ABSTRACT The macro and microanatomy of thymus gland in Aplocheilus panchax (Hamilton, 1822)  [a larvivorous fish] belonging to the Order: Cyprinidontiformes has been studied under light microscope (LM), scanning and transmission electron microscope (SEM + TEM) respectively. A. panchax possess apparently triangular shaped thymus gland which is located within the branchial cavity at the base of third and fourth gill arches. The surface structure of thymus gland shows numerous pores of varying diameter (0.75μm. to 1.5μm.) under scanning electron microscope (SEM). Microanatomically, the thymus is not well demarcated into cortex and medulla. This gland is composed of different types of lymphoid and non-lymphoid cellular components. The thymic epithelial cells and thymic trabeculae are also identified. The heterogeneity of thymic cells is need to further study for exploring the multifunctional aspects of this concerned lymphoid organ as a whole

Gopal Pal

Sarkar &amp

Subrata Kumar De

Suvendu Samanta

Swaraj Kumar

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G.J.B.B., VOL.4 (2) 2015: 199-202 ISSN 2278 – 9103
199
ANATOMY AND MICROSCOPICAL STUDIES ON THYMUS OF A
LARVIVOROUS FISH [APLOCHEILUS PANCHAX (HAMILTON, 1822)]
Subrata Kumar De*, Suvendu Samanta, Swaraj Kumar Sarkar & Santi Gopal Pal1
Ultrastructure and Fish Biology Research Unit, Department of Zoology, Vidyasagar University,
Midnapore (West) – 721102, West Bengal
1Vill. - Kadarat, Narendrapur Station Road, P.O. R. K. Pally, Kolkata - 700 150
*Corresponding author’s e-mail: skdvu@yahoo.co.in
ABSTRACT
The macro and microanatomy of thymus gland in Aplocheilus panchax (Hamilton, 1822) [a larvivorous fish] belonging to
the Order: Cyprinidontiformes has been studied under light microscope (LM), scanning and transmission electron
microscope (SEM + TEM) respectively. A. panchax possess apparently triangular shaped thymus gland which is located
within the branchial cavity at the base of third and fourth gill arches. The surface structure of thymus gland shows
numerous pores of varying diameter (0.75μm. to 1.5μm.) under scanning electron microscope (SEM). Microanatomically,
the thymus is not well demarcated into cortex and medulla. This gland is composed of different types of lymphoid and
non- lymphoid cellular components. The thymic epithelial cells and thymic trabeculae are also identified. The
heterogeneity of thymic cells is need to further study for exploring the multifunctional aspects of this concerned lymphoid
organ as a whole.
KEYWORDS: A. panchax, thymus, trabeculae, macrophage, etc.
INTRODUCTION
Thymus is a primary lymphoid organ of fish (Bowden et
al., 2005). This gland plays a crucial role in development
of functional immune system and acts as an important site
for T-cell development (Nakanishi, 1986; Tatner, 1996).
The gross anatomy of this gland in teleosts was first
studied by Stannius (1850) in angler fish (Lophius
piscatorius). Generally this gland is situated superficially
in contact with the epithelium of the branchial cavity
(Fänge and Pulsford, 1985). The thymus gland is
surrounded by a capsule (Chilmonczyk, 1992). This gland
is composed of different types of lymphoid cells within a
network of epithelial cells (Manning, 1994). However, the
cortex and medulla are the most notable parts of thymus
but hardly demarcated in teleosts (Trede et al., 1998).
Apart from that, the structural details of thymus gland in
most of the Indian teleosts are still less studied so far.
Larvivorous fishes are the most significant teleostean
group that can control population of mosquito larva in
urban and periurban areas of developing countries (Talwar
and Jhingran, 1991; Chandra et al., 2008). In India,
Aplocheilus panchax (Hamilton, 1822) [Order:
Cyprinidontiformes] is commonly used as a potential bio-
controlling agent for mosquito larvae (Kumar et al., 1998).
This freshwater species mostly consume substantial
amount of mosquito larva from variable environmental
conditions of sewage drain water, tap water, etc. (Manna
et al., 2011). This study emphasizes on the microscopical
details on thymus gland in A. panchax inhabiting sewage
drain water to highlights its structural components as well
as their probable functions in comparison with other
freshwater teleosts.
MATERIALS & METHODS
i) Experimental specimen
Aplocheilus panchax (Hamilton, 1822) [Order-
Cyprinidontiformes and Family-Aplocheilidae] is an
indigenous larvivorous fish of South East Asia (fig 1).
This species is widely distributed in all over South-East
Asia. According to the International Union for
Conservation of Nature (IUCN), there are no known
threats to the population of this species (i.e., ‘Least
Concern’).
ii) Anatomy
For anatomical studies, live and healthy A. panchax
specimens were directly collected from the various ponds
and sewage drain water of different area of Howrah and
East Midnapore districts of West Bengal, India. The
collected specimens were brought to the laboratory and
acclimatized with the laboratory conditions [temperature:
20˚ to 25˚C, humidity: >40%] for 24 hours. The specimens
having total body length (3 - 6) cm. and body weight (5 -
6) gm were sorted out and anaesthetized by using MS–
222 (dose: 100mg/L – 200mg/L). The thymus glands of A.
panchax were dissected out from the opercular cavity and
immediately fixed in aqueous Bouin’s fluid [75 ml of
saturated aqueous picric acid solution is added with 25ml
of 40% formaldehyde and 5 ml of glacial acetic acid is
mixed to make fresh aqueous Bouin’s fluid] for overnight.
The glands were mounted by glycerine on grease-free
glass slides and examined under light microscope (LM).
iii) Microanatomy
For microanatomical study, the procured thymus glands of
A. panchax were separately fixed in aqueous Bouin’s
solution for (6-10) hours at room temperature (25˚C -
30˚C). The tissues were then washed and subsequently
Thymus of a larvivorous fish
200
dehydrated through graded ethanol. The dehydrated tissues
were immediately transferred into Cedar Wood Oil for (24
–48) hours. The tissues were then cleared in xylene and
embedded in graded paraffin - xylene mixture under a
thermostat vacuum paraffin-embedding bath [temperature
58°C – 60°C] for a period of 60 minutes each respectively.
The serial thin sections (about 5μm) of the thymus gland
were cut by using rotary microtome and stretched on
Mayer’s albuminised glass slide. The sections were then
stained with haematoxylin - eosin and examined under
light microscope (LM).
iii) Scanning electron microscopical (SEM) study
For scanning electron microscopical (SEM) study, the
thymus gland of A. panchax specimens were fixed in 2.5%
glutaraldehyde in 0.1 (M) phosphate buffer (pH. 7.2 – 7.4)
at 4° C for 1 –2 hours. After completion of the fixation,
the samples were then rinsed in same buffer i.e., 0.1 (M)
phosphate buffer (pH 7.2 – 7.4) for 3 changes at a regular
interval of 15 minutes and dehydrated in graded chilled
acetone. The dehydrated specimens were critically dried
(CPD) with liquid carbon dioxide (CO2) and then carefully
placed on aluminium stub and coated with gold (thickness:
20nm–30nm). The prepared samples were examined under
scanning electron microscope (SEM: LEO - 435) at 0° tilt
angle, operated at 15 – 20 kV.
iv) Transmission electron microscopical (TEM) study
For transmission electron microscopical study, the thymus
glands of A. panchax were separately dissected out and
immediately fixed in 2.5% glutaraldehyde in 0.1(M)
phosphate buffer (pH 7.2–7.4) at 4°C for 1– 2 hours. After
completion of the primary fixation, the tissues were rinsed
in the same buffer and further fixed in 1% osmium
tetraoxide (OsO4) in 0.1 (M) phosphate buffer (pH 7.2–
7.4) for 1hour at 4°C. The thymus glands were then
dehydrated in graded, chilled acetone (at 4°C). The
dehydrated tissues were embedded in araldite mixture and
incubated at 60° C for 48–72hours. Ultrathin sections
(70nm –90nm) were cut with the help of ultramicrotome
(Leica Ultracut UC6) and collected on copper grids;
stained with uranyl acetate solution and lead citrate;
examined under transmission electron microscope [TEM:
MORGAGNI – 268D] operated at 40kV.
RESULTS
Anatomically, the thymus gland of A. panchax is
bilaterally placed and apparently triangular in shape,
observed under optical light microscope (LM).
Microanatomically, this gland is located at the uppermost
part of the mid dorso-lateral region on the either side of
opercular cavity at the base of third and fourth gill arches
(Fig. 2) which is also very close to the head kidney (Fig.
3). The thymus gland is hardly characterized into cortex
and medulla. A large number of lymphoid cells are
frequently noted within the thymic stroma of A. panchax
(Fig. 4). Under scanning electron microscope (SEM), the
thymus gland in A. panchax also shows clusters of cells
and mucous secretory pores with variable diameter
ranging from 0.75μm to 1.5μm. (Fig.5). The polygonal
microridges (thickness: 0.2μm) are also well demarcated
on the surface of the said tissue (Fig. 5). Under
transmission electron microscope (TEM), the thymo
pharyngeal epithelial cells are frequently observed at
cortical region of thymus (Fig. 6). This cell possess
prominent chromatinized nucleus. Heterochromatin
components are evenly distributed within the peripheral
part of the nucleoplasm (Fig. 6). Thymocytes and
thymoblasts are distributed in throughout the thymic
stroma (Fig. 7). Thymocytes are showing chromatinized
nucleus with distinct nucleolus at the central part of the
cytoplasm (Fig. 7). This cell is also observed within the
thymic trabeculae (Fig. 7). The shape, size and diameters
of trabeculae are variable within the thymic tissue. The
basement membrane, blood capillaries, peri-trabecular
epithelial cells, endothelial cells and connective tissue are
the key component of thymic trabeculae (Fig. 7). Apart
from that, free granulocytes and macrophages are also
distinctly identified within the trabeculae (Fig. 7).
FIGURE 1: Aplocheilus panchax (Hamilton, 1822); a teleostean larvivorous fish of South East Asia.
FIGURE 2: The photograph indicates true position of thymus gland (→) at the base of gill arches (G). [Not to Scale]
G.J.B.B., VOL.4 (2) 2015: 199-202 ISSN 2278 – 9103
201
FIGURE 3: The anatomical photomicrograph (from temporary preparation) shows that triangular thymus gland (T) of A.
panchax is closely associated with head kidney (HK). [Mag: x 100 (approx.)]
FIGURE 4: The microanatomy (from paraffin sections) of thymus gland shows large number of thymocytes within the stroma.
FIGURE  5: The mucous secretory pores (→) and polygonal microridges (>) are distinctly identified at the surface of thymus
gland of A. panchax.
FIGURE  6: The transmission electron micrograph shows prominent thymopharyngeal epithelial cell within thymus stroma of A.
panchax which is located very close to the pharynx. This cell shows broad apical part with several microvilli (arrows). The
spherical, chromatinized nucleus (N) is also noted at the central part of this epithelial cell.
FIGURE 7: The thymocyte (T) is distinctly marked within the thymic trabeculae. Granulocyte, macrophage, etc. are also noted.
The peritrabecular epithelial cell (ptec), thymocytes (T) and thymoblast cell (Tb) are also observed at the peripheral part of
thymic trabeculae.
DISCUSSION
The present study represents the macro- and
microanatomy of thymus gland in larvivorous teleost (A.
panchax) inhabits in different freshwater bodies. This
species can tolerate a wide range of polluted
environmental conditions (Talwar and Jhingran, 1991).
The thymus gland in fish is commonly considered as
‘being well protected against antigenic stimulation’
(Chilmonczyk, 1985). The presence of numerous pores on
the surface of thymus gland may facilitate the passage of
bioactive compounds from the external environment
(Chilmonczyk, 1985). Therefore, the specific cell
mediated protective measures of thymus gland against
invading pathogens in this fish would be an interesting
question to explore. Apart from their variability in
occurrences of different environmental conditions, the
structural as well as cellular organization of thymus gland
in A. panchax shows remarkable similarities with other
telelostean species. This gland may acts as a major site for
differentiation, maturation and storage of T-cell (Manning,
1994). It is also regarded as the first organ that acquires
lymphocytes during histogenesis of lymphoid tissue
(Zapata et al., 1996). The differentiating lymphoid cells
and epithelial cells within the stroma are generally
organized in cortical and medullary zones of thymus gland
(Manning, 1994). This type of demarcation is noted under
light microscopical studies (Zapata et al., 1996) but it is
not very distinct in fish including A. panchax. The
presence of lymphoid cells within the epithelial framework
is an indicative of interaction between ectoderm and
endoderm at the time of development (Schuurman et al.,
1997). The thymic epithelial cells are one of the most
important cellular components that are significantly
involved in development of thymocyte through
intercellular communication (Sun et al., 2013). The other
thymic components i.e., the capsule, trabeculae and
perivascular components, etc. are believed to be
mesenchymal in origin (Schuurman et al., 1997). The
arrangement of trabeculae in higher vertebrates is also
very unique which extends from cortex to medulla of
thymic stroma (Bowden et al., 2005). This structure in A.
panchax, is extending towards the interior part of the
organ and subdivided the tissue into an extensive
interconnecting space. Several authors have reported that,
the thymic trabeculae are significantly acts as a ‘blood-
thymic barrier’ which may regulate the exchange of
substances between circulatory system and thymus (Ranga
et al., 1982; Henry et al., 1992; De and Pal, 2000). In
consequence to that, the cellular association within and
peripheral part of thymic trabeculae may denote as an
important cytological feature regarding development of
Thymus of a larvivorous fish
202
thymocytes. Thus, an extensive study on the basis of the
present base line data is needed to explore multiple aspects
of thymic biology in this experimental species (A.
panchax).
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ANATOMY AND MICROSCOPICAL STUDIES ON THYMUS OF A LARVIVOROUS FISH [APLOCHEILUS PANCHAX (HAMILTON, 1822)]

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ANATOMY AND MICROSCOPICAL STUDIES ON THYMUS OF A LARVIVOROUS FISH [APLOCHEILUS PANCHAX (HAMILTON, 1822)]

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