g/L and under zero feeding. Salinities were combined under each
temperature (Fig. 7a), while temperatures were combined under each salinity (Fig. 7b).
Bars within a cluster with different letters are significantly (P < 0.05, Tukey’s test)
xi
different. Dagger (†) indicates a significant (P < 0.05) difference from the time 0 value
............................................................................................................................... 37
xii
Introduction
1.1 Red Porgy
Red porgy (RP), Pagrus pagrus, is a demersal finfish that inhabits sandy and hard
bottoms over the eastern and western Atlantic Ocean continental shelves (Manooch and
Huntsman, 1977; Potts and Manooch, 2002) ranging in depth from 18-185 m (Manooch and
Hassler, 1978). RP, also referred to as silver or pink snapper, can reach an age of 18 years, a size
of 733 mm total length and 5.9 kg wet weight (Potts and Manooch, 2002), and become sexually
mature at three years, around 364 mm TL (Manooch, 1976). Juvenile RP are found in shallower
waters than their adult counterparts and over sandy substrates with seagrass and algae patches
(Labropoulou et al., 1999). Their diet consists of approximately 90% benthic organisms,
including crustaceans, mollusks, and echinoderms (Manooch, 1977). RP are classified as
protogynous hermaphrodites, starting life as females and changing to males around 400-450 mm
total length (Kentouri et al., 1995). Maturation of gonads is correlated with an increase in
photoperiod during late winter and early spring. Spawning occurs between January and April
(Manooch, 1976).
RP are sparids (family Spariade) and are grouped with the snapper-grouper complex of the
southeastern United States. Since 1991, RP populations have declined severely (SAFMC, 2006)
mainly due to overfishing (Vaughan and Prager, 2002). Average annual total recreational and
commercial landings from 1977 to 1991 were over 1 million pounds with a peak in 1982 of over 2
million pounds compared to 104,000 pounds in 2004, as managed by recreational and commercial
quotas (SAFMC, 2006). From 1973 to 1997, there was an estimated 97% decline in RP recruitment
to age one from three million to 94,000 fish, respectively (Vaughan and Prager, 2002), as well as a
large difference in recruitment to age 2 from 1973 (1.34 million) to 2006 (547,700 fish) (SAFMC,
2006). There has also been an 89% decline in spawning stock biomass from 1979 to 1997, from
3530 tons to 397 tons, respectively, a biomass that is below what could support maximum
sustainable yield (Vaughan and Prager, 2002). Management measures include 14 inch total length
size limit, a bag limit of 1 RP/person/day, seasonal closures between January and April, and a 50
pound commercial bag limit during the rest of the year (SAFMC, 2000). While these management
practices have reduced overfishing in the past decade (SAFMC, 2006), a further reduction in fishing
mortality would be necessary to rebuild U.S stocks (Vaughan and Prager, 2002).
1.2 Red Porgy Aquaculture
RP has been found to be a viable aquaculture candidate in the Mediterranean (Kentouri et
al., 1995; Kolios et al., 1997; Hernandez-Cruz et al., 1999; Conides and Glamuzina, 2001;
Mihelakakis et al., 2001; Machinandiarena et al., 2003), and Argentina (Aristizabal, 2005;
Aristizabal and Suarez, 2006; Aristizabal, 2007; Aristizabal et al., 2009), with high market value.
More recently, RP are being considered as a candidate for aquaculture in North America (Morris
et al., 2008). Much of the available literature on RP aquaculture describe larval development
(Kolios et al., 1997; Roo et al., 1999a; Conides and Glamuzina, 2001; Mihelakakis et al., 2001;
Machinandiarena et al., 2003; Mylonas et al., 2004; Aristizabal, 2005; Büke et al., 2005; Darias
et al., 2005; Darias et al., 2007; Suzer et al., 2007; Morris et al., 2008; Aristizabal et al., 2009),
effects of diet on skin color (Cejas et al., 2003; Chatzifotis et al., 2005; Kalinowski et al., 2005;
Kalinowski et al., 2007), or background tank color effects on stress (Rotllant et al., 2003; Van
der Salm et al., 2004; Van der Salm et al., 2006). While a significant amount of work has been
conducted in the Mediterranean on RP culture, much of the literature is anecdotal, experimental
data is scarce, and protocols for raising larvae and juveniles are not well defined. In Japan, the
2
red sea bream Pagrus major, a congener of RP, is the second most commonly cultured marine
fish (Foscarini, 1988) after yellow tail Seriola quinqueradiata with an average of 67 million
individuals being produced for aquaculture purposes (Fushimi, 2001) weighing a total of 81,000
tons (Kato et al., 2002). Due to suitability for intensive culture, high market demand for reef fish
species, and similar culture attributes to red sea bream, optimal rearing environments for
larviculture are being developed.
1.3 Larval Development
1.3.1 Yolk Sac larvae
Hatching of RP embryos begins about 50 hours after fertilization at 18ºC and produces
yolk sac larvae with notochord lengths ranging from 1.92 to 3.00 mm (Conides and Glamuzina,
2001; Machinandiarena et al., 2003) and yolk sac volumes ranging from 0.07-0.1 mm3 (Conides
and Glamuzina, 2001; Mihelakakis et al., 2001; Machinandiarena et al., 2003). During the yolk
sac phase, which lasts from day 0 post-hatching (d0ph) to d3ph (Machinandiarena et al., 2003),
pectoral fins begin to develop and are functional by d3ph (Conides and Glamuzina, 2001).
Larvae are buoyant and face downward in the water column (Machinandiarena et al., 2003).
Newly hatched yolk sac larvae display low heartbeats (80/minute) but quickly increase to
170/minute on d2 and 3ph (Conides and Glamuzina, 2001).
Eye development occurs between d2 to d3ph and is important in exogenous feeding. At
hatching, the eyes are unpigmented (Conides and Glamuzina, 2001), but develop nonfunctional
visible lenses and pigmentation at d2ph (Conides and Glamuzina, 2001; Machinandiarena et al.,
2003). By d3ph, lenses are well defined by a single epithelial cell layer and are fully pigmented,
making larval eyes functional (Roo et al., 1999b; Conides and Glamuzina, 2001).
3
Digestive tract development coincides with eye development. At hatching, the mouth is
unformed and, while the digestive tract is visible as a straight thin tube over the yolk sac, the
anus is closed (Conides and Glamuzina, 2001; Mihelakakis et al., 2001). During this time, the
larvae utilize the lipids and proteins within the yolk sac for energy and development. By d2ph
the digestive tract develops a small curvature in the posterior region (Roo et al., 1999a) and the
mouth is opened (Mihelakakis et al., 2001). By d3ph, when eyes are fully functionally, the
digestive system can be divided into several functional parts: an esophagus, a foregut, a midgut,
hindgut (Roo et al., 1999a), and an anus (Mihelakakis et al., 2001).
1.3.2 Preflexion larvae
This pelagic larval stage lasts from d4-17ph and has a notochord length of 3.00-4.5 mm
(Machinandiarena et al., 2003). Several features begin to form and become noticeable including:
a urinary vesicle, traces of a tail fin (d6ph), occipital spines (d10ph), preopercular spines, and the
olfactory organ on the snout of the larvae (Machinandiarena et al., 2003).
During the preflexion larval stage, the larvae switch completely from endogenous to
exogenous feeding and the yolk sac and oil globule disappear by d7ph (Conides and Glamuzina,
2001; Mihelakakis et al., 2001). By d4-5ph, the mouth and digestive tract are functional and
food particles appear for the first time in most fish (Roo et al., 1999a; Conides and Glamuzina,
2001). There is also a clear differentiation of the retina layers, pigmentation is completed, and
the eyes appear like those of adult fish. The increase in eye diameter is significantly related to
larval total length (Roo et al., 1999b; Conides and Glamuzina, 2001).
4
1.4 Effects of temperature and salinity on marine finfish larvae
1.4.1 Temperature
Temperature and salinity are dominant environmental factors affecting marine finfish
larvae (Blaxter, 1969; Brett, 1970). Temperature can have a significant impact on development,
growth, and survival of marine finfish embryos and early larval stages. For RP, water
temperatures as low as 14 and as high as 22ºC were found to support development (fertilization
to hatching), but temperatures ranging between 16 and 18ºC appeared optimal for development
(Saka et al., 2005). Development from fertilization to hatching had a positive relationship with
temperature. Hatching in RP was delayed (70-115 h) at low temperatures (14-15ºC), faster (4044 h) at moderate temperatures (20ºC), and accelerated (28-30 h) at high temperatures (24-25ºC)
(Radonic et al., 2005; Saka et al., 2005). At extreme temperatures, development was abnormal:
at 8ºC, cleavage did not occur, while at 24ºC, cell division was either absent, highly
asynchronous, or produced irregularly sized cells (Saka et al., 2005). Mortality was also
observed to be high (100%) outside the range of 14-22ºC (Saka et al., 2005). Higher
temperatures also accelerate embryo development and yolk sac absorption of summer flounder
Paralichthys dentatus through first feeding stages (Johns and Howell, 1980; Watanabe et al.,
1999). Similar findings were reported in yellowtail flounder Pleuronectes ferruginea (Howell,
1980) and bairdiella Bairdiella icistia (May, 1974).
In general, larval growth increases with increasing rearing temperatures. For example, in
black sea bass Centropristis striata, growth was most favorable at temperatures >18ºC, with
growth through first feeding increasing with increasing rearing temperatures within the range 1230ºC (Berlinsky et al., 2004). Similar results were observed for snapper Pagrus auratus larvae:
growth increased as temperature increased, with larvae reared at 24 ºC six times heavier than
5
larvae reared at 15ºC (Fielder et al., 2005). No studies to date have been completed on the
effects of temperature on RP larval development.
1.4.2 Salinity
Salinity also has clear effects on the structures of marine larvae (Holliday, 1988), and
these effects are dependent on species. For example, eggs of pomfret Pampus punctatissimus did
not develop when exposed to low salinities (<10 g/L), but were not affected at higher salinity
levels up to 40 g/L (Shi et al., 2008). Hatching rates, on the other hand, decreased substantially
at salinities < 25 and > 40 g/L, with the highest hatching rate and the lowest percentage of
abnormalities occurring at 29-32 g/L (Shi et al., 2008). In summer flounder, salinities ranging
from 26 to 36 g/L did not influence development or growth from yolk sac through first feeding
stages (Watanabe et al., 1998). After first feeding, lower salinities (25 g/L) stunted growth rates
(notochord length, wet and dry weights) compared to larvae reared at 34 g/L (Watanabe et al.,
1998). Reduced buoyancy could have been a factor in decreased growth at lower salinities
(Watanabe et al., 1998; Henne and Watanabe, 2003). Southern and summer flounder are
negatively buoyant at 25 g/L and neutrally or positively buoyant at 34 g/L (Henne and Watanabe,
2003). This suggested that in the lower salinity, energy was used for swimming and maintaining
position in the water column rather than for growth (Watanabe et al., 1998; Moustakas et al.,
2004). No studies to date have been completed on the effects of salinity on RP larval
development.
Due to its effects on swimming and osmoregulation, salinity can have a major impact on
growth and survival of marine finfish larvae. In addition to the energy used for swimming,
osmoregulation can consume a large percentage (20-50+ %) of energy resources in fishes (Boeuf
6
and Payan, 2001). In some species, such as seabream Sparus sarba (Tandler et al., 1995),
summer flounder Paralicthys dentatus (Specker et al., 1999), southern flounder Paralichthys
lethostigma (Smith et al., 1999), milkfish Chanos chanos (Swanson, 1996), and red tilapia
Oreochromis sp (Watanabe et al., 1989), better larval growth rates were observed between 8-20
g/L due to lower standard metabolic rates at near isoosmotic salinities (see review by Boeuf and
Payan, 2001). Snapper Pagrus auratus larvae showed decreased growth rates in higher salinities
(45 g/L) for the same reason (Fielder et al., 2005) since the isosmotic value for snapper is
approximately 12 g/L (Woo and Fung, 1981). Energy demand for osmoregulation increases in
hyperosmotic environments as fish attempt to maintain homeostasis (Fielder et al., 2005). Larval
flatfishes, including summer flounder, can tolerate wider salinity regimes throughout their larval
and juvenile life stages than the adults due in part to the undeveloped gills and thus the reduced
surface area for osmotic water loss (Schreiber, 2001).
Whole body osmolality (WBO) is a measure of the concentration of undissociated
molecular ions (sodium, chloride, potassium, glucose, and other ions) in the whole body fluids.
In marine fish larvae, WBO is maintained within a relatively small range in the face of a high
osmotic pressure gradient relative to the external environment and can be indicative of osmotic
stress. For example, in larval southern flounder, whole-body osmolality was lower in 25 g/L
(296 mOsm/kg) than in 34 g/L (344 mOsm/kg) (Moustakas et al., 2004), suggesting
osmoregulatory stress at 24 g/L and that these larvae are relatively stenohaline. Henne and
Watanabe (2003), observed lower whole-body osmolality in larval southern flounder at 24 g/L
(304 mOsmol/kg) than at 34 g/L (343 mOsmol/kg) as well as when compared to other marine
fish larvae in full strength seawater (320-400 mOsmol/kg), including Pacific Herring Clupea
7
pallasii (Alderdice et al., 1979), Cod Gadus morphua (Davenport et al., 1981), and Halibut
Hippoglossus hippoglossus (Riis-Vestergaard, 1982; Hahnenkamp et al., 1993).
Osmoregulation in larval fish is accomplished by Na+/K+ ion-transport pumps within
cells throughout their bodies until the gills and intestinal osmoregulatory systems are developed,
which is species dependent (Rombough, 2007). Na+/K+ ATPase (NKA) are at the center of
active secretory functions of chloride cells in juvenile fish by generating a chemical gradient for
which ions will move. Osmoregulatory ability can be evaluated by measuring the messenger
RNA (mRNA) expression of Na+/K+ ATPase enzyme, with a higher mRNA level likely
indicating the elaboration of the ion transfer pump, . However, little or no published data is
available on measuring mRNA levels of Na+/K+ ATPase in larval fish. In black sea bass, mRNA
levels of Na+/K+ ATPase increased 24 h after transfer from 35 g/L to 43 g/L in larvae fed 6-12%
(% Total Fatty Acid) dietary arachidonic acid (ARA), but not in those fed 0% ARA, suggesting
that ARA affected Na+/K+ ATPase gene expression and ultimately the osmotic stress response in
fish (Carrier, 2006).
1.4.3 Temperature and Salinity Interactions
Species-specific differences make it difficult to generalize temperature and salinity
interactive effects on marine finfish embryo and larval development and survival. For example,
the effects of temperature (15-33ºC) and salinity (5-45 g/L) on larval growth (total length, wet
and dry weight) of snapper Pagrus auratus were independent (Fielder et al., 2005). An
interaction between temperature and salinity on hatching rate was observed. Summer flounder
development was accelerated by higher temperatures and salinities, but higher temperatures and
lower salinities inhibited development and hatching (Watanabe et al., 1999).
8
At 24°C, hatch
rate was highest at 34 g/L, but greatly reduced at 28 and 22 g/L. However, at 16 and 20°C,
hatching rates were moderate to high under all salinities (Watanabe et al., 1999). Interactive
effects of temperatures and lower salinities on growth in yellowtail flounder (Howell, 1980) and
winter flounder Psuedopleuronectes americanus (Rogers, 1976) have also been reported. In
Atlantic halibut, larvae experienced high mortality due to deformities and injury when raised in
high and low temperatures and high salinities (Ottesen and Bolla, 1998).
Successful incubation of embryos is possible in marine finfish aquaculture occurs over a
wider salinity range than observed variation were spawning occurs naturally, however
temperature range suitable for successful development coincides with natural conditions. For
example, successful hatching of red sea bream Pagrus major embryos occurred over a broad
salinity range of 18-36 g/L, but over a narrow temperature range of only 15-22 ºC (Mihelakakis
and Yoshimatsu, 1998). This is similar to the greenback flounder in which hatching and
development occurred over broad salinity range (15-45 g/L), but over a narrow temperature
range (12-15 ºC) (Hart and Purser, 1995). In contrast, in the black sea bass, optimal
development and hatching occurred over a broad range of salinities (≥ 15 g/L) and temperatures
(15-27 ºC) (Berlinsky et al., 2004). No studies have been completed on the combined effects of
temperature and salinity on larval development in RP.
1.5 Objectives
The objectives of this study were to determine under controlled laboratory conditions, the
effects of temperature and salinity on growth, survival, osmoregulatory ability, and stress
resistance in RP from embryo to day 16 post hatch. Based on these results, we hope to
9
recommend an optimum temperature and salinity combination to be applied in commercial
larviculture of RP.
1.5.1 Null hypothesis
There will be no differences in growth, survival, whole-body osmolality, mRNA
expression of the Na+/K+ ATPase, and stress resistance in RP larvae raised in four temperatures
(17, 19, 21, and 23°C) and two salinities (24 and 34 g/L).
10
Methods
2.1 Experimental Animals
Eggs were collected from RP broodstock held at the Center for Coastal Fisheries and
Habitat Research, National Oceanic and Atmospheric Administration (Beaufort, NC, U.S.A).
The broodstock (n = 20, total length (mean ± SE) = 306 ± 4.7 mm, weight = 373 ± 2 g) were
collected by hook and line off the coast of North Carolina during March 2004 and maintained in
a recirculating 31-m3 round tank under conditions simulating seasonal offshore bottom
temperatures off North Carolina from 27-33 m (Parker and Dixon, 1998) and under ambient
photoperiod. RP broodstock spawn volitionally in captivity (Morris et al., 2008) and eggs were
collected by filtering surface water from the spawning tank through a 505 µm nitex mesh net.
Eggs were transferred to the University of North Carolina Wilmington Center for Marine
Science (UNCW-CMS) Aquaculture Facility on Wrightsville Beach, NC, U.S.A. in oxygen filled
plastic bags. Eggs were placed in a conical plexiglass tank (15 L volume) in 32 g/L seawater
where viable (buoyant) eggs were separated from nonviable eggs and removed for use in the
experiment.
2.2 Experimental System
RP larvae were reared in 15-L cylindrical, black polyethylene tanks held in four
temperature controlled 213-L water baths in a controlled-environmental laboratory. Air was
supplied to each tank through an air diffuser positioned at the bottom center of each tank and
maintained at 90 mL/minute (Rezek, personal communication). Light intensity at the surface of
each tank was 500 lx (492.26 ± 3.94) (Watanabe et al., 1998) supplied by two-40 watt full
11
spectrum fluorescent light bulbs suspended over each table. Photoperiod was controlled at
16L:8D starting at 0600h (Biswas et al., 2005), with extraneous light excluded by black curtains
surrounding the water bath. Light intensity and photoperiod were determined to be optimal for
other marine finfish.
2.3 Experimental Design
A 4 x 2 factorial design was used to compare the effects of four temperatures and two
salinity levels on the growth, survival, and whole body osmolality of larval RP from d1ph to
d16ph. Each water bath was adjusted to 17 (17.3 ± 0.10), 19 (18.9 ± 0.02), 21 (21.4 ± 0.09), or
23°C (22.9 ± 0.19), within the temperature ranges used by previous investigators (Kentouri et al.,
1995; Kolios et al., 1997; Roo et al., 1999b; Conides and Glamuzina, 2001; Mihelakakis et al.,
2001; Machinandiarena et al., 2003; Morris et al., 2008). For each temperature, salinities of 24
(24.3 ± 0.04) or 34 g/L (33.5 ± 0.03) were maintained for a total of eight treatments, with four
replicate tanks per treatment. Full strength seawater was pumped from the Atlantic Intracoastal
Waterway, Wrightsville Beach, North Carolina U.S.A., while brackish water (24 g/L) was
prepared by mixing full strength seawater with dechlorinated tap water using a carbon filter.
To begin the experiment, embryos were stocked into each tank at a density of 60/L (900
total eggs) at 19°C. Temperature was adjusted 2°C per day after hatching until treatment
temperatures were reached. To maintain eggs in suspension, airflow was maintained at 150
mL/min in the 34 g/L treatments and 350 mL/min in the 24 g/L treatments. After hatching (2448 h post fertilization), the airflow in all tanks was decreased to 90 mL/min for the remainder of
the experiment to avoid injury from excessive turbulence (Mangino and Watanabe, 2006).
12
2.4 Experimental Conditions
Water was exchanged daily (33%) in each tank by siphoning 5 L from each tank and then
replacing with water of appropriate salinity. At d1ph, a non-viable condensed microalgae
Nannochloropsis oculata paste (Reed Mariculture, Campbell, California U.S.A.) was added to
each tank at a concentration of 750,000 cells/mL as background. The microalgae was added to
provide rotifer nutrition. Beginning d2ph, larvae were fed s-type rotifers (5-20 rotifers/mL)
cultured in a continuous culture system (Bentley et al., 2008). Rotifers were enriched with
Algamac 2000 (Aquafauna, Bio-Marine Inc. Hawthorne, California U.S.A.) to increase
docosahexaenoic acid (DHA) at a concentration of 300 mg/ 106 rotifers at 16 and 6 h before
being fed to the larvae.
Temperature (± 0.1 °C), salinity (± 0.1 g/L), D.O. (7.21 ± 0.08 mg/L), pH (8.31 ± 0.01)
(YSI 560, Yellow Springs, OH), and light intensity (492.26 ± 3.94 lx) were measured twice daily
(0900 and 1600 h) from alternating replicate tanks per treatment. Ammonia (0.02 ± 0.005 mg/L)
was tested twice a week (HACH DR 850, Loveland, CO. USA) and airflow (±1 ml/min) to each
tank was tested daily using a Gilmont flow meter (Gilmont Instruments Inc, Barrington, Illinois
U.S.A.) and adjusted as necessary.
2.5 Experimental Procedures
2.5.1 Growth and Survival
To measure growth and survival, 10 larvae were randomly sampled from each replicate
tank on days 1, 4, 10, and 16 ph at approximately 0730 h. Prior to sampling, airflow was
increased to uniformly distribute the larvae in tanks. The number of fish were counted and
divided by water volume removed during sampling to find sample density. Survival was
13
assessed for each tank by dividing sampling density by initial densities. Using 0.5 mg/L of 2phenoxyethanol to anesthetize the larvae, notochord lengths (NL) were measured using an ocular
micrometer on a dissecting microscope (Meiji Techno Co., Ltd., Japan). Wet weight was
determined using an electrobalance (± 0.01 mg) (Sartorius, Goettingen, Germany) by placing 10
larvae on a preweighed microscope slide after they were blotted dry. The slides were then
placed into a laboratory oven at 60°C for 72 h and then reweighed to determine dry weights of
larvae.
2.5.2 Whole-body Osmolality
To measure osmoregulatory ability, whole-body osmolality (mOsm/kg) was measured on
d2-3, 5-6, 11-12, and 17-18ph larvae using a vapor pressure osmometer (Wescor Vapor Pressure
Osmometer 5520, Logan, Utah, U.S.A.). Larvae (n = 3-5) were removed from each tank, gently
rinsed with deionized water on a nitex screen, and transferred to a sample disc before being
placed into the osmometer chamber. Measurements were taken after a 25 min equilibrium
period (Henne and Watanabe, 2003; Mangino and Watanabe, 2006).
2.5.3 Survival Activity Index
At the end of the experiment, a stress test was performed to measure the larvae’s ability
to withstand an abrupt increase in salinity to 40 g/L under conditions of no feeding. A survival
activity index (SAI) to test larvae quality was calculated using the following equation:
k
SAI = 1 Σ (N – hi) x i
N i:1
14
where N is the total number of initial larvae, hi is the cumulative mortality by the ith hour, and k
is the number of hours that have elapsed until all larvae have died or the experiment ended
(Matsuo et al., 2006). Using volumetric methods, survival was monitored every 12 h for 48h,
when cannibalism was observed in some treatment tanks.
2.5.4 mRNA expression of Na+,K+, ATPase
In a separate, but similar experiment, fish on d16ph were subjected to a sub-lethal
hyperosmotic salinity challenge of 44 g/L to measure the mRNA expression of Na+/K+ ATPase.
Fish were sampled prior to the increase in salinity (time 0 h) and every 12 h thereafter for a total
of 48 h. Fish were pooled at each sample and stored in RNAlater (Ambion, Woodward Texas,
USA) at -20ºC to preserve the integrity of the RNA within the larval tissues.
2.5.5 RNA extraction and Reverse Transcription
Total RNA were extracted using a RNeasy Mini Kit (Qiagen, Valencia, California, USA)
modified by using Trizol (Invitrogen, Carlsbad, California, USA). RNA was bound to a column
with 70% ethanol and eluted with 30 ul of nuclease-free water with maximum RNA yield
obtained by a second elution with the 30 ul eluant. An Agilent 2100 Bioanalyzer (Agilent, Palo
Alto, California, USA) was used to assess the quality and the quantity of each RNA extract.
Superscript III reverse transcriptase (Invitrogen) was used for cDNA synthesis, employing the
manufacturer’s protocol modified by the inclusion of betaine and higher reaction temperature to
ensure that the RNA remained denatured. The reaction was primed with oligo(dT). Differences
in RNA concentration were normalized by using 0.2 ug of total RNA in each first-strand cDNA
reaction.
15
2.5.6 Sequencing of Na+/K+ ATPase
Larvae were used to obtain a partial sequence of the alpha (α) subunit of the RP Na+/K+
ATPase. Degenerate primers were designed from known NKA sequences obtained through
GenBank (NCBI) and aligned using ClustalW (EMBL-EBI, European Bioinformatics Institute).
PCR fragments and the predicted lengths were verified by agarose gel electrophoresis and were
produced using these degenerate primers. The products were purified using a Wizard SV Gel
and Cleanup Kit (Promega, Madison, Wisconsin, USA). Dideoxynucleotide sequencing was
done using Big-Dye Terminator Ready Reaction Mix (Applied Biosystems) and purified with an
Exterminator Kit (Applied Biosystems). The eluants were dried on a speed-vac, resuspended in
10 ul of Hi-Dye (Applied Biosystems, Foster City, California, USA) and transferred to a
sequencing plate. Forward and reverse sequences were obtained from a capillary DNA
sequencer, ABI 3100 (Applied Biosystems, Foster City, California, USA). They were checked
and assembled using Vector NTI (Invitrogen, Carlsbad, California, USA) to obtain a partial
sequence of α-subunit NKA Pagrus pagrus, which was verified using BLAST (NCBI).
(Genbank accession no. EU884411)
2.5.7 Quantitative PCR
Quantitative real time PCR was accomplished using qPCR primers designed in Primer
Express (Applied Biosystems, Foster City, California, USA) from the partial sequence of the RP
α-NKA. All samples were analyzed in the presence of the SYBR green fluorescent Dye
(Applied Biosystems, Foster City, California, USA) using an Agilent 7500 quantitative PCR
system (Agilent, Palo Alto, California, USA). To quantify the relative expression of NKA
between samples, the same standard was used for all sample periods (0, 12, 24, and 48 h).
16
2.6 Analytical methods
Quantitative values were expressed as treatment means ± standard error. Arcsine
transformation was performed for percentage data and log transformation was performed for SAI
data before analysis. To meet the assumptions of ANOVA, all data was analyzed using Levene’s
Homogeneity of variance test prior to a two-way ANOVA. A Kruskal Wallis test was run if the
data violated the assumptions of the two-way ANOVA to compare the independent effects of
temperature and salinity. The effects of temperature, salinity, and their interaction on survival,
growth, osmoregulatory ability, and stress resistance were analyzed using a two-way ANOVA.
When no interactive effects were observed, temperature treatments were combined across
salinities and salinity treatments were combined across temperatures. A Tukey-Kramer honestly
significant difference test was used for multiple comparisons among means. All statistical
analyses were performed using SAS statistical software (version 9.1.3, SAS Institute, Cary,
North Carolina U.S.A.) and a P-value ≤ 0.05 was considered significant.
17
Results
3.1 Growth
3.1.1 Notochord Length
No significant interactive effects between temperature and salinity on notochord length
were observed on any sampling day throughout the study. Hence, the effects of temperature on
NL were compared by combining data from both salinities (Figure 1a, Table 1a), while the
effects of salinity were compared across all temperatures (Figure 1b, Table 1b).
Under both salinities, highly significant (P < 0.001) effects of temperature on NL (mm)
were observed on d1, d4, d10, and d16ph (Figure 1a, Table 1a). On d1ph, notochord lengths
ranged from 2.61 to 3.15 among temperature treatments and generally increased with
temperature from a minimum value of 2.61 at 19ºC and 2.89 at 17ºC, to a maximum of 3.15 at
23ºC. Mean NL at 21ºC and 23ºC were significantly (P < 0.05) greater than at 17 and 19ºC
(Table 1a). On d4ph, mean NL at 17ºC (3.36) was significantly higher than at 19-23ºC (3.083.15). On d10ph, a clear departure among treatments was observed, with NL increasing with
temperature from a minimum of 3.28 at 17ºC to a maximum of 4.68 at 23ºC, with significant
differences among all temperature treatments. This trend was maintained on d16ph, when NL
ranged from 3.65 at 17ºC to a maximum of 5.8 at 23ºC (Fig. 1a). NL at 21 and 23ºC were
significantly higher than at 17 and 19ºC (Fig. 1a).
Under all temperatures, no significant salinity effects were observed on d1ph (Fig. 1b).
NL were significantly greater at 24 g/L than at 34 g/L on d4ph (3.24 vs. 3.12) (P < 0.01) and
d10ph (3.91 vs. 3.78) (P < 0.05). However, by d16ph, mean NL was 4.77 for both salinities,
with no significant differences (Fig. 1b, Table 1b).
18
Figure 1: Growth in notochord length (mm) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were
combined under each temperature (Fig. 1a), while temperatures were combined under each
salinity (Fig. 1b). Asterisks indicate significant treatment differences and probability levels
(*0.05, **0.01, ***0.001, ANOVA).
A
7
17
19
21
23
6
°C
°C
°C
°C
***
5
***
4
Notochord Length (mm)
3
2
0
2
4
6
8
10
12
14
16
18
14
16
18
Age (dph)
5.5
34 g/L
24 g/L
5.0
Notochord Length (mm)
B
***
***
4.5
*
4.0
3.5
**
3.0
2.5
0
2
4
6
8
10
Age (dph)
19
12
Table 1: Growth in notochord length (mm) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23ºC, n = 8) and salinities (24 and 34 g/L, n = 16). In Table 1a,
both salinities were combined under each temperature. In Table 1b, all temperatures were
combined under each salinity. At each age, probability values are shown (ANOVA). Means in
the same row followed by the same letter are not significantly different (Tukey’s, P < 0.05). NS=
not significant (P > 0.05).
A
Temperature (°C)
Age (dph)
17
19
21
23
P-Value
1
2.89 ± 0.08
a
4
3.36 ± 0.03
a
3.13 ± 0.07
b
3.08 ± 0.06
b
3.15 ± 0.02
b
< 0.001
10
3.28 ± 0.05
a
3.48 ± 0.05
b
3.94 ± 0.05
c
4.68 ± 0.06
d
< 0.0001
16
3.65 ± 0.04
a
4.32 ± 0.12
b
5.31 ± 0.12
c
5.80 ± 0.20
c
< 0.0001
2.61 ± 0.05
a
3.12 ± 0.09
b
B
Salinity (g/L)
Age (dph)
24
34
P-Value
1
2.99 ± 0.08
2.90 ± 0.07
NS
4
3.24 ± 0.02
3.12 ± 0.05
< 0.01
10
3.91 ± 0.14
3.78 ± 0.14
< 0.05
16
4.78 ± 0.23
4.75 ± 0.24
NS
20
3.15 ± 0.03
b
< 0.0001
3.1.2 Wet Weight
No significant interactive effects between temperature and salinity on wet weight were
observed, except on d10ph (Fig. 2a). The effects of temperature on wet weight were further
compared by combining both salinities (Fig. 2a, Table 2a) and the effects of salinity were
compared across all the temperatures (Fig. 2b, Table 2b).
Significant effects of temperatures on wet weight (mg) were not observed until d10ph
(Fig. 2a). On d1ph and d4ph, wet weights averaged 0.185 mg and 0.14 mg, respectively. (Table
2a). There was a clear divergence in wet weight on d10ph (Fig. 2a, Table 2a) and a significant
(P < 0.05) interaction between temperature and salinity was observed. At 17ºC, fish decreased in
wet weight to 0.12, while at 19, 21, and 23ºC, fish grew to 0.21, 0.43, and 0.84, respectively
(Table 2a). On d16ph, fish at 21 and 23ºC continued to show an increase in wet weight (2.03
and 2.91, respectively) that was significantly higher than 17ºC (0.20) and 19ºC (0.69) (Fig. 2a).
No significant salinity effects on wet weight were observed on d1 or d4ph (Fig. 2b, Table
2). Differences between salinities on d10ph increased with increasing temperatures (i.e
interaction), with 24 g/L being significantly (P < 0.05) larger than 34 g/L (0.97 versus 0.71 mg)
at 23°C. When temperatures were combined across salinities, fish at 24 g/L (0.45) were
significantly (P < 0.001) heavier than at 34 g/L (0.34). On d16ph, wet weights increased to 1.58
in 24 g/L and 1.34 in 34 g/L but with no significant difference (Table 2b).
21
Figure 2: Growth in wet weight (mg) (mean ± s.e) of larval red porgy at different temperatures
(17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were combined
under each temperature (Fig. 2a), while temperatures were combined under each salinity (Fig.
2b). Asterisks indicate significant treatment differences and probability levels (*0.05, **0.01,
***0.001, ANOVA) while i denotes a significant interaction.
A
3.5
17C
19C
21C
23C
3.0
***
Wet Weight (mg)
2.5
2.0
1.5
*** i
1.0
0.5
0.0
0
2
4
6
8
10
12
14
16
18
12
14
16
18
Age (dph)
B
2.0
34 ppt
24 ppt
1.8
1.6
Wet Weight (mg)
1.4
1.2
1.0
0.8
0.6
***
0.4
0.2
0.0
0
2
4
6
8
10
Age (dph)
22
Table 2: Growth in wet weight (mg) (mean ± s.e) of larval red porgy at different temperatures
(17, 19, 21, and 23ºC, n =8) and salinities (24 and 34 g/L, n =16). In Table 2a, both salinities
were combined under each temperature. In Table 2b, all temperatures were combined under
each salinity. At each age, probability values are shown (ANOVA). Means in the same row
followed by the same letter are not significantly different (Tukey’s, P < 0.05). NS= not
significant (P > 0.05). Asterisk (*) indicates interaction.
A
Temperature (°C)
19
Age (dph)
17
21
23
P-Value
1
0.17 ± 0.01
0.19 ± 0.01
0.18 ± 0.002
0.20 ± 0.01
NS
4
0.18 ± 0.01
0.15 ± 0.002
0.11 ± 0.003
0.11 ± 0.01
NS
10
0.12 ± 0.01
a
0.21 ± 0.02
a
0.43 ± 0.03
b
0.84 ± 0.07
c
< 0.0001*
16
0.20 ± 0.01
a
0.69 ± 0.05
a
2.03 ± 0.07
b
2.91 ± 0.11
c
< 0.0001
B
Salinity (g/L)
Age (dph)
24
34
P-Value
1
0.19 ± 0.01
0.19 ± 0.002
NS
4
0.15 ± 0.01
0.13 ± 0.01
NS
10
0.45 ± 0.09
0.34 ± 0.07
< 0.001
16
1.58 ± 0.33
1.34 ± 0.29
NS
23
3.1.3 Dry Weight
There were no significant interactive effects between temperature and salinity on dry
weight observed on any sampling day throughout the study. Therefore, the effects of
temperature on dry weight were combined across both salinities (Fig. 3a, Table 3a). Likewise,
the effects of salinity were combined across all temperatures (Fig. 3b, Table 3b).
Under both salinities, highly significant (P < 0.001) temperature effects on dry weight
(mg) were observed on d1, 10, and 16ph (Fig. 3a, Table 3a). On d1ph, dry weight ranged from
0.025 to 0.038 among treatments and was significantly lower at 23ºC (0.025) than at 17ºC
(0.038) and 19ºC (0.036) (Table 3a). On d4ph, dry weights ranged from 0.022 to 0.028, with no
significant differences. On d10ph, dry weight at 23ºC (0.121) was significantly higher than at
17ºC (0.036), 19ºC (0.032), and 21ºC (0.076); dry weight at 21ºC was also significantly greater
than 17 and 19ºC (Table 3a) on d10ph. A clear trend toward faster growth with increasing
temperature was observed on d16ph, with dry weights increasing from 0.026 at 17ºC to 0.451 at
23ºC (Fig. 3a, Table 3a). Under all temperatures, there were no significant salinity effects on dry
weight on any sampling days during this experiment (Fig. 3b, Table 3b).
24
Figure 3: Growth in dry weight (mg) (mean ± s.e) of larval red porgy at different temperatures
(17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were combined
under each temperature (Fig. 3a), while temperatures were combined under each salinity (Fig.
3b). Asterisks indicate significant treatment differences and probability levels (*0.05, **0.01,
***0.001, ANOVA).
A
0.5
17
19
21
23
Dry Weight (mg)
0.4
***
°C
°C
°C
°C
0.3
0.2
***
0.1
***
0.0
0
2
4
6
8
10
12
14
16
18
12
14
16
18
Age (dph)
B
0.30
34 g/L
24 g/L
Dry W eight (mg)
0.25
0.20
0.15
0.10
0.05
0.00
0
2
4
6
8
10
Age (dph)
25
Table 3: Growth in dry weight (mg) (mean ± s.e) of larval red porgy at different temperatures
(17, 19, 21, and 23 ºC, n =8) and salinities (24 and 34 g/L, n =16). In Table 3a, both salinities
were combined under each temperature. In Table 3b, all temperatures were combined under
each salinity. At each age, probability values are shown (ANOVA). Means in the same row
followed by the same letter are not significantly different (Tukey’s, P < 0.05). NS= not
significant (P > 0.05).
A
Age (dph)
Temperature (°C)
19
17
a
a
21
23
ab
P-Value
b
1
0.038 ± 0.001
4
0.022 ± 0.001
10
0.036 ± 0.001
a
0.032 ± 0.011
a
0.076 ± 0.008
b
0.121 ± 0.003
c
< 0.0001
16
0.026 ± 0.007
a
0.099 ± 0.019
a
0.305 ± 0.000
b
0.451 ± 0.093
c
< 0.0001
Age (dph)
24
34
P-Value
1
0.19 ± 0.009
0.03 ± 0.002
NS
4
0.15 ± 0.01
0.02 ± 0.002
NS
10
0.45 ± 0.09
0.07 ± 0.01
NS
16
1.58 ± 0.33
0.21 ± 0.05
NS
0.036 ± 0.001
0.031 ± 0.00
0.020 ± 0.003
0.026 ± 0.005
B
Salinity (g/L)
26
0.025 ± 0.002
0.028 ± 0.018
< 0.0001
NS
3.2 Survival
No significant interactive effects between temperature and salinity on survival were
observed throughout this study. Temperature effects were compared by combining salinities
(Fig. 4a, Table 4a), while salinity effects were compared across all temperatures (Fig. 4b, Table
4b).
Under both salinities, there were no significant temperature effects on survival (%)
through d4ph. Survival fell abruptly to 42.2 - 54.0 on d1ph and then more gradually to 35.7 41.9 on d4ph (Fig. 4a, Table 4a) and to 12.1-23.9% on d10ph. Significant (P < 0.05)
temperature effects on survival were first observed on d10ph, with higher survival at 23ºC (23.9)
than at 17-21ºC (12.1-14.9) (Table 4a). On d16ph, survival was significantly higher (P < 0.01) at
23ºC (22.0) than at 17-21°C (8.11-11.2) (Fig. 4a , Table 4a).
When the effects of salinity were compared across all temperatures, highly significant
effects were observed from d1ph (Fig. 4b, Table 4b), with larvae reared at the lower salinity (24
g/L) showing a higher survival rate than the larvae reared at 34 g/L for the duration of the
experiment. On d1ph, survival at 24 g/L was 57.2 compared to 41.7 at 34 g/L. Survival
remained stable at 54.7 at 24 g/L on d4ph, compared to a large decline to 21.3 at 34 g/L (Table
4b). On d10ph, survival in 24 g/L (22.4) continued to be more than double that of the 34 g/L
treatment (10.8). On d16ph, survival was 18.4 at 24 g/L versus 6.77 at 34 g/L (Table 4b).
27
Figure 4: Survival (%) (mean ± s.e) of larval red porgy at different temperatures (17, 19, 21, and
23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were combined under each
temperature (Fig. 4a), while temperatures were combined under each salinity (Fig. 4b).
Asterisks indicate significant treatment differences and probability levels (*0.05, **0.01,
***0.001, ANOVA).
A
100
Survival (%)
80
17
19
21
23
60
°C
°C
°C
°C
40
*
**
20
0
0
2
4
6
8
10
12
14
16
18
Age (dph)
B
100
Survival (%)
80
**
60
34 g/L
24 g/L
***
40
***
***
20
0
0
2
4
6
8
Age (dph)
28
10
12
14
16
18
Table 4: Survival (%) (mean ± s.e) of larval red porgy at different temperatures (17, 19, 21, and
23 ºC, n =8) and salinities (24 and 34 g/L, n =16). In Table 4a, both salinities were combined
under each temperature. In Table 4b, all temperatures were combined under each salinity. At
each age, probability values are shown (ANOVA). Means in the same row followed by the same
letter are not significantly different (Tukey’s, P < 0.05). NS= not significant (P > 0.05).
A
Temperature (°C)
19
Age (dph)
17
21
1
48.6 ± 5.01
53.2 ± 6.24
4
35.7 ± 7.23
40.1 ± 11.24
10
12.1 ± 1.86
a
15.7 ± 3.99
ab
14.9 ± 2.65
ab
23.9 ± 3.15
b
< 0.05
16
8.99 ± 1.87
a
11.2 ± 3.42
a
8.11 ± 1.98
a
22.0 ± 4.31
b
< 0.01
23
P-Value
42.2 ± 0.07
54.0 ± 4.29
NS
36.5 ± 6.77
41.9 ± 7.18
NS
B
Salinity (g/L)
Age (dph)
24
34
P-Value
1
57.2 ± 2.63
41.7 ± 0.72
< 0.01
4
54.7 ± 1.49
22.3 ± 1.03
< 0.0001
10
22.5 ± 1.52
10.8 ± 1.15
< 0.0001
16
18.4 ± 2.14
6.77 ± 1.1
< 0.0001
29
3.3 Whole-body Osmolality
There were no significant interactive effects between temperature and salinity on wholebody osmolality on any sampling day throughout this study. The effects of temperature were
compared by combining data for both salinities (Fig. 5a, Table 5a), while the effects of salinities
were compared across all temperatures (Fig. 5b, Table 5b).
Under both salinities, there were no significant temperature effects observed on d1ph,
with whole-body osmolality (mOsm/kg) ranging from 390 at 17ºC to 457 at 19 and 23ºC (Fig 5a,
Table 5a). Whole-body osmolality was significantly (P < 0.05) different on d6ph, with a marked
increase at 19 and 21ºC, which was not observed in 17 or 23ºC. At 19 and 21ºC, whole body
osmolality increased to 667 and 683, respectively. Whole body osmolality was significantly
higher at 21ºC than at 23ºC (Table 5a). On d10ph, whole-body osmolality were near initial
levels for all temperature treatments, with no significant treatment differences. On d16ph, there
was an apparent increase at 17ºC, but no significant differences were observed (Table 5a).
When salinities were compared across all temperatures, mean osmolality values were
generally higher at 34 g/L (441-613 mOsm/kg) than at 24 g/L (342-486 mOsm/kg) for the
duration of the study. On d2ph, whole body osmolality was significantly (P < 0.05) higher at 34
g/L (522) than at 24 g/L (342) (Table 5b). On d6ph, whole-body osmolality increased from 522
to 613 mOsm/kg in 34 g/L and from 342 to 486 mOsm/kg in 24 g/L, but with no significant
difference between salinities. However, whole-body osmolality at 24 g/L was significantly
higher (P < 0.05) on d6ph than on d2ph. On d10ph, whole-body osmolality decreased under
both 24 g/L (347) and 34 g/L (411), but with no significant differences (Table 5b). At 34 g/L,
whole body osmolality was significantly lower than on d2ph. On d16ph whole-body osmolality
was significantly (P < 0.05) higher at 34 g/L (481) than at 24 g/L (412).
30
Figure 5: Whole body osmolality (mOsm/kg) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). Salinities were
combined under each temperature (Fig. 5a), while temperatures were combined under each
salinity (Fig. 5b). Asterisks indicate significant treatment differences and probability levels
(*0.05, **0.01, ***0.001, ANOVA). Dagger (†) indicates a significant (P < 0.05) difference
from the d2ph value.
800
*
Whole body Osmolality (mOsm/kg)
A
17
19
21
23
700
°C
°C
°C
°C
600
500
400
300
0
2
4
6
8
10
12
14
16
18
Age (dph)
B
Whole body Osmolality (mOsm/kg)
700
†
650
34 g/L
24 g/L
600
550
**
*
500
450
†
400
350
300
0
2
4
6
8
10
Age (dph)
31
12
14
16
18
Table 5: Whole body osmolality (mOsm/kg) (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). In Table 5a,
both salinities were combined under each temperature. In Table 5b, all temperatures were
combined under each salinity. At each age, probability values are shown (ANOVA). Means in
the same row followed by the same letter are not significantly different (Tukey’s, P < 0.05). NS=
not significant (P > 0.05). A dagger (†) indicates means that were significantly (t-test, P < 0.05)
different from the d2ph values.
A
Temperature (°C)
Age (dph)
17
19
21
23
P-Value
457 ± 146
427 ± 111
457 ± 115
NS
2
390 ± 137
6
446 ± 18.6
11
398 ± 108
349 ± 24.4
385 ± 14.1
383 ± 33.6
NS
17
573 ± 133
408 ± 9.37
411 ± 23.5
396 ± 30.4
NS
ab
667 ± 270
ab
683 ± 121
B
Salinity (g/L)
Age (dph)
24
34
2
342 ± 34.6
6
486 ± 79.6
11
347 ± 24.4
411 ± 46.0
17
412 ± 46
482 ± 124
†
P-Value
522 ± 32.7
< 0.005
613 ± 234
NS
†
NS
< 0.05
32
a
402 ± 11.5
b
< 0.05
3.4 Survival Activity Index (SAI)
There were no significant temperature or salinity effects (P > 0.05) on SAI at 48h
following an abrupt salinity challenge, but a significant (P < 0.01) interactive effect was
observed (Figs. 6a and 6b). In general, SAI decreased with increasing temperature in 24 g/L
from 25.18 at 17°C to 0 at 23°C, but increased with increasing temperature in 34 g/L treatments
from 1.7 at 17°C to 60.4 at 23°C (Figs. 6a and 6b).
33
Figure 6: Survival Activity Index at 48h (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16) following a
salinity challenge to 40 g/L and under zero feeding conditions. Salinities were combined under
each temperature (Fig. 6a), while temperatures were combined under each salinity (Fig. 6b). A
significant (P < 0.01, Two-way ANOVA) temperature x salinity interaction was observed.
A
100
17 °C
19 °C
21 °C
23 °C
Survival Activity Index
80
60
40
20
0
24
34
Salinity (g/L)
B
100
24 g/L
34 g/L
Survival Activity Index
80
60
40
20
0
17
19
21
Temperature (°C)
34
23
3.5 mRNA Expression of Na+/K+ ATPase
Two partial RP α Na+/K+ ATPase sequences were observed. Na/K-ATPase alpha subunit
isoform A (Genbank accession no. EU884411) and Na/K-ATPase alpha subunit isoform B
(Genbank accession no. EU884412) were assembled. Na/K-ATPase alpha subunit isoform A
was used in this experiment due to its close relation to black porgy Acanthopagrus schlegeli.
There were no significant interactive effects between temperature and salinity on the
mRNA expression of Na+/K+ ATPase on any sampling day throughout the study. The effects of
temperatures were compared by combining data for both salinities (Fig. 7a, Table 6a), while the
effects of salinities were compared across all temperatures (Fig. 7b, Table 6b).
Under both salinities, there were no significant temperature effects observed at any
sampling period after the hypersaline stress test (P > 0.05) (Fig.7a, Table 6a). At time 0h, the
mRNA expression of Na+/K+ ATPase ranged from 7.48-14.7 among temperatures. There was a
decrease in mRNA expression of Na+/K+ ATPase after 12h, ranging from 2.47-5.59 among
temperatures, with only the value at 21°C being significantly (P < 0.05) lower than time 0 levels.
At 24h, mRNA expression of Na+/K+ ATPase increased (13.4-24.5), but with no significant
differences among treatments and to time zero treatment levels. After 48h, mRNA expression of
Na+/K+ ATPase ranged from 18.1-26.0, with no significant differences (Table 6a).
When salinities were compared across all temperatures, there were no significant
differences observed at any sampling time except for 24h (Fig. 7b, Table 6b). At time 0h,
mRNA expression of Na+/K+ ATPase for both salinities ranged from 10.1 to 10.7. A significant
(P<0.05) decrease was observed at 12h for both 24 and 34 g/L (3.99 and 4.42). mRNA
expression of Na+/K+ ATPase increased at 24h to 24.3 at 24 g/L and to 11.5 at 34 g/L, with a
35
significant difference (P < 0.05) between salinities (Fig. 7b). A similar trend was observed at
48h for both salinities (10.8 and 22, respectively), but was not significant (Table 6b).
36
Figure 7: mRNA expression of Na+/K+ ATPase (mean ± s.e) of larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16) after a salinity
challenge to 44 g/L and under zero feeding. Salinities were combined under each temperature
(Fig. 7a), while temperatures were combined under each salinity (Fig. 7b). Bars within a cluster
with different letters are significantly (P < 0.05, Tukey’s test) different. Dagger (†) indicates a
significant (P < 0.05) difference from the time 0 value.
A
Na+K+ ATPase qPCR expression
50
17
19
21
23
40
°C
°C
°C
°C
30
20
10
†
0
0
12
24
48
Hours from start
B
Na+K+ ATPase qPCR expression
35
34 g/L
24 g/L
30
b†
a
25
20
15
a
a
a
a
10
a†
5
0
0
a†
12
24
Hours from start
37
48
Table 6: mRNA expression Na+/K+ ATPase (mean ± s.e) of d16ph larval red porgy at different
temperatures (17, 19, 21, and 23 ºC, n = 8) and salinities (24 and 34 g/L, n = 16). In Table 6a,
both salinities were combined under each temperature. In Table 6b, all temperatures were
combined under each salinity. At each time period, probability values are shown (ANOVA).
NS= not significant (P > 0.05). Dagger (†) indicates a significant (P < 0.05) difference from the
time 0 value.
A
Temperature (°C)
Time
17
19
21
23
P value
0
10.85 ± 2.44
7.48 ± 2.03
14.69 ± 3.00
9.12 ± 2.33
NS
12
5.59 ± 1.00
4.15 ± 0.73
2.47 ± 0.38
†
3.84 ± 1.41
NS
24
24.55 ± 10.08
13.36 ± 3.95
18.11 ± 4.40
15.39 ± 2.66
NS
48
15.76 ± 5.03
12.14 ± 5.06
26.03 ± 12.22
12.84 ± 4.16
NS
B
Salinity (g/L)
Time
24
34
P value
0
10.1 ± 1.37
10.72 ± 2.17
NS
12
3.99 ± 0.76
†
NS
24
24.33 ± 4.99
48
21.96 ± 6.39
†
†
4.42 ± 0.62
11.52 ± 2.66
< 0.05
10.77 ± 2.59
NS
38
Discussion
4.1 Growth
In this study, NL, wet weight, and dry weight of larval RP during the exogenous feeding
period showed clear trends toward faster growth with increasing temperature within in the range
of 17-23°C, and with highly significant differences among treatments from d10ph through
d16ph. Salinity did not have an effect on overall growth, except for NL on d4 and 10ph and wet
weight on d10ph, which an interaction occurred. In both cases, 24 g/L were larger within a
temperature treatment most likely due to lower metabolic rates as described later, but overall
growth was more affected by temperature. Higher growth was likely related to accelerated
metabolism and feeding at higher temperatures (Johns and Howell, 1980; Watanabe et al., 1995;
Watanabe et al., 1999; Fielder et al., 2005; Yoseda et al., 2006; Bustos et al., 2007). Faster
growth with increasing temperatures within a geographical range has been reported for many
other marine finfish species including: summer flounder Paralichthys dentatus (Johns and
Howell, 1980; Watanabe et al., 1999), southern hake Merluccius australis (Bustos et al., 2007),
brown sole Pseudopleuronectes herzensteini (Aritaki and Seikai, 2004), Nassau grouper
Epinephelus striatus (Watanabe et al., 1995), Malabar grouper Epinephelus malabaricus
(Yoseda et al., 2006), greenback flounder Rhombosolea tapirina (Hart and Purser, 1995),
Australian Snapper Pagrus auratus (Fielder et al., 2005), and black sea bass Centropristis striata
(Berlinsky et al., 2004). The authors of these studies suggested that the increased growth at
higher temperatures was due to increased metabolism (Johns and Howell, 1980; Watanabe et al.,
1995; Watanabe et al., 1999; Fielder et al., 2005; Yoseda et al., 2006; Bustos et al., 2007),
efficiency of transformation of energy to body tissue (Berlinsky et al., 2004), ability to absorb
39
endogenous and exogenous nutrition more efficiently (Yoseda et al., 2006), and similarity to
natural temperatures within the range tested (Fielder et al., 2005).
This is the first study to compare the effects of temperature on growth of larval RP.
Other studies have reported larval RP performance at specific temperatures but many different
rearing conditions (e.g., feed rate, tank size, rearing density) were used in those studies, making
comparisons difficult. Growth rates of RP in this study were generally lower than reported for
this species in the Mediterranean, indicating that genetic differences among Atlantic RP and
Mediterranean strains of RP may exist, as previously suggested by Morris et al. (2008). Largescale genetic differences between Eastern (Mediterranean) and Western (North and South
American) Atlantic strains of RP have been reported based on microsatellite and mitochondrial
DNA markers (Ball et al., 2003; Ball et al., 2007). Previous studies at 19 and 21°C have
reported Mediterranean RP lengths at the time of yolk exhaustion (d3-d5ph) of 3.32-3.95 TL and
3.5 mm SL, respectively (Hernandez-Cruz et al., 1999; Mihelakakis et al., 2001; Papandroulakis
et al., 2004), early feeding larvae (d10ph) of 4.3-5 mm TL and 4.74 mm SL, respectively
(Hernandez-Cruz et al., 1999; Mihelakakis et al., 2001; Papandroulakis et al., 2004), and late
feeding larvae (d16ph) of 5.75-6 mm SL and 5.75 mm SL, respectively (Hernandez-Cruz et al.,
1999; Mihelakakis et al., 2001; Papandroulakis et al., 2004), which are all larger than results of
this study (3.13, 3.48, and 4.32 mm NL at 19°C, respectively and 3.08, 3.94, and 5.31 mm NL at
21°C, respectively). Differences between TL and NL reported in these studies are relatively
minor in fish of this size and should not affect these comparisons. Genetic differences may
contribute to growth differences between strains, but precise comparisons between studies are
not possible because of differences in experimental conditions used (e.g. stocking densities, food
availability, tank sizes). No studies have reported growth of RP at 17°C or 23°C.
40
Early larval growth can be described in three growth phases: an early rapid phase after
hatching, slow growth phase near completion of yolk sac, and subsequent self-metabolism and
loss of weight without successful feeding (Blaxter, 1969). Transition to exogenous feeding of
larval RP in this study at all temperatures occurred after d4ph, when their endogenous reserves
were exhausted and no growth occurred, a pattern typical of marine finfish (Blaxter, 1969).
Morphologically, the RP were ready to begin exogenous feeding by d3ph (eyes are formed and
mouth open), but few fish had food in the digestive tract, as they were learning to capture living
prey (Roo et al., 1999a). Larval Australian snapper Pagrus auratus, which belong to the same
genus as RP, showed comparable patterns in the utilization of yolk reserves. Snapper showed an
initial rapid length increase (2.8 mm to 3.2 mm SL) during yolksac absorption and an
intermediate phase of no growth during transition from endogenous to exogenous feeding
(Pankhurst et al., 1991; Battaglene and Talbot, 1992).
In this study, higher temperatures caused faster exhaustion of yolk reserves for RP larvae
at 19, 21, and 23°C, which exhibited initial rapid growth and subsequent decrease in notochord
length on d4ph when the yolk reserves were completely used up. In contrast, yolk sac larvae at
17°C continued to gain weight on d4ph, indicating that rate of yolk-sac absorption was reduced
at 17°C. Similar results of higher temperatures causing faster exhaustion of yolk reserves were
reported in newly hatched larval summer flounder (Johns and Howell, 1980; Watanabe et al.,
1999), southern hake (Bustos et al., 2007) and Nassau grouper (Watanabe et al., 1995). This is
consistent with the decrease in weights for fish in 19, 21, and 23°C on d4ph (Figs. 3-4).
Development of the visual and digestive systems occur concurrently to yolksac absorption (Roo
et al., 1999a) and was accelerated by higher temperatures and metabolism (Watanabe et al.,
1995). The point of no return, which is the point in which fish are unable to feed on exogenous
41
food even when food is available, is also highly affected by increasing water temperature with
increasing temperatures decreasing the time of the point of no return (McGurk, 1984; Dou et al.,
2002, 2005).
When compared to fish at 19°C, which showed significant growth, RP larvae reared at
17°C in this experiment showed minimal growth in terms of NL (Fig. 1a, Table 1a), wet weight
(Fig. 3a, Table 2a), and dry weight (Fig. 4a, Table 3a). This suggested that 17°C was near the
lower limit for RP larval rearing in the laboratory. RP spawn from 16-22°C (Manooch, 1976) so
16°C may be the lower temperature limit of their ecological range (Manooch, 1975), making the
growth results at 17°C not surprising.
There is a temperature limit above which growth rate will not increase and which can be
detrimental to the larvae. The upper rearing temperature for larval RP is not known and was not
determined in this experiment, since maximum growth was obtained at the highest temperature
treatment (23°C). In brown sole larvae, growth rates increased from 6-15°C, but were similar to
15°C at 21°C, indicating that larvae do not need to be raised above 15°C to achieve optimal
growth (Aritaki and Seikai, 2004). Similarly, the largest greenback flounder larvae and fastest
growth rate were observed at 15°C when compared to 6-18°C (Hart and Purser, 1995). Since the
23°C treatment fish grew the largest in this experiment, it is possible that they can be raised at
even higher temperatures. Preliminary experiments in our laboratory showed that 25°C
produced higher growth in larval RP compared to 16, 19, and 22°C (A. Ostrowski, unpublished
data). Ecologically, wild larval RP may experience temperatures of 23-25°C in warmer areas of
their geographical range, or if transported into marsh areas or nursery habitats. Further studies
over higher temperature ranges are needed to determine the optimum and maximum temperature
for rearing RP larvae.
42
4.2 Survival
RP early survival during the yolk sac stage was not affected by temperature in this study.
Survival among temperature treatments was similar, ranging from 42-54% on d1ph and 36-42%
on d4ph. This was likely due to the fact that final temperature treatments were not applied until
d2ph. Salinity, on the other hand, had a much greater influence on early survival, which was
higher at 24 g/L than at 34 g/L on d1ph (57% vs. 42) and more than twice as high on d4ph (55%
vs. 22%) (Fig. 6, Table 4). The lower salinity treatment (24 g/L = 614 mOsm/kg) was closer to
the isoosmotic conditions (WBO = 342-412 mOsm/kg) (Table 5) than the high salinity treatment
(34 g/L = 911 mOsm/kg) and may have reduced osmoregulatory stress and increased survival as
reported for gilthead seabream (Woo and Fung, 1981). In contrast, RP reared in 34 g/L seawater
(WBO = 411-613 mOsm/kg) (Table 5) in this study showed a significant decline in survival from
d1 to d4ph (42-22%), possibly due to osmoregulatory stress at this hyperosmotic salinity.
While survival of RP in this study from yolk sac to feeding stage (d4-d10ph) remained
relatively high (36-15%) at the higher temperatures of 21 and 23°C, survival dropped
considerably in the lower temperature treatments of 17 and 19°C, declining from 42% on d4ph to
8% on d10ph (Table 4). This is similar to what Conides and Glamuzina (2001) reported for RP
reared at 18°C, and is possibly due to slower development of hunting capabilities (Büke et al.,
2005) and transition to exogenous sources of food (Kolios et al., 1997) at lower water
temperatures.
The effects of salinity on larval marine finfish survival are species dependent. Survival
of larval gilthead seabream to d32ph increased as salinity decreased from 40 to 25 g/L (Tandler
et al., 1995), similar to the observations in this study. In gilthead seabream, the increased
survival at reduced salinities (25 g/L) was believed to be associated with the energy saving costs
43
for osmoregulation since the lower salinities were closer to isoosmotic conditions in gilthead
seabream, and hyperosmotic environments divert more energy into metabolism rather than
growth (Tandler et al., 1995). In contrast, southern flounder could not tolerate lower salinities
until post metamorphosis, with early survival higher in 34 g/L than 24 g/L (Henne and Watanabe
2003; Moustakas et al. 2004) possibly due to full strength seawater optimizing buoyancy,
vertical positioning, and feeding, while conserving energy (Moustakas et al. 2004). After
metamorphosis, however, southern flounder survived in salinities ranging from 5-30 g/L (Smith
et al. 1999), which is when these flounder are found in brackish inshore waters in nature (Burke
et al., 1991). While this study has demonstrated that RP can be raised in 24 g/L brackish water
during the early larval periods, the effects of salinity on growth and survival of juveniles are
unknown and warrants further investigation.
In contrast to RP in this study, salinity did not affect survival in some larval marine
finfish, such as Atlantic halibut, Australian snapper, summer flounder, or southern flounder. In
Atlantic halibut incubated between 27-34 g/L, survival of larvae to d49ph (69-90%) was not
significantly different (Lein et al., 1997). No survival differences (18-33%) were also observed
for Australian snapper larvae to d21ph reared from 20-35 g/L (Fielder et al., 2005). However,
salinity effects on larval growth and survival are also dependent on other rearing conditions. For
example, the effects of salinity on survival were influenced by temperature in larval summer
flounder (Watanabe et al., 1999), and by water turbulence in larval southern flounder (Mangino
and Watanabe, 2006). In southern flounder, higher turbulence improved buoyancy, preventing
sinking in early stage larvae, but was detrimental to feeding stage larvae since it decreased prey
encounters and feeding efficiency (Mangino and Watanabe, 2006). In RP, turbulence also
influenced survival at different salinities, with the higher survival in the lower turbulence levels
44
(20 and 90 mL/min) compared to the highest turbulence levels (150 and 250 mL/min) at 24 and
34 g/L (Rezek, pers. comm.). Since survival is cumulative, it is possible that the initial
turbulence levels when eggs were stocked out could have impacted survival throughout this
study, however, the turbulence levels were adjusted as necessary so that the eggs were
maintained in suspension.
4.3 Whole Body Osmolality
Whole body osmolality of larval RP was significantly different between 21 and 23°C on
d6ph, but was not significantly different among temperatures different during the remainder of
the experiment (Fig. 7). The difference on d6ph is difficult to explain since there were no clear
temperature related trends and may have been an artifact of the sampling method. On the other
hand, whole-body osmolality was lower at 24 g/L (WBO = 342-486 mOsm/kg) than at 34 g/L
(WBO = 411-613 mOsm/kg) throughout the experiment. Assuming that whole body osmolality
of marine finfish body fluids are generally within the range of 320-400 mOsm/kg (Alderdice et
al., 1979; Davenport et al., 1981; Riis-Vestergaard, 1982; Hahnenkamp et al., 1993), this
suggested that osmotic stress was lower in brackish water than in full strength seawater,
consistent with the idea that isoosmotic rearing salinities reduce osmoregulatory stress (Woo and
Fung, 1981). In larval southern flounder, whole body osmolality was 320-343 in 34 g/L,
compared to only 288-304 mOsmol/kg in 24 g/L (Henne and Watanabe, 2003; Moustakas et al.,
2004). These authors suggested that greater osmoregulatory stress at 24 g/L was not likely, since
the fish in 24 g/L seawater (614 mOsm/kg) were closer to isoosmotic conditions compared to
fish in 34 g/L seawater (960 mOsm/kg). These authors further suggested that reduced buoyancy
45
in brackish water caused feeding larvae to use energy for vertical positioning at the expense of
energy for growth and survival (Henne and Watanabe, 2003; Moustakas et al., 2004). The fact
that RP maintain near normal osmotic pressure at 24 g/L suggests that these RP larvae do not
need to expend excessive energy on vertical positioning, possibly related to the fact that RP
larvae have a swim bladder by d7ph (Mihelakakis et al., 2001; Morris et al., 2008), whereas
flounder larvae lack or have a greatly reduced swim bladder (Helfman et al., 1999).
4.4 Survival Activity Index
In this study, survival Activity Index (SAI) was used as a practical indicator of larval
stress resistance to an abrupt salinity increase and to starvation. SAI at 48 h post transfer to 40
g/L increased with increasing temperatures for fish reared in 34 g/L, but not for 24 g/L, which
showed a decrease in SAI with increasing temperature (i.e., temperature x salinity interaction)
(Fig. 8b). This was not surprising because fish from 24 g/L experienced a higher salinity
increase (∆ 16 g/L) compared to those from 34 g/L (∆ 6 g/L). For fish raised at 24 g/L, this large
salinity increase was apparently too high to allow physiological adaptation to the increased
salinity, causing death. Fish from 24 g/L were also burdened by the higher metabolism
requirements at the higher temperatures, consistent with the decrease in SAI with increasing
temperature. Hence, the combined effects of excessive salinity stress under conditions of
starvation caused relatively quick mortality for fish reared at 24 g/L. For fish raised at 34 g/L,
higher SAI at higher temperatures was likely related to larger energy reserves accumulated by
higher metabolisms, feeding, and growth, in the higher temperature treatments. More
comparable results would have been obtained if the salinity increase was the same for each
46
treatment. Carrier (2006) used an acute salinity challenge in larval black sea bass causing rapid
mortality which did not allow dietary arachidonic acid treatment effects on SAI to be resolved.
This author suggested a lower sublethal salinity challenge (from 45 to 50 g/L) to discern these
effects. In other studies, SAI was influenced by broodstock nutrition, especially dietary EFA
levels (Furuita et al., 2003). Nutritional effects, however, were unlikely in this study because
larvae came from broodstock fed the same diets.
4.5 mRNA expression of Na+/K+ ATPase
In this study, larval whole body mRNA expression of Na+/K+ ATPase generally
decreased in all treatments after 12 h following a hypersaline challenge prior to increasing after
24 h (Fig 9). Carrier (2006) also found an increase in relative mRNA expression of Na+/K+
ATPase in black sea bass larvae fed a high dietary ARA levels (> 6.7% TFA) 24 h after exposing
larvae to an sublethal hypersaline challenge (43 g/L), but did not determine mRNA expression of
Na+/K+ ATPase at 12 h post transfer. Caberoy and Quinitio (2000), on the other hand, observed
a decrease in mRNA expression of Na+/K+ ATPase 12 and 24 h after transfer from 30 g/L to 40
g/L followed by an increase after 48 hours in d20ph grouper, Epinephelus coidoides. The
authors suggested that following salinity transfer, mRNA expression reached a threshold and
cells were no longer able to sustain activity, resulting in death of Na+/K+ ATPase enzyme units
within 12-24 h of the change in salinity, followed within 48 h by an increase in fractional area of
chloride cells, where Na+/K+ ATPase are expressed (Caberoy and Quinitio, 2000). The results of
this study with larval RP are consistent with the idea (Caberoy and Quinitio, 2000), that
following a salinity challenge, there is a decrease in cell activity (chloride cell death), followed
47
by regrowth of new chloride cells and subsequent increase in mRNA expression of Na+/K+
ATPase. Euryhaline milkfish juveniles also showed an increase in mRNA expression of Na+/K+
ATPase at 48 h after transfer from seawater to freshwater, but only after a small decrease at 24 h
(Lin et al., 2006).
Juvenile marine fish also show a similar time course of adjustments to salinity change.
Juvenile silver sea bream were capable of coping with hypoosmotic changes (from 33 g/L to 6
g/L), making a majority of readjustments within the first 24 h, and most measured parameters
(serum chemistry, stored metabolites and gill morphology) returning to new steady state values
by 5 d (Kelly and Woo, 1999). Two osmoregulatory periods have been suggested: an adjustment
period and a chronic regulatory period (Mancera et al., 2002; Arjona et al., 2007). Within the
sampling period of this study, larval RP in 24 g/L were in the adjustment period throughout the
time of post transfer because relative expression of Na+/K+ ATPase never returned to initial (0 h)
levels, unlike fish in the 34 g/L, which returned to initial (0 h) levels by 24 h post transfer (i.e.,
regulatory period). This was likely due the relatively large salinity increase for fish from 24 g/L
(∆ salinity = 20 g/L) compared to those from 34 g/L (∆ salinity = 10 g/L).
Temperature did not affect osmoregulation (i.e. mRNA expression of Na+/K+ ATPase) in
larval RP at any time following a sublethal salinity challenge, suggesting that temperature had a
larger effect on larval metabolism feeding and growth than on Na+/K+ ATPase activity of RP
larvae. Similar results were observed for the orange-spotted grouper Epinephelus coioides
(Caberoy and Quinitio, 2000). Although no studies have reported temperature effects on Na+/K+
ATPase in larval fish, temperature has been shown to affect osmoregulatory ability in juvenile
marine finfish. For example, a cold tolerant northern killifish (Fundulus heteroclitus) strain
48
showed a quicker mRNA expression of Na+/K+ ATPase than a warm tolerant southern strain
following abrupt transfer to seawater (Scott and Schulte, 2005). Within the range of 10-33°C,
the highest gill enzyme (Na+/K+ ATPase) activity in juvenile turbot (Scophthalmus maximus)
was at 22°C, and there was a positive correlation between temperature and enzyme activity
(Imsland et al., 2003). On the other hand, Na+/K+ ATPase activity was not affected by rearing
temperatures between 20-35°C in juvenile tilapia (Oreochromis mossambicus) (Fiess et al.,
2007).
Rearing salinity can also affect Na+/K+ ATPase activity in juvenile marine finfish. Low
and moderate salinities (15 and 25 g/L, respectively) reduced gill Na+/K+ ATPase activity in
juvenile turbot (Imsland et al., 2003) held at the various salinities. These authors suggested that
reduced energy expenditures contributed to higher growth rates for fish reared at these salinities.
Relative abundance of Na+/K+ ATPase activity was higher in gills of the euryhaline spotted
green pufferfish reared in seawater (35 g/L) compared to those raised in freshwater (Tang and
Lee, 2007), although these workers did not compare growth or survival under these different
salinity conditions.
Conclusions
Within the range of 17-23°C and at salinities of 24 and 34 g/L, larval RP growth to d16ph
was improved with increasing temperature. Survival was affected by both temperature and
salinity with the highest survival (under both salinities) observed at the highest temperature of
23°C (S = 22%) and (under all temperatures) at the lower salinity of 24 g/L (S = 18.4%) by
49
d16ph. Larval whole body osmolality values indicated less osmotic stress throughout the
experiment in 24 g/L than at 34 g/L. SAI following abrupt transfer to 40 g/L was higher in 34
g/L treatment fish than those from 24 g/L at 48h post transfer due to the lower salinity challenge
those treatment fish experienced. For fish reared at 34 g/L, SAI increased with increasing
temperature, consistent with better growth at higher temperature for fish at 34 g/L. However,
SAI decreased with increasing temperatures for fish at 24 g/L, which could not adjust to this
acute salinity challenge (i.e. temperature x salinity interaction). Results suggest that SAI was
better for fish raised in SW; however, fish in 24 g/L were challenged to a greater delta salinity.
Following a sub-lethal (44 g/L) hypersaline challenge, mRNA expression of Na+,K+, ATPase
decreased at 12 h and then increased and was higher for larvae reared at 24 g/L than at 34 g/L at
24 h. This suggested greater chloride cell activity and elaboration of the Na+, K+ transport pump
for larvae at 24 g/L. This is consistent with a higher salinity change for larvae at 24 g/L than at
34 g/L. Future studies should use the same salinity change to provide meaningful comparisons
of stress resistance for fish reared at different salinities. Based on maximum growth, survival,
and osmoregulatory abilities in this study, 23°C and 24 g/L are recommended for larval RP
culture, but additional studies to evaluate performance at higher temperatures are warranted.
50
LITERATURE CITED
Alderdice, D.F., Rao, T., Rosenthal, H., 1979. Osmotic responses of eggs and larvae of the
Pacific herring to salinity and cadmium. Helgolander wiss. Meersunters 32, 508-538.
Aristizabal, E., 2005. Morphological development of the mouth and improvement in feeding
ability in the early larval stages of red porgy, Pagrus pagrus. Rev. Invest. Desarr. Pesq.
17, 43-53.
Aristizabal, E., 2007. Energy investment in the annual reproduction cycle of female red porgy,
Pagrus pagrus (L.). Marine Biology 152, 713.
Aristizabal, E., Suarez, J., 2006. Effeciency of co-feeding red porgy (Pagrus pagrus L.) larvae
with live and compound diet. Revista de Biologia Marina y Oceanografia 41, 203-208.
Aristizabal, E., Suárez, J., Vega, A., Bargas, R., 2009. Egg and larval quality assessment in the
Argentinean red porgy (Pagrus pagrus). Aquaculture 287, 329-334.
Aritaki, M., Seikai, T., 2004. Temperature effects on early development and occurrence of
metamorphosis-related morphological abnormalities in hatchery-reared brown sole
Pseudopleuronectes herzensteini. Aquaculture 240, 517-530.
Arjona, F.J., Vargas-Chacoff, L., Ruiz-Jarabo, I., Martin del Rio, M.P., Mancera, J.M., 2007.
Osmoregulatory response of Senegalese sole (Solea senegalensis) to changes in
environmental salinity. Comparative Biochemistry and Physiology - Part A: Molecular &
Integrative Physiology 148, 413.
Ball, A.-O., Sedberry, G.-R., Wessel, J.-H., III, Chapman, R.-W., 2003. Large-scale genetic
differentiation of Pagrus pagrus in the Atlantic. Journal of Fish Biology 62, 1232-1237.
Ball, A., Beal, M., Chapman, R., Sedberry, G., 2007. Population structure of red porgy, Pagrus
pagrus, in the Atlantic Ocean. Marine Biology 150, 1321.
Battaglene, S.C., Talbot, R.B., 1992. Induced spawning and larval rearing of snapper, Pagrus
auratus, (Pisces: Sparidae), from Australian waters. New Zealand Journal of Marine and
Freshwater Research 26, 179-183.
51
Bentley, C., Carroll, P., Watanabe, W., 2008. Intensive rotifer production in a pilot-scale
continuous culture recirculating system using nonviable microalgae and an ammonia
neutralizer. Journal of the World Aquaculture Society 39, 625-635.
Berlinsky, D.L., Taylor, J.C., Howell, R.A., Bradley, T.M., Smith, T.I.J., 2004. The Effects of
Temperature and Salinity on Early Life Stages of Black Sea Bass Centropristis striata.
Journal of the World Aquaculture Society 35, 335-344.
Biswas, A.K., Seoka, M., Inoue, Y., Takii, K., Kumai, H., 2005. Photoperiod influences the
growth, food intake, feed efficiency and digestibility of red sea bream (Pagrus major).
Aquaculture 250, 666.
Blaxter, J., 1969. Development: eggs and larvae. In: Hoar, W., Randall, D. (Eds.), Fish
Physiology. Academic Press, New York, pp. 178-252.
Boeuf, G., Payan, P., 2001. How should salinity influence fish growth? Comparative
Biochemistry and Physiology. C, Toxicology & Pharmacology, 411-423.
Brett, J., 1970. Temperature-Fishes. In: Kinne, O. (Ed.), Marine Ecology. Wiley-Interscience,
New York, pp. 515-560.
Büke, E., Akpinar, Z., Ayekin, B., Dereli, H., 2005. Spawning performance and larval rearing of
red porgy (Pagrus pagrus, L., 1758) under culture conditions. E.U. Journal of Fisheries
and Aquatic Sciences 22, 303-309.
Burke, J.S., Miller, J.M., Hoss, D.E., 1991. Immigration and settlement pattern of Paralichthys
dentatus and P. lethostigma in an estuarine nursery ground, North Carolina, U.S.A.
Netherlands Journal of Sea Research 27, 393-405.
Bustos, C.A., Landaeta, M.F., Bay-Schmith, E., Lewis, R., Moraga, X., 2007. Effects of
temperature and lipid droplet adherence on mortality of hatchery-reared southern hake
Merluccius australis larvae. Aquaculture 270, 535-540.
Caberoy, N., Quinitio, G., 2000. Changes in Na+,K+ -ATPase activity and gill chloride cell
morphology in the grouper Epinephelus coioides larvae and juveniles in response to
salinity and temperature. Fish Physiology and Biochemistry 23, 83-94.
52
Carrier, J.K., 2006. Growth, survival, and resistance to hypersaline stress in larval black sea bass
(Centripristis striata) fed varying levels of dietary arachidonic acid (20:4n-6). Center for
Marine Science. University of North Carolina-Wilmington, Wilmington, NC, pp. 105.
Cejas, J.R., Almansa, E., Tejera, N., Jerez, S., Bolanos, A., Lorenzo, A., 2003. Effect of dietary
supplementation with shrimp on skin pigmentation and lipid composition of red porgy
(Pagrus pagrus) alevins. Aquaculture 457-469.
Chatzifotis, S., Sterioti, A., Divanach, P., Vardanis, G., Pavlidis, M., Jimeno, C.D., 2005. The
effect of different carotenoid sources on skin coloration of cultured red porgy (Pagrus
pagrus). Aquaculture research 36, 1517-1525.
Conides, A.J., Glamuzina, B., 2001. Study on the early larval development and growth of the red
porgy, Pagrus pagrus with emphasis on the mass mortalities observed during this phase.
Scientia Marina 65, 193-200.
Darias, M.J., Cardenas, S., Yufera, M., Murray, H.M., Martinez-Rodriguez, G., 2005. Gene
expression of pepsinogen during the larval development of red porgy (Pagrus pagrus).
Aquaculture 248, 245-252.
Darias, M.J., Murray, H.M., Gallant, J.W., Douglas, S.E., Yúfera, M., Martínez-Rodríguez, G.,
2007. Ontogeny of pepsinogen and gastric proton pump expression in red porgy (Pagrus
pagrus): Determination of stomach functionality. Aquaculture 270, 369-378.
Davenport, J., Lønning, S., Kjørsvik, E., 1981. Osmotic and structural changes during early
development of eggs and larvae of the cod, Gadus morhua L. Journal of Fish Biology 19,
317-331.
Dou, S.Z., Masuda, R., Tanaka, M., Tsukamoto, K., 2002. Feeding resumption, morphological
changes and mortality during starvation in Japanese flounder larvae. Journal of Fish
Biology 60, 1363-1380.
Dou, S.Z., Masuda, R., Tanaka, M., Tsukamoto, K., 2005. Effects of temperature and delayed
initial feeding on the survival and growth of Japanese flounder larvae. Journal of Fish
Biology 66, 362-377.
Fielder, D.S., Bardsley, W.J., Allan, G.L., Pankhurst, P.M., 2005. The effects of salinity and
temperature on growth and survival of Australian snapper, Pagrus auratus larvae.
Aquaculture 250, 201.
53
Fiess, J.C., Kunkel-Patterson, A., Mathias, L., Riley, L.G., Yancey, P.H., Hirano, T., Grau, E.G.,
2007. Effects of environmental salinity and temperature on osmoregulatory ability,
organic osmolytes, and plasma hormone profiles in the Mozambique tilapia
(Oreochromis mossambicus). Comparative Biochemistry and Physiology - Part A:
Molecular & Integrative Physiology 146, 252.
Foscarini, R., 1988. A review: Intensive farming procedure for red sea bream (Pagrus major) in
Japan. Aquaculture 72, 191.
Furuita, H., Yamamoto, T., Shima, T., Suzuki, N., Takeuchi, T., 2003. Effect of arachidonic acid
levels in broodstock diet on larval and egg quality of Japanese flounder Paralichthys
olivaceus. Aquaculture 220, 725-735.
Fushimi, H., 2001. Production of juvenile marine finfish for stock enhancement in Japan.
Aquaculture 200, 33.
Hahnenkamp, L., Senstad, K., Fynn, H.J., 1993. Osmotic and ionic regulation of yolksac larvae
of Atlantic halibut (Hippoglossus hippoglossus). In: Walther, B., Fynn, H. (Eds.),
Physiological and biochemical aspects of fish development. University of Bergen,
Norway, pp. 259-262.
Hart, P.R., Purser, G.J., 1995. Effects of salinity and temperature on eggs and yolk sac larvae of
the greenback flounder (Rhombosolea tapirina Günther, 1862). Aquaculture 136, 221230.
Helfman, G.S., Collette, B.B., Facey, D.E., 1999. The Diversity of Fishes. Blackwell Science,
Malden, Ma, 528 pp.
Henne, J.P., Watanabe, W.O., 2003. Effects of light intensity and salinity on growth, survival,
and whole-body osmolality of larval sourthern flounder Paralicthys lethostigma. Journal
of the World Aquaculture Society 34, 450-465.
Hernandez-Cruz, C.-M., Salhi, M., Bessonart, M., Izquierdo, M.-S., Gonzalez, M.-M.,
Fernandez-Palacios, H., 1999. Rearing techniques for red porgy (Pagrus pagrus) during
larval development. Aquaculture 179, 489-497.
Holliday, F., 1988. The effects of salinity on the eggs and larvae of teleosts. In: Hoar, W.,
Randall, R. (Eds.), Fish Physiology. Academic Press, New York, pp. 293-311.
54
Howell, W., 1980. Temperature effects on growth and yolk utilization in yellowtail flounder,
Limanda ferruginea, yolk-sac larvae. Fishery Bulletin 78, 731-739.
Imsland, A.K., Gunnarsson, S., Foss, A., Stefansson, S.O., 2003. Gill Na+, K+-ATPase activity,
plasma chloride and osmolality in juvenile turbot (Scophthalmus maximus) reared at
different temperatures and salinities. Aquaculture 218, 671.
Johns, D.M., Howell, W.H., 1980. Yolk Utilization in Summer Flounder (Paralichthys dentatus)
Embryos and Larvae Reared at Two Temperatures. Marine Ecology Progress Series 2, 18.
Kalinowski, C.T., Izquierdo, M.S., Schuchardt, D., Robaina, L.E., 2007. Dietary
supplementation time with shrimp shell meal on red porgy (Pagrus pagrus) skin colour
and carotenoid concentration. Aquaculture 272, 451-457.
Kalinowski, C.T., Schuchardt, D., Izquierdo, M.S., Robaina, L.E., Fernandez-Palacios, H., 2005.
Effect of different carotenoid sources and their dietary levels on red porgy (Pagrus
pagrus) growth and skin colour. Aquaculture 244, 223-231.
Kato, K., Hayashi, R., Yuasa, D., Yamamoto, S., Miyashita, S., Murata, O., Kumai, H., 2002.
Production of cloned red sea bream, Pagrus major, by chromosome manipulation.
Aquaculture 207, 19-27.
Kelly, S.P., Woo, N.Y.S., 1999. The response of sea bream following abrupt hyposmotic
exposure. Journal of Fish Biology 55, 732-750.
Kentouri, M., Pavlides, M., Papandroulakis, N., Divanach, P., 1995. Culture of the red porgy,
Pagrus pagrus, in Crete. Present knowledge, problems and perspectives. Cahiers Options
Mediterraneennes 16, 65-78.
Kolios, P., Kiritsis, S., Katribusas, N., 1997. Larval-rearing and growout of the red porgy (Pagrus
pagrus) in the Riopesca hatchery (Greece). Hydrobiologia 358, 321-325.
Labropoulou, M., Machias, A., Tsimenides, N., 1999. Habitat selection and diet of juvenile red
porgy, Pagrus pagrus (Linnaeus, 1758). Fishery Bulletin (Washington D C).
Lein, I., Tveite, S., Gjerde, G., Holmefjord, I., 1997. Effects of salinity on yolk sac larvae of
Atlantic halibut (Hippoglossus hippoglossus L). Aquaculture 156, 291-303.
55
Lin, Y.M., Chen, C.N., Yoshinaga, T., Tsai, S.C., Shen, I.D., Lee, T.H., 2006. Short-term effects
of hyposmotic shock on Na+/K+-ATPase expression in gills of the euryhaline milkfish,
Chanos chanos. Comparative Biochemistry and Physiology - Part A: Molecular &
Integrative Physiology 143, 406.
Machinandiarena, L., Muller, M., Lopez, A., 2003. Early life stages of development of the red
porgy Pagrus pagrus (Pisces, Sparidae) in captivity, Argentina. Invest. Mar. 31, 5-13.
Mancera, J., Carrión, R.L., del Pilar Martín del Río, M., 2002. Osmoregulatory action of PRL,
GH, and cortisol in the gilthead seabream (Sparus aurata L.). General and Comparative
Endocrinology 129, 95-103.
Mangino, J., Adam, Watanabe, W.O., 2006. Combined effects of turbulence and salinity on
growth, survival, and whole-body osmolality of larval southern flounder. Journal of the
World Aquaculture Society 37, 407-420.
Manooch, C.S., 1975. A study of the taxonomy, exploutation, life history, ecology and tagging of
the red porgy, Pagrus pagrus, Linnaeus off the Caolinas. North Caolina State University,
Raleigh, pp. 275.
Manooch, C.S., 1976. Reproductive cycle , fecundity, and sex ratios of the red porgy, Pagrus
pagrus (Pisces: Sparidae) in North Carolina. Fish. Bull. NMFS/NOAA 74, 775-781.
Manooch, C.S., 1977. Foods of Red Porgy, Pagrus-Pagrus Linnaeus (Pisces Sparidae), from
North-Carolina and South-Carolina. Bulletin of Marine Science 27, 776-787.
Manooch, C.S., Hassler, W.W., 1978. Synopsis of biological data on the red porgy, Pagrus
pagrus (Linnaeus). . NOAA Technical Report, 1-19.
Manooch, C.S., III, Huntsman, G.R., 1977. Age, growth, and mortality of the red porgy, Pagrus
pagrus. Transactions of the American Fisheries Society 106, 26-33.
Matsuo, Y., Kasahara, Y., Hagiwara, A., Sakakura, Y., Arakawa, T., 2006. Evaluation of larval
quality of viviparous scorpionfish Sebastiscus marmoratus. Fisheries Science 72, 948954.
May, R.C., 1974. Effects of temperature and salinity on yolk utilization in Bairdiella icistia
(Jordan and Gilbert) (Pisces:Sciaenidae). Journal of Experimental Marine Biology and
Ecology 16, 213-225.
56
McGurk, M.D., 1984. Effects of delayed feeding and temperature on the age of irreversible
starvation and on the rates of growth and mortality of Pacific herring larvae. Marine
Biology 84, 13-26.
Mihelakakis, A., Yoshimatsu, T., 1998. SHORT COMMUNICATION Effects of salinity and
temperature on incubation period, hatching rate and morphogenesis of the red sea bream.
Aquac. Int. 6, 171-177.
Mihelakakis, A., Yoshimatsu, T., Tsolkas, C., 2001. Spawning in captivity and early life history
of cultured red porgy, Pagrus pagrus. Aquaculture 199, 333-352.
Morris, J.A., Rezek, T.C., McNeill, N.A., Watanabe, W.O., 2008. Aquaculture of the Atlantic
Red Porgy. North American Journal of Aquaculture 70, 184-191.
Moustakas, C., Watanabe, W.O., Copeland, K.A., 2004. Combined effects of photoperiod and
salinity on growth, survival, and osmoregulatory ability of larval southern flounder
Paralichthys lethostimga. Aquaculture 229, 159-179.
Mylonas, C.C., Papadaki, M., Pavlidis, M., Divanach, P., 2004. Evaluation of egg production
and quality in the Mediterranean red porgy (Pagrus pagrus) during two consecutive
spawning seasons. Aquaculture 232, 637-649.
Ottesen, O.H., Bolla, S., 1998. Combined effects of temperature and salinity on development and
survival of Atlantic halibut larvae. Aquac. Int. 6, 103-120.
Pankhurst, P.M., Montgomery, J.C., Pankhurst, N.W., 1991. Growth, development and
behaviour of artificially reared larval Pagrus auratus (Bloch and Schneider,
1801)(Sparidae). Australian Journal of Marine and Freshwater Research 42, 391-398.
Papandroulakis, N., Kentouri, M., Divanach, P., 2004. Biological performance of red porgy
(Pagrus pagrus) larvae under intensive rearing conditions with the use of an automated
feeding system. Aquacultural International 12, 191-203.
Parker, R.O., Dixon, R.L., 1998. Changes in a North Carolina Reef Fish Community after 15
Years of Intense Fishing&#8212;Global Warming Implications. Transactions of the
American Fisheries Society 127, 908-920.
57
Potts, J.C., Manooch, C.S., III, 2002. Estimated ages of red porgy (Pagrus pagrus) from fisherydependent and fishery-independent data and a comparison of growth parameters. Fishery
Bulletin 100, 81-89.
Radonic, M., Lopez, A., M, O., E.O, A., 2005. Effect of the incubation temperature on the
embryonic development and hatching time of eggs of the red porgy, Pagrus pagrus
(Linne, 1758)(Pisces:Sparidae). Revista de Biologia Marina y Oceanografia, 91-99.
Riis-Vestergaard, J., 1982. Water and salt balance of halibut eggs and larvae (Hippoglossus
hippoglossus). Marine Biology 70, 135-139.
Rogers, C.A., 1976. Effects of temperature and salinity on the survival of winter flounder
embryos. Fishery Bulletin 74, 52-58.
Rombough, P., 2007. The functional ontogeny of the teleost gill: Which comes first, gas or ion
exchange? Comparative Biochemistry and Physiology - Part A: Molecular & Integrative
Physiology 148, 732-742.
Roo, F.J., Socorro, J., Izquierdo, M.S., Caballero, M.J., Hernández-Cruz, C.M., Fernández, A.,
Fernández-Palacios, H., 1999a. Development of red porgy Pagrus pagrus visual system in
relation with changes in the digestive tract and larval feeding habits. Aquaculture 179,
499-512.
Roo, F.J., Socorro, J., Izquierdo, M.S., Caballero, M.J., Hernandez-Cruz, C.M., Fernandez, A.,
Fernandez-Palacios, H., 1999b. Development of red porgy Pagrus pagrus visual system
in relation with changes in the digestive tract and larval feeding habits. Aquaculture 179,
499-512.
Rotllant, J., Tort, L., Montero, D., Pavlidis, M., Martinez, M., Wendelaar Bonga, S.E., Balm,
P.H.M., 2003. Background colour influence on the stress response in cultured red porgy
Pagrus pagrus. Aquaculture 223, 129-139.
SAFMC, 2000. Final, amendment number 12 to the fishery management plan for the snapper
grouper fishery of the South Atlantic region. South Atlantic Fishery Management
Council, Charleston, SC, 159 + appendices.
SAFMC, 2006. Stock assessment of red porgy off the southeastern USA. Southeast Data,
Assessment, and Review (SEDAR). Report of Assessment Workshop. Beaufort, North
Carolina. April 4-5 2006.
58
Saka, S., Firat, K., Kamaci, H.O., Buke, E., 2005. The effect of temperature on embryonic
development of the red porgy (Pagrus pagrus) eggs. E.U. Journal of Fisheries and
Aquatic Sciences 22, 95-99.
Schreiber, A.M., 2001. Metamorphosis and early larval development of the flatfishes
(Pleuronectiformes): an osmoregulatory perspective. Comparative Biochemistry and
Physiology Part B: Biochemistry and Molecular Biology 129, 587-595.
Scott, G.R., Schulte, P.M., 2005. Intraspecific variation in gene expression after seawater
transfer in gills of the euryhaline killifish Fundulus heteroclitus. Comparative
Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 141, 176.
Shi, Z., Huang, X., Fu, R., Wang, H., Luo, H., Chen, B., Liu, M., Zhang, D., 2008. Salinity stress
on embryos and early larval stages of the pomfret Pampus punctatissimus. Aquaculture
275, 306-310.
Smith, T.I.J., Denson, M., R., Heyward Sr., L.D., Jenkins, W., E., Carter, L.M., 1999. Salinity
Effects on Early Life Stages of Southern Flounder Paralichthys lethostigma. Journal of
the World Aquaculture Society 30, 236-244.
Specker, J.L., Schreiber, A.M., McArdle, M.E., Poholek, A., Henderson, J., Bengtson, D.A.,
1999. Metamorphosis in summer flounder: effects of acclimation to low and high
salinities. Aquaculture 176, 145-154.
Suzer, C., Kamaci, H.O., Çoban, D., Saka, E., Firat, K., Özkara, B., Özkara, A., 2007. Digestive
enzyme activity of the red porgy (Pagrus pagrus, L.) during larval development under
culture conditions. Aquaculture research 38, 1778-1785.
Swanson, C., 1996. Early development of milkfish: effects of salinity on embryonic and larval
metabolism, yolk absorption and growth. Journal of Fish Biology 48, 405-421.
Tandler, A., Anav, F.A., Choshniak, I., 1995. The effect of salinity on growth rate, survival and
swimbladder inflation in gilthead seabream, Sparus aurata, larvae. Aquaculture 135, 343353.
Tang, C.H., Lee, T.H., 2007. The effect of environmental salinity on the protein expression of
Na+/K+-ATPase, Na+/K+/2Cl- cotransporter, cystic fibrosis transmembrane conductance
regulator, anion exchanger 1, and chloride channel 3 in gills of a euryhaline teleost,
Tetraodon nigroviridis. Comparative Biochemistry and Physiology - Part A: Molecular &
Integrative Physiology 147, 521.
59
Van der Salm, A.L., Martinez, M., Flik, G., Wendelaar Bonga, S.E., 2004. Effects of husbandry
conditions on the skin colour and stress response of red porgy, Pagrus pagrus.
Aquaculture 241, 371-386.
Van der Salm, A.L., Pavlidis, M., Flik, G., Wendelaar Bonga, S.E., 2006. The acute stress
response of red porgy, Pagrus pagrus, kept on a red or white background. General and
Comparative Endocrinology 145, 247-253.
Vaughan, D.S., Prager, M.H., 2002. Severe decline in abundance of the red porgy (Pagrus
pagrus) population off the southeastern United States. Fishery Bulletin 100, 351-375.
Watanabe, W.O., Ellis, S.C., Ellis, E.P., 1999. Temperature effects on eggs and yolk sac larvae
of the summer flounder at different salinities. North American Journal of Aquaculture 61,
267-277.
Watanabe, W.O., Lee, C.-S., Ellis, S.C., Ellis, E.P., 1995. Hatchery study of the effects of
temperature on eggs and yolksac larvae of the Nassau grouper Epinephelus striatus.
Aquaculture 136, 141-147.
Watanabe, W.O., Feeley, M.W., Ellis, S.C., Ellis, E.P., 1998. Light Intensity and salinity effects
on eggs and yolk sac larvae of the summer flounder. The Progressive Fish-Culturist 60,
9-19.
Watanabe, W.O., French, K.E., Ernst, D.H., Olla, B.L., Wicklund, R.I., 1989. Salinity During
Early Development Influences Growth and Survival of Florida Red Tilapia in Brackish
and Seawater. Journal of the World Aquaculture Society 20, 134-142.
Woo, N.Y.S., Fung, A.C.Y., 1981. Studies on the biology of the red sea bream, Chrysophrys
major,--II. Salinity Adaptation. Comparative Biochemistry and Physiology - Part A:
Molecular & Integrative Physiology 69, 237-242.
Yoseda, K., Dan, S., Sugaya, T., Yokogi, K., Tanaka, M., Tawada, S., 2006. Effects of
temperature and delayed initial feeding on the growth of Malabar grouper (Epinephelus
malabaricus) larvae. Aquaculture 256, 192-200.
60