Int. J. Aquat. Biol. (2016) 4(4): 295-300DOI:
ISSN: 2322-5270; P-ISSN: 2383-0956
Journal homepage: www.ij-aquaticbiology.com
© 2016 Iranian Society of Ichthyology
Original Article
Histological and allometric growth analysis of eye in Caspian kutum, Rutilus kutum
Kamensky, 1901 (Teleostei: Cyprinidae) during early developmental stages
Shaghayegh Hasanpour1, Soheil Eagderi*1, Seyed Valli Hosseini1, Mohamad Hasan Jafari Sayadi2
1Department of Fisheries, Faculty of Natural Resources, University of Tehran, P.O. Box: 4111, Karaj, Iran.
2Department of Agriculture and Natural Resources, University of Payam Noor, Karaj, Iran.
Article history:
Received 7 April 2016
Accepted 22 June 2016
Available online 2 5 August 2016
Keywords:
Vision
Retina
Ontogeny
Growth pattern
Abstract: Fish larvae have several sensory systems that are functional at or soon after hatching and
then are developed further during larval and juvenile stages. This study was conducted to investigate
development of the eye in Rutilus kutum, based on histological and allometric growth analysis during
early developmental stages up to 35 day post hatching with emphasis on retinal morphology. For this
purpose, the histological sections were prepared and allometric growth pattern of the eye was
calculated. The results showed that the most eye’s structures along with the retina of the newly
hatched larvae, as the inner sensory (photosensitive) tissue were completely differentiated.
Allometric growth pattern of the eye diameter up to the inflexion point (7 dph) was somewhat positive
and then it became negative. The results revealed that the Caspian kutum is dependence on visual
capability as visual feeder during their larval period which itself explains completion of eye structures
and the high growth rate of eye before 3 dph i.e. beginning of mixed feeding.
Introduction
Fish larvae have several sensory systems that are
functional at or soon after hatching and then are
developed further during larval and juvenile stages
(Wahl et al., 1993; Hall and Wake, 1999; Loosey et
al., 2000). The feeding habits of fishes are reflected
on the structure and size of the sense organs
particularly the eyes (Atta, 2013). Eyes are among
the major sensory organs in fishes for detecting
photic stimuli and forming images of the
environment (Chai et al., 2006). Fish visual
capability is highly related to the eye structure,
therefore study of the eye structure and its retinal
morphology can provide insight to the visual
capability of fish (Lim et al., 2014). In addition, the
development of the functional eye is correlated with
the feeding habits of fishes for instance distinct
changes in the retinal morphology occur
concomitant with a shift from pelagic to a benthic
habitat (Hall and Wake, 1999).
Different growth rate of various parts of the body
* Corresponding author: Soheil Eagderi
E-mail address: soheil.eagderi@ut.ac.ir
or allometric growth is a common phenomenon
during early development of fishes (Osse and van
den Boogart, 1995), which it is responsible for a
progressive transformation of recently hatched
specimen from a larval body shape to juvenile or
adult form in a relatively short time (Khemis et al.,
2013). Hence, understanding normal growth pattern
and morphological changes are crucial to reduction
of the hatchery losses (Khmis et al., 2013; Pena and
Dumas, 2009).
Caspian kutum, Rutilus kutum is an important
commercial and edible fish in the southern Caspian
Sea distributed from the southwest (the Atrak River)
to northward (the Volga River) (Abdolhay et al.,
2011). Due to over fishing and deterioration of its
spawning grounds and natural habitats, this species
has experienced a dramatic reduction in its fishing
yields. Therefore, its artificial propagation in
hatcheries was established to recruit its natural
stocks by releasing its fingerlings into rivers that
drain to the Caspian Sea basin (Jafari et al., 2010).
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Hasanpour et al./ Histological and allometric growth analysis of Caspian kutum during early development
Since in restocking programs, providing basic
biological information is crucial for breeding and
rearing of larvae; therefore, this study was conducted
to investigate the development of the eye in the
Caspian kutum based on histological and allometric
growth analysis during early developmental stages
from hatching up to 35 day post hatching (dph) with
emphasis on retinal morphology. Its ontogeny will
provide insight on the visual capability of this
species and can help to optimize its larval culture
conditions.
Materials and Methods
Specimens rearing and sampling: Larval specimens
were obtained from artificial propagation of 15
female and 30 male broodstocks in April-May 2012,
in Dr. Yousef-pour Fish Hatchery Center (Siahkal,
Guilan, Iran). The eggs were incubated in 10 L vase
incubators with flow-through system at 22°C. After
six days of incubation, eggs were hatched and
transferred to a large larval collector tanks (200 L).
After 3 days, at the beginning of exogenous feeding,
30,000 larvae were transferred to an earthen pond
(0.1 ha) with a flow-through system with a mean
temperature, pH and dissolved oxygen of 25±2.2°C,
8.1±0.5 and 7.4±1.1 mg L-1, respectively. The
natural water flow provided some natural prey but
additional artificial feed was supplied from 7 dph.
The feed was a specialized feed for Caspian kutum
larvae and juveniles based on a mix of protein and
cereal meals. Larval specimens were randomly
sampled from 1-20 dph every days and then every 5
days up to 35 dph (n=30) from the same larval batch
prior to feeding, in the morning.
Allometric analysis: The samples were sacrificed by
overdose of MS222 (Sigma-Aldrich), weighed to the
nearest 0.0001 mg and fixed in 5% phosphate
buffered formaldehyde solution. The left side of the
fresh larvae, aged 1-12 dph, were photographed
using a dissecting microscope equipped with a
Cannon camera (5MP resolution) and the older
specimens were photographed using a Copy-stand
equipped to the camera. Total length (TL: from tip
of the snout to the end of the caudal fin) and eye
diameter were measured from the digital images to
the nearest 0.01 mm using the ImageJ software
(version 1.240). TL was measured as the reference
point in the description of the ontogeny because it is
a proper measure of ontogenetic state than age
(Hasanpour et al., 2015, 2016; Saka et al., 2008;
Sfakianakis et al., 2004, 2005).
Allometric growth pattern was calculated as a
power function of TL using non-transformed data:
Y=αXb. Where Y is the dependent variable, X: the
independent variable (TL), 𝛼 the intercept and b the
growth coefficient. Allometric growth pattern is
considered as positive, when b is larger than the
isometric value (b=1) and as negative when b>1
(Gisbert, 1999). The inflexion point of growth curve
was determined according to van Sink et al (1997).
Both preparation of the plots and data analysis were
performed using Manitab (version 16), PAST and
Microsoft Excel (version 2013) softwares.
Histological analysis: Six fixed specimens per
sampling day were randomly selected and
subsequently dehydrated in a graded series of
ethanol (70-100%) and cleared with Xylene and
finally embedded into paraffin. The histological
sections were prepared with 6 µm thickness,
mounted on the glass slides and stained with
hematoxylin and eosin (Hewitson and Darby, 2010;
Eagderi et al., 2013). The sections were examined
under a light microscope and photographed by a
Nicon camera (13MP resolution).
Results
At 1 dph, the eye was developed and had almost
similar structures as adults showing importance of
this organ during eleuthero-embryonic stage. In
addition, the retina was completely differentiated as
inner sensory (photosensitive) tissue of eye at this
stage. The pigment epithelium layer of the retina as
a non-nervous area, was thin. The inner nervous area
of the retina is composed of 9 layers, including
(definitions are according to Atta, 2013): (1)
Photoreceptor cell layer (Ph), (2) Outer or the
external limiting membrane (OM), (3) Outer nuclear
layer (ON) representing the nuclei of the
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Int. J. Aquat. Biol. (2016) 4(4): 295-300
photoreceptor cells, (4) Outer plexiform layer (OP)
i.e. the location of synaptic relationship between
photoreceptor, bipolar, amacrine and horizontal cells
as well as mullers cells, (5) Inner nuclear layer (IN)
contains the nuclei of several types of neurons
mainly of bipolar, amacrine and horizontal cells as
well as mullers cells, (6) Inner plexiform layer (IP),
the location of synaptic relationship between the
bipolar and ganglionic cells, (7) Ganglionic layer (G)
that is composed of a narrow chain of granular and
spherical cells surrounded by a fine connective tissue
network, (8) Nerve fiber layer (N) represents axons
of the ganglionic layer, and (9) Inner limiting
membrane (IM) (Fig. 1). In addition, the diameter of
the inner nuclear layer was significant compared to
the ganglionic layer.
The median uveal layer had not been completely
developed at 1 dph. The lens as an avascular
spherical ball was made up of 4 tissue layers,
including an extra cellular matrix (capsule), a
monolayer of nucleated flattened or cuboidal cells
capable of division and secretion, hyaline layer, and
fourth layer consisting long, slender, transparent,
non-nucleated fibrous cells that are arranged as
parallel rows (Fig. 1).
At 2 dph, the blood vessels of the rete choroid was
strongly developed to support the retina. There was
no significant structural changes in the eye expect
their size from 3 dph onward that is coincided with
starting mixed feeding, i.e. development of the retina
and choriocapillary layer is completed before
exogenous feeding. After 3 dph, the only noticeable
changes in the retina were increasing some layers’
diameter and the density of the rod, cone and
Figure 1. Eye development of Rutilus kutum. a-b: 1-dph, c: 2-dph, d-e: 3-dph, f: 4dph, g-h: 5-dph, j: 35-dph. N: nerve fiber layer, G: ganglionic
layer, IP: inner plexiform layer, IN: inner nucleus layer, OP: outer plexiform layer, ON: outer nucleus layer, OM: outer membrane, Ph:
photoreceptor cell layer, PE: pigment epithelium, BM: brush membrane, CL: choriocapillar layer, L: lens, I: iris, and C: cornea (scale bar = 100
µm).
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Hasanpour et al./ Histological and allometric growth analysis of Caspian kutum during early development
ganglion cells. During this period, the pigment
epithelium was gradually thickened.
The eye was externally unpigmented at hatching
and pigmented at the beginning of the mixed feeding
stage (3 dph). Allometric growth pattern of the eye
diameter up to the inflexion point (7 dph,
TL=12.48±0.67) was somewhat positive (b = 1.05,
r2=0.59), and it became negative after this point
(b=0.79, r2=0.95) (Fig. 2).
Discussion
The eye determines the ability to feed, search,
distinguish objects and orient in a three dimensional
light environment (Chai et al., 2006). Therefore,
major events in the functional ontogeny of the visual
system are closely correlated with life history events
where fish experiences changes in the photic
environment due to a change in vertical or horizontal
position or changes in behavioral repertoire (Blaxter
and Stains, 1970; Hall and Wake, 1999).
Several studies found that many fishes possess
only cone photoreceptor cells at the onset of
exogenous feeding, as the larvae live near the surface
of the water where sun light penetrates (Blaxter and
Stains, 1970; Hall and Wake, 1999; Lenkowski and
Raymond, 2014). The appearance of rod
photoreceptor cells in the retina delay until the larvae
move to deeper water (Chai et al., 2006; Ebbessen et
al., 2007; Hall and Wake, 1999; Lenkowski and
Raymond, 2014). At hatching, the retina of the
Caspian roach larvae had composed of well-
differentiated photoreceptor cells in contrast to many
fish species (Chai et al., 2006), showing their
dependence on visual capability as visual feeder
during early development. Prior to initiation of
exogenous feeding at 3-5 dph, whole layers of the
retina were present in the Caspian roach larvae,
illustrating the importance of visual sense for its
exogenous feeding. In addition, the complete
development of the choriocapillar layer, which
Figure 2. The allometric growth pattern of the eye of Rutilus kutum (The dashed line represents the inflexion points of growth).
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Int. J. Aquat. Biol. (2016) 4(4): 295-300
supply the high oxygen demand of the eye, up to 3
dph was in accordance with formation of other
structures of the eye in this species. Increasing of the
rod photoreceptor cells of in older Caspian roach
specimens i.e. about 35 dph shows switching its
habitat preference to deep water like many bottom
feeders e.g. yellow perch (Perca flavescens) (Whal
et al., 1993).
Allometric growth pattern of the eye diameter up
to the inflexion point was somewhat positive, and it
became negative after this point i.e. the most of the
ontogenesis and differentiation of the eye structure
had been completed before 3 dph. Larger eye
diameter commonly accommodates the larger eye
lens. Therefore, more light can be gathered and a
higher resolution image can be generated in the
brain. The greater eye size also provides the better
visual sensitivity to the fish especially under dim
light conditions with its better light gather feature
(Lim et al., 2014). In addition, changes in allometry
of morphometric characters are hypothesized to be
related to many functions such as predator avoidance
and feeding (Yúfera and Darias, 2007). After
exhaustion of the yolk-sac, larvae need to shift from
endogenous to exogenous feeding, as a result, the
positive allometry of those structures involved in
exogenous feeding i.e. eye is predicted. Positive
allometry of eye diameter in early larvae (0-7 dph)
confirmed this hypothesis and its critical role in
feeding, prey detection, schooling behavior and
predator avoidance (Rodríguez and Gisbert, 2001;
2002). Our results shows that the Caspian kutum is
mainly eye-dependent during their larval period
which itself explains completion of eye structure
particularly the retina and the high growth rate of eye
before starting mixed feeding.
Acknowledgments
This study was financially supported by the
University of Tehran.
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