E-mail: aclee@mail.ncyu.edu.tw Abstract The dissolved oxygen DO concentration that induces the onset of anaerobic metabolism in hard clams was found to be 1.11mg O2L 1, at which time, th
Trang 2Effects of low dissolved oxygen on the digging
behaviour and metabolism of the hard clam
(Meretrix lusoria)
An-Chin Lee, Yu-Ching Lee & Tzong-Shean Chin
Department of Aquatic Biosciences, College of Life Science, National Chiayi University, Chiayi, Taiwan
Correspondence: A-C Lee, Department of Aquatic Biosciences, College of Life Sciences, National Chiayi University, 300 University Road, Chiayi 600, Taiwan E-mail: aclee@mail.ncyu.edu.tw
Abstract
The dissolved oxygen (DO) concentration that induces
the onset of anaerobic metabolism in hard clams was
found to be 1.11mg O2L 1, at which time, the
con-centration of succinate in the body £uid was
4.4mmol mL 1 When the DO concentration was
o1.11mg O2L 1, the burial depth was signi¢cantly
reduced, and succinate signi¢cantly accumulated in
the body £uid After 24 h of anoxic exposure,
succi-nate had accumulated in the gills and equal amounts
of succinate and alanine had accumulated in the foot
This indicates that carbohydrates in the gills and
ami-no acids in the foot contribute to anaerobic energy
production The accumulation rates of succinate and
propionate in the body £uid were the highest
com-pared with those in other tissues, while no
accumula-tion of alanine in the body £uid was found The
recovery rates of succinate in the body £uid and
ala-nine in the foot were the highest compared with those
in other tissues The results of this study suggest that
the DO concentration in the bottom water of clam
ponds should be maintained at 1.11mg O2L 1,
and the anoxia-tolerant ability of hard clams can be
assessed by the contents of carbohydrates
Keywords: hard clam, critical concentration, DO,
digging behaviour, anoxia
Introduction
Hard clams (Meretrix lusoria) are widely found
throughout Asia, in Japan, Korea, China,Taiwan and
elsewhere They naturally reside in the estuary of theTanshui River in northern Taiwan and along thewest coast of Taiwan Most hard clams sold com-mercially are cultured in ponds in Taiwan, as localestuaries are polluted by industrial pollutantsduring the early rainy season (Jeng & Wang 1976).However, massive die-o¡s of hard clams have oc-curred in ponds in times of high water tempera-tures (Tseng 1976) High stocking densities(1^2 106seeds ha 1) and supplementation ofpowdered ¢sh meal and fermented organic matter inponds can result in a reducing layer in the sedimentsand, possibly, in low dissolved oxygen (DO) concen-trations in the bottom waters of ponds (Hon 1988).Low DO may decrease the burial depth, alter the me-tabolism and ultimately cause massive die-o¡s ofhard clams Clarifying the cause of these massivedie-o¡s of hard clams is very important for the clamculture industry as they are an important economicbivalve, and their production ranked ¢rst among themalacofauna in Taiwan in 2008 (Fisheries Agency2008/2010) Hard clams are commonly found eitherpartially or completely buried in bottom sediments oftheir marine habitat, suggesting that they have devel-oped the metabolic capacity to survive periods ofhypoxia and anoxia When anaerobic conditionsbecome severe, physiological and behaviouralchanges occur that are associated with high clammortality The in£uence of environmental oxygen onadaptive biochemical and physiological survival stra-tegies is well documented for a variety of facultativeanaerobic mollusks, and data indicate that criti-cal oxygen levels that trigger hypoxic and anoxic
Trang 3energy-producing pathways di¡er among species
(Cheng, Chang & Chen 2004; Long, Brylawski & Seitz
2008) The current research concerns the
relation-ship between environmental oxygen availability, and
the burial behaviour and metabolism of hard clams
DO concentrations considerably a¡ect the survival
and behaviour of aquatic organisms (Long et al
2008) The DO concentration that induces the onset
of anaerobic metabolism is de¢ned as the critical
con-centration, which varies with di¡erent bivalves (de
Zwaan, Cortesi, van der Thillart, Brooks, Storey,
Roos, van Lieshout, Cattani & Vitali 1992; Le Moullac,
Que¤au, Le Souchu, Pouvreau, Moal, Le Coz & Samain
2007) When hard clams experience hypoxia, they
usually emerge from the bottom sediment to stay at
a higher position in search of oxygen (Lee, Lin, Lin,
Lee & Chen 2007) After emerging from the bottom,
clams usually die within a short time if extra oxygen
is not found Once environmental hypoxia occurs,
the ability to obtain chemical energy through
anae-robic metabolism a¡ects their survival
The strategy adopted by marine mollusks exposed
to hypoxia/anoxia requires a decrease in ATP
turn-over that involves coordinated down-regulation of
ATP consumption and production (i.e through
de-pression of the metabolic rate) In mollusks,
meta-bolic rates decline by a factor of 10^20 or more
(Storey & Storey 1990; Brooks & Storey 1997) The
re-maining lowATP demand is covered by the chemical
energy produced through anaerobic metabolism
Anaerobic metabolism has been widely studied in
many kinds of invertebrates and vertebrates (de
Zwaan 1983; Storey & Storey 1990; de Zwaan &
Eer-tman 1996; Larade & Storey 2002) Under hypoxia/
anoxia, invertebrates can obtain energy by anaerobic
metabolism that occurs under functional and
envir-onmental anoxia (Livingstone 1991; P˛rtner 2002)
The lactate and opine pathways play important roles
in functional anoxia, while the aspartate^succinate
and glucose^succinate pathways are utilized during
environmental anoxic survival (Livingstone 1991;
Lee, Lee, Lee & Lee 2008; Lee & Lee 2010)
Anoxic-tolerant mollusks exposed to hypoxia meet
their energy demands mostly by relying on glycolysis
However, alternative substrate-level phosphorylation
with the accumulation of succinate or propionate
oc-curs when the concentration of DO is lower than a
critical concentration The initial responses to anoxia
are coupled to the fermentation of glycogen and
as-partate, causing the accumulation of succinate and
alanine In the aspartate^succinate pathway, amino
groups from aspartate are transferred to pyruvate to
form alanine, and their carbon skeleton, tate, is then transformed to succinate in hypoxia.When aspartate pools are depleted, succinate ismostly formed from glycogen, which involves the
oxaloace-£ow of carbon through the phosphoenolpyruvate(PEP) branch point of glycolysis This is accomplishedvia inhibition of pyruvate kinase due to the accumu-lation of alanine and protons This favours thecarboxylation of PEP as mediated by the PEP carbox-ykinase reaction to produce oxaloacetate that feedsthe reaction of succinate synthesis (Bacchiocchi &Principato 2000; Larade & Storey 2002) Under pro-longed anoxia, succinate is converted to propionate
by a pathway inside the mitochondria that produces
an extra mole of ATP per unit of succinate (Schulz,Kluytmans & Zandee 1982; Isani, Cattani, Zurzolo,Pagnucco & Cortesi 1995) A hallmark of anoxicadaptation is an evolved capacity for the rapid rever-sible entry into and return from metabolically de-pressed steady states This involves speci¢c control ofkey regulatory enzymes to reorganize metabolismand allow entry and arousal from anoxia (Larade &Storey 2002, 2009)
The critical concentration of DO is an importantparameter for managing clam culture However, in-formation on the relationship between the DO con-centration of seawater and the burial behaviour ofhard clams is scant In addition, the study of anaero-bic energy and its sources in tissues of hard clams sofar has been super¢cial, although some results werereported (Lee et al 2008) Therefore, the aims of thisstudy were to examine the e¡ects of the DO concen-tration on the burial behaviour of hard clams, to de-termine the critical DO concentration inducing theonset of anaerobic metabolism, and ¢nally to exam-ine anaerobic energy sources in tissues of hard clams
Material and methodsAnimals
Hard clams (14.6 1.1g) were purchased from clamfarms in the Taishi area (Yunlin County, southwes-tern Taiwan) Arti¢cial seawater (ASW) (20 g L 1)was prepared by dissolving 1:50 (w/v) synthetic seasalts (Meersaltzs, Heinsberg, Germany) in tap waterthat was strongly aerated for at least 2 weeks beforeuse (Taiwan Water Corporation 2003) No chlorinewas detected in the aerated water Three hundredclams were acclimated at room temperature (23^
28 1C) in two 2000 L tanks, each of which contained
1300 L of 20 g L 1ASW with a 10 cm depth of sand
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Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 201 , 43, 1–13
3 2
Trang 4on the bottom Clams that did not burrow into the
sand were excluded Clams were fed the microalgae
Isochrysis a¡ galbana, Chaetoceros muelleri and
Tetra-selmis chui at the respective ¢nal concentrations of
was replaced with clean 20 g L 1ASW No food was
provided 1 day before the experiments
Design for water sampling
The design for water sampling is presented in Fig 1
The DO concentration of the sample was determined
using the Winkler method (azide modi¢cation)
(APHA, AWWA & WEF 1995) The end (opening) of a
tube was placed on the top of the sediment in an
aqua-rium.Water sampled from the top of the sediment was
termed ‘bottom water’ in this study In order to avoid
oxygen molecules in the tube and DO bottle with this
design, nitrogen was used to £ush the tube and DO
bottle when switch I was turned on and switch II was
turned o¡ After nitrogen £ushing, a hand-operated
vacuum pump was used to aspirate water through
the tube when switch II was turned on and switch I
was turned o¡ The DO bottle was used to collect
sam-ple water to determine the DO concentration
Determination of the burial depth and
succinate concentrations in the body £uid of
hard clams
Experiment I: Aquarium with an open-water surface
Twelve aquaria [60 (L) 30 (W) 35 cm (H)], each of
which contained 40 L of 20 g L 1ASW with a 6 cm
depth of sand on the bottom, were used in this study.Thirty hard clams were placed in each aquarium.Mild aeration was applied to the aquarium overnight
at 25 1C The experiment began on day 0, and after, no aeration was applied The assessment of dig-ging indices of the hard clams and the determination
there-of DO concentrations in the bottom water were ried out every other day After assessing the diggingindices and determining the DO concentration, 30hard clams from one aquarium were sampled to de-termine the concentration of succinate in the body
car-£uid This experiment was performed for 20 days.Body £uid (extracellular £uid) from ¢ve clams wascollected as a replicate Therefore, there were six repli-cates for each aquarium The digging index of theclams was scored as follows (Lee et al 2007): 1, com-pletely dug into the sand; 0.9, completely dug into thesand but the top of the shell was exposed; 0.33 and0.67, only 1/3 and 2/3, respectively, of a clam was cov-ered by sand; and 0, the clam was on top of the sand.Therefore, the burial depth of hard clams could be in-dicated by a digging index: a smaller value of the dig-ging index indicated a shallower burial depth
Experiment II: Aquarium with 50% of the surfacecovered
In order to limit the in£ux of oxygen molecules in theair into the water, 50% of the water surface in anaquarium was covered by a Styrofoam board Thir-teen aquaria were used in this study Seven of themwere used to study changes in the concentration ofsuccinate in the body £uid The other six were used
to study changes in the digging indices of hard clamsand the DO concentration of bottom water in thetank The following conditions were similar to those
Figure 1 A design for water sampling
Trang 5in experiment I In the group used to determine the
concentration of succinate in the body £uid, 50% of
the water surface of six aquaria was covered by a
Styr-ofoam board after the hard clams from an aquarium
were sampled to determine the concentration of
suc-cinate on day 0 Thereafter, hard clams from an
aqua-rium were sampled to determine the concentration of
succinate every day In the group used to determine
the digging indices of hard clams and the DO
concen-tration of bottom water, 50% of the water surface of
six aquaria was covered by a Styrofoam board after
both parameters were determined on day 0
There-after, these parameters were determined every day
Normoxic and anoxic exposure of hard clams
In total, 275 clams were placed in a tank with 275 L of
20 g L 1aerated seawater overnight The DO was
maintained at 7 ppm Five clams were sampled as
a replicate, and ¢ve replicates served as a normoxic
group (0 h exposure) Twenty litres of 20 g L 1ASW
placed in a plastic bag with 50 glass jars ( 130 mL)
was bubbled with N2 for 2 h After bubbling, 250
clams were placed in the plastic bag with N2 gas
blowing over them Five clams were sealed in a glass
jar inside the plastic bag Fifty glass jars were placed
at 25 1C Five glass jars were, respectively, sampled at
8, 24, 48 and 64 h After 64 h of anoxic exposure, 20
glass jars with no dead clams were sampled as the
materials for the recovery experiment The other jars
were not used further in the experiment
Recovery after 64 h of anoxic exposure
Clams in 20 glass jars after 64 h of anoxic exposure
were released into 100 L of ASW with aeration at 25 1C
Twenty-¢ve clams were, respectively, sampled at 0,8, 24
and 48 h Five clams were pooled together as a replicate
Their body £uid, digestive gland, gills, foot, adductor
and mantle were sampled and, respectively, pooled
There were ¢ve replicates for each sampling time
Digging indices during 48 h of recovery after
64 h of anoxic exposure
Clams in 18 glass jars after 64 h of anoxic exposure
were released and washed as soon as was possible
with clean ASW Three aquaria as described above
were used in this study Thirty hard clams were
placed in each aquarium Mild aeration was applied
to the aquaria at 25 1C The digging indices of thehard clams were assessed at 0, 8, 24 and 48 h
Collection of specimens, and extraction anddetermination of anaerobic end products intissues
Collection of specimensAfter the seawater in the mantle cavity had beendrained out, the body £uid (extracellular £uid) wascollected from shucked clams as quickly as possible.After the body £uid of the clams had been sampled,the foot, digestive gland, adductor, gills and mantle
of the ¢ve clams in the glass jar were, respectively, sected and stored in liquid nitrogen until used
dis-Extraction of anaerobic end products in tissuesThe extraction of anaerobic end products was based
on the methods described by Lee et al (2007, 2008).Frozen tissues were powdered in a steel mortarcooled with dry ice One gram of powdered tissuewas homogenized in a homogenizer (PRO Scienti¢c,Oxford, CT, USA) with 4.5 mL of 6% cooled perchloricacid (PCA) on ice water The homogenate was centri-fuged at 10 000 g for 20 min at 4 1C The precipitatewas extracted with a further 3 mL of 6% cooledPCA After centrifugation, both supernatants werecombined and neutralized with 2.08 mL of a 3.6 MKOH solution The contents were continuously mixedduring neutralization to avoid the production of localalkalinity in the solution, and then the solution waskept on ice for 30 min to allow the KClO4to precipi-tate The precipitate was washed with 2 mL of deio-nized water Both supernatants were combined andstored at 20 1C for later analysis of anaerobic endproducts Nine millilitres of 6% cooled PCA wasadded to 3 mL of body £uid The mixture was centri-fuged at 10 000 g for 20 min at 4 1C The supernatantwas neutralized with 2.5 mL of 3.6 M KOH and placed
on ice for 30 min Subsequent steps were conductedsimilar to those described above
Determination of anaerobic end productsSuccinate and propionate in tissues were analysed byhigh-performance liquid chromatography (HPLC)using a Transgenomic ICSep ICE-ION-300 column(7.8 300 mm) using 0.001 N sulphuric acid as themobile phase following the method described byChiou, Lin and Shiau (1998) The £ow rate was0.3 mL min 1
The column temperature was set
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Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 2012, 43, 1–13
3
Trang 6to 52 1C, and detection was monitored at 210 nm.
Succinate and propionate in the body £uid were
determined by HPLC using a Mightysil RP-C18
GP column (4.6 250 mm, 5 mm) using 10 mM
H3PO4 as the mobile phase The £ow rate was
1mL min 1 The concentration of alanine was
deter-mined using an enzymatic method (Grassl & Supp
1984)
Analysis of dataAmong-treatment di¡erences in data from Table 1,and Figs 3, 4, and 5 were determined using Duncan’scomparison test to determine where signi¢cant dif-ferences occurred The signi¢cance of all tests wasaccepted at Po0.05 The accumulation and recoveryrates of anaerobic end products in tissues were calcu-
Table 1 Concentrations of succinate, alanine and propionate in tissues of hard clams after 0, 8, 24, 48 and 64 h of anoxic exposure
Tissue
Time of anoxic exposure (h)
Anaerobic end products (lmol g 1 wet weight)
Accumulation is expressed as the di¡erence in the concentrations of anaerobic end products in tissues of hard clams between at 0 and
at 64 h of anoxic exposure Data are given as the mean the standard error for ¢ve replicates (¢ve clams per replicate) Data of a speci¢c end product in a speci¢c tissue with di¡erent superscript letters signi¢cantly di¡er among di¡erent exposure times (P o0.05) Data of a speci¢c end product at 0 or 64 h anoxic exposure with di¡erent subscript letters signi¢cantly di¡er among tissues (P o0.05).
In a speci¢c tissue at a speci¢c exposure time, data of an end product with ‘’are signi¢cantly greater than those with‘’, which are in turn signi¢cantly greater than those with no asterisks (P o0.05).
wUnits of body £uid are mmol mL 1
.
Trang 7lated by linear regressions using SigmaPlot software
vers.10 from SPSS (Chicago, IL, USA) The simple
cor-relation coe⁄cient of the straight line was
deter-mined
Results
Determination of burial depth and succinate
level in the body £uid of hard clams
The digging index of a hard clam that had completely
dug into the sand was scored as1 Therefore, the
max-imum score for the digging index of an aquarium
containing 30 clams was 30 Although the DO
con-centration in the bottom water dramatically
de-creased from 6.2 mg O2L 1on day 0 of exposure to
3 mg O2L 1at 2 days of exposure, it was slightly
higher at 4.33 mg O2L 1after 20 days of exposure
at 25 1C (Fig 2) The depth of the water body in the
aquaria was only 20 cm Water £ow produced by the
clams might have been su⁄cient to have created local
circulation, which would have enhanced the
di¡u-sion of oxygen molecules in the air into the water
This level of DO does not cause stress that would
af-fect the behaviour and metabolism of hard clams
After 20 days of exposure, no signi¢cant changes in
the digging indices or the concentration of succinate
in the body £uid of hard clams were observed (Fig 2)
No mortality was found during the experiment
The DO concentration in the bottom water of the
aquaria with 50% of the water surface covered
decreased sharply from 6.31mg O2L 1 on day 0
of exposure to 3.22 mg O2L 1 at 1 day and1.34 mg O2L 1at 2 days of exposure at 25 1C (Fig.3) After 6 days of exposure, the DO concentration ofthe bottom water had gradually decreased to0.28 mg O2L 1 Although no signi¢cant change inthe digging indices of hard clams before 3 days ofexposure was found, the digging index (27.7) at 3days of exposure had dramatically decreased (22.2)
at 4 days of exposure This means that hard clamswere obviously observed to have emerged from thesediment to some extent At the same time, the con-centration of succinate in the body £uid of hard clamshad greatly increased from 4.39mmol mL 1at 3 days
of exposure to 9.54mmol mL 1at 4 days of exposure.Therefore, the third day of exposure was when anae-robic metabolism began in hard clams The DO con-centration of 1.11mg O2L 1 on the third day ofexposure can be de¢ned as the critical concentration
of DO for hard clams During the experiment, only sixdead clams were found
Anaerobic end products under normoxia andanoxia
The presence of anaerobic end products was ined in the body £uid and ¢ve tissues of M lusoria.Under normoxia (day 0), the concentration of succi-nate was much higher in the digestive gland than in
exam-Exposure time (days)
Dissolved oxygenDigging indiceSuccinate(12) (11) (10) (9) (8) (7) (6) (5) (4) (3) (2)
Figure 2 E¡ects of the dissolved oxygen (DO) concentration on the digging index and concentration of succinate in thebody £uid of hard clams in aquaria (30 clams per aquarium) with an open surface at 25 1C Numbers in parentheses in-dicate the numbers of replicates for measurements of DO (n 5 2^12 aquaria) and the digging index (n 5 2^12 aquaria).There were six replicates in the measurement of succinate in the body £uid (¢ve clams per replicate) Data are given as themean with the standard error
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–1 6
Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 201 , 43, 1–13
3 2
Trang 8the body £uid and other tissues (Table 1) However,
the concentrations of alanine were signi¢cantly
higher in the foot and adductor muscle than in the
body £uid and other tissues After 64 h of anoxic
ex-posure, increases in the concentrations of anaerobic
end products were found in all tissues of the hard
clam The greatest accumulations of succinate and
propionate were found in the body £uid, while those
of alanine were observed in the foot The
concentra-tion of alanine in the body £uid was minor compared
with those in other tissues Among succinate,
ala-nine and propionate, the accumulation of succinate
was greater than those of alanine and propionate in
all tissues, except the foot However, the
accumula-tion of succinate in the foot was comparable to that
of alanine
Accumulation rate of anaerobic end products
under anoxia
Data from Table 1 were used to calculate the
accumu-lation rates of anaerobic end products in tissues
Be-fore 48 h of anoxic exposure, huge proportions of
succinate and alanine had accumulated in the
var-ious tissues However, the accumulation of
propio-nate in the tissues of hard clams before 24 h of
anoxic exposure was minor Therefore, the anoxic
period of 0^48 h was used to calculate the tion rates of succinate and alanine, while 24^64 hwas used for propionate All of the simple correlationcoe⁄cients (g) in the calculation of accumulationrates of anaerobic end products in the tissues were
accumula- 0.94, except those for alanine accumulations inthe foot (g 50.82) and adductor (g 50.80) This wasdue to large amounts of alanine accumulating in thefoot and adductor at 8 h of anoxic exposure Amongthese six tissues, the accumulation rate of succinate inthe body £uid was the highest (0.99mmol h 1mL 1),while those in the other ¢ve tissues were comparable(0.19^0.27mmol h 1
g 1wet weight) (Table 2) Morethan half of the alanine had accumulated in the foot andadductor muscle at 8 h of anoxic exposure (Table 1).Therefore, accumulation rates of alanine inthe foot and adductor muscle were di⁄cult to com-pare with those in the other three tissues The order
of the accumulation rates of alanine in tissues wasthe mantle (0.18mmol h 1g 1wet weight)4digest-ive gland (0.13mmol h 1g 1 wet weight) 4gills(0.04mmol h 1g 1wet weight) The order of the accu-mulation rates of propionate in tissues was body
£uid (0.22mmol h 1mL 1)4gills (0.12 mmol h 1g 1wet weight)4mantle (0.08 mmol h 1g 1wet weight)4foot (0.07 mmol h 1g 1 wet weight) 5 adductormuscle (0.07mmol h 1g 1wet weight) 4digestivegland (0.04mmol h 1
30
Dissolved oxygenDigging indiceSuccinatea
aa
a
dde
ee
Figure 3 E¡ects of the dissolved oxygen (DO) concentration on the digging index and concentration of succinate in thebody £uid of hard clams in aquaria (30 clams per aquarium) in which 50% of the water surface was covered at 25 1C Therewere six replicates in the measurements of DO (an aquarium per replicate), the digging index (an aquarium per replicate)and succinate in the body £uid (¢ve clams per replicate) Data are given as the mean with the standard error Data withdi¡erent letters signi¢cantly di¡er at di¡erent exposure times (Po0.05) The arrow indicates the position at the critical DOconcentration
Trang 9Succinate and alanine in tissues and digging
indices of clams during the recovery period
Rapid declines in the concentrations of succinate and
alanine were observed in these tissues after 8 h of
re-covery, except for alanine in the adductor (Fig 4)
During aerobic recovery, the concentrations of
meta-bolites returned to basal levels: succinate within 8 h
in the digestive gland and foot, and 24 h in the
adduc-tor, mantle and body £uid, while restoration in the
gills required 48 h (Fig 4) In the digestive gland, foot
and mantle, alanine concentrations had returned to
basal levels within 8 h, while 48 h was required in
the gills The fastest recovery rate for succinate was
in the body £uid (2.84mmol h 1
mL 1), followed bythe digestive gland (2.05mmol h 1g 1wet weight),
with the slowest rate in the foot (0.56mmol h 1g 1
wet weight) (Table 2) However, the recovery rate of
alanine in the foot was the highest compared with
those in other tissues No recovery rate of alaninewas found in the adductor The pro¢le of digging in-dices of clams was similar to that of succinate concen-trations in the body £uid After 8 h of recovery, theconcentration of succinate in the body £uid had de-creased signi¢cantly (Fig 4f), and the digging indices
of clams had increased signi¢cantly (Fig 5) cant changes in the succinate concentration and dig-ging indices were also observed at 8^24 h of recovery
Signi¢-DiscussionTissue-speci¢c substrates of anaerobicmetabolism
The accumulation of succinate was accompanied bythat of alanine in all tissues of the hard clam (M lusor-ia), except the gills, after 24 h of anoxic exposure A ra-pid increase in the concentration of succinate withoutalanine accumulation was found in the gills This indi-cates that carbohydrates contribute to anaerobic en-ergy production in the gills However, equal amounts
of succinate and alanine had accumulated in the foot
of clams after 64 h of anoxic exposure, indicating thatamino acids might contribute to anaerobic energyproduction in the foot, as the succinate formed was de-rived from the carbon skeletons of amino acids The di-gestive gland, adductor and mantle of hard clamsseem likely to utilize both carbohydrates and aminoacids as energy sources during anaerobic metabolism
In general, aspartate pools represent the major lar energy source mobilized in early anoxia (Zurburg
cellu-& de Zwaan1981; Eberlee, Storey cellu-& Storey1983; stone 1983), and other amino acids, such as alanineand glycine, acquire importance as energy substratesduring subsequent recovery in normoxia (Eberlee et al.1983) However, Chiou et al (1998) found a 24% de-crease in glutamate and a 60% increase in alanine aswell as no signi¢cant change in aspartate in the ediblemeat of hard clams after 5 days of aerial exposure at
Living-20 1C Therefore, glutamate may play a certain role inthe anaerobic metabolism of hard clams
Unique anaerobic metabolism in gillsThe succinate that accumulated in the gills is not anintermediate of the glucose^succinate or aspartate^succinate pathways Succinate might be derived frommalate, a product of malic enzymes This speculationwas supported by the observation that high activities
of malic enzymes were found in the gills of the ribbed
Table 2 Accumulation and recovery rates of anaerobic
end products calculated from data in Table 1 and Fig 4
g 1wet weight) c
Recovery rate after 8 h of normoxic exposure (lmol h 1
g 1wet weight)
The accumulation of anaerobic end products was calculated by
subtracting the mean value of ¢ve replicates at 0 h from those at
each sampling time The time period for calculating the
accumu-lation rates of succinate and alanine was 0^48 h, and that for
propionate was 24^64 h.
Units of body £uid are mmol h 1
mL 1
g is the simple correlation coe⁄cient for the calculation of the
Trang 10mussel Modiolus demissus, Mytilus edulis and
Crassos-trea virginica (Paynter, Karam, Ellis & Bishop 1985;
Brodey & Bishop 1992) Optimum pH values for
ma-late and pyruvate utilization by malic enzymes in
the gills of M demissus were 8.5 and 6 respectively
(Brodey & Bishop 1992) Therefore, the direction of
the reaction catalysed by malic enzymes in gill
tis-sues of clams under anoxia would favour the
produc-tion of malate, which is then reduced to succinate
(Skorkowski 1988) The accumulation of succinate
was also solely observed in the gills of the commoncockle Cardium edule after anoxic exposure (Meinar-dus & Gde 1981)
Relationships among the DO concentration,digging behaviours and anaerobic metabolism
A low DO (o2 mg O2L 1) was found to reduce theburial depth of Macoma balthica (Long et al 2008)
b
bb
Recovery after 64 h anoxic exposure (h)
010
20
30
40
ba
b
c
SuccinateAlanine
010
Units of body £uid aremmol mL 1
Trang 11Our previous study showed that magnesium ions are
important when hard clams bury themselves in
sedi-ments (Lee et al 2007) In Mg21-free ASW, an
in-crease in the concentration of succinate in the body
£uid of hard clams indicated that they were
experien-cing hypoxic stress This stress causes a decrease in
the burial depth of hard clams that emerge from the
sediment to a higher position in search of oxygen In
this study, we provide more evidence to show the
strong relationships between DO concentrations in
the seawater, and burial behaviour and anaerobic
metabolism of hard clams.When hard clams were
ex-posed to a DO of 1.11mg O2L 1, anaerobic
meta-bolism was initiated, succinate began to accumulate
in the body £uid and the clams eventually emerged
from the sediment Once hard clams had been
ex-posed to anoxia for 64 h and began to recover, the
concentration of succinate that had accumulated in
the body £uid began to decline, and burial behaviour
was again observed The phenomenon of a decrease/
increase in the concentration of succinate in the body
£uid coinciding with an increase/decrease in the
burial depth of clams suggests that the burial
beha-viour of hard clam is controlled by the concentration
of succinate in the body £uid
Occurrence of low DO in bottom water
Generally, the extensive cultivation of ¢lter-feeding
bivalves is considered to have low impacts on the
en-vironment, as their production is dependent on in
situ phytoplankton productivity (Naylor, Goldburg &Primavera 2000) However, intense ¢ltration result-ing in the production and subsequent deposition
of faeces and pseudofaeces increases inputs of labileorganic matter to the super¢cial sediments (Graf &Rosenberg 1997) Furthermore, mussel farmingwas reported to induce intense biodeposition oforganic matter onto underlying sediments, whichconsiderably stimulates the sediment oxygen de-mand (Nizzoli,Welsh, Bartoli & Viaroli 2005; Manga-naro, PulicanoØ, Reale, San¢lippo & SaraØ 2009) The
DO concentration in the bottom layer of clam pondscan decrease due to the respiration of benthic organ-isms and microorganisms and the oxidation oforganic matter in the sediments, while it can be in-creased by the photosynthesis of microalgae and bycurrents created by the wind However, most microal-gae in the bottom layer are taken up by the intense
¢ltration of bivalves Therefore, oxygen consumption
in the bottom layer of a clam pond cannot be fullycompensated for by the input of oxygen moleculesfrom photosynthesis and from the air Indeed, a low
DO concentration range of 1.5^3.5 mg O2L 1was served in the bottom water of a clam pond at 24 1C inthe early morning (unpubl obs.) The concentration of
ob-DO in the bottom water can be determined using thewater sampling design presented in this study Thisdesign allows one to easily sample water of the bot-tom layer and at other depths in a pond Flushingnitrogen through the tubes and DO bottles is abso-lutely necessary when the concentration of DO is atthe level of 1mg O2L 1 Without nitrogen £ushing,50^70% higher DO concentrations may be obtained
Application of this study to the management
of clam cultureThe results of this study reveal the relationship be-tween digging behaviour and anaerobic metabolism
of hard clams, and the DO concentration of seawater.Based on the occurrence of hypoxia in the bottomwater, ¢ve suggestions are provided for clam culturemanagement
Maintaining the DO of the bottom water at41.11mg O2L 1
According to the anoxic indicator of succinate in thebody £uid, the critical concentration of DO for hardclams is 1.11mg O2L 1 Anaerobic metabolism is in-itiated at a DO ofo1.11mg O2L 1 Therefore, theconcentration of DO in the bottom water of clam
Figure 5 Digging indices of hard clams during 48 h of
recovery after 64 h of anoxic exposure Hard clams were
kept in aquaria (30 clams per aquarium) with aeration
and an open surface at 25 1C There were three replicates
in the experiment (an aquarium per replicate) Data are
gi-ven as the mean with the standard error Data with
di¡er-ent letters di¡er signi¢cantly at di¡erdi¡er-ent exposure times
Trang 12ponds should be maintained above this critical
con-centration Increasing the air^water exchange rate
can elevate the DO concentration in the bottom layer
of pond water (Broecker & Peng 1974)
Managing clam culture in times of high water
temperatures
Although metabolic rate depression is adopted by
mollusks after anoxic exposure, an increase in the
temperature can disturb this depression The
phe-nomenon of depression being relieved by high
tem-peratures is supported by the observation that the
median lethal time (Lt50) of hard clams (M lusoria)
ex-posed to anoxia decreased considerably with
increas-ing temperature The Lt50values at 15, 20, 25, 30 and
34 1C were 15.7, 9.7, 3.8, 2.8 and 2.5 days respectively
(Lee et al 2008) The fact that the di¡erence in the
Lt50(at 6 days) between 15 and 20 1C is much greater
than that (at 0.3 days) between 30 and 34 1C indicates
that metabolic rate depression is relieved by high
tem-peratures The anoxia-tolerant ability of hard clams
should decrease dramatically at high temperatures
When a DO ofo1.11mg O2L 1in the bottom water
of clam pond is found, e¡orts to elevate the DO
con-centration should be made in a timely manner
Monitoring the glycogen content in tissues of the hard
clam
Even though the aspartate pools are a major cellular
energy source of clams under anoxia, their amino
groups need to be transferred to pyruvate produced
by glycolysis and to form alanine Therefore, the
en-ergy demands of anoxia-tolerant mollusks are mostly
met by relying on the degradation of glycogen
Signif-icant mortalities of juvenile Mercenaria mercenaria
occurred when the carbohydrate content declined to
a level at or below 10% of their total dry weight
(Zar-noch & Schreibman 2008) The survival of mollusks
under anoxia increases with increasing content of
glycogen in their tissues The content of glycogen in
tissues can be used as an indicator of the vitality of
hard clams, although data on the content of glycogen
that allows them to survive are scant
Monitoring the concentration of succinate in the body
£uid
A concentration of 4.4mmol mL 1succinate can be
used as an indicator of anoxic stress Determination
of the concentration of succinate in the body £uid that
indicates anoxic stress of clams is an alternative way of
determining the DO concentration in the bottomwater The former more clearly shows the in situ an-oxic condition of clams than the latter, because a DOconcentration at 1.11mg O2L 1needs to last for someperiod before anaerobic metabolism is initiated.We re-cently developed a kit with a colour reaction to deter-mine the concentration of succinate in the body £uid
of clams (Pan 2009) This kit is very easy to use at clamponds The body £uid is collected from a clam and di-luted 50-fold with 100 mM glycyl glycine bu¡er at pH8.0 Fifty microlitres of the diluent is added to the kit,and the reaction takes 15 min The blue colour shown
by the kit indicates that a hard clam is experiencingnormoxia However, a light-yellow colour indicatesthat the hard clam is exposed to hypoxia This work
in the laboratory has been straightforward so far
Maintaining the sediment in as ‘young’a condition aspossible
Large amounts of organic matter and many bacteriaaccumulate in ‘aged’ sediments, and they consumemuch of the available oxygen This consumption canresult in low DO in the bottom waters of ponds.Therefore, the surface (10 cm in depth) of the sedi-ment needs to be renewed as frequently as possible
In addition, the addition of nitrate, an electron tor, was reported to protect the sediment surfacelayer against reducing conditions in the sediment(Meijer & Avnimelech 1999)
accep-AcknowledgmentsThe authors are grateful for the ¢nancial supportfrom the National Science Council (NSC97-2313-B-415-003-MY3) and the National Chia-Yi University(NCYU 97T001-05-06-001) of Taiwan The authorsalso thank Mr R.S Tai at the Mariculture ResearchCenter, Taiwan Fisheries Research Institute, Taishi,Taiwan, for assistance in preparing this manuscript
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Trang 15Hatch rate of channel catfish Ictalurus punctatus
sulphate pentahydrate
David L Straus1, Andrew J Mitchell1, Ray R Carter1& James A Steeby2
1 US Department of Agriculture, Agricultural Research Service, Harry K Dupree ^ Stuttgart National Aquaculture Research Center, Stuttgart, AR, USA
2 Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Belzoni, MS, USA
Correspondence: D L Straus, US Department of Agriculture, Agricultural Research Service, Harry K Dupree ^ Stuttgart National Aquaculture Research Center, Post O⁄ce Box 1050, Stuttgart, AR 72160, USA E-mail: dave.straus@ars.usda.gov
Abstract
Cat¢sh hatcheries use copper sulphate pentahydrate
(CuSO4 5H2O) as an economical control for
sapro-legniasis on eggs This study determines hatch rate
of channel cat¢sh, Ictalurus punctatus (Ra¢nesque
1818), eggs in hatching troughs containing 23.8 1C
£ow-through well water when treated with
100 mg L 1 CuSO4 5H2O (10 times the proposed
therapeutic dose) Eggs were treated daily until the
embryos reached the eyed stage Fry survival in the
control and 100 mg L 1 CuSO4 5H2O treatments
was signi¢cantly di¡erent (15% and 71%
respec-tively) This study demonstrates that there is a
consid-erable margin of safety in using CuSO4 5H2O as a
cat¢sh egg treatment to control saprolegniasis
Keywords: channel cat¢sh eggs, copper sulphate
pentahydrate, hatch rate
Introduction
Copper sulphate pentahydrate (CuSO4 5H2O) has a
multitude of uses in agriculture It is used as a
micro-nutrient in feed (swine, bovine and poultry), as a
component of fertilizers for copper-de¢cient soils, as
a footbath for dairy cattle, and as a fungicide for
crops such as grapes and potatoes It is commonly
used in earthen pond culture of the channel cat¢sh,
Ictalurus punctatus (Ra¢nesque 1818), to control
o¡-£avor caused by blue-green algae (Tucker &
Har-greaves 2004), to control the protozoan parasite
Ichthyophthirius multi¢liis (Straus 1993; Wise, Camus,
Schwedler & Terhune 2004), to control snails ing trematodes (Wise et al 2004), and to control sa-prolegniasis on cat¢sh eggs (Steeby & Avery 2005).Saprolegnia and Achlya spp are the typical water-molds or fungi responsible for saprolegniasis (Neish
vector-& Hughes 1980; Tucker vector-& Robinson 1990), whichcauses severe losses in cat¢sh hatcheries (Rogers1979) E¡ective rates of CuSO4 5H2O for treating sa-prolegniasis on cat¢sh eggs have been established(Straus, Mitchell, Carter, Radomski & Steeby 2009;Straus, Mitchell, Carter & Steeby 2009; Straus, Mitchell,Carter & Steeby 2011); an optimum treatment of
10 mg L 1CuSO4 5H2O was determined and
con-¢rmed in these studies
Copper sulphate is not approved by the US Food andDrug Administration (FDA) for use in aquaculture,but regulatory action has been deferred pending theoutcome of ongoing research to gain this approval.The e¡ectiveness and safety of CuSO4 5H2O to con-trol the given indication must be demonstrated andthe data must be submitted, reviewed and accepted
by FDA to complete each technical section of a NewAnimal Drug Approval Safety of the proposed dose(10 mg L 1 CuSO4 5H2O) to channel cat¢sh eggshas been accepted by FDA and the e¡ectiveness tech-nical section has been submitted and is under review.The purpose of the current study was to determinethe hatch rate of channel cat¢sh eggs exposed to 10times the therapeutic dose of 10 mg L 1 Cu-
SO4 5H2O in hatching troughs with £ow-throughwell water The current study provides supportivedata to FDA about the wide margin of safety of thiscompound
Aquaculture Research, 201 , 43, 14–18 doi: 10.1111/j.1365-2109.2010.02791.x
[ ] 2
Trang 16Materials and methods
Channel cat¢sh were spawned at the Harry K
Du-pree ^ Stuttgart National Aquaculture Research
Cen-ter (SNARC); the experimental hatching trough
system and methods have been described previously
(Straus, Mitchell, Carter, Radomski et al 2009; Straus,
Mitchell, Carter & Steeby 2009; Straus et al 2011)
Brie£y, troughs were divided into nine separate
com-partments each containing 35 L of ¢ltered (75mM)
well water that £owed through each compartment
continuously at a rate of 1.25 L min 1(28 min
ex-change) Each compartment had a rotating paddle to
provide water circulation, a plastic mesh basket to
hold the eggs until hatching, and a water inlet and a
stand-pipe covered with a deep-water release drain
Similar portions (69.2 13.0 g; mean SD) were
obtained from egg masses (o24 h old) and were
placed in baskets of individual compartments and
ac-climated for 1h The adhesive matrix on the eggs
re-mained intact Smaller samples (15.4 3.7 g) from
the original egg mass were weighed, and the
adhe-sive matrix was dissolved with 1.5% sodium sul¢te
(Sigma Chemical Co., St Louis, MO, USA) in order to
count eggs These counts and weights were used to
estimate the number of eggs in each 69.2 g portion
Triangle Brandscopper sulphate (CuSO4 5H2O;
Freeport-McMoRan Copper & Gold, Phoenix, AZ,
USA) was used in this study There was one
concen-tration of CuSO4 5H2O (100 mg L 1) and a control
treatment (no CuSO4 5H2O added) A 140 g L 1
Cu-SO4 5H2O stock solution was prepared with
deio-nized (DI) water; amber vials were prepared for
dosing with either a 25 mL aliquot of stock solution
or DI water (control) Doses were administered at
24 h intervals until any embryo in the experiment
de-veloped eyes
Saprolegniasis occurred through natural
infesta-tion and daily growth (cm2) was recorded Fungi
were veri¢ed by the presence of typical mycelia,
hy-phae or zoosporangia Samples from the controls
were cultured on yeast peptone sucrose media
(con-taining 68mg mL 1streptomycin and
chloramphe-nicol) and were identi¢ed by PCR ampli¢cation and
sequencing of the rRNA gene internal transcribed
spacer (ITS) region (White, Bruns, Lee & Taylor
1990; Straus, Mitchell, Carter, Radomski et al 2009)
Hatching took a total of 3 days, and when
com-plete, all fry were siphoned from each compartment,
preserved in 70% ethanol, and counted within 1
week to establish per cent hatch Copper sulphate
pentahydrate concentrations were determined from
water samples taken at mid-water depth 30 s afterdosing and were ¢ltered (0.22mM), acidi¢ed to 1%with concentrated nitric acid, and stored for lateranalysis Samples were analyzed with an OptimaDV2000 Perkin-Elmer Inductively Coupled PlasmaOptical Emission Spectrometer (ICP-OES; PerkinEl-mer,Waltham, MA, USA) according to Eaton, Clesceri,Rice, Greenberg and Franson (2005) standard meth-ods for Cu analysis
The pH (Orion Research 720A meter;Thermo tron, Beverly, MA, USA), total alkalinity and totalhardness (Eaton et al 2005) were measured at the be-ginning and end of the study Dissolved oxygen andwater temperature were monitored daily in eachcompartment with a YSI Model 95 meter (YSI Envir-onmental,Yellow Springs, OH, USA)
Elec-The study was conducted in a randomized blockdesign There were four troughs and each troughcontained a control and CuSO4 5H2O treatment inseparate compartments (n 5 4); replicates were fromdi¡erent egg masses Survival was analyzed using ageneralized linear mixed model inSAS(Proc GLIM-MIX; version 9.1.3, SAS Institute, Cary, NC, USA) totest for a group e¡ect The model ‘treatment’ used the
¢xed e¡ect and ‘trough’ was the random e¡ect withinthe model statement A binomial error and logit linkwere used, and the response was entered as the ratio
of the number of hatched fry divided by the lated number of eggs Treatment e¡ects were consid-ered signi¢cant at P 0.05
calcu-Results and discussionWater chemistry parameters for this study were typi-cal to that of commercial cat¢sh hatcheries Totalalkalinity and total hardness (as CaCO3) were198.0 0.0 and 106.5 1.6 mg L 1, respectively,and pH was 7.5 0.1 Temperature and DO were23.8 0.2 1C and 66.7 2.2% saturation respec-tively This was the typical seasonal temperature ofSNARC well water and is at the lower end of recom-mended hatching temperatures Temperatures in thisrange are conducive to fungal growth, but also result
in longer incubation times (Avery & Steeby 2004).Eye pigment was ¢rst seen in the embryos duringafternoon observations on day 6 and treatmentswere discontinued Fry started to hatch on day 8,and hatching was ¢nished by day 11 Two of the repli-cates were mostly hatched (480%) by day 10, butthe other two replications were only 20%hatched; the latter replicates were obviously younger
Trang 17eggs Fry survival in the control treatment was
14.5 16.0% and 70.5 16.4% in the 100 mg L 1
CuSO4 5H2O treatment There was a signi¢cant
di¡erence in fry survival between treatments
(P 5 0.0113)
The survival of each replicate of the control
treat-ment (30.2%, 0.1%, 1.4% and 26.4%) and the
Cu-SO4 5H2O treatment (62.7%, 94.9%, 59.7% and
64.8%) clearly show variability, and such variability
would be expected in commercial hatcheries Several
reasons can be given for variable hatch rates in
cat-¢sh hatcheries including: (1) susceptibility of an
indi-vidual spawn to saprolegniasis; (2) the number of
unfertilized eggs in an individual spawn and (3)
other pathogens a¡ecting an individual spawn (e.g.,
bacteria, ciliates) In a 2003 survey of the cat¢sh
in-dustry (USDA, APHIS, VS, CEAH 2003), the typical
survival in hatcheries was 79.3%.Wolters (2001) had
previously estimated egg survival in the cat¢sh
in-dustry to be 60% when formalin and iodine were
used as needed to control fungal infections
A large amount of fungus on two of the
replica-tions (11cm2) of control treatment eggs resulted in
hatch rates ofo2%; the remaining two replications
had 6 and 8 cm2of fungus and hatch rate averaged
28% In previous e¡ectiveness studies under similar
conditions, control treatments had hatch rates of 2%
(Straus, Mitchell, Carter & Steeby 2009), 8% (Straus ,
Mitchell, Carter, Radomski et al 2009) and 4%
(Straus et al 2011) In the present study, very small
amounts of fungus were present in two of the
replica-tions in the 100 mg L 1CuSO4 5H2O-treated eggs
and this was attributed to unfertilized eggs The
other two replications did not have any fungus, but
one of these replications had a large proportion of
un-fertilized eggs which resulted in poor hatch rate
There were two sequences identi¢ed from the four
fungus samples through PCR and ITS sequence
char-acterization These sequences were 100% and 99%
homologous with Saprolegnia spp UNCW377 and
UNCW315, respectively, which are listed in GenBank
(http://www.ncbi.nlm.nih.gov/Genbank/)
The control treatments did not contain any
measured dissolved Cu and the 100 mg L 1
Cu-SO4 5H2O treatments contained 9.9 1.5 mg L 1
dissolved Cu The calculated concentration of Cu in
the CuSO4 5H2O treatment was 25.4 mg L 1;
there-fore, the dissolved Cu concentration of the treatment
was 39% of the calculated concentration The per
cent of recovered Cu was expected based on
Straus, Mitchell, Carter and Steeby (2009) where a
di-minishing amount of dissolved Cu is recovered as the
CuSO4 5H2O treatment concentration increases Aquadratic equation of this data (Straus, Mitchell, Car-ter & Steeby 2009 and the present study) is displayed
in Fig.1; this calculation is speci¢c to the study tions and water source of SNARC Copper stabilityconstants are discussed in Stumm and Morgan(1970) to describe the stability of complexes in transi-tion metal cations; copper has a high a⁄nity and willbind before other commonly present cations This ex-plains why the per cent of dissolved copper decreases
condi-as the dose is increcondi-ased As other authors have plied, the speciation and solubility of Cu complexesare di⁄cult to determine and dependent on waterchemistries (Stumm & Morgan 1996)
im-The margin of safety when using CuSO4 5H2O totreat saprolegniasis on cat¢sh eggs appears to bemuch greater than the margin of safety when usingthe FDA-approved hydrogen peroxide and formalin-containing compounds The proposed treatment ratefor CuSO4 5H2O is 10 mg L 1, so the present studyindicates it has at least a 10-fold margin of safety.The label rate for hydrogen peroxide (H2O2) is 750^
1000 mg L 1and Mitchell, Radomski, Straus andCarter (2009) reported that treatments of eggs with
2000 mg L 1H2O2and higher caused complete eggmortality, while treatment with 1000 mg L 1H2O2resulted in 11% survival However, Rach, Gaikowski,Howe and Schreier (1998) reported survivals were78%, 68% and 0% at 1000, 3000 and 6000mL L 1
H2O2, respectively, under di¡erent study conditions.Therefore, the approximate margin of safety is 2- to
6-fold The label rate for formalin is 1000^
2000mL L 1, and Rach, Howe and Schreier (1997)reported that treatments of 1500, 4500 and
7500mL L 1resulted in survivals of 100%, 25% and
Figure 1 Relationship between measured dissolved Cuand total Cu added when treating with 2.5, 5, 10, 20, 40and 100 mg L 1CuSO4 5H2O The quadratic equation is
y 5 0.0171x210.8056x10.4455; r2599.3%
Hatch rate of eggs treated with CuSO4 DL Straus et al Aquaculture Research, 201 , 43, 14–18
[ ]
2
0246810
0 5 10 15 20 25
Total Cu (mg / L)
Trang 180%; therefore, the approximate margin of safety is
o3-fold
Current approved treatments for fungus control on
cat¢sh eggs are expensive and have potential human
health issues for hatchery personnel (see Material
Safety Data Sheets) Copper sulphate pentahydrate is
an inexpensive treatment that is safe to use and
e¡ec-tive In addition to the low cost of CuSO4 5H2O, it
does not have the human health implications or
sto-rage precautions associated with hydrogen peroxide
or formalin
Savings on production costs are critical to the US
farm-raised cat¢sh industry because of increased
costs of feed, fuel and labour, and the competition in
domestic markets with low-priced imported ¢sh The
di¡erence in cost of treating eggs to control fungus
with the recommended rate of 10 mg L 1
Cu-SO4 5H2O and the FDA-approved label rate (FDA
2008) of 750^1000mL L 1 hydrogen peroxide or
1000^2000mL L 1formalin compounds is
consider-able Based on current prices (August 2010; USD) and
using the lowest concentration, a typical 378 L
(100 gallon) hatching trough would cost US$0.01 per
treatment with CuSO4 5H2O, but US$0.45 and
US$0.72 per treatment with hydrogen peroxide and
formalin respectively Savings to the cat¢sh industry
make CuSO4 5H2O a very advantageous option
In conclusion, treating channel cat¢sh eggs with
10 times the recommended dose of 10 mg L 1
Cu-SO4 5H2O did not appear to adversely a¡ect the
hatch rates in this study Therefore, the present study
demonstrates that there is a considerable margin of
safety in using CuSO4 5H2O as a cat¢sh egg
treat-ment to control saprolegniasis
Acknowledgments
We thank Freeport-McMoRan Copper & Gold
(for-merly Phelps Dodge) for supplying the Triangle
BrandsCopper Sulphate Cindy Ledbetter and
sum-mer student Christen Proctor set up the experiment,
took care of the daily activities and counted fry at the
end of the study Special thanks to Dale Jamison and
George Huskey for maintaining the channel cat¢sh
brood-stock for this study Larry Dorman, Bill
Hem-street, Jerry Ludwig, Bart Green and Les Torrans
pro-vided critical reviews of the manuscript Mention of
trade names or commercial products in this article is
solely for the purpose of providing speci¢c
informa-tion and does not imply recommendainforma-tion or
endorse-ment by the US Departendorse-ment of Agriculture
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on channel cat¢sh eggs at a commercial hatchery Journal
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on channel cat¢sh eggs Journal of Aquatic Animal Health
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J.A Hargreaves), pp 215^278 Elsevier Science Publishers
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ky & T.J White), pp 315^322 Academic Press, New York,
NY, USA.
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Hatch rate of eggs treated with CuSO4 DL Straus et al Aquaculture Research, 201 , 43, 14–18
[ ]
2
Trang 20Heritability for growth traits in giant freshwater prawn,
best linear unbiased prediction methodology
Nissara Kitcharoen1, Wikrom Rungsin2, Skorn Koonawootrittriron3& Uthairat Na-Nakorn4
1 Department of Aquaculture, Graduate School, Kasetsart University, Bangkok,Thailand
2 Department of Zoology, Faculty of Science, Kasetsart University, Bangkok,Thailand
3 Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok,Thailand
4 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok,Thailand
Correspondence: U Na-Nakorn, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand E-mail: ⁄surn@ku.ac.th
Abstract
In this study, heritability was estimated for
growth-re-lated traits of giant freshwater prawn (Macrobrachium
rosenbergii) before and after morphological sexual
dif-ferentiation Estimation was made on data from16
full-sib and eight half-full-sib families The variance estimation
was performed using a univariate mixed linear animal
model and variance components were analysed
fol-lowing an animal model using restricted maximum
likelihood procedure using average information
algo-rithm Heritability estimates (h2) varied considerably
with ages At 2 months old, h2for carapace length
(CL; 0.35 0.15) and body weight (BW; 0.26 0.13)
were higher than those estimated at 5 months old,
based on mixed sex data However, when data were
sorted by sex, h2calculated from data of females were
higher than those of males for CL (0.26 0.16 vs
0.10 0.06), BW (0.28 0.17 vs 0.12 0.08), body
length (0.40 0.17 vs 0.11 0.07), total length
(0.47 0.18 vs 0.11 0.07) and claw length
(0.29 0.16 vs 0.03 0.04) The same trend was
ob-served for traits at 6 months old in both bulk and
indi-vidual rearing
Keywords: heritability, growth, Macrobrachium
rosenbergii
Introduction
Aquaculture of giant freshwater prawn (GFP)
(Macrobrachium rosenbergii de Mann 1879) is of
eco-nomically important (e.g average annual production
of 4200 000 tonnes as of 2007 (FAO 2009) withthree major contributors, China, Thailand and India(New 2005) Despite a long culture history, only afew genetically improved strains have been estab-lished (e.g Charoenpokphand strain, Anonymous2001)
Heritability (h2), which refers to the proportion ofadditive genetic variance to phenotypic variance of atrait, is important for the success of genetic improve-ment programme (Falconer & Mackay 1996) Previouspublications have demonstrated sex-di¡erentiatedheritability estimates for growth of juvenile GFP,whereby h2of 11 months old prawn was higher for fe-males (h250.35 0.15) than for males of which h2
was not di¡erent from zero (Po0.05, Malecha,
Masu-no & Onizuka 1984); hSD2 estimated in 5 months oldprawn were 0.018 0.014 and 0.122 0.074 re-spective to length and weight of males, while it was0.060 0.054 and 0.030 0.041respective to lengthand weight of females (Uraiwan, Sumanojitraporn &Ampolsak 2002) As such, e⁄ciency of selection per-formed after sex di¡erentiation may be compromiseddue to the confounding of male morphotypes There-fore, selection to improve these traits should be per-formed before sexual di¡erentiation and thus theestimation of h2at this stage is necessary Moreover,the estimation of h2mentioned previously, used statis-tical models disregarding systematic errors (e.g ef-fects of age, sex, pond-cage, farm, feed, Gjedrem &Olesen 2005) which are di⁄cult to avoid
Trang 21The present study used a mixed model equation
(MME, Henderson 1975) for the estimation of h2
for growth-related traits of GFP To elucidate
con-founding e¡ect of morphotypes, the estimations were
made at three stages; before sex di¡erentiation
(2 months old), after sex di¡erentiation but before
morphotype di¡erentiation (5 months old) and at
commencement of morphotype di¡erentiation (6
months old) The MME model is considered as best
linear unbiased prediction (BLUP, Henderson 1975)
capable of separating random and systematic errors
and hence is expected to give a precise estimation of
h2 Besides, we examined impacts of two rearing
con-ditions (RC), pooled full-sib family and individual
rearing, on h2values
Materials and methods
Preparation of brooders and mating
The 200 GFP brooders were collected from Pasaak
Chonlasidth Dam, Lop Buri Province They probably
originated from the domesticated stock released by
the Department of Fisheries (Panuwat Kamutchart,
pers com.) They were checked for two virus strains,
MrNV (M rosenbergii nodavirus) and XSV
(extra-small virus) using reverse transcriptase polymerase
chain reaction and only the virus-free individuals
were used A total of eight males was successfully
mated to 16 females at a ratio of one male to two
females following a single pair mating scheme
resulting in 16 full-sib families and eight half-sib
families
Larval rearing
Each family of larvae was separately reared in 250 L
tanks, fed with brine shrimp (Artemia spp.) three
times daily until reaching the post-larva (PL) stage
At 2 weeks after reaching the PL stage, they were
transferred to two types of rearing
(communal-with-in each family, and (communal-with-individual rear(communal-with-ing) The pooled
full-sib family rearing was performed in concrete
tanks (2 1 0.5 m3
) wherein each tank was domly stocked with 200 prawns family 1with two
ran-replications Two replications of individual rearing
were performed at 5 months old when 20 individuals
(10 males and 10 females) per replication were
solita-rily randomly stocked in a round plastic box (20 cm
40 prawns were randomly sampled from eachreplication and measured for carapace length(CL: from an ocular lobe to a carapace groove), bodylength (BL: from ocular lobe to telson), total length(TL: from an ocular lobe to a tail), claw length (ClL)and body weight (BW)
All individually reared GFP were measured at 6months old
Data analysesThe dataset composed of individual pedigree andgrowth traits measured from 1280 GFP at 2 and 5months old, and 1824 GFP at 6 months old (progeny
of 16 full-sib and eight half-sib families, pooled andindividually reared) Descriptive statistics of thedataset (Table 1) were calculated using the program
SAS(SAS 2003) Variance component estimation wasperformed using a univariate mixed linear animalmodel The model is written in matrix notation
as follows:
Y¼ Xb þ Za þ Wc þ ewhere y is a vector of observations (at 2 monthsold: CL and BW; at 5 and 6 months old: CL BW, BLand ClL); b is a vector of ¢xed e¡ects [at 2months old: hatching months (HM); at 5 months old:
HM and sex; at 6 months old: HM, sex and RCs]; c is avector of random e¡ect of common environment
full-sib family; a is a vector of animal additive genetice¡ects; e is a vector of residual e¡ects; X, Z and
W are incident matrices assigning the observations
to levels of b, a and c respectively The assumptionwas as follows:
y a c e
2 6 4
3 7
5 NID;
Xb 0 0 0
2 6 4
3 7 5;
3 7 5
0 B
@
1 C A
when G 5 Asa2, A is a numerator relationship matrix(Henderson 1975), C ( 5Isc2) is a common environ-mental matrix and R ( 5Ise2) is a residual variancematrix
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 19–25 20
Heritability for growth traits in GFP N Kitcharoen et al Aquaculture Research, 201 , 43, 19–25 2
Trang 22Variance components for each trait were estimatedfollowing the animal model using a restricted maxi-mum likelihood procedure (REML) using average in-formation algorithm Then the variance componentestimates were used for calculation of heritability fol-lowing the formula h25sa2/sp2 Breeding value foreach trait of the individual GFP was predicted usingBLUP The calculation was facilitated by a computerpackageASREML (Gilmour, Gogel, Cullis, Welham &Thompson 2002).
ResultsOverall, the length of incubation and larval develop-ment were very consistent among families Descrip-tive statistics for CL, BL, TL, ClL and BW at di¡erentstages are shown in (Table 1) It was obvious that var-iance on growth traits increased with ages Maleprawns grew faster than female and the variance ongrowth traits was larger In general, the pooled full-sibs prawn, which was directly stocked in tanks, grewfaster than individually reared prawns It was note-worthy that the latter group showed larger variationthan the communally reared prawns except for BW
Heritability and correlationHeritability of growth-related traits varied consider-ably with ages At 2 months after reaching the PLstage, h2of CL (0.35 0.15) and BW (0.26 0.13)was high At 5 months, h2of CL and BW, based onmixed sex data, was lower than those estimated at 2months old (Table 2) However, when data weresorted by sex, h2calculated from females data was as
0.26 0.16 for CL, 0.28 0.17 for BW, 0.40 0.17for BL, 0.47 0.18 for TL and 0.29 0.16 for ClL)and was especially higher than those of males (h2
was for 0.10 0.06 for CL, 0.12 0.08 for BW,
0.03 0.04 for ClL) The same trend was observed at
6 months for both rearing methods (bulk and dual rearing), whereby, h2 estimated from femaledata was higher than those of males However, the es-timation made from individual rearing showed highand unacceptable standard errors (SE) Notably, theproportion of c2to total variance for all estimationwas small (ranged 0^0.08)
indivi-Most of the phenotypic correlations of all related traits at 2, 5 and 6 months were high and sta-tistically signi¢cant (0.54^0.97; Table 3)
Trang 23Although the h2of growth-related traits of
crusta-cean tremendously varied, but the majority indicated
intermediate heritabilities in the range of 0.20^0.84
(e.g in Penaeus vannamei, Perez-Rostro & Ibarra
2003; Argue, Arce, Lotz & Moss 2005; Gitterle, Rye,
Salte, Cock, Johansen, Lozano, Suarez & Gjerde 2005;
De Donato, Manrique, Ramerez, Mayer & Howell
2005; Castillo-Juarez, Casares,
Campos-Montes,Ville-la, Ortega & Montaldo 2007; Penaeus japonicas, Hetzel,
Crocos, Davis, Moore & Preston 2000; Penaeus
mono-don, Benzie, Kenway, Trott & 1997; Kenway, Macbeth,
Salmon, McPhee, Benzie,Wilson & Knibb 2006) This
was true for the estimations made at 2 months
(mixed sex) and 5 and 6 months estimated from
fe-males GFP in the present study, despite of low h2
esti-mates in males The h2estimates of growth-related
traits of GFP reported here together with those
reported by Malecha et al (1984) (h250.35 0.15),
Uraiwan et al (2002) (h250.254 0.080 and
0.272 0.210) and a report on the high additive
ge-netic variance of GFP based on a diallel crossing
(Thanh, Ponzoni, Nguyen, Vu, Barnes & Mather
2010) may imply that the h2for growth of GFP is
mod-erate to high in general
Heritability of growth-related traits of mixed sexGFP declined with age (e.g hF1M2 for CL at 2 and 5months of PL stage 5 0.35 0.15 and 0.12 0.75 re-spectively), which was in accordance with those re-ported by Malecha et al (1984); Meewan (1993) andUraiwan et al (2002) This may be partly explained
by an aggressive behaviour of particularly maleprawn, which di¡erentiates into three morphotypes(Meewan 1993; Ranjeet & Kurup 2002; Karplus & Hu-lata 2005; Thanh, Ponzoni, Nguyen, Vu, Barnes &Mather 2009) at about 5 months after reaching the
PL stage The di¡erentiation of morphotypes hanced variation in growth capacity, especially ofmales
en-Heritability of GFP was sexually dimorphic, high
in females and low in males (e.g h2of CL at 5 months
of PL stage was 0.10 0.06 for male and 0.26 0.16for female) Similar ¢nding was also reported byMalecha et al (1984) Likewise, Thanh et al (2009)reported that growth variation, and relative frequen-cies among male morphotypes of GFP masked theamong-strain growth di¡erence for male while itwas pronounced in females Despite limited numbers
of studies, sexual dimorphic heritabilities were alsoreported for other crustaceans, [e.g h2for growth-related traits was higher for the immature male than
Table 2 Heritability (h 2
SE) and ratio of common environmental variance/total variance (cratio SE) for carapace length, body length, total length, claw length and body weight of Macrobrachium rosenbergii at 2, 5 and 6 months after reaching the post-larva stage, by sex and rearing conditions
Age
(months) Sex
Rearing conditions
Carapace length
Body length
Total length
Claw length
Body weight
5 Mixed sexes 0.12 0.75 0.18 0.12 0.16 0.08 0.02 0.05 0.11 0.08
(0.022 0.017) (0.018 0.016) (0.007 0.012) (0.033 0.022) (0.043 0.024) Male – 0.10 0.06 0.11 0.07 0.11 0.07 0.03 0.04 0.12 0.08
(0.000 0.000) (0.003 0.017) (0.000 0.000) (0.000 0.000) (0.011 0.022) Female – 0.26 0.16 0.40 0.17 0.47 0.18 0.29 0.16 0.28 0.17
(0.080 0.050) (0.029 0.035) (0.007 0.029) (0.053 0.041) (0.104 0.058)
6 Mixed sexes 1 0.24 0.13 0.20 0.11 0.18 0.10 0.15 0.10 0.20 0.71
(0.000 0.000) (0.000 0.000) (0.000 0.000) (0.000 0.000) (0.000 0.000)
2 0.15 0.08 0.16 0.08 0.15 0.08 0.01 0.02 0.07 0.04 (0.006 0.012) (0.001 0.010) (0.003 0.011) (0.015 0.023) (0.003 0.010) Male 1 0.10 0.10 0.03 0.06 0.01 0.05 0.05 0.09 0.20 0.49
(0.000 0.000) (0.000 0.000) (0.000 0.000) (0.000 0.000) (0.000 0.000) Female 1 0.42 0.19 0.46 0.20 0.47 0.20 0.27 0.15 0.26 3.09
(0.000 0.000) (0.000 0.000) (0.000 0.000) (0.000 0.000) (0.000 0.000) Male 2 0.05 0.05 0.05 0.05 0.06 0.06 0.00 0.00 0.03 0.04
(0.008 0.021) (0.014 0.022) (0.015 0.023) 0.011 0.017) (0.002 0.018) Female 2 0.54 0.19 0.52 0.19 0.51 0.19 0.10 0.07 0.33 0.14
(0.000 0.000) (0.000 0.000) (0.000 0.000) (0.0100 0.071) (0.000 0.000)
1, individual rearing; 2, bulk rearing; SE, standard error.
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 19–25 22
Heritability for growth traits in GFP N Kitcharoen et al Aquaculture Research, 201 , 43, 19–25 2
Trang 24female in Red Swamp craw¢sh, Procambarus clarkii
(Girard), Lutz & Woltress 1989], and ¢sh (El-Ibiary &
Joyce 1978; Crandell & Gall 1993)
Despite the well-documented advantage of BLUP
over the analysis of variance (ANOVA)-based analysis
(Gjedrem & Olesen 2005), the application of BLUP to
aquatic animals was well documented back to only
the last decade [e.g Chinook salmon, Oncorhynchus
tshawytscha (Winkelman & Peterson 1994a) Nile
tilapia, Oreochromis niloticus (Gall & Bakar 2002;
Charo-Karisa, Komen, Rezk, Ponzoni, van Arendonk
& Bovenhuis 2006), Paci¢c white shrimp, Litopenaeus
vannamei (Gitterle et al 2005; Castillo-JuaŁrez et al
2007), Coho salmon, Oncorhynchus kisutch (Neira
et al 2006), common carp, Cyprinus carpio L (Wang
& Li 2007), Atlantic salmon, Salmo salar (Powell,
White, Guy & Brotherstone 2008)] The present studywas the ¢rst attempt to use BLUP for estimating h2inthe GFP According to the prior information obtainedfromANOVA(data not shown), major factors a¡ectingthe present estimation of h2were HMs, tanks, e¡ect
of sex at 5 and 6 months and RCs (bulk and dual rearing) Therefore, these factors were in-corporated into the model in order to separate thee¡ects of these factors from genetic e¡ect (Gjerdrem
indivi-& Olesen 2005) and thus enhanced precision ofthe estimation It is noteworthy that, in our study,the variation of hatching time (months) waslarge (covering a period of 5 months), but with theBLUP approach, precision of the study was improvedover the studies with less variation of spawning time[e.g within a week (Malecha et al 1984; Meewan1993)]
It was our concern that the number of families(16 full-sib families) used in the present study wassmaller than those used in a majority of the studies[e.g 50 full- and half-sib families nested with 16 sires(Malecha et al 1984); 430 full-sib families in Paci¢cwhite shrimp, L vannamei (Gitterle et al 2005)].However, it meets the range of the recommendednumbers [e.g 20^30 families (Robertson 1959);
n 5 2/h2for full-sib families and n 5 4/h2for half-sibfamilies (Falconer & Mackay 1996)] The weaknessdue to a consequence of small number of familiescould be partly compensated by relatively largefamily size (80 prawns family 1for the pooled rearedGFP140 prawns family 1 for the individuallyreared GFP) As a result, regarding SE, we obtainedbetter precision of h2 of female and male BW at
5 months (h250.28 0.17 and 0.12 0.08 tively) than Malecha et al (1984) based on 50 full-sib families (hS250.34 0.24 and 0.24 0.11 forfemale and male respectively)
respec-Based on our experimental design that eachfamily was reared in duplicate, our animal modelconsidered the random common environmentale¡ect in addition to the random additive genetice¡ect and they were assumed to have no covariation
As such, the e¡ect of common environment wastheoretically removed and hence resulted in amore congruent estimation (Winkelman & Peterson1994b) Thus, h2 in this present study can beconsidered without any confounding e¡ect ofcommon environmental e¡ect (c2) However, it is ofconcern that the limited population size andthe small genetic relationship available for the ana-lyses may cause high SEs of the h2estimates in thepresent study
Table 3 Phenotypic correlation (rP , below diagonal) for
car-apace length (CL), body length (BL), total length (TL), claw
length (ClL) and body weight (BW) of Macrobrachium
rosen-bergii at 2, 5 and 6 months after reaching the post-larva
BW 0.58 0.57 0.57 0.54 –
Trang 25Implications for selective breeding
programmes
1 High heritability of the growth-related traits
sug-gested that a simple mass selection, in which
selection is up on individual’s performance,
traits of the GFP with a concern on high
probabil-ity of inbreeding accumulation (Falconer &
Mackay 1996) Nonetheless, this method has been
e⁄ciently used in marine shrimp (e.g 17% and
14% increase respective to survival rate and
weekly weight gain, FCR reduced by 19%,
over 11 generations of the Paci¢c white shrimp,
L vannamei, De Donato et al 2005) However, the
empirical data showed that the Paci¢c white
shrimp su¡ered from inbreeding accumulated
over 22 years of family selection (Moss, Arce,
Otoshi, Doyle & Moss 2007) which, theoretically,
is a better selection method to control
accumula-tion of inbreeding than mass selecaccumula-tion (Falconer &
Mackay 1996) Therefore, inbreeding depression
should be concerned if a mass selection is used to
improve GFP
2 Selection may be performed on mixed sexes
prawn at 2 months after reaching the PL stage
because heritability is relatively high However, it
is of concern that the growth pro¢le of the selected
individuals may change as they grow up
According to our unpublished data, we observed
high correlation between growth-related traits
at 2 and 7 months after reaching the PL
stage (r 5 0.70 for CL, Po0.001; r 5 0.60 for BW,
reared prawns Therefore, it is also recommended
that a selection to improve growth of GFP may be
performed at 2 months after reaching the PL
stage
3 Selection performed after sex di¡erentiation
should be exerted on females, by selecting
on either CL, BL or TL According to the
present study, male BW is not a good target for
selection due to relatively low h2 It is of
concern that the correlation of CL and other
traits was high Therefore, selection for increasing
BL or TL may increase the CL, which is
unde-sirable
4 High h2estimate for CL indicated a possibility of
selection for smaller head However, other
growth-related traits may also decline as
sug-gested by the high positive correlation between
these traits and CL
AcknowledgmentsThis research was supported by three fundingsources, Thailand Research Fund (the Senior Re-search scholar Program 2007: project RTA 5080013awarded to U Na-Nakorn), the Royal Golden JubileePhD scholarship awarded to Nissara Kitcharoenand Kasetsart University We would like to thankmembers of Laboratory of Aquatic Animal GeneticsKasetsart University and Mr Suwinai Pannkao fortechnical assistances Finally, we thanked the anon-ymous referees whose comments signi¢cantly im-proved the manuscript
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Trang 27Preliminary trials on the use of large outdoor tanks for
Pedro Domingues1, Nelda Lo¤pez2& Carlos Rosas2
1
Centro OceanograŁ¢co de Vigo, Instituto Espanol de Oceanograf|¤a, Cabo Estai, Canido,Vigo, Spain
2
Unidad Multidisciplinaria de Docencia e Investigacio¤n, Facultad de Ciencias UNAM,YucataŁn, Me¤xico
Correspondence: P Domingues, Centro OceanograŁ¢co deVigo, Instituto Espanol de Oceanograf|¤a, Cabo Estai, Canido,36390 Vigo, Spain E-mail: pedro.domingues@vi.ieo.es
Abstract
Octopus maya has high growth rates, direct
embryo-nic development and high hatchling survival, making
it a good candidate for aquaculture diversi¢cation
The present study was designed to evaluate growth
rate, survival and food conversion of O maya juveniles
cultured in outdoor tanks Octopuses were captured
from the wild during the ¢shing season, and fed
dis-carded ¢sh heads and whole crabs Three trials were
conducted between 23 and 32 days, in September
(trial1), October (trial 2) and November (trial 3) where
a decrease in sea water temperature was registered
(29^24 1C, from September to November respectively)
Octopuses were held in three outdoor tanks of 5 m2of
bottom area and 0.5 m deep, aerated sea water and
water £ow allowing 10% of water exchange per day
Initial density was between 2.9 and 3.8 kg m 3with
di¡erent initial mean weight of 542.3 18.8,
493 11.9 and 321 7.8 g, for trials 1, 2 and 3
re-spectively Speci¢c growth rate varied between 1.8
and 2.7% BWday 1with no apparent relation with
the culture temperature These results put in
evi-dence that tanks used are adequate for the ongrowing
of O maya juveniles, with commercial size being
at-tained in a few weeks
Keywords: cephalopods, culture, growth rate, food
conversion, Octopus maya, outdoor tanks
Introduction
The study of Octopus spp culture has recently
under-gone a signi¢cant increase throughout the world,
particularly in Spain, Italy, Portugal and Greece
(Vaz-pires, Seixas & Barbosa 2004; Iglesias, Sanchez,Bersano, Carrasco, Dhont, Fuentes, Linares, Munoz,Okumura, Roo, Van Der Meeren, Vidal & Villanueva2007) The need for diversi¢cation, taking into con-sideration biological and economical aspects, makescephalopods in general and octopuses in particular,potential candidates for industrial culture
TheYucatan octopus, Octopus Maya, is an endemicspecies from theYucatan peninsula, with distributionranging from the Campeche bay, in the North of theYucatan peninsula, to Isla Mujeres (Solis1967) Lately,the range has been widened in the Northern limit toCiudad del Carmen, Campeche (Solis 1997,1998) Thisspecies accepts dead prey or prepared diets immedi-ately after being born, and easily adapts to laboratoryconditions (Rosas, Tut, Baeza, SaŁnchez, Sosa, Pasc-ual, Arena, Domingues & Cuzon 2008) Octopus mayahas been cultured in the laboratory (Van Heukelem
1976, 1977; Derusha, Forsythe, Dimarco & Hanlon1989; Solis 1998; Domingues, Lo¤pez, Munoz, Maldo-nado, Gaxiola & Rosas 2007; Rosas, Cuzon, Pascual,Gaxiola, Lo¤pez, Maldonado & Domingues 2007; Ro-sas,Tut, Baeza, SaŁnchez, Sosa, Pascual, Arena, Dom-ingues & Cuzon 2008) up to four (Hanlon & Forsythe1985) or ¢ve (Van Heukelem 1983) generations Thisspecies has fast growth rates up to 8% BWday 1,due to high feeding rates and food conversions thatvary between 30% and 60% (Van Heukelem 1983;Hanlon & Forsythe 1985; Domingues et al 2007) Oc-topus maya can grow to 1kg in 4 months at 25 1C,reaching a maximum weight at 9 months (Van Heu-kelem 1983) Also, it has direct embryonic develop-ment with high hatchling survival (Van Heukelem1976; Domingues et al 2007) These characteristicsmake this species the better candidate for commer-
© 2011 Blackwell Publishing Ltd 26
Aquaculture Research, 201 , 43, 26–31 2 doi: 10.1111/j.1365-2109.2011.02797.x
Trang 28cial aquaculture, after O bimaculoides and O digueti
(Hanlon & Forsythe 1985)
Tanks (open and close system) and £oating cages
have been proposed for O vulgaris culture in an
at-tempt to propose a standardized technology for
bio-mass production Rodr|¤guez, Carrasco, Arronte and
Rodr|¤guez (2006) showed the validity of £oating cages
to produce O vulgaris biomass at a commercial level
Nevertheless, the continental shelf of the YucataŁn
Pe-ninsula is shallow, with water depths ranging from 4
to 8 m up to 5 km from the shoreline, which does not
allow cultivation of octopuses in £oating cages
There-fore, other culture facilities should be considered
Juveniles used in these trials, weighing less than
the legal capture weight (650 g) were collected from
the wild during the ¢sheries season This is a problem
in this region, as ¢sherman frequently keep those
in-dividuals for own consumption Thus, the idea to
cre-ate a ¢sherman’s cooperative for the ongrowing of
this species with animals captured under minimum
legal emerged For this reason, the present study was
designed to evaluate variations on growth rate,
survi-val and gross food conversion of O maya juveniles
fat-tened in tanks The octopuses were fed with discarded
¢sh heads and crabs (Callinectes sp.) following the
same idea proposed for O vulgaris All of the trials
were conducted until all the octopuses in the tanks
reached the legal weight and higher market price
Material and methods
Animals
The octopuses were caught in front of Sisal harbour
(YucataŁn, Me¤xico) using artisan line with crab
(Calli-nectes sp.) as bait, between September and November
Animals weighing o650 g were separated on the
boat, and transported to the laboratory situated
300 m inland in 120 L circular tanks with sea water
No mortality was observed during the several
collect-ing trips
Tanks
The octopuses were held in a £ow-through system
composed of three outdoor tanks of 5 m2of bottom
area and 0.5 m of water depth Water £ow allowed a
10% water exchange per day Two hundred and forty
polyvinyl chloride (PVC) tubes (4 in in diameter) were
placed into the tanks as refuges The aeration was
pro-vided with six airlifts placed in the centre of the tank
(Fig 1) Salinity, temperature and dissolved oxygenwere measured every day, and total ammonia was re-corded on a weekly basis The system allowed a highdissolved oxygen concentration (45.0 mg L 1; 80%saturation level), with salinity ranging between 35.4and 36 g L 1, pH between 7.8 and 8.2 and a total am-monia concentration between 0.8 and 2.75 mg L 1.Food was provided once a day (at 08:00 hours).Tanks were cleaned and uneaten remains were re-moved and weighed once a day by siphoning at16:00 hours
Three trials were conducted: September 19 to tober 11, October 11 to November 9 and November 16
Oc-to December 16 The Oc-total number of animals usedwas 734, distributed by the three trials (234, 218 and
282 for trials 1, 2 and 3 respectively)
Octopuses were stoked in three tanks at a density
of 78, 72 and 94 octopuses per tank in trials 1, 2 and
3, respectively, with average weights of 542.3 18.8,
493 11.9 and 321 7.8 g, for trials 1, 2 and 3 spectively Initial densities were between 2.9 and3.8 kg m 3, which are considerably lower than thosereported for O vulgaris by Otero, Moxica, SaŁnchezand Iglesias (1999) and Domingues, Garcia andGarrido (2010), or Sepia o⁄cinalis (Domingues &Marque¤z 2010)
re-A mixed diet of ¢sh heads (Lutjanus spp.:16% of tal diet) and crabs (Callinectes spp.: 84% of total diet)was used during each trial, according to the follow-ing sequence: crab (100%) during the ¢rst week, crab(50%) and ¢sh heads (50%) during the second weekand crab (100%) until the end of the trial Chemicalcomposition of both ¢sh heads and crabs is shown inTable 1 Water content was 71.0 2.4% for the ¢shheads and 76.0 3.0% for the crabs
to-AB
C
Figure 1 Five-metre-diameter round outdoor tank usedfor Octopus maya experimental ongrowing A, airlift; B,PVC 4 in refuges; C, drainage
Trang 29Crude protein levels were calculated from the
de-termination of total nitrogen using LECO
auto-analy-ser, based on N 6.25 ([Association of O⁄cial
Analytical Chemists (AOAC) 1990; No Aa 5^91)
Crude fat content of the diets was determined
gravi-metrically following extraction of lipids according to
the Soxhlet method (AOAC, No.920.39) Standard
pro-cedures were used for moisture (AOAC, No 934.01),
crude ¢bre (AOAC, No 962.09) and ash (AOAC, No
942.05)
Octopuses were fed between 15% and 5% body
weight day 1, at the start and the end of each trial
respectively The feeding ratio was adjusted every
week using the living weight of 15 octopuses
ran-domly collected from each tank
Growth rate was calculated as absolute growth
rate [AGR (g day 1) 5 Wt W0/t; where Wtis the
¢-nal living weight in g,W0the initial living weight in g
and t the culture time in days], and speci¢c growth
rate [SGR (% day 1) 5 lnWt lnW0)/t] Gross food
conversion factor was calculated as GFCF 5
Deliv-ered food (g by pond)/Total biomass production per
pond Survival was obtained as the di¡erence
be-tween the number of octopuses at the beginning
and at the end of each trial
Statistical di¡erences between trials were obtained
using anANOVA(Zar 1999) A transformation arcsin
was used before the analysis of percentage values
(Zar 1999)
Results
During the three trials, living weight followed a
logis-tic curve (Fig 2) As a result of growth and survival,
the ¢nal density was 6.1, 5.0 and 4.3 kg m 3for trials
1to 3, respectively, and di¡erent (Po0.05) for all trials
(Table 2) Those densities were equivalent to 3.7, 3.2
and 3.7 octopuses m 2 for trials 1^3 respectively
(Table 2) Final average weight was 952.0 66.5 g
for trial 1, 839.5 34.9 for trial 2 and 652.0 29.3for trial 3 (Table 2), and were di¡erent (Po0.05)among all trials
Variation in water temperature during the threetrials is shown in Fig 3 The higher temperature dur-ing trial 1 could explain the similar growth rate(P40.05) obtained with these larger animals(2.4% day 1), and those smaller from trial 3(2.2% day 1), but cultured at lower temperatures Infact, because growth rates are related to animalweight, they are usually higher for smaller animals,
in similar culture conditions The SGR (% day 1)was similar for octopuses from trials 1 and 3, andhigher (Po0.05) compared with octopuses from trial
2 (Table 2)
Absolute growth rate (AGR; g day 1) was higher(Po0.05) in octopuses from trial1, which were larger,compared with trials 2 and 3 (Table 2)
A total of 111, 128 and 136 kg of food per tank wereused in trials 1, 2 and 3, respectively, with an average
of 60, 49 and 42 kg tank 1cropped, being higher(Po0.05) for trial 1 This corresponded to gross foodconversions of 1.9:1, 2.6:1 and 3.3:1 for trials 1, 2 and 3respectively (Po0.05; Table 2) Survival was similar(P40.05) between trials, and was 92 5% fortrial 1, 94 5% for trial 2 and 84 6% for trial 3(Table 2)
DiscussionThe tanks used in the present study were adequatefor the ongrowing of O maya juveniles The growthrate and survival observed revealed that this species
Table 1 Chemical characteristics of crab (Callinectes spp.)
and ¢sh heads (Lutjanus spp.) used to fatten juvenile Octopus
maya during the three trials
0 7 14 21 28
Time, days
Trial 1 Trial 2 Trial 3
Figure 2 Octopus maya growth (g) during the three perimental trials.Values as mean SE
ex-© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 26–31 28
Ongrowing of Octopus maya in large outdoor tanks P Domingues et al Aquaculture Research, 201 , 43, 26–31 2
Trang 30is a good candidate for tropical aquaculture Octopus
maya, like O vulgaris, is highly territorial, and
re-quires space for growth Therefore, 240 shelters of
4 in PVC per pond were used, allowing between two
and three refuges per octopus, and o¡ering the
ani-mals the opportunity to choose between several
re-fuges Octopus vulgaris has been grown in £oating
cages, where researchers and producers also
success-fully use a proportion between two and three refuges
per octopus (Rodr|¤guez et al 2006) Although we can
conclude that initial and ¢nal densities close to 4 and
6 kg m 3, respectively, and a proportion of three
re-fuges per animal provide good results on growth and
survival of O maya, further studies on the e¡ect of
oc-topus density and refuges (number, form, position on
tank, etc.) could be useful to determine a proportion
between both variables that might improve the
ongrowing of this species In fact, Otero et al (1999)
suggests much higher initial (between 10 and
20 kg m 3) and ¢nal stocking densities (43 kg m 3)for O vulgaris fattened in inland tanks Similarly, re-cent results for O vulgaris ongrowing in concretetanks report adequate growth (with similar survival)
at initial culture densities of 8 and 15 kg m 3ingues et al 2007), which are considerably higherthan those initial stocking densities used in the pre-sent study This suggests that further research on
(Dom-O maya density studies should be conducted, in order
to optimize production
A density of 10 kg m 3 that is presently mended for O vulgaris ongrowing, is based on £oat-ing cages in the ocean (Vaz-Pires et al 2004) For
recom-O maya fattened in tanks, density could also be ciated to the bottom area (m2) rather than volume,considering the low water depth (0.5 m) In this envir-onment, water quality, type of food and biologicalinteractions as a result of territorial behaviour aremore strongly related to the surface of tank than towater depth The water quality in the culture tankswas maintained thanks to partial daily recycling.Although there are no formal studies related toammonia toxicity for O maya, it appears that totalammonia levels registered during the trials (0.8^2.75 mg L 1) were not toxic for animals maintained
asso-in these conditions Further studies on the e¡ect ofionized and non-ionized ammonia on survival andgrowth of O maya, particularly upper sustainablelimits should be addressed to establish the tolerancelimits of this species in tanks, because ammonia is alimiting factor for invertebrate culture (Chen, Cheng
Table 2 Growth, survival and food conversion of Octopus maya ongrowing in outdoor tanks
Di¡erent letters indicate signi¢cant di¡erences between trials.
AGR, absolute growth rate; SGR, speci¢c growth rate; FC, food conversion (weight gain/food eaten).
Figure 3 Maximum and minimum temperature
varia-tions on tanks during the three experimental Octopus
maya ongrowing trials.Values as mean SE
Trang 31& Chen 1994; Alcaraz, Espinoza, Vanegas & Carrara
1999; Lin & Chen 2001)
A high SGR% was observed in the three trials, with
values that varied between 1.8 and 2.4 These results
were higher than those obtained for O vulgaris by
Iglesias, SaŁnchez, Otero and Moxica (2000), between
0.77% and 0.95%, and by Rodr|¤guez et al (2006),
between 0.54% and 1.54%, and those obtained for
Enteroctopus megalocyathus by Pe¤rez, Lo¤pez, Aguila
and GonzaŁlez (2006), between 1.1% and 1.3%
In those trials, octopuses were fed exclusively on
crustaceans, or mixed diets of crustaceans, ¢sh and
mussels The O maya SGR% obtained in outdoor
tanks was lower than that reported in indoor
tanks by Domingues et al (2007) using a monodiet of
crab and individualized octopuses Although a
monodiet of crab (Callinectes spp.) could be the best
for O maya, it is not economically feasible inYucataŁn,
due to the high production costs associated to this
practice
Growth is dependent on body weight, diet,
repro-ductive condition and temperature, and seems to
de-crease when body weight inde-creases During the
present study, the growth rate followed a logistic
curve in the three trials, indicating that octopuses
were in the last part of the growth curve phase
(Bri-ceno-Jacques, Mascaro¤ & Rosas 2010) Considering
all the trials together, it is evident that the initial
liv-ing weight did not itself explain the di¡erences in
growth rates between trials Time of the year and/or
the quality and composition of the food provided also
seemed to a¡ect octopus growth (Vaz-Pires et al
2004; Rodr|¤guez et al 2006)
A reduction on water temperature was observed
during the trials, with high temperatures in trial 1
(28 1 1C), followed by trial 2 (26 2 1C) and trial
3 (24 0.8 1C) Although actual results showed that
this range of temperatures did not a¡ect growth rate
of O maya, further research should be conducted to
elucidate the temperature range that promotes the
maximum growth rate for O maya
A relatively high survival was observed in the trials
with values between 84% and 94%, indicating that
in juveniles larger than 300 g, O maya cannibalism
is not a major constraint, although mortality
ob-served was related to cannibalism or injuries
pro-voked by ¢ghting
In the three trials, relatively high GFCFs (1.9^3.3)
were obtained, corresponding to conversion rates
be-tween 30% and 50% This indicates that this type of
food was highly assimilated by O maya Domingues
et al (2007) report lower conversion rates (between
20% and 40%) for O vulgaris, but in this case muchhigher culture densities were used
Results obtained with O maya juveniles showedthat crabs are highly digestible (85%) for this species(Domingues et al 2007) Similar results were ob-tained for O vulgaris fed crabs (Boucher-Rodoni &Mangold 1985; Aguado Gime¤nez & Garc|¤a Garcia2002; Garc|¤a Garc|¤a & Aguado Gime¤nez 2002; Igle-sias, Otero, Moxica, Fuentes & SaŁnchez 2004; Pe¤rez
et al 2006) Several researchers have demonstratedthat chemical diet characteristics, in particular pro-tein, have an important role on cephalopod growth.Under experimental conditions, Aguado Gime¤nezand Garc|¤a Garcia (2002) and Garc|¤a Garc|¤a andAguado Gime¤nez (2002) reported better growth in
O vulgaris when fed frozen crabs compared with ¢sh,evidencing that crustaceans covered better nutri-tional requirements In contrast, when cephalopodswere fed on high-fat meals (e.g sardines) theyproduced faeces that £oat indicating the limited ca-pacity of O vulgaris to metabolize lipids Fish heads
of Lutjanus spp that had only 12% of total lipids and
a protein level of 61% were used in this study Thechemical composition of Lutjanus spp head is close
to crab composition, with relatively low lipid content,with the advantage of these ¢sh heads being aby-product from ¢sh industry For these reasons, this
is a good candidate to be used as food for O mayaongrowing
AcknowledgmentsThe present study was partially ¢nanced by DGAPA-UNAM project no: IN216006-3, Fundacio¤n ProduceYucataŁn and CONACYT-BaŁsico 24743 The authorsare grateful to Moluscos del Mayab group for themaintenance of octopus during the experiments
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Trang 33Ultrastructural changes in the hepatopancreas
Radhika Gopinath1, Rajain Paul Raj2, Kizhakkayil Chandy George3& Nandiath Karayi Sanil3
1 Department of Marine Biology, Microbiology and Biochemistry, PDF, CUSAT, Kochi, Kerala, India
2 Coastal Aquaculture Authority, Chennai,Tamilnadu, India
3 Central Marine Fisheries Research Institute (CMFRI), Kochi, Kerala, India
Correspondence: R Gopinath, Department of Marine Biology, Microbiology and Biochemistry, PDF, CUSAT, Kochi, Kerala 682 022, India E-mail: radhikacpg@gmail.com
Abstract
Fungal contamination of shrimp feeds a¡ects the
shelf life leading to substantial economic losses
Ul-trastructural changes in Penaeus monodon sub-adults
fed three di¡erent doses (50, 1000 and 2000 ppb) of
a£atoxin B1(AFB1) were studied At the end of 4 and
8 weeks of experiment, the hepatopancreas of
shrimps were observed for ultrastructural changes
The prominent ultrastructural changes in
hepato-pancreas of the shrimps fed 1000 and 2000 ppb
AFB1were rupture of cell membrane and microvillus
border, damage and swelling of mitochondria,
frag-mentation of endoplasmic reticulum, nuclear
vacuo-lation, chromatin condensation and autophagy This
study helps to decipher the ultrastructural changes
and relate the e¡ects on biochemical, pathological,
immunological and histological architecture of the
shrimps fed AFB1-incorporated feed The observed
ul-trastructural changes could serve as indicators in
shrimps exposed to toxicants
Keywords: a£atoxin B1, AFB1-incorporated feed,
Penaeus monodon, hepatopancreas, ultrastructural
changes
Introduction
The culture of Penaeus monodon (Fabricius 1798) is
constantly hindered by outbreaks of bacterial, viral
and parasitic diseases and also by
environmental-and nutrition-related diseases One such constraint
is the disease caused by fungal contamination of feed
that often invites secondary infections
Among the mycotoxins, A£atoxins are extremelybiologically active secondary metabolites produced
by the fungi, Aspergillus species These toxicants areparticularly important in aquaculture since theirpresence exerts a negative economic impact on rele-vant commerce as well as severe health problemsafter exposure to infected food and feed Toxic feedcontaminants can cause abnormalities such as poorgrowth, physiological imbalances and histologicalchanges that result in yield reduction and pro¢tabil-ity of shrimp culture A£atoxins are polycyclic unsa-turated compounds with a coumarin molecule
£anked on one side by a bisfuran moiety and on theother side by either a pentanone for B series or a six-membered lactone for G series (Coulombe 1991).The toxicity of a£atoxin B1 (AFB1) has been re-ported for poor growth, hepatotoxic, nephrotoxic,mutagenic, teratogenic and cytotoxic properties(Halver 1969; Jantrarotai & Lovell 1990; Chavez-San-chez, Palacios & Moreno 1994; Sahoo, Mukherjee,Nayak & Dey 2001; Santacroce, Conversano, Casali-
no, Lai, Zizzadoro, Centoducatti & Crescezenso 2008;Han, Xie, Zhu,Yang & Guo 2009; Raghavan, Zhu, Lei,Han,Yang & Xie 2009)
Experimental studies of a£atoxicosis in shrimpsare reported in Penaeus vannamei (Lightner, Redman,Price & Wiseman 1982), Penaeus stylirostris (Wise-man, Price, Lightner & Williams 1982; Ostrowski-Meissner, LeaMaster, Duerr & Walsh 1995), P monodon(Bautista, Lavilla-Pitogo, Subosa & Begino1994; Diva-karan & Tacon 2000; Boonyaratpalin, Supamattaya,Verakunpiriya & Suprasert 2001; Bintvihok, Pon-pornpisit, Tangtrongpiros, Panichkriangkrai, Ratta-napanee, Doi & Kumagai 2003) and Litopenaeus
© 2011 Blackwell Publishing Ltd 32
Aquaculture Research, 201 , 43, 32–43 2 doi: 10.1111/j.1365-2109.2011.02798.x
Trang 34vannamei (Burgos-Hernandez, Farias, Torres-Arreola
& Ezquerra-Brauer 2005)
Electron microscopic studies of a£atoxicosis were
reported by Scarpelli (1967) and Nunez, Hendricks
and Duimstra (1991) in rainbow trout and by Sahoo,
Mukherjee, Jain and Mukherjee (2003) in rohu, Labeo
rohita Kalaimani, Ali, Shanmugasundaram and
Sar-athchandra (1998) have reported the presence of
a£a-toxin in imported and indigenous shrimp feeds in the
range of 10^130 ppb collected from shrimp farms in
Andhra Pradesh in India
The objective of this study was to determine the
ul-trastructural changes in the hepatopancreas of P
monodon fed di¡erent doses of AFB1 Cellular assay
techniques serve as reliable biological indicators of
stress and disease The crustacean hepatopancreas
displays considerable cytological, cytochemical and
ultrastructural alterations at chronic exposure to
xe-nobiotics Hence it has been identi¢ed as a target
or-gan of interest in toxicity investigations (Bryan 1976;
Buss & Gibson 1979; Livingstone 1985; Moore 1990;
Sandbacka, Part & Isoma 1999) Hepatopancreas was
selected for detailed study owing to severe e¡ect as
revealed by histological changes (Radhika 2006;
Radhika & Paul Raj 2009) The doses of AFB1for the
experiment were selected on the basis of ¢eld surveys
of shrimp feeds from local farms, standardization
trials and previous works (Lightner et al 1982;
Wise-man et al 1982; Ostrowski-Meissner et al 1995;
Boo-nyaratpalin et al 2001)
Materials and methods
Experiment protocol
Sub-adult P monodon (200 numbers) of size 7.5
0.72 g brought from a farm at Narakkal,
Erna-kulam district, Kerala were acclimatized to
0.5 g L 1salinity for 1 week in holding tanks of
2 tonne capacity in the Marine Hatchery complex of
Central Marine Fisheries Research Institute (CMFRI),
Kochi, Kerala, India One control and three treatment
groups were selected for the experiment of 60 days
duration The doses of AFB1selected were 0, 50, 1000
and 2000 ppb Shrimps were weighed and about 26
numbers were segregated into separate 1tonne
¢bre-glass-reinforced plastic tanks of 2 1 0.5 m The
shrimps stocked in one tank were taken as one
treat-ment, and after 4 weeks of feeding AFB1, 13 shrimps
were sacri¢ced for analysis and the rest13 were
sacri-¢ced at the end of the experiment Three shrimps
from each group were taken for normal
ultrastruc-tural analysis at the start of the experiment, andthree shrimps from each treatment group weresampled at 4 and 8 weeks
Experimental dietsPreparation of stock solution and working solution of AFB1Pure crystalline powder of AFB1was obtained fromSigma chemical company (Product Name A6636; StLouis, MO, USA) 50 mg of AFB1was dissolved in
5 mL of chloroform to form a stock solution ing 10 mg AFB1/mL of chloroform From this, a work-ing solution (WS) was prepared by adding 1mL of thestock solution to 49 mL of chloroform (10 mg AFB1/
contain-50 mL of chloroform 0.2 ppm AFB1) Before tion of the toxin into experimental feeds, the requiredamount of toxin dissolved in chloroform from theworking solution was taken in a glass beaker, evapo-rated in a water bath and replaced with equal vo-lumes of ethanol
addi-Processing of feed ingredients and feed preparationThe feed ingredients chosen for preparing the experi-mental diets were ¢sh meal, shrimp meal, clam mealand soyabean meal as protein sources, where wheat
£our as carbohydrate source and cod liver oil as lipidsource Moisture, crude protein and crude ¢bre levels
in the feed and feed ingredients were determined asper AOAC (1990) Shrimp feed formulation contain-ing 38% crude protein was used to make experimen-tal diets All the feed ingredients were taken toprepare 500 g each of four types of feed, viz, control,and three test diets ^ 50 ppb (0.125 mL of WS),
1000 ppb (2.5 mL of WS) and 2000 ppb (5 mL of WS).The formulation of the basal diet and the determinedAFB1levels are represented in Tables 1and 2 The feedingredients and feeds were analysed for the presence
of AFB1using a £urometer (VICAM 1 Series 4)
Data collectionDoses of a£atoxin and days of experiment were main-tained Feeding rate was 4.2% for the ¢rst 30 days and3.5% for next 30 days Small amounts of feed wereadded at a time to ensure complete feeding to avoidfeed wastage and contamination Water exchangewas carried out daily at 50% level and individualtanks were provided proper aeration Water quality,temperature, dissolved oxygen and salinity weremonitored daily The salinity was maintained at
Trang 3520 0.5 g L 1, temperature at 26 2 1C and
dis-solved oxygen at 5 1mg L 1for the entire
experi-mental duration The hepatopancreas from each
group was taken for ultrastructural study
The tissue preparation and processing for
ultra-structure were carried out as described by Dawes
(1988) The ultrastructural studies were carried out
using a Hitachi ^ H-600 Transmission Electron
mi-croscope (Hitachi, Tokyo, Japan) Following primary
¢xation, washing and post-¢xation, dehydration was
carried out in ascending grades of acetone (Analar)
and the tissues were embedded in Spurr’s resin as
per the method described by Spurr (1969) Ultra-thin
sections of blocks were taken using an LKB Nova
Ul-tra microtome (LKB ^ Producter AB, Bromma,
Swe-den) The ultra-thin sections were stained withuranyl acetate and lead citrate, dried and observedunder a Hitachi H-600 transmission electron micro-scope (Hitachi)
ResultsHepatopancreas of shrimps from the control group,
50 ppb group, 1000 and 2000 ppb AFB1treatmentsafter 4 and 8 weeks, were taken for ultrastructuralexamination The control group showed normal ar-chitecture of the cells with nucleus, well-developedsmooth endoplasmic reticulum (SER) and rough en-doplasmic reticulum (RER) along with round androd-shaped mitochondria with numerous cristaeand granules The golgi bodies and microvilli werenormal The nucleus appeared spherical with abun-dant euchromatin and well-developed nucleolus(Figs 1^3)
Hepatopancreas sections from 50 ppb group at 4weeks were similar to the control group But therewere some modi¢cations in the con¢guration of mi-tochondria at few focal areas in this group after 8weeks (Fig 4)
Hepatopancreas of the shrimp exposed to the
1000 ppb AFB1group for 4 weeks revealed tation of endoplasmic reticulum (ER) with small dila-
fragmen-Table 1 Feed formulation of shrimp feeds for sub-adults of
chole-ascorbic acid, 200 mg; thiamine hydrochloride, 60 mg;
ribo£a-vin, 40 mg; calcium pantothenate, 60 mg; pyridoxine
hydro-chloride, 40 mg; nicotinic acid, 200 mg; D-Biotin, 1mg; choline
zOil: A combination of 1:1 cod liver oil and vegetable oil.
Table 2 Supplemented and determined AFB 1 levels of the
experimental diets
Supplemented AFB 1 (mg kg 1 ) ppb 1 0 50 1000 2000
Determined AFB 1 (mg kg 1) ppb 1 0 56 1029 1973
Figure 1 Electron micrograph depicting the gross view
of cells in the hepatopancreas of control Penaeus monodon
Trang 36tions and accumulation of densities Changes wereobserved in the nucleus with the condensation ofchromatin to nuclear membrane and vacuolation inthe cytoplasm The nucleus contained chromatingranules and electron-dense inclusions The cellmembrane was broken abruptly Shapes of mitochon-dria were a¡ected by change in the structure of thecristae Microvilli were broken at few places Exten-sive vacuolation also was observed (Figs 5 and 6).After 8 weeks, the hepatopancreas revealed nuclearvacuolation and condensation, appearance of elec-tron dense material, irregular shape of nucleus andloss of nuclear membrane Fragmentation of ER wasextensive There was swelling of mitochondria, va-cuolation and loss of organelles in few areas Forma-tion of vesicle in the cytoplasm and loss of microvilliwere observed Cell rounding and extensive necrosiswere also observed (Figs 7^10).
In 2000 ppb group at 4 weeks, the electron scopic view of hepatopancreas revealed completefragmentation of ER and degranulation Mitochon-drial damage was severe and cristae almost disap-peared Nuclear vacuolations were extensive Therewas autophagic vesicle, lipid droplets and accumula-tion of densities in the cytoplasm Cell membrane andmicrovilli were broken in many areas (Figs 11^13)
micro-Figure 2 The ultrastructural view of hepatopancreas of
control shrimp showing intact cell membrane (CM), large
number of mitochondria (M) and microvilli (mv)
project-ing into the lumen 15 000
Figure 3 Electron micrograph of the hepatopancreas of
control Penaeus monodon depicting nucleus (N) with
eu-chromatin (EC) and heteroeu-chromatin (HC) attached to
the nuclear membrane Note ER cisternae (ER) and cell
membrane (CM) 10 000
Figure 4 Electron micrograph depicting change in thecon¢guration of mitochondria (SM) and intact endoplas-mic reticulum (ER) in hepatopancreas of shrimp given
50 ppb AFB1after 8 weeks 20 000
Trang 37Figure 5 Electron micrograph of hepatopancreas of
1000 ppb AFB1-fed group after 4 weeks Note the
endo-plasmic reticulum fragmentation with small dilations
(ERFD), swollen mitochondria (M) condensation of
mitochondria (CM) and broken cell membrane (BC)
20 000
Figure 6 Electron micrograph of hepatopancreas of
1000 ppb AFB1-dosed shrimp after 4 weeks showing
con-densation of chromatin (C) in the nucleus 10 000
Figure 7 Electron micrograph depicting extensive cuolation (V) in the hepatopancreas of shrimp fed
Trang 38Figure 9 Electron micrograph depicting the rounding of
cells (RC) in the hepatopancreas of shrimp dosed 1000 ppb
AFB1at 8 weeks 8000
Figure 10 Electron micrograph of shrimp
hepatopan-creas given 1000 ppb AFB1at 8 weeks revealing nuclear
condensation (NC) and granulation of endoplasmic
reti-culum (ERG) 15 000
Figure 11 Electron micrograph of hepatopancreas ofshrimp fed 2000 ppb AFB1at 4 weeks showing lipid dro-plet (LD), granulation of endoplasmic reticulum (ERG)and loss of nuclear contents (LN) 6000
Figure 12 Electron micrograph showing the rupture ofmicrovillus border (lmb) into the lumen of the hepatopan-creas of shrimp fed 2000 ppb AFB1at 4 weeks Note theswollen mitochondria (SM) in the section 5000
Trang 39The hepatopancreas cells were mostly necrosed
in the ultrastructural view of shrimps fed diets
with 2000 ppb AFB1 after 8 weeks There was
complete loss of cell membrane and structure of cells
Microvilli were ruptured in many places with empty
spaces Mitochondria became swollen and cristae
disappeared along with granules The amount of
het-erochromatin in the nucleus increased Autophagy
and whorl formation were also observed The notable
changes were fragmentation of ER, whorl formation,
autophagy, vesicle formation, broken cell membrane
and loss of cell organelles (Figs 14^16) The frequency
of ultrastructural changes of each group is presented
in the Table 3
Discussion
Ultrastructural alterations are used as e¡ective
in-dices of physiological and biochemical changes
caused by toxicant stress These biological indices
provide insight into cellular injuries (Moore 1990)
The hepatopancreas is a vital organ with secretory,
absorptive, digestive and excretory functions
(Al-Mo-hanna & Nott 1986) This has been identi¢ed as a
tar-get organ for xenobiotic attack because it shows
Figure 13 Electron micrograph of the hepatopancreas of
shrimp given 2000 ppb AFB1at 4 weeks Note the broken
cell membrane (CM), swollen mitochondria (SM) and
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 32–43 38
Ultrastructural changes caused byAFB1 in P monodon R Gopinath et al Aquaculture Research, 201 , 43, 32–43 2
Trang 40critical ultrastructural variations at very early stages
of exposure, even before morphological
manifesta-tions This study elucidates the major ultrastructural
changes brought about by dietary AFB1in the vital
cellular organelles of hepatopancreas of P monodon
The electron microscopic view of the
hepatopan-creas section in the control shrimps revealed normal
structure of di¡erent cells and cell organelles and is
in agreement with the study by Vogt, Storch, Quinitioand Pascual (1985) In the group given 50 ppb AFB1,the ultrastructural view of the hepatopancreas at 4weeks was similar to the control group But after 8weeks, the mitochondrion has undergone changes
in the con¢guration of mitochondria This could bedue to the toxic e¡ect of AFB1, which reveals that thelowest dose of 50 ppb could a¡ect the structure of themitochondrion The alteration of the con¢guration ofmitochondria would naturally modify functions ofthe organelle In the shrimps given 1000 ppb AFB1at
4 and 8 weeks, the ultrastructural analysis of topancreas revealed changes in the cell membrane,mitochondria, nucleus and ER.While in the group gi-ven 2000 ppb AFB1, alterations were more severe likeloss of cellular organelles, vesicle formation, autop-hagy and necrosis
hepa-The peculiar features of the ultrastructuralchanges in hepatopancreas of shrimps in AFB1-trea-ted groups were intense fragmentation of SER andRER, chromatin condensation, electron dense inclu-sions, broken cell membrane, change in shapes of mi-tochondria and structure of the cristae, loss ofmicrovilli and extensive vacuolation, cell rounding,extensive necrosis, autophagy and whorl formation.Thus, AFB1has severely a¡ected all the cellular orga-nelles of hepatopancreas like microvillus border, cellmembrane, ER, mitochondria and the nucleus of P.monodon These observations are similar to the re-ports on a£atoxicosis in livestock (Rajan, Maryamma
& Gopalakrishnan Nair 1989) and ¢sh (Sahoo et al.2003)
Toxicants are known to a¡ect the structure andfunctions of cellular components leading to impair-ment of vital functions of many marine organisms(Baticados, Coloso & Duremdez 1987) A£atoxinsmay be considered as biosynthetic inhibitors both invivo and in vitro, with large doses causing total inhi-bition of biochemical systems and lower doses a¡ect-ing di¡erent metabolic systems Biochemically,a£atoxins can a¡ect energy metabolism, carbohy-drate metabolism, nucleic acid and protein metabo-lism (Ellis, Smith & Simpson 1991)
The cell membrane controls the movement intoand out of the cell and in particular controls the os-motic gradients involving £uids (Thomson 1984);hence, breakage of the cell membrane as observed inthe present study would a¡ect the normal cell func-tions and structure Peroxidation of unsaturated li-pids in the bio-membranes by free radicals may lead
to con¢gurational changes or breakdown The free
Figure 16 Electron micrograph of hepatopancreas of
shrimp fed 2000 ppb AFB1at 8 weeks Note the beginning
of autophagy formation (AF) and condensation of
mito-chondria (CM) 17 000
Table 3 Frequency of Ultrastructural changes in Penaeus
monodon after feeding with AFB 1 in di¡erent concentrations
Ultrastructural changes included:
Changes in the con¢guration of mitochondria in the
hepatopan-creas cells.
Broken cell membrane, swollen mitochondria, ER fragmentation,
cell rounding and nuclear vacuolation.
Whorl formation, autophagy, loss of cellular organelles, vesicle
and vacuole formation and necrosis.
Figures in parentheses indicate number of shrimp in which
changes were detected/total number of shrimp examined.