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Journal of Entomology and Zoology Studies 2016; 4(6): 46-49
E-ISSN: 2320-7078 P-ISSN: 2349-6800 JEZS 2016; 4(6): 46-49 © 2016
JEZS Received: 08-09-2016 Accepted: 09-10-2016
Bhavna Prishnee Baishya Department of Biotechnology, Gauhati
University, Guwahati, Assam, India Sunayan Bardoloi Department of
Zoology, B. Borooah College, Guwahati, Assam, India Rupjyoti
Bharali Department of Biotechnology, Gauhati University, Guwahati,
Assam, India Correspondence Bhavna Prishnee Baishya Department of
Biotechnology, Gauhati University, Guwahati, Assam, India
Morphological changes in the hemocytes of Antheraea assama
(Lepidoptera: Saturniidae) upon
bacterial infection
Bhavna Prishnee Baishya, Sunayan Bardoloi and Rupjyoti Bharali
Abstract Giemsa stained hemocytes of bacteria-infected 5th instar
larvae of Antheraea assama were studied under 100X magnification by
light microscopy. Bacterial infection was observed to induce
distinct morphological changes in the various hemocyte types,
including enlarged or swollen cells, highly vacuolated cytoplasm,
severe nuclear fragmentation, loss of cytoplasmic compactness or
cytoplasmic lysis, blebbing of plasma membrane or completely lysed
plasma membrane, and sometimes aggregation of hemocytes to one
another leading to encapsulation of the bacterial products
Keywords: Cytoplasmic extensions, hemocytes, plasmatocytes,
granulocytes, phagocytosis, vacuolization 1. Introduction Hemocytes
are the cellular inclusions present in the hemolymph of insects and
other invertebrate groups. They play important role in the
physiology of the organism to which they belong [1]; thus, they are
responsible for coagulation of hemolymph [2, 3], connective tissue
synthesis [4-6], wound healing, self-recognition, general and
specific immune response, including opsonisation, phagocytosis,
encapsulation and nodulation [7-11]. Therefore, any stress
condition resulting in changes in hemocyte population, morphology
and function would ultimately have an adverse effect on the overall
physiology and survival of the affected insect or organism. This
study aims to investigate the changes in hemocyte morphology of
Muga silkworm Antheraea assama upon induced infection with the
bacteria E. coli with the help of light microscopy. A. assama has
been reported to have basically five distinct hemocyte types, viz.
Prohemocytes (PR), Plasmatocyte (PL), Granulocyte (GR),
Spherulocyte (SP) and Oenocytoid (OE) [1]. Insects do not have an
acquired immune system but possess well-developed innate immune
responses, comprising of both cellular and humoral immune
reactions; the cellular immune defense being provided by the
hemocytes. Infection itself is a stress condition whereby
hemocytes, along with other components of the insect immune system,
work to eliminate the stress. In doing so, they undergo various
morphological changes as evident from works of various workers.
These structural alterations sometimes include excessive changes in
hemocyte cell contour leading to cytoplasmic lysis and finally cell
death which decreases the circulating hemocyte population in the
hemolymph available for eliminating the stress condition. 2.
Materials and methods 2.1 Insects: 5th instar larvae (48-hr post
moult) reared on some plants were directly collected from the State
Sericulture farm, Khanapara, and brought to the laboratory for
conduction of the experiments. 2.2 Infection of larvae: 5th instar
larvae were infected with the bacteria E. coli, cultured in the
laboratory in Luria Broth, incubated overnight at 28 ºC,
centrifuged and the pellet suspended in physiological saline (final
concentration of bacteria 1.9x108 cells/ml approx.). 10 µl of this
bacterial suspension was injected sub-dorsally between the prolegs
at the seventh segment of the larvae using a Hamilton
microsyringe.
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Journal of Entomology and Zoology Studies
2.3 Slide preparation: Hemolymph drops were obtained on clean
and dry glass slides by severing the tip of the proleg of larvae,
12 hrs after infection and smears were made. Slides with uniform
hemolymph smears were air-dried, fixed in methanol, stained with
Giemsa, mounted with DPX and observed under light microscope (make:
Labomed) at 100X magnification. 3. Results The structural
alterations and malformations of the A. assama hemocytes upon
induced bacterial infection are quite evident from figure 1.
Hemocytes, particularly PLs and GRs, were observed to adhere to one
another forming aggregates in the smears (Fig 1a, 1b, 1c, 1d). This
aggregation of hemocytes could enclose bacterial products present
in the hemolymph, which may later lead to encapsulation and
melanization reactions. Most hemocytes were seen with distorted
cell contour with swollen or enlarged cells with loss of
cytoplasmic as well as organelle compactness. The cytoplasm in
most hemocytes had extensive vacuolization, leading to cytoplasmic
lysis and disintegration of overall hemocyte morphology.
Vacuolization was also observed in both PRs and GRs (Fig. 1e, 1f,
1g, 1h); their vacuoles probably enclosing the invading bacteria or
products of bacterial degradation, thereby suggesting their role in
phagocytosis. The participation of these two hemocytes, viz. PLs
and GRs in phagocytic activity was confirmed by the presence of
extended cytoplasmic processes arising from their cell surface
(Fig. 1e, 1f, 1g). Moreover, nuclear disintegration was observed to
be more pronounced in the GRs (Fig. 1g, 1h). PRs generally have
distinctive large nucleus with a thin band of cytoplasm surrounding
it, however, in the altered morphology they appeared to be much
enlarged and swollen (Fig. 1i). Few malformed SPs (Fig. 1i) were
also observed; however, disintegrated oenocytoids were not
seen.
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Journal of Entomology and Zoology Studies
Fig 1: (a, b, c, d, e, f, g, h, i, j) showing changes in
hemocyte morphology upon bacterial infection- swollen enlarged
hemocytes with cell membrane lysis, hemocyte aggregation, extensive
cytoplasmic vacuolization,
cytoplasmic degradation and cell lysis, extended cytoplasmic
processes in plasmatocytes and granulocytes citing phagocytic
behaviour.
4. Discussion The altered hemocyte morphology observed in this
study were found to be in accordance with those of other workers,
viz. Silva, et al, 2002; Sahayarij, et al, 2007; Pandey et al,
2010; Awad, 2012 etc [12-15]. In this study, hemocyte aggregation
was a common observation in A. assama hemocytes upon bacterial
infection, suggesting that encapsulation of bacterial product may
be a subsequent step in this reaction, which was also reported in
hemocytes of parasitized Anastrepha obliqua larvae which surround
its endoparasite in layers forming capsules, which later lead to
encapsulation/ nodule formation and elimination of the target [12].
Moreover, structural malformations of hemocytes evident in this
study were similar to that reported in Helicoverpa armigera
hemocytes which showed cytotoxic responses, together with cell
lysis or aggregation, on treatment with fern phytoecdysterone [13].
Hemocytes of Agrotis epsilon larvae when treated with Bacillus
thuringiensis showed numerous structural alterations including
swollen and enlarged PRs, extensive vacuolization of the cytoplasm
of PLs and GRs, deformed or lysed cell membrane leading to
cytoplasmic lysis and cell death [15] which were also evident in A.
assama hemocytes. Moreover, Awad (2012) [15] clearly stated that
vacuolization of cytoplasm upon bacterial infection as well as
presence of cell surface projections may be indicative of
phagocytosis of the
bacterial products by the hemocytes, as clearly evident in the
cytoplasm of PLs and GRs in this study. Thus, structural
alterations in the hemocytes of Antheraea assama during bacterial
infection or stress were compared to available literature in this
regard and the results were found to be similar to those of other
insects. Such studies provide an insight into the physiological
functions of the various cell types, specially the phagocytic
activity as well as encapsulation by the PLs and GRs in A. assama,
which in turn confirm their role as immunocytes in such insects. 5.
Acknowledgement We would like to thank the Institutional Biotech
Hub, B. Borooah College for permitting us to utilize their
instruments during this work. 6. References 1. Baishya BP, Bardoloi
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