Fish Physiol Biochem (2014) 40:607–624
DOI 10.1007/s10695-013-9870-y
Ultrastructure of the anterior intestinal epithelia
of the orange-spotted grouper Epinephelus coioides larvae
under different feeding regimes
Y. H. Primavera-Tirol • R. M. Coloso •
G. F. Quinitio • R. Ordonio-Aguilar •
L. V. Laureta Jr.
Received: 11 July 2012 / Accepted: 23 September 2013 / Published online: 2 October 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Enterocytes of the anterior to midsection
of the intestine in grouper Epinephelus coioides larvae
were compared among different treatments: unfed to
the point-of-no-return (PNR), fed natural food only,
and co-fed natural food and artificial diet. On day 3,
the nutritional condition of unfed grouper larvae
regressed with its reduced enterocyte heights which
were further degraded on day 4, the PNR, when all the
enterocytes were in advanced stages of apoptosis. The
apoptosis appeared to be internally directed via the
mitochondria. Among day 3 fed larvae, enterocyte
heights of those fed artificial diet did not differ from
those fed natural food only. Dietary phospholipid
deficiency was indicated in larvae co-fed artificial diet
on day 3 with an unusually large chylomicron opening
into the inter-enterocyte space, and on days 6 and 33
by intestinal steatosis. On day 19, scant to absent lipid
droplets in enterocytes of larvae disclosed heightened
nutritional requirement preparatory to metamorphosis.
Y. H. Primavera-Tirol (&)
R. M. Coloso
Southeast Asian Fisheries Development Center-
Aquaculture Department, 5021 Tigbauan, Iloilo,
Philippines
e-mail: yhprimavera@gmail.com
Y. H. Primavera-Tirol
G. F. Quinitio

R. Ordonio-Aguilar
L. V. Laureta Jr.
College of Fisheries and Ocean Sciences, University of
the Philippines Visayas, 5023 Miagao, Iloilo, Philippines
As observed in unfed day 3 and premetamorphic day
19 E. coioides, larvae undergoing critical periods and
starvation during development employ apoptosis to
dispose of degenerated enterocytes that are phagocy-
tosed by adjacent healthy enterocytes without causing
inflammatory distress. Upon metamorphosis, grouper
larval gut develops better immunity fitness with
eosinophilic granule cells observed in the intestinal
epithelia of day 33 larvae. Future studies on grouper
larval nutrition may consider the appropriate dietary
phospholipid levels and larval competence to biosyn-
thesize highly unsaturated fatty acid from linoleic acid
vis-à-vis the use of plant ingredients in artificial diet
formulations. In vivo challenge tests may validate
appropriate dietary nutrient supplementation and lead
to better feed formulation, matching the varying
energetic demands and digestive capacities of devel-
oping E. coioides larvae.
Keywords Grouper larvae
Digestive
ontogeny
Anterior intestine
Apoptosis

Ultrastructure
Nutritional condition
Introduction
The grouper (family Serranidae, subfamily Epinephe-
linae) is popular as medium- to high-priced live food
fish in the Asian market. Because of increasing market
demand and live reef food fish trade, wild capture
fisheries and aquaculture production of grouper have
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expanded in many Southeast Asian and Pacific
countries (Sadovy et al. 2003). The Asian grouper
aquaculture production reached a total of about 1.5
million t valued at more than USD 5 billion and
comprising 77.5 % of the global grouper aquaculture
production in 2009 (FAOSTAT 2009). Despite the
continuing expansion of grouper aquaculture in the
Asia–Pacific region, the supply of fingerlings for
grow-out culture from grouper hatcheries is still
unreliable (Rimmer et al. 2004; Toledo et al. 1999).
Nevertheless, a better survival rate was reported in the
larval rearing of the orange-spotted grouper Epinephe-
lus coioides (Hamilton 1822, Toledo et al. 1999)
corresponding to higher larval feeding preference for
copepods. The copepods had higher phospholipid
content than the rotifer (Brachionus plicatilis) and
brine shrimp (Artemia salina) which were also fed to
the larvae. Given the importance of dietary lipid
utilization for larval rearing success (Izquierdo et al.
2000; Tocher 2003) and that lipid metabolism is
known to take place in the anterior intestine in fish
larvae (Iwai and Tanaka 1968; Zambonino Infante and
Cahu 2007), taking a closer look at the anterior
intestine enterocytes in grouper larvae may lead to a
better understanding of their digestive physiology.
Assessing the digestive capacity of fish larvae is
important to develop dependable and sustainable larval
rearing techniques where knowledge on larval nutri-
tion in relation to the development of digestive and
metabolic systems is required (Lazo et al. 2010;
Zambonino Infante and Cahu 2007). Nutrition can
affect many mechanisms that determine the morpho-
logical and functional development of fish larvae
(Zambonino Infante and Cahu 2007). This relationship
has been observed in hormonal and molecular mech-
anisms controlling the expression of proteolytic and
lipolytic enzymes modulated by the dietary protein
content and lipid fraction, respectively (Zambonino
Infante and Cahu 2007). Also, the expression of
specific nuclear receptors is modulated by the dietary
level of their specific ligands (e.g., peroxisome prolif-
erator-activated receptors by polyunsaturated fatty
acids, retinoic acid receptors, and retinoid 9 receptors
by vitamin A), which strongly impact other signaling
pathways regulating larval development (Zambonino
Infante and Cahu 2007).
Moreover, fish larvae are especially sensitive to
nonoptimal feeding conditions or nutritional stressors
(dietary imbalances), because most tissues and organs
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Fish Physiol Biochem (2014) 40:607–624
are in progression and intense differentiation and
development and larvae do not have enough reserves
(yolk and oil globule) stored to withstand starvation
(Gisbert et al. 2008). The development and organiza-
tion of the enterocyte ultrastructure with respect to the
digestive ontogeny in marine fish larvae (Elbal et al.
2004; Qu et al. 2012) during start-feeding and starva-
tion (Kjørsvik et al. 1991) and when fed artificial diet
(Mani-Ponset et al. 1994; Segner et al. 1993) have been
previously reported. Other studies focused on the
anterior intestine and the absorption of lipid and
protein during early development (Diaz et al. 1997,
2002; Iwai 1968, 1969; Iwai and Tanaka 1968; Mani-
Ponset et al. 1994) and in immature fish with adultlike
alimentary tract (Deplano et al. 1989; Olsen et al. 1999,
2000; Sire et al. 1981). Recent work on the ultrastruc-
ture of the digestive tract of fish larvae investigated
intestinal microflora (Ringø et al. 2003) and its role in
promoting different aspects of fish larval gut differen-
tiation (Bates et al. 2006), as well as bacterial
translocation and pathogenesis in the digestive tract
of fish larvae and fry (Ringø et al. 2007). Favorable gut
microbiota has also been reported to improve growth in
grouper juveniles (Sun et al. 2009).
Previous studies characterizing the digestive capacity
of E. coioides larvae have been conducted, including the
development of the gastrointestinal (GI) tract (Quinitio
et al. 2004a, b) and the ontogeny of digestive enzymes
(Eusebio et al. 2004; McBride 2004). This study aims to
describe the ultrastructure of the anterior intestine
enterocytes in E. coioides larvae fed natural food alone
and co-fed natural food and artificial diet at critical
periods in larval rearing. Unfed larvae to the point-of-no-
return (PNR) (Blaxter and Hempel 1963) were also
evaluated. Results may help elucidate how aspects of the
digestive ontogeny of E. coioides may be related to the
high mortalities experienced in larval rearing (Liao et al.
2001). The first aim of the present study may contribute to
our understanding of fish larvae survival and lead to the
optimization of the larval feeding regime and artificial
diet formulation for early-developing grouper.
Materials and methods
Spawning and incubation
Larvae from the same cohort of naturally spawned
eggs of E. coioides from tank-reared broodstock were
Fish Physiol Biochem (2014) 40:607–624
sourced from the Marine Finfish Hatchery of the
Aquaculture Department of the Southeast Asian
Fisheries Development Center (SEAFDEC/AQD) at
Tigbauan, Iloilo, Philippines. Grouper eggs were
incubated at an average density of 500 eggs/L in
500-L circular fiberglass tanks with mildly aerated
seawater at ambient salinity (33–35 ppt) and temper-
ature (28–30 °C). The eggs hatched about 18–20 h
after spawning. The density of hatchlings was esti-
mated from five water samples taken from different
sections of the tank (Duray et al. 1996).
Natural food and artificial diet
The rotifer B. plicatilis was inoculated into algal
tanks (Nannochlorum sp. cultured in 9-ton circular
concrete tanks) when algal population density
reached minimum of 107 cell ml-1. After 5–6 days
when the density reached at least 120 ind/ml,
rotifers were harvested using a 42-lm-mesh-size
plankton bag according to Duray et al. (1997). Cysts
of brine shrimp A. salina (Ocean Star International)
were incubated for 24 h in 500-L fiberglass tanks
(100 g/tank). The SEAFDEC/AQD standard enrich-
ment emulsion was allowed to drip for 16 h
overnight into the aerated tank where first-harvest
rotifer or newly hatched A. salina were collected.
The following morning, the rotifer or brine shrimp
were rinsed and fed to the grouper larvae. The
enrichment emulsion included cod liver oil
(95.681 %), lecithin (0.383 %), vitamin C as ascor-
byl phosphate Mg (0.095 %), b-carotene (0.009 %),
and a-tocopherol (0.004 %).
The microbound artificial diet for grouper larvae
produced by SEAFDEC/AQD was used for the co-
fed treatment. This diet included the ingredients
Danish fish meal (72 % protein of dry matter), squid
meal, Acetes spp., soy protein concentrate (60 %
protein), bread flour, vitamin and mineral mix, a-
tocopherol, cod liver oil, soybean lecithin, b-caro-
tene, and carrageenan (O.M. Reyes, unpublished
data). Larvae were fed pellets 150–200 lm size
from day 2 to 15 and [500 lm size from day 16 to
45. The microbound diet was analyzed to determine
crude protein (Kjeldahl method, AOAC 1980), crude
fat (Soxtec method, AOAC 1980), and nitrogen-free
extract (by difference) content (Table 1). No
enzyme assays were conducted for the natural food
and artificial diet.
609
Table 1 Proximate analyses results of SEAFDEC/AQD
grouper larval diet
Larval Crude Crude Crude Nitrogen- Ash
diet protein fat (%) fiber free extract (%)
(%) (%) (%)
Bday 15 47.23 18.16 1.93 17.23 15.13
[day 15 47.16 16.69 1.87 19.08 15.51
Larval rearing, treatments, and sampling
Newly hatched larvae were reared indoor in ten 1.5-
ton capacity fiberglass tanks for 35 days. Four tanks
were used for the natural food treatment, another four
tanks for the co-fed natural food and artificial diet
treatment, and two tanks for the unfed to PNR
treatment. Grouper larvae were stocked at a density
of 20/L and water maintained at 33–35 ppt salinity
measured using an Atago refractometer, ambient
temperature (25.5–29.5 °C), and with mild aeration.
Dissolved oxygen monitored daily (using YSI DO200)
ranged from 5.0 to 6.5 ppm. Nannochlorum sp. was
supplied with green water beginning day 2 at 1–3 9
105cells/ml following the larval rearing protocol of
Duray et al. (1997). The larvae were fed natural food
that included enriched B. plicatilis (day 2 to 4 at 3/ml,
day 5 to 35 at 10/ml) and enriched newly hatched A.
salina (day 15 to 35 at 3–5/ml) following Duray et al.
(1997). SEAFDEC/AQD microbound artificial diet for
grouper larvae was also fed to the larvae in the co-fed
treatment (day 2 to 14—0.5–1 g/ton, 150–200 lm and
day 15 to 35—2–5 g/ton, [ 500 lm), in the morning
and afternoon (SEAFDEC/AQD Marine Fish Hatch-
ery Protocol, 2009; see Fig. 1, modified from Duray
et al. 1997). The tank bottom was siphoned to remove
debris, fecal material, and dead fish starting on day 7
when *10 % water volume was changed and then
increased to 50 % daily water change on day 10, and
50–70 % from day 20 to 35.
About 10–20 larvae were collected per tank for
transmission electron microscopic (TEM) analysis on
days 0, 3, 4, 6, 19, and 33 to monitor the critical
periods reported by Liao et al. (2001) in grouper fry of
0–7 and 18–19 days after hatching, and during meta-
morphosis. Samples from day 3 included the treat-
ments natural food only, co-fed artificial diet, and
unfed to PNR. Only the unfed to PNR treatment was
sampled on day 4.
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Fig. 1 Feeding and water

management scheme in Water management:
rearing E. coioides larvae
(SEAFDEC/AQD Marine
Siphoning of tank bottom (every other day)
Fish Hatchery Protocol, Water volume change
2009, modified from Duray
et al. 1997) 20-30% 50-70% 70-90%

Feeding Scheme:
Brachionus plicatilis
3 ind/ml 10 ind/ml
Nannochlorum sp.
5
(1-3x10 cells/ml)

Grouper larval diet

0.5-1 g/ton 2-5 g/ton 5-10 g/ton
Artemia salina
3-5 ind/ml (increasing sizes)
minced trash fish
(ad libitum)
0 5 10 15 20 25 30 35 40 45 50 55 60
Culture Period (day)

Transmission electron microscopy
Three larvae sampled from each treatment were
processed for TEM analyses. For samples taken on
days 0, 3, and 6, the whole larvae were fixed and
sectioned. For samples taken on days 19 and 33, the
head and tail were removed prior to fixation. Larvae
were fixed in 2 % glutaraldehyde, postfixed in 2 %
osmium tetroxide, dehydrated in a series of graded
ethanol, infiltrated with ethanol/Epon mixture,
pre-embedded in Epon 812, and polymerized in
silicon mold at 72 °C for 16 h. Ultrathin sections
(*70–90 nm) from the anterior to midportion of the
intestine were cut (microtome Leica Ultracut UCT),
stained with lead citrate, counterstained with uranyl
acetate, and examined (JEOL Transmission Electron
Microscope JEM-1010).
Enterocyte ultrastructure measurement
enterocyte height (lm), mitochondrial size (lm2) and
relative abundance (no/lm2), and lipid droplet size
(lm2) and relative abundance (no/lm2) were deter-
mined using the software Gatan Digital Micrograph v.
3.7.1. Microvilli relative density (no/lm) was the
number of microvilli counted for a given length of the
apical membrane perpendicular to the microvilli.
Enterocyte height (lm) was measured from the basal
membrane to the tip of the microvillus. Mitochondrial
and lipid droplet sizes (lm2) were computed as the
area of an ellipse (A = p ab, where A = area of the
ellipse, a and b = radii of the x- and y-axes of the
ellipse, respectively). Relative abundance was com-
puted as the number of mitochondria and lipid droplets
counted for a given area (lm2) in the enterocyte.
Diameter of lipid inclusions was as follows: lipid
droplets C0.50 lm, chylomicron 0.07–0.50 lm, and
very low-density lipoprotein (VLDL) 0.02–0.07 lm
(Diaz et al. 1997).

All measurements were made from three replicate fish
samples for each treatment with a minimum of five
(for small larvae—days 0, 3, and 6) and a maximum of
20 (for big larvae—days 19 and 33) random sections
from the anterior to midportion of the intestine per fish
sample. Microvilli relative density (no/lm length),
Statistical analyses
Data were tested for normality using the Kolmogorov–
Smirnov test. One-way ANOVA and Tukey’s post hoc
test were used to compare feed treatments (unfed,
natural food only, and co-fed) during day 3. The t test

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Fish Physiol Biochem (2014) 40:607–624
Fig. 2 TEM micrograph of sections from the anterior to
midsection of the intestine of day 0 E. coioides. a Periblast
with lipoprotein particles (LP) and mitochondria (M), scale bar
0.5 lm, magnification = 915,000. b Enterocyte with develop-
ing microvilli (MV), basolateral chylomicrons (C) adjacent to
the inter-enterocyte space (IES), and basal lamina (BL), scale
was applied to compare the co-fed and natural food-
only treatments separately during days 6, 19, and 33.
In case normality was not achieved with transforma-
tion, the Mann–Whitney test was used in place of the
t test. All statistical analyses were done using SPSS
v.16.0. Size and relative abundance measurements
were randomly determined from equally random
sections of the grouper larvae anterior intestine, using
5–20 random sections per fish and three replicate fish
per treatment. Values were expressed as mean ± stan-
dard deviation for samples (days 0 and 4) and
mean ± standard error after statistical tests had been
performed (days 3, 6, 19, and 33). Differences in
means were considered significant at P \ 0.05.
Results
Qualitative observations
The periblast, or syncytial layer surrounding the yolk
and oil globule, of newly hatched day 0 grouper larvae
had mitochondria and electron-lucent lipoprotein
particles (Fig. 2a). In the enterocytes, chylomicrons
(0.12–0.22 lm diameter) were observed in the cyto-
plasm, inter-enterocyte space (IES), and basal lamina
(Fig. 2b). Microvilli started to develop as granular
cytoplasmic extensions from the apical membrane of
611
bar 1 lm, magnification = 915,000. c Columnar enterocytes
delineated by evenly structured apical MV, tight junctions (TJ),
IES, and BL developed within 24 h in undifferentiated gut, scale
bar 5 lm, magnification = 93,000. Lumen (L), nucleus (N),
terminal web (TW)
enterocytes (Fig. 2b) and were fully differentiated
within 24 h after hatching with evenly structured
microvilli in columnar enterocytes (Fig. 2c). The tight
junctions, inter-enterocyte space, basal lamina, and
apical microvilli delineated the enterocytes (Fig. 2c).
Mucosal folds started to form (Fig. 3a) on day 3
in unfed larvae. A closed (not exposed to the lumen)
entero-endocrine cell (EC) was observed in the
intestinal mucosa (Fig. 3a). Many mitochondria
were observed (Fig. 3a, b). A number of enterocytes
were undergoing apoptosis (Jones and Gores 1997)
as shown by their pyknotic and karyorrhexic nuclei
(Fig. 3a, b). An enterocyte with a pyknotic nucleus
appeared to phagocytose an apoptotic body from an
adjacent enterocyte (Fig. 3b). Electron-lucent chylo-
microns (0.08–0.52 lm diameter) associated with
lamellar structures were observed beside the inter-
enterocyte space and the basal lamina (Fig. 3b).
Nuclear and cell fragments were observed in
apoptotic bodies (Fig. 3b). Enterocytes in advanced
apoptosis were observed to have more numerous
mitochondria, a disorganized terminal web, and
sloughing off microvilli (Fig. 3c). The lumen was
contracted and filled with cilia and sloughed off
microvilli (Fig. 3c).
On day 4, the PNR, all the enterocytes of all unfed
larvae samples were in various stages of apoptosis.
Gaps were observed in the microvilli as well as
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Fig. 3 TEM micrograph of sections from the anterior to
midsection of the intestine of unfed day 3 E. coioides.
a Apoptotic enterocytes with pyknotic (PN) and karyorrhexic
nuclei (KN), a mucosal fold (MF) beginning to form and a
‘closed’ entero-endocrine cell (EC), scale bar 2 lm, magnifi-
cation = 96,000. b Section showing well-organized microvilli
(MV), a thin terminal web (TW), apoptotic bodies (AB)
phagocytosed by adjacent enterocyte having PN, abundant
mitochondria (M) associated with lamellar structures (LS), and
chylomicrons (C) near the IES and BL, scale bar 2 lm,
magnification = 96,000. c Section showing apoptotic entero-
cyte with KN, many M associated with LS, degraded TW, MV
mitochondrial fission and fragmentation of their inner
and outer membranes (Fig. 3d). The enterocytes had
collapsed with sloughing off of the microvilli and
degraded basal lamina (Fig. 3e). Only mitochondria
remained associated with lamellar structures while
other enterocyte organelles and structures were disor-
ganized or in apoptotic bodies (Fig. 3e). Advanced
enterocyte disintegration before the death of the larvae
was observed when microvilli were completely
sloughed off and no organelles were discernible other
than apoptotic bodies and disorganized lamellar
structures (Fig. 3f).
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Fish Physiol Biochem (2014) 40:607–624
sloughing off, and collapsed lumen (L) with cilia (CI), and
detached MV in the middle, scale bar 1 lm, magnifica-
tion = 915,000. Unfed day 4 E. coioides. d Apoptotic entero-
cyte with gaps between MV and fission (arrow) of M with inner
and outer membranes (asterisk) fragmented, scale bar 0.2 lm,
magnification = 940,000. e Section showing collapsed entero-
cyte with MV sloughing off, AB, and degraded BL, a
mitochondrion (M) remained intact associated with LS, scale
bar 0.5 lm, magnification = 925,000. f Section showing
advanced enterocyte disintegration and autolysis with the MV
totally sloughed off and cell organelles undiscernible other than
LS and AB, scale bar 2 lm, magnification = 915,000
In day 3 larvae fed solely natural food, an open EC
was observed releasing cell components to the lumen
(Fig. 4a). Lipid droplets (0.02–0.14 no./lm2) were
observed in the enterocytes (Fig. 4a). An unusually
large chylomicron (0.99–1.45 lm diameter) was
transported into the lateral IES (Fig. 4b). Enterocytes
of larvae co-fed artificial diet had more abundant
(0.08–0.33 no./lm2), infranuclear, and clustered lipid
droplets (Fig. 4c). Spherical particles were observed
in between the microvilli and above the inter-micro-
villi invagination like the one shown in Fig. 4d.
Electron-lucent lipidic inclusions in the terminal web
Fish Physiol Biochem (2014) 40:607–624
Fig. 4 TEM micrograph of sections from the anterior to
midsection of the intestine of day 3 E. coioides fed natural
solely food. a Section showing an ‘open’ entero-endocrine cell
(EC) releasing cell components to the lumen (L), enterocytes
with well-organized microvilli (MV), nucleus (N), some
mitochondria (M), lipid droplets (LD), and chylomicrons
(C) near the basal lamina (BL) and inter-enterocyte space
(IES), scale bar 2 lm, magnification = 96,000. b Section
showing N, M, C and one unusually large chylomicron
transported into the IES, scale bar 1 lm, magnifica-
tion = 915,000. Day 3 E. coioides co-fed artificial diet.
and apical cytoplasm were observed above and beside
multivesicular bodies (MVBs), while the latter con-
tained intraluminal vesicles (Fig. 4d, e).
On day 6, enterocytes in the anterior intestine of E.
coioides larvae fed solely natural food had electron-
lucent spherical material in between the microvilli, as
well as lipidic inclusions of the same material below
the terminal web associated with the endoplasmic
reticulum (Fig. 5a). These inclusions appeared to be
endocytosed through the inter-microvilli invagin-
ations (Fig. 5b). Vesicles were observed inside an
MVB located below the terminal web and beside the
613
c Section showing columnar enterocytes with MV, M, N,
infranuclear clustered LD, and basal C, scale bar 2 lm,
magnification = 94,000. d Section showing spherical particle
(asterisk) between MV above the inter-MV invagination (IMVI),
tight junction (TJ) across the terminal web (TW), electron-lucent
lipidic inclusions (LI) in the apical cytoplasm and beside a
multivesicular body (MVB) with vesicles (V) inside, scale bar
1 lm, magnification = 980,000. e. Section showing electron-
lucent LI through the TW to the apical cytoplasm and an MVB
below with many intraluminal V inside, scale bar 0.5 lm,
magnification = 940,000
lipidic inclusions (Fig. 5b).Very large supra- and
infranuclear lipid droplets were observed together
with the small mitochondria (Fig. 5c). Differently
stained vesicles were observed in the perinuclear
Golgi apparatus (Fig. 5c). Lateral chylomicrons asso-
ciated with lamellar structures were observed near the
IES and beside the basal lamina (Fig. 5c, d). Larger
lipid droplets with small mitochondria were observed
in the enterocytes of larvae co-fed artificial diet
crowding out the nucleus (Fig. 5d).
Enterocytes in day 19 E. coioides larvae fed solely
natural food had regular mitochondria (Fig. 6a).
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Fig. 5 TEM micrograph of sections from the anterior to
midsection of the intestine of day 6 E. coioides fed natural food
only. a Section showing enterocyte with electron-lucent lipidic
material (asterisk) in between microvilli (MV) and lipidic
inclusions (LI) of the same material below the terminal web
(TW) associated with endoplasmic reticulum (ER), scale bar
0.5 lm, magnification = 915,000. b Section showing inter-
microvilli invaginations, electron-lucent LI through the TW and
beside a multivesicular body (MVB), scale bar 0.5 lm,
Apoptotic bodies and pyknotic nuclei were also
observed (Fig. 6a). The apoptotic bodies appeared to
be exocytosed apically from the enterocyte to the
lumen through the terminal web and microvilli
(Fig. 6b). Apical mucus granules secreted into the
lumen were first observed on day 19 (Fig. 6c).
Enterocytes of larvae co-fed artificial diet had MVBs
in the apical cytoplasm (Fig. 6d), but the mitochondria
were swollen and irregular with translucent matrices
and fragmented cristae (Fig. 6e). No lipid droplets
were observed.
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Fish Physiol Biochem (2014) 40:607–624
magnification = 925,000. c Section showing very large supra-
and infranuclear lipid droplets (LDs), nucleus (N), mitochondria
(M), perinuclear Golgi apparatus (G) with differently stained
vesicles (GV), chylomicron (C) near the inter-enterocyte space
(IES) associated with lamellar structures (LS), scale bar 2 lm,
magnification = 95,000. Day 6 E. coioides co-fed artificial
diet. d Section showing small M, very large LD crowding out the
N, C beside the basal lamina (BL), scale bar 2 lm,
magnification = 94,000
On day 33, enterocytes in larvae fed solely natural
food had many supranuclear mitochondria and mostly
infranuclear lipid droplets about as big as the nuclei
(Fig. 7a). Secretory vesicles were also observed
(Fig. 7a). Apical mucus granules were observed
through the terminal web (Fig. 7b). Differently stained
vesicles (0.51–0.83 lm diameter) were observed in
the Golgi apparatus (Fig. 7c). Chylomicrons were also
observed near the Golgi body (Fig. 7c). On the other
hand, enterocytes in larvae co-fed artificial diet were
filled with supra- and infranuclear lipid droplets much
Fish Physiol Biochem (2014) 40:607–624 615

Fig. 6 TEM micrograph of

sections from the anterior to
midsection of the intestine
of day 19 E. coioides fed
solely natural food.
a Section showing regular
mitochondria (M), pyknotic
nuclei (PN), and apoptotic
bodies (AB), scale bar 2 lm,
magnification = 96,000.
b Inset showing enlarged
view of ABs evacuated
through the terminal web
(TW) and microvilli (MV),
scale bar 1 lm,
magnification = 915,000.
c Section showing apical
mucus granules (MG)
secreted to the lumen (L),
scale bar 2 lm,
magnification = 94,000.
Day 19 E. coioides co-fed
artificial diet. d Section
showing multivesicular
bodies (MVB) in the apical
cytoplasm, scale bar 2 lm,
magnification = 93,000.
e Section showing irregular
M with translucent matrices
and fragmented cristae,
N and lamina propria (LP),
scale bar 5 lm,
magnification = 92,500

bigger than the nucleus together with smaller mito-
chondria (Fig. 7d). Thinning and irregular microvilli
were observed in enterocytes with very large apical
lipid droplets (Fig. 7e). Apical mucus granules and an
EC were also observed (Fig. 7e). An eosinophilic
granule cell (EGC) with dark-stained cytoplasmic
granules was observed as well (Fig. 7f). A goblet cell
with apical mucus granules and infranuclear lipid
droplets beside and below the nucleus was also
observed (Fig. 7g).
Quantitative observations
Among the days sampled, enterocyte microvilli
density (2.75 ± 0.60 no./lm) was lowest on day 0
comparable to that of unfed day 4 grouper larvae

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616
Fig. 7 TEM micrograph of sections from the anterior to
midsection of the intestine of day 33 E. coioides fed solely
natural food. a Section showing enterocytes with many
supranuclear mitochondria (M), mostly infranuclear lipid
droplets (LD) about as big as the nuclei (N) and secretory
vesicles (SV), scale bar 5 lm, magnification = 93,000. b Sec-
tion showing apical microvilli (MV) and mucus granules (MG)
through the terminal web (TW), N, and many supranuclear M,
scale bar 2 lm, magnification = 98,000. c Golgi apparatus
(G) with differently stained vesicles (GV) and adjacent
chylomicron (C), scale bar 0.5 lm, magnification = 925,000.
(Fig. 8a) at PNR. On day 3, enterocytes in unfed
larvae had significantly higher microvilli density
(12.06 ± 0.51 no./lm; P \ 0.05) than those of
larvae fed solely natural food. On day 19,
123
Fish Physiol Biochem (2014) 40:607–624
Day 33 E. coioides co-fed artificial diet. d Section showing
enterocytes filled with supra- and infranuclear LD much bigger
than N and smaller than M, scale bar 5 lm, magnifica-
tion = 92,500. e Section showing thinning and irregular MV
with very large apical LD, apical MG, and an entero-endocrine
cell (EC), scale bar 4 lm, magnification = 94,000. f Section
showing eosinophilic granule cell (EGC) with dark-stained
cytoplasmic granules (CG) and surrounding LD, scale bar 2 lm,
magnification = 912,000. g Section showing goblet cell (GC)
with apical MG and infranuclear LD beside and below the N,
scale bar 2 lm, magnification = 95,000
enterocytes in larvae fed solely natural food had
significantly higher microvilli density (10.25 ± 0.41
no./lm2, P \ 0.01) than those of larvae co-fed
artificial diet.
Fish Physiol Biochem (2014) 40:607–624 617

a 14 natural food only size was greater than those of larvae co-fed artificial
b
12 ab co-fed artificial diet
a diet (1.00 ± 0.11 lm2, P \ 0.001).
b
Microvilli density

a unfed aa
(no./ length-µm)

a On the other hand, mean mitochondrial relative

10
a abundance in enterocytes of day 0 larvae (0.76 ± 0.10
8
no./lm2) was comparable with those of day 3 fed
6
larvae (Fig. 9b). On day 3, unfed larvae had signifi-
4 cantly higher mean mitochondrial relative abundance
2 (1.14 ± 0.08 no./lm2; P \ 0.001). This peaked on
0 day 4, the PNR, to about triple (3.53 ± 1.37 no./lm2;
Day 0 Day 3 Day 4 Day 6 Day 19 Day 33 Fig. 9b) from the day earlier. On days 19 and 33,
mitochondrial relative abundance was significantly
b 20 b higher in enterocytes of larvae fed solely natural food
a (day 19 0.68 ± 0.04 no./lm2, P \ 0.001; day 33
15
b a 1.51 ± 0.36 no./lm2, P \ 0.05; Fig. 9b).
Enterocyte ht (µm)

a On day 0, no lipid droplets were observed in the

b a
10
b
a larvae on days 3 and 4 (Fig. 9c, d). Lipid droplets were
5 first observed in the enterocytes of day 3 fed larvae and
0 abundance between the two feed treatments (Fig. 9c,
Day 0 Day 3 Day 4 Day 6 Day 19 Day 33
metamorphic
endotrophic | endo-exotrophic | exotrophic
Fig. 8 Differences in a microvilli density and b enterocyte
height in E. coioides larvae reared under different feeding
regimes. Bars of a given day with the same letter are not
significantly different at P [ 0.05. Values are mean ± SEM
Mean enterocyte height (7.83 ± 1.79 lm) in day 0
grouper larvae was comparable with those of day 3 fed
larvae (Fig. 8b). Among day 3 treatments, the unfed
larvae had significantly lower mean enterocyte height
(4.71 ± 0.32 lm; P \ 0.001) compared to their fed
counterparts (Fig. 8b). This was further reduced on
day 4 (3.64 ± 1.22 lm), the PNR. On day 6, mean
enterocyte height was significantly greater in larvae
fed solely natural food (11.12 ± 1.05 lm, P \ 0.05)
than those of larvae co-fed artificial diet. This was
reversed on day 19 when enterocyte height was
significantly greater in larvae co-fed artificial diet
(17.47 ± 0.37 lm, P \ 0.001, Fig. 8b).
Mitochondrial size was relatively high in entero-
cytes of day 0 larvae (0.94 ± 0.06 lm2) compared to
those of larvae sampled on other days (Fig. 9a). On
day 3, mean mitochondrial size in the enterocytes of
unfed larvae (0.36 ± 0.05 lm2; P \ 0.05) was
reduced from day 0 and smaller than those of the fed
larvae. This was further contracted (0.13 ± 0.06 lm2)
on day 4, the PNR. On day 19, mean mitochondrial
enterocytes of newly hatched larvae and in unfed
did not significantly differ in size and relative
d). On day 6, larvae from the two feed treatments had
very large lipid droplets, but those in enterocytes of
larvae co-fed artificial diet had greater mean size
(4.02 ± 2.42 lm2, max. diameter 9.06 lm) than that
of larvae fed solely natural food (1.27 ± 0.42 lm2,
max. diameter 4.81 lm, Fig. 9c). On the other hand,
lipid droplets had significantly higher relative abun-
dance (0.24 ± 0.04 no./lm2, P \ 0.05) in enterocytes
of larvae fed solely natural food than those co-fed
artificial diet (Fig. 9d). On day 19, lipid droplets were
scant in enterocytes fed solely natural food
(0.01 ± 0.01 no./lm2) and absent in those of larvae
co-fed artificial diet (Fig. 9c, d). On day 33, entero-
cytes of larvae co-fed artificial diet had larger mean
lipid droplet sizes (3.27 ± 0.89 lm2, P \ 0.05) and
greater lipid droplet relative abundance (0.39 ± 0.06
no./lm2, P \ 0.05) than those fed solely natural food
(Fig. 9c,d).
Summary
The qualitative differences and developmental
changes in enterocyte ultrastructure with particular
attention to apoptosis, mitochondria, lipid droplets,
and MVBs in the anterior intestine of E. coioides can
be summarized as shown in Fig. 10. On day 0, no lipid
droplets were observed, and mitochondria were largest
in enterocytes among the days sampled. On day 3
when larvae go through the transition from

123
618 Fish Physiol Biochem (2014) 40:607–624

a endogenous to exogenous feeding, enterocytes in

1.5 unfed larvae showed pyknosis, an early stage of
natural food only
Mitochondria size (µm2)

co-fed artificial diet apoptosis, which progressed to karyorrhexis accom-

b
unfed panied by the sloughing off of microvilli on day 4
1 b (PNR). There was no notable difference in the sizes
and relative abundance of lipid droplets and mito-
ab a
a a chondria in the enterocytes of larvae fed solely natural
0.5
aa a food and co-fed artificial diet. On day 6, when larvae
depended solely on exogenous food, enterocytes of
0 larvae co-fed artificial diet had significantly larger
Day 0 Day 3 Day 4 Day 6 Day 19 Day 33
lipid droplet sizes but of lower density than those of
b larvae fed solely natural food. Mitochondrial size and
Mitochondria rel.abundance

4 density did not vary between the two feed treatments.

On day 19, preparatory to metamorphosis, lipid
3 droplets were scant to absent in enterocytes of larvae
(no./µm2)

b
in both feed treatments while mitochondria were more
2 abundant in enterocytes of larvae fed solely natural
a
b a food where some enterocytes were also apoptotic.
1 aa b
a Upon metamorphosis on day 33, the enterocytes in
a
larvae co-fed artificial diet had much larger and more
0 abundant lipid droplets than those of larvae fed solely
Day 0 Day 3 Day 4 Day 6 Day 19 Day 33
natural food, while the latter had more abundant
c mitochondria. MVBs were observed in the apical
7 a cytoplasm of enterocytes in day 3 and 19 larvae co-fed
Lipid droplet size (µm2)

6 artificial diet and in day 6 larvae fed solely natural

5 b food (Fig. 10).
4
b
3 ab
a
2
Discussion
1 a aa a
0 0 0 0
0
Day 0 Day 3 Day 4 Day 6 Day 19 Day 33
Endogenous feeding

d Newly hatched day 0 E. coioides larvae had an

0.5 b undifferentiated gastrointestinal tract comprised of
simple cuboidal enterocytes (Quinitio et al. 2004a)
abundance (no./µm2)

0.4
Lipid droplet rel.

that developed into columnar enterocytes within 24 h

b
0.3 b a after hatching. The lipoprotein particles in the peri-
0.2
blast may have been derived from the yolk sac and oil
a
ab globule and hydrolyzed by acid phosphatase and
a
0.1
0 a nonspecific esterase, respectively, in newly hatched E.
0 0 a0
0
coioides larvae (Quinitio et al. 2004b). Diaz et al.
Day 0 Day 3 Day 4 Day 6 Day 19 Day 33 (2002) reported that lipoprotein particles in the
metamorphic
endotrophic| endo-exotrophic | exotrophic
periblast were transported as complex lipids to early
postembryonic fish larvae (Dicentrarchus labrax,
Fig. 9 Differences in a mitochondrial size and b relative Sparus aurata, and Stizostedion lucioperca). Ordo-
abundance, and c lipid droplet size and d relative abundance in
nio-Aguilar (1995) reported that endotrophic E. coio-
enterocytes of E. coioides larvae under different feeding
regimes. Bars of a given day with the same letter are not ides larvae catabolized mainly triglycerides (41 %)
significantly different at P [ 0.05. Values are mean ± SEM and phospholipids (47 %).

123
Fish Physiol Biochem (2014) 40:607–624
Fig. 10 Diagram of changes in anterior intestine enterocytes in E. coioides larvae from day 0 to 33 under different feeding regimes
(cells not drawn to scale, but relative proportions of mitochondria and lipid droplet sizes to enterocyte height have been approximated)
Mitochondrial size of newly hatched larvae was
generally larger than that of older larvae and may
indicate intensified energy production to support accel-
erated growth immediately after hatching (Ordonio-
Aguilar et al. 1995). Kjørsvik et al. (1991) also reported
greatest area of mitochondria in the anterior intestine
enterocytes of Atlantic cod (Gadus morhua L.) in the
earliest stages. In E. coioides, this was supported by a
strong endogenous growth hormone (GH) mRNA signal
at this stage (Li et al. 2005).
Transition from endogenous to exogenous feeding
Unfed larvae
The reduced enterocyte height indicated poor nutri-
tional status (Theilacker and Watanabe 1989) in unfed
619
larvae. Starved cod and turbot (Scophthalmus maxi-
mus) larvae had shrunk and thin epithelial cells of the
midgut (Kjørsvik et al. 1991; McFadzen et al. 1994).
Despite the impaired status of the anterior intestinal
epithelia in unfed day 3 E. coioides larvae, signs of
continued digestive function were observed. The
higher microvilli density increased the surface area
for food absorption (Kuz’mina and Gelman 1997),
suggesting a survival mechanism at the cellular level.
Apoptotic enterocytes phagocytosed by adjacent
healthy ones demonstrated apoptosis as another
mechanism to overcome nutritional deficit. Chylomi-
crons were observed adjacent to the basal lamina and
IESs, demonstrating the transport of lipids from the
intestine (Tocher 2003). A ‘closed’ EC (Sternini et al.
2008) was observed, indicating the regulation of
postprandial secretion and motility (Reinecke et al.
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620
1997). ECs were also reported in the midgut of newly
hatched sea bream (Sparus aurata, Calzada et al.
1998). Thus, E. coioides larvae employ survival
mechanisms and continued digestive function to thrive
when food is captured at this time.
The PNR for unfed E. coioides larvae recorded on
day 4 coincided with the exhaustion of the yolk sac and
oil globule (Ordonio-Aguilar et al. 1995; Quinitio
et al. 2004a). Enterocyte heights were severely
reduced. Microvilli were markedly reduced and
sloughing off, same as that of starved cod (Kjørsvik
et al. 1991) and turbot (McFadzen et al. 1994) larvae.
Nuclei fragmented into apoptotic bodies and were
phagocytosed by adjacent epithelial cells also reported
for cod larvae (Kjørsvik et al. 1991). Multiple small
units of fragmented mitochondria denoted mitochon-
drial regulation of apoptosis (Suen et al. 2008). The
PNR demonstrated an irreversibly compromised
digestive function that even if food is ingested at this
time, the damaged enterocytes will not be able to
digest and absorb the nutrients and the larvae will die.
Fed larvae
On day 3 when the larvae undergo the transition from
endotrophy to exotrophy, data on enterocyte heights in
the anterior intestine suggest that supplementation
with the larval artificial diet did not improve the
nutritional status of grouper larvae. This may be
related to the preference of first-feeding larvae for
mobile live prey over inert artificial diet. Biochemical
attractants may be present in zooplankton that lack
artificial diets (Rust 2002). However, other factors
such as the relationship between mouth size and prey
size (Russo et al. 2009) may also play a role.
Lipid droplets and chylomicrons in the enterocytes
demonstrate functional lipid metabolism in fed larvae.
The same was reported in first-feeding larvae of other
fish species (Calzada et al. 1998; Diaz et al. 2002;
Kjørsvik et al. 1991; Sarasquete et al. 1995). However,
an unusually large chylomicron (0.99–1.45 lm diame-
ter) was observed opening toward the IES. Its large size
indicated a storage function as a lipid droplet (Diaz et al.
1997), but its position denoted a transport function as a
chylomicron. The peculiar behavior of this particular
lipid inclusion with respect to morphology and function
indicates an inadequate supply of phosphatidylcholine
(PC). Large-sized lipoprotein granules in the intestinal
mucosa related to lecithin deficiency were also reported
123
Fish Physiol Biochem (2014) 40:607–624
for farm-reared sea bass (Deplano et al. 1989). Studies
indicate that a lack of choline, lysoPC, or PC may impair
the clearance of fat from the intestinal mucosa (Niu et al.
2008; Tocher et al. 2008).
An ‘open’ EC (Sternini et al. 2008) in the intestinal
epithelia that acts as sensors of luminal contents
(Reinecke et al. 1997) indicated a functional digestive
system in first-feeding grouper larvae. Inter-microvilli
invaginations (IMVIs), inclusions, and MVBs
observed in the apical cytoplasm of enterocytes on
day 3, and also on days 6 and 19 denote functional
intracellular digestion (Piper and Katzmann 2007).
The observed IMVI closely resembled the apical pits/
vesicles reported by Sire et al. (1981) in rainbow trout
(Salmo gairdneri Rich.), which may also be related to
intracellular lipid metabolism.
Exogenous feeding
The greater enterocyte height in day 6 E. coioides
larvae fed solely natural food denoted better nutri-
tional condition (Theilacker and Watanabe 1989) than
in those co-fed artificial diet. Enterocytes of larvae co-
fed artificial diet with large lipid droplet sizes
(4.02 ± 2.42 lm2) in smaller enterocytes were the
characteristics of intestinal steatosis (Deplano et al.
1989; Gisbert et al. 2005). This may indicate subop-
timal dietary lipid class composition (Morais et al.
2007) attributed to a deficiency in phospholipids
(Hamre 2006; Morais et al. 2007).
The accumulation of lipid droplets in the intestinal
mucosa of fish larvae indirectly leads to positive
buoyancy (Phleger 1998). This may have contributed
to the very high mortalities of epinepheline serranids
reported at this early stage (Liao et al. 2001; Toledo
et al. 1999). Grouper larvae exhibit positive phototac-
tic behavior with eye pigmentation and actively swim
to the water surface (Ordonio-Aguilar et al. 1995).
This is also the time when mucus production peaks
which acts as glue that traps the larvae in the water
surface-tension resulting in mortalities (Yamaoka
et al. 2000). Its positive buoyancy may make it more
difficult for the larvae to detach from the water
surface-tension particularly when it first inflates its
swim bladder on days 6–7, although mucus production
stops at this time (Yamaoka et al. 2000). Toledo et al.
(1999) reported very high mortalities of E. coioides on
day 7, which coincides with the first inflation of the
swim bladder.
Fish Physiol Biochem (2014) 40:607–624
Limited lipid metabolism and the consequent
intestinal steatosis increased the risk of entrapment
to the water surface-tension both undermine the
growth and survival of early E. coioides larvae. This
may be related to the limited ability of early devel-
oping stages of fish to biosynthesize phospholipids,
leading to impaired lipoprotein synthesis, hindering
lipid transport from the intestine, and reducing lipid
utilization (Cahu et al. 2003, 2009; Tocher et al. 2008).
Increased growth, normal morphogenesis, and sur-
vival were reported in first-feeding and older larval
fish fed diets with 3–12 % phospholipids (Cahu et al.
2003; Cahu et al. 2009; Niu et al. 2008). First-feeding
E. coioides larvae had improved larval growth and
survival when fed copepods, which had 2 to 3 times
higher phospholipid (PC) content than that of rotifers
(Toledo et al. 1999). Grouper larval diets require
appropriate levels of phospholipid supplementation.
Preparatory to metamorphosis
Day 19 E. coioides larvae showed suboptimal nutri-
tional condition with scant to absent lipid droplets,
smaller enterocyte heights (Theilacker and Watanabe
1989), and some apoptotic enterocytes. Passage of
disintegrating midgut enterocytes into the lumen was
also described in fed northern anchovy (Engraulis
mordax) larvae that failed to capture food (O’Conell
1976). The same was reported in starved turbot larvae
(McFadzen et al. 1994).
The amount of feed applied in this study (about 10
rotifers/ml and 5 brine shrimp/ml) especially prepara-
tory to metamorphosis may have been insufficient
given the relatively high stocking density used. Cunha
et al. (2009) reported that the calculated minimum
daily food requirement (day 15—about 67 ind.
prey day-1) of Epinephelus marginatus larvae prepa-
ratory to metamorphosis increases exponentially
during metamorphosis (day 25—about 513 ind.
prey day-1). Increased assimilation and energy trans-
formation efficiencies have also been reported for
metamorphic Japanese flounder (Paralichthys oliva-
ceus) larvae (Sumule et al. 2003). Duray et al. (1996)
reported higher survival of grouper larvae when fed
more rotifers ((20/ml, Duray et al. 1996).
Another nutritional concern may be indicated in
day 19 E. coioides larvae co-fed artificial diet as
shown by the enterocytes having swollen mitochon-
dria with translucent matrices and fragmented cristae.
621
The same irregular mitochondrial morphology was
reported in anterior intestine enterocytes in turbot
larvae fed diets with soybean phospholipid (Mac-
Queen Leifson et al. 2003). In contrast, these irregu-
larities were not observed in larvae fed solely natural
food as were those in turbot larvae fed live feed or
diets with marine phospholipid (MacQueen Leifson
et al. 2003). MacQueen Leifson et al. (2003) associ-
ated this with the high levels of linoleic acid in the diet
not normally found in natural food of marine fish
larvae. Although the phospholipid and linoleic acid
contents of the two feed treatments in this study were
not quantified, the observed irregular mitochondria
may also be associated with the linoleic acid content of
the soybean lecithin in the artificial diet. Further
investigations on the genome complement of desatur-
ase and elongase genes in E. coioides larvae may
validate its competence to biosynthesize highly unsat-
urated fatty acid (HUFA) from linoleic acid, which
may vary during the growth cycle, with source of oil in
the diet and environmental salinity (Sarker et al. 2011;
Tocher 2010). In vivo challenge tests may also be
conducted for postflexion E. coioides larvae fed diet
containing soybean lecithin to assess the fitness of the
larvae at this critical period.
Metamorphosis
Anterior intestine enterocytes in day 33 E. coioides
larvae co-fed natural food and artificial diet had
abundant and large lipid droplets with few mitochon-
dria, while those of larvae fed solely natural food had
smaller and less abundant lipid droplets but more
abundant mitochondria. This observed inverse behav-
ior of lipid droplet and mitochondrial size and relative
abundance shows the direct role of mitochondrial b-
oxidation in the catabolism of fatty acids (Frøyland
et al. 1997) in enterocytes of metamorphic grouper
larvae. Intestinal steatosis was again evident with the
greater size and relative abundance of lipid droplets in
enterocytes of larvae co-fed artificial diet, same as in
day 6 and day 33 larvae. This indicates a deficiency in
dietary phospholipid (Hamre 2006; Tocher et al. 2008)
and the need for increased phospholipid supplemen-
tation (Cahu et al. 2009; Tocher et al. 2008) in E.
coioides larvae. Teleost larval stages are extremely
sensitive to dietary phospholipid deficiency and
require higher levels than juveniles (Dapra et al.
2011) and adults (Tocher et al. 2008). The thinning
123
622
and irregular microvilli structures found in some
enterocytes merit further study as these may lead to an
induced pathogenic state where the epithelial barrier is
weakened (Deplano et al. 1989; Olsen et al. 1999,
2000).
Eosinophilic granule cells (EGCs), analogous to
mammalian mast cells, were observed in the intestinal
epithelia of metamorphic grouper larvae signifying
increased immunity capability at this time (Mulero
et al. 2007). As histamine was found to be biologically
active in EGCs of perciform teleost (Mulero et al.
2007), histidine supplementation may be investigated
to validate its role in the development of immunity
fitness in metamorphic grouper larvae. In addition, the
associated role of gut microbiota in the differentiation
(Bates et al. 2006) of grouper larval gut represents a
relevant but less studied subject that also merits further
investigation.
Acknowledgments The authors are grateful to the Commission
on Higher Education, the Philippine Council for Agriculture,
Aquatic Natural Resources Research and Development and the
Science Education Institute of the Department of Science and
Technology, and the Bureau of Agricultural Research-
Department of Agriculture of the Government of the Philippines
for financial support (extended to Y.H. Primavera-Tirol) and to the
Southeast Asian Fisheries Development Center-Aquaculture
Department for the use of its aquaculture facilities. Thanks are
given to V. Balinas for statistical advice, to T. Billena-Hagy, C.
Sombito, and N. Bautista for technical assistance with the
transmission electron microscopy, and to I. Tendencia for the
artwork. Helpful advice from two anonymous reviewers that
improved the manuscript is gratefully acknowledged.
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