Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol184.Issue1-2.Apr2000:

Aquaculture 184 Ž2000. 45–66
www.elsevier.nlrlocateraqua-online

Ultrastructure and pathogenesis of Monodon
baculoÕirus žPm SNPV / in cultured larvae and
natural brooders of Peneaus monodon
P. Ramasamy a , P.R. Rajan a , V. Purushothaman b,
G.P. Brennan c,)
a

Department of Zoology, Life Sciences Building, UniÕersity of Madras, Chennai 600 025, India
Department of Microbiology, Madras Veterinary College, TanuÕas, Chennai 600007, India
School of Biology and Biochemistry, Medical Biology Centre, The Queen’s UniÕersity of Belfast, Belfast,
BT9 7BL Northern Ireland, UK
b

c

Accepted 8 September 1999

Abstract

Monodon baculoÕirus ŽMBV. occurred in the hepatopancreas of cultured larvae, zoea, mysis
and post-larvae and in wild caught juveniles, sub-adults and brood stocks of Penaeus monodon.
Infected larvae exhibited lethargy and reduced feeding and preening activities while infected
juveniles, sub-adults and brooders exhibited normal behaviour. MBV-infected hepatopancreatic
cells exhibited a 300% volume increase in the hypertrophied nuclei, which contained eosinophilic,
spherical, intranuclear occlusion bodies. At the apical cell surface, the number of microvilli were
greatly reduced by cytolysis. The cytoplasm contained numerous vacuoles and few mitochondria.
Two types of occlusion bodies were observed. Type 1 comprised a paracrystalline array of
polyhedrin sub-units with a lattice spacing of 5–7 nm, with numerous occluded virions and a few
non-occluded virions in the periphery within a double envelope measuring 267 " 2 nm long and
78 " 3 nm wide. Type 2 occlusion bodies consisted of non-crystalline, granulin-like sub-units
each measuring 12 nm in diameter and contained mostly non-occluded virions measuring 326 " 4
nm long and 73 " 1 nm wide. In the zoea, mysis and post-larvae, the average infection rates for

)

Corresponding author. Tel.: q44-1232-272083; fax: q44-12342-236505; e-mail: g.brennan@qub.ac.uk

0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 1 7 - 8

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P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

MBV were 28% " 2%, 57% " 3% and 91% " 1%, respectively. Mortality rates were greater in
the infected than in the uninfected post-larvae, at 81% " 0% and 11% " 0%, respectively. The
mortality rate in MBV infected zoea and mysis was 23% " 3% and 49% " 2%, respectively,
while in the uninfected larvae, it was 5% " 0% and 8% " 0%, respectively. The mortality rate for
the infected and uninfected nauplii was similar at 3% " 0%. In the brood stocks, the infection rate
was 21% " 1%, while in the juveniles and sub-adults, it was lower at 4% " 0% and 5% " 1%.
MBV infected larvae harboured 10 times more bacteria than uninfected larvae. q 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Monodon baculoÕirus; Penaeus monodon; Hepatopancreas

1. Introduction
Viral diseases are recognized as a major factor limiting the future of the shrimp
aquaculture industry in India with consequent lost productivity estimated at 10,000–
12,000 metric tonnes at a cost of US$6–8 billion per annum ŽAlagarswami, 1995;
Ramasamy, 1995, 1999.. It was not until 1980, with the aid of electron microscopy, that

research was directed at identifying and locating the infecting organisms. Numerous
invertebrate viruses were identified, many of them in shrimp which were previously
considered disease-free ŽRamasamy, 1995, 1996; Ramasamy et al., 1995.. Monodon
baculoÕirus ŽMBV. was first described by Lightner and Redman Ž1981. in Taiwanese
Penaeus monodon and was thought to be specific to this species. However, it has since
been identified in several penaeid shrimp from different geographic regions. To date, 21
different viral species have been described from cultured penaeid shrimp ŽRamasamy,
1999.. Only a few of these have been implicated in cases of heavy shrimp losses, while
many remain a curiosity ŽSindermann, 1990; Spann and Lester, 1996, 1997; Ramasamy,
1999.. From 1994 to 1999, aquaculture farms on the east and west coasts of India
experienced repeated outbreaks of viral disease identified as a single outbreak of MBV
present in post-larval P. monodon ŽRamasamy, 1995.. The distribution, pathogenesis
and morphology of MBV have been reviewed ŽBrock et al., 1983; Lightner, 1983, 1993;
Lightner et al., 1983, 1985, 1990; Fukuda et al., 1988; Johnson and Lightner, 1988;
Hung et al., 1991; Vega-Villasante and Puente, 1993; Chen et al., 1995; Flegel, 1997;
Ramasamy, 1999.. The prevalence and ultrastructure of MBV from Žpost-larval. PL 20to 37-day old cultured Indian penaeid shrimp was recorded by Ramasamy et al. Ž1995.,
while Sundararaj et al. Ž1996. reported MBV in 35- to 60-day old shrimp as determined
at the light microscope level. Studies by Ramasamy et al. Ž1995. showed that MBV
infections peaked in the post-larval stage with a mortality rate of 90% with subsequent
heavy losses in early juveniles and senescent adults. At that time, however, MBV had

not been demonstrated in the zoea, mysis, sub-adults or brooders from either wild or
cultured P. monodon ŽLin, 1989; Tangtongpiroj, 1989; Thikiew, 1990; Lightner, 1993;
Lightner et al., 1983.. The aim of our study was to determine the prevalence and
pathogenesis of MBV in cultured zoea, mysis, post-larvae and natural brooders and to
clarify whether MBV alone, was responsible for the high mortality rates observed in the
larval shrimp.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

47

2. Materials and methods
Eggs, larvae Žnauplii, mysis., post-larvae, juveniles, sub-adults and brooders of the
penaeid shrimp, P. monodon, were collected from hatcheries, aquaculture farms and
natural sources in the coastal regions of Andhra Pradesh and Tamil Nadu, India Žsee
Scheme 1., where outbreaks of viral disease had been reported ŽTables 1 and 3..
The hepatopancreas was removed and dissected from post-larvae, juveniles, sub-adults
and brood stocks of P. monodon. A squash preparation, stained with 0.05% aqueous
malachite green, was used to identify the presence of hypertrophied nuclei with
spherical, multiple occlusion bodies. Whole mount squash preparations were also made

from the eggs and larvae Žnauplii, mysis..

Scheme 1.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

48

Table 1
Prevalence of MBV infection in captured juveniles, sub-adults and brood stocks of P. monodon
Note the date, locality, growth stage, total number of specimens, number of MBV-infected specimens and
prevalence of MBV infection.
Date of
collection

Source of
collection

Growth
stage

October
1994
January
1995
May 1995

Muttukadu
Estuary, TN
Muttukadu
Estuary, TN
Muttukadu
Estuary, TN
Muttukadu
Estuary, TN
Muttukadu
Estuary, TN
Muttukadu
Estuary, TN
Muttukadu

Estuary, TN
Muttukadu
Estuary, TN
Kalpakkam,
TN
Gudur,
AP
Kovalam,
TN
Nellore,
AP
Tuticorin,
TN
Gudur,
AP

Juvenile

25

1

4.0

Juvenile

30

1

3.3

Juvenile

35

2

5.7

Juvenile

45

2

4.4

Subadult

30

2

6.7

Subadult

40

2

5.0

Subadult

30

1

3.3

Subadult

50

3

6.0

Brood stocks

75

16

21.3

Brood stocks

55

19

20.0

Brood stocks

73

14

19.18

Brood stocks

85

19

22.89

Brood stocks

100

21

21.0

Brood stocks

48

9

18.75

January
1996
November
1994
March 1995
June 1996
February
1997
May 1995
May 1995
June 1995
June 1995
May 1996
May 1996

Total no.
of shrimp
collected

No. of shrimp
infected with
MVB

Percentage
of infection

Mean percentage
of infection"SE

4.4"0.4

5.3"0.6

20.5"0.6

TNs Tamil Nadu. APs Andhra Pradesh.

2.1. Histopathology
For histopathology, the tissues selected for examination were hepatopancreas, midgut,
hindgut, heart, haemopoetic tissue, nerve cord, lymphoid organs and muscle tissue from
infected and uninfected shrimp. The tissues were fixed in Davidson’s fixative ŽBell and
Lightner, 1988. for 24–72 h, routinely processed for light microscopy and stained with
haematoxylin and eosin. Changes in volume of MBV infected hepatopancreatic cells
was calculated by measuring the diameter of the nucleusrcell ratio using the formula
4r3r 3 and compared to the uninfected cells.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

49

2.2. Ultrastructure
For transmission electron microscopy ŽTEM., MBV-infected tissues of P. monodon
were fixed in 4% glutaraldehyde buffered to pH 7.2 with 0.1 M sodium cacodylate–HCl
containing 3% sucrose and 0.5% sodium chloride for 24 h at 48C. The tissues were
washed in buffer, post-fixed in 1% aqueous osmium tetroxide ŽOsO4 . for 1 h, dehydrated through an ascending series of ethanols, infiltrated and embedded in Agar 100
resin ŽAgar Scientific, Stansted, England. and polymerized for 48 h at 608C. Ultrathin
sections, 60–70 nm in thickness, were double stained with uranyl acetate and lead citrate
and examined in a JEOL 100CX TEM for the presence of viral inclusions.
2.3. Field studiesr hatcheries
Various stages of ŽPL 7–20. P. monodon were periodically collected from four
different hatcheries in Tamil Nadu between 1996 and 1997 ŽTable 3. and examined for
the presence of MBV infection, using a squash preparation. The post-larvae were
maintained in well-aerated, filtered sea water and fed with Artemia nauplii. Their initial
size was recorded, and abnormalities in behaviour or morphology were noted in
individual shrimp, together with the mortality rate, over a 48-h period. The hepatopancreas was dissected out and examined for histopathological changes and screened for
MBV to determine the percentage infection. Similarly, wild caught juveniles and
sub-adults of P. monodon collected from the Muttukadu estuary and brooders collected
from various sites in Tamil Nadu and Andhra Pradesh, India were examined for the
occurrence of MBV ŽTable 1..
Using Bergey’s Manual of Determinative Bacteriology ŽHolt et al., 1994., the total
viable bacteria and Vibrio per post-larvae Žinfectedruninfected. were determined. The
post-larvae were washed in several changes of autoclaved seawater, dipped in 70%
ethanol, rinsed in autoclaved seawater Ž1218Cr15 min. and homogenised using a sterile
homogeniser. The samples were then serially diluted and plated on Zobell’s marine
2216E agar ŽHi Media, Bombay. or thiosulphate–citrate–bile salt–sucrose agar ŽTCBS.
ŽHi Media, Bombay., incubated for 24 h at 288C and the total colony forming units
ŽCFUrper larva. of the heterotrophic bacteria and Vibrio were determined.
2.4. Experimental trials with nauplii, zoea, mysis and post-larÕae
The mortality rates for MBV-infected and uninfected nauplii, zoea, mysis and
post-larvae from cultured P. monodon, recovered from a hatchery in Tamil Nadu were
determined. Nauplii were stocked at a concentration of 100 nauplii ly1 , in concrete tanks
containing 5000 l of filtered, UV-irradiated water. Zoea were fed Chaetocerus, at a
density of 1–1.3 million cells mly1 tank water every 24 h. Mysis were fed Chaetocerus
at a density of 65,000–80,000 cells mly1 and 0.25 Artemia nauplii mly1 or microencapsulated pellet feed ŽArgent Chemicals, Redmond, WA, USA.. The water was
exchanged twice daily and maintained at 268C with a salinity of 28 ppm at pH 8.3. The

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P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

density of the zoea, mysis and post-larval populations in each tank was estimated daily
from aliquots removed from the tanks ŽRamasamy et al., 1996.. Dead and moribund
larvae and post-larvae were removed from each tank and counted. Samples of nauplii,
zoea, mysis and post-larva were collected routinely and examined for MBV infection.
Mortalities and behavioural abnormalities, if any, were determined by observing the
actions of the shrimp. In addition, squash preparations were also made to determine their
infection status.

3. Results
MBV infection was recorded in the hepatopancreas of cultured larvae Žzoea and
mysis., post-larvae, wild caught juveniles, sub-adults and brood stocks. Infected larvae
and post-larvae exhibited lethargy and reduced feeding and preening activities, while
juveniles, sub-adults and brooders showed no abnormal behaviour. In the later stages of
infection MBV and bacteria occurred concurrently.
3.1. Histopathology
Within MBV-infected hepatopancreatic epithelial cells, the nuclei contained from 1 to
12 spherical, refractile occlusion bodies which varied in staining intensity from weak to
strongly eosinophilic ŽFigs. 1–5.. Many epithelial cells exhibited extensive degeneration
characterised by an increased ratio in the nuclear volume to the cell cytoplasmic volume
Ž3:1., and the presence of numerous vacuoles, 15 " 1 mm in diameter ŽFigs. 2–5.. In
heavy infections, necrosis and lysis of the epithelial cells occurred with sloughing-off of
Fig. 1. Light photomicrograph of mysis P. monodon showing the nucleus of an MBV infected hepatopancreatic cell containing an eosinophilic occlusion body ŽOB.. Two uninfected cells ŽUF. are also present. Nucleus
Žn.. Scale bar s 5 mm.
Fig. 2. Light photomicrograph of mysis P. monodon. An hypertrophied nucleus ŽN. within an hepatopancreatic cell containing intensely stained eosinophilic bodies ŽOB.. Numerous large vacuoles ŽV. are present in the
cytoplasm. Scale bar s 4 mm.
Fig. 3. Light photomicrograph of post-larval P. monodon. Section through the anterior midgut cells of P.
monodon showing an MBV occlusion body ŽOB.. Numerous vacuoles ŽV. are present within the cytoplasm of
the infected cells. Gut lumen ŽL.. Scale bar s15 mm.
Fig. 4. Light photomicrograph showing infected cells containing MBV occlusion bodies ŽOB. within the nuclei
of the of juvenile P. monodon anterior midgut cells. Numerous uninfected cells ŽUF. are also present. Scale
bar s15 mm.
Fig. 5. Section through a region of the anterior midgut of post-larval P. monodon showing MBV occlusion
bodies ŽOB. within an hypertrophied nucleus. Gut lumen cells that have been sloughed off Žcl. are lying free in
the lumen ŽL.. Scale bar s 5 mm.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

51

infected and uninfected cells into the tubule lumen ŽFig. 5.. Numerous hepatopancreatic
cells were infected. Pathological changes normally associated with MBV infections were
absent in tissues other than the hepatopancreas and the anterior midgut.

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P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

3.2. Ultrastructure
TEM studies of uninfected anterior midgut cells and hepatopancreatic epithelial cells
showed numerous mitochondria, endoplasmic reticulum ŽER. and vesicles ŽFigs. 6 and
7.. Infected hepatopancreatic cells exhibited a thin band of cytoplasm, numerous
vacuoles, Golgi, ER, polyribosomes and few mitochondria, with a considerable loss of
microvilli from the apical cell surface ŽFigs. 8–10.. The nuclei were hypertrophied and
contained from one to three ovoid, crystalline, occlusion bodies, the nucleoplasm was
less electron-dense while the chromatin appeared to disintegrate and electron-lucent
areas were widespread ŽFigs. 8–11.. The nucleolus was peripherally located, the nuclear
membrane was dilated and nuclear pores were abundant. Two types of occlusion bodies
were identified. Type 1 contained an electron-dense matrix with a paracrystalline array
of polyhedrin subunits, 14.5 nm in diameter, with a lattice spacing of approximately 5–7
nm. The virions were rod-shaped, occluded and non-occluded with a double-layered
envelope ŽFigs. 10, 12 and 13. which measured 78 " 3 nm wide and 276 " 2 nm long,
with a nucleocapsid 54 " 2 nm long and 161 " 3 nm wide Ž n s 100. ŽFig. 14.. Within
the virions, electron-dense nucleoproteins were present. In some larval stages, occluded
virions were embedded within the paracrystalline matrix however, virions could not
always be observed within the occlusion bodies ŽFig. 15.. The type 2 occlusion bodies
consisted of non-crystalline, granulin-like sub-units, each measuring 12 nm in diameter
ŽFigs. 16 and 17. and contained numerous non-occluded virions aggregated close to the
nuclear membrane, with only a few occluded virions present in the matrix. The virions
possessed a single-layered envelope and measured 326 " 4 nm long and 73 " 1 nm wide
with a nucleocapsid 295 " 1 nm long and 67 " 1 nm wide Ž n s 100..
Three distinct phases of MBV infection were recognized, an early eclipse phase, a
middle-developing phase and a final degenerative phase ŽTable 2.. In the eclipse phase,
no cytopathological changes were observed. The nucleus was slightly hypertrophied
Ž44%. with the nucleolus towards the periphery. The chromatin was mostly disintegrated
but the nucleoplasm appeared normal. Virions and aggregations of polyhedrins, in the
form of small occlusion bodies were occasionally observed. Bacterial infections were
absent. In the middle-developing phase ŽFigs. 1, 4 and 9., the chromatin had diminished
considerably and small occlusions were apparent in the center of the nucleus. The
nucleolus had disintegrated, the nuclear volume had increased by 116%, and occlusion
Fig. 6. Transmission electron micrograph of post-larval P. monodon, a section through an uninfected anterior
midgut cell showing the cell organelles, nucleus ŽN., microvilli Žmv., mitochondria ŽM., endoplasmic
reticulum ŽER. and tight junctions Žt.. Gut lumen ŽL.. Scale bar s1 mm.
Fig. 7. Transmission electron micrograph, post-larval P. monodon, of a section through an uninfected
hepatopancreatic cell showing the cell organelles, nucleus ŽN., nucleolus Žn., heterochromatin Žh., nuclear
membrane Žnm.. Scale bar s1 mm.
Fig. 8. Transmission electron micrograph of a section through MBV infected hepatopancreatic cells of mysis
P. monodon showing a number of type 2 occlusion bodies ŽOB. within denatured, electron lucent nuclei ŽN..
Numerous vacuoles ŽV. are present within the cells and microvilli Žmv. have been lost from the apical cell
surface Žunlabeled arrows.. Hepatopancreas lumen ŽL.. Scale bar s 2 mm.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

53

bodies, with or without virions, had developed. Within the cell, the cytoplasm contained
large vacuoles and the organelles appeared disorganized. Bacterial infections were
absent. In the final degenerative phase ŽFigs. 8 and 18., the nucleus was hypertrophied,

54

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

the cytoplasm was granular and the cells had undergone cytolysis. Occlusion bodies and
free virions, along with the remaining cellular contents were released into the space
previously occupied by the MBV-infected cells.
Secondary invaders, such as bacteria, also infected the larvae and post-larvae
increasing cytolysis and sloughing-off of the infected cells. In the terminal stages of
infection, only the boundaries of the hepatopancreatic and anterior midgut cells were
present enclosing occlusion bodies, free or occluded virions, and bacteria which entered
the intestinal tract via the hepatopancreatic lumen ŽFigs. 15 and 18.. MBV occlusion
bodies and virions were absent in P. monodon eggs and nauplii.
3.3. Field studiesr hatcheries
Histopathological examination of hepatopancreas tissues from P. monodon postlarvae, recovered from four hatcheries ŽA, B, C and D. revealed the frequent occurrence
of MBV occlusion bodies in post-larvae from hatcheries C and D. Hatcheries A and B
were infection-free ŽTable 3.. The percentage occurrence of MBV-infected post-larvae
averaged 84% " 2%, with an average mortality rate of 55% " 6% and a survival rate of
45% " 6%, over a 48-h period. For the uninfected larvae, the mortality rate was less,
while the survival rate was 99% " 0% ŽTable 3.. The total viable heterotrophic bacterial
countsrMBV infected post-larva Ž7.0 " 0.6 = 10 5 . were 10 times greater than in
uninfected larva Ž0.7 " 0.1 = 10 5 ., though the counts were variable. Vibrio counts were
also greater in MBV infected larvae Ž5 " 0.7 = 10 3 . than uninfected larvae Ž0.3 " 0.1 =
10 3 ..
Examination of captured P. monodon juveniles and sub-adults collected from the
Muttukadu Estuary, and brooders collected from various sites in Tamil Nadu and
Andhra Pradesh, showed the widespread occurrence of MBV infected shrimp in India
Žsee Scheme 1.. The brood stocks displayed the highest infection rates, 21% " 0.6%,
while in juveniles and sub-adults, the infection rates were relatively low, 4.4% " 0.4%
and 5.3% " 0.6%, respectively ŽTable 1..
3.4. Experimental trials with nauplii, zoea, mysis and post-larÕae
Mortality rates for the larvae Žnauplii, zoea and mysis. and post-larvae of cultured P.
monodon infected with MBV were observed in six separate hatchery tanks with different
stocking densities. MBV infections recorded in the zoea, mysis and post-larvae was
28 " 2%, 57 " 3% and 91% " 1%, respectively. The highest recorded mortality rates
Fig. 9. Transmission electron micrograph of a section through an hepatopancreatic cell of P. monodon mysis
stage showing crystalline type 1 occlusion bodies ŽOB. and hypertrophied nuclei ŽN.. Within the nucleus,
chromatin is absent and the density of the nucleoplasm is greatly reduced. Nuclear membrane Žnm.,
polyribosomes Žarrow.. Scale bar s1 mm.
Fig. 10. Transmission electron micrograph of post-larval P.monodon showing uninfected ŽUF. and an infected
hepatopancreatic cell nuclei ŽN.. The infected cell nucleus contains a type 1 MBV occlusion body ŽOB., The
nucleus is enlarged and less electron dense. The uninfected cell nucleus ŽN. appears normal. Scale bar s1 mm.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

55

occurred in MBV-infected post-larvae, 81% " 0%, with lower rates in the zoea and
mysis, 23% " 3% and 49% " 2%, respectively ŽTable 4a.. In all cases, the mortality
rates in the uninfected larvae and post-larvae were lower ŽTable 4b.. The mortality rates
of infected and uninfected nauplii was similar Ž3% " 0%. ŽTable 4a,b..

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P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

The occurrence of MBV infections in hatchery-reared mysis and post-larvae at,
57% " 3% and 91% " 1%, respectively, was higher than in the zoea at 28% " 2%. In
hatchery-reared larval and post-larval shrimp, the prevalence of MBV infections increased with increasing age and peaked in the post-larvae.

4. Discussion
The occurrence of MBV has been identified and described in each life cycle stage of
the Indian penaeid shrimp, P. monodon. Visible indications of MBV infected P.
monodon included, lethargy with reduced feeding and preening activities. Internally, the
presence of hypertrophied nuclei with intranuclear spherical occlusion bodies confirmed
the presence of MBV as described by Lightner and Redman Ž1981., Lightner et al.
Ž1983., Doubrovsky et al. Ž1988., Baticados et al. Ž1991. and Ramasamy et al. Ž1995..
MBV occlusion bodies have been identified only in the nuclei of the hepatopancreatic
and anterior midgut epithelial cells of larval and post-larval P. monodon, and only in the
hepatopancreas of juveniles, sub-adults and adults. MBV was absent in all other tissues
examined supporting the observations of Lightner et al. Ž1983. and Vogt Ž1992..
Whether these cells have specific surface receptors that bind to the MBV virions and
promote rapid invasion, division and proliferation is unknown and requires further
investigation, which may help clarify the mode of entry, replication and pathogenesis of
MBV in the host cells. MBV-infected cells exhibited necrosis, lysis and sloughing-off of
cells into the tubule lumen as described in previous studies ŽLightner and Redman, 1981;
Baticados et al., 1991.. Infected cells also underwent partial or complete disintegration,
releasing free virions and occlusion bodies, and this together with the cannibalistic
behaviour of the shrimp greatly enhances transmission of MBV infection.
At the sub-cellular level, MBV-infected hepatopancreatic cells exhibited a reduced
cytoplasmic volume, an increased number of vacuoles and free ribosomes with a few
dilated ER and Golgi complexes and reduced numbers of mitochondria, as reported in
previous studies ŽLightner and Redman, 1981; Lightner et al., 1983; Couch, 1989;
Halder et al., 1989; Vogt, 1992; Lu et al., 1995, 1996.. The increased numbers of
vacuoles may indicate the degree of infection and degeneration or cytolysis of the
Fig. 11. Enlarged image of an MBV type 2 crystalline occlusion body ŽOB. showing a number of occluded
virions Žunlabeled arrows.. The density of the nucleoplasm is reduced compared to uninfected nuclei. Nucleus
ŽN.. Scale bar s 0.5 mm.
Fig. 12. Magnified image of a section through a type 1 MBV crystalline occlusion body Žc. showing numerous
occluded virions ŽVi.. Scale bar s 0.2 mm.
Fig. 13. Type 1 MBV crystalline occlusion bodies ŽOB. containing numerous occluded virions ŽVi., while
some non-occluded virions lie free at the periphery of the occlusion body. Occluded virions cut in longitudinal
section ŽLS.. Scale bar s1 mm.
Fig. 14. Magnified image of a cross-sectional view of non-occluded type 2 MBV virions showing the outer
Žoe. and inner envelope Žie. and the nucleocapsid Žnc.. Scale bar s 0.1 mm.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

57

58

Table 2
Cytopathological changes in the development of MBV infection in cells of the hepatopancreas in post-larval P. monodon

Normal
uninfected cell
ŽFigs. 7 and 11.
MBV infection
during early
eclipse phase
MBV infection
during middle
developing
phase
ŽFigs. 1, 4 and 9.
MBV infection
during final
degenerative
phase
ŽFigs. 8 and 18.

% Volume
changes in
infected nuclei

Nuclear characteristics

Cytoplasmic characteristics

Normal. Nucleus with nucleolus,
heterochromatin and euchromatin

Normal. Intact cell organelles,
ER, mitochondria, ribosomes
and Golgi
Normal cytoplasm

6.2"1.3 mm

0

6.9"0.6 mm

44

Slight hypertrophy, nucleus
marginalised, disintegrated chromatin

7.96"0.6 mm

116

Diminished chromatin. Nucleolus
disintegrated, hypertrophied nucleus
th small occlusion bodies with or
without virions

Large vacuoles, cytoplasmic
organelles appeared
disorganised

9.75"0.3 mm

297

Nuclear hypertrophy, nuclei were lysed,
occlusion bodies, free virions were
released into the gut lumen

Granular cytoplasm, increased
free ribosomes, decreased
granular ER, large abnormal
mitochondria, cytolysis,
disintegrationrdegeneration

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

Nuclear dimensions in
diameter Ž X "SE. of
hepatopancreatic
cells Ž ns 20.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

59

infected cells or perhaps the lack of lipid reserves necessary for viral replication. Vogt
Ž1992. showed that three types of hepatopancreatic cells ŽR, F and B. and the anterior
midgut cells, infected by MBV, exhibited similarities in nuclear alterations namely,
nuclear hypertrophy, chromatin marginalisation, the formation of occlusion bodies and
virion assembly.
Two types of MBV-infections were identified in P. monodon. Type 1 consisted of
polyhedrin sub-units. Type 2 consisted of granulin-like sub-units and is the first known
report of this type of occlusion body associated with MBV although, its significance is
unknown. Also present were occlusion bodies in which virions were few or absent.
These variations may occur because the different occlusion bodies may release different
virions, which may represent different species. Further molecular biological characterization studies may help to clarify this situation. P. monodon infected with MBV virions
with a single-layered envelope have been reported in the Indopacific region ŽLightner et
al., 1983., while those with double-layered envelopes have been reported in Asia
ŽHalder et al., 1989. and Australia along with P. merguiensis ŽDoubrovsky et al., 1988..
However, this is the first record of the presence of rod-shaped MBV virions with both
single- and double-layered envelopes in the Indian shrimp, P. monodon.
Polyhedrin is a crystalline protein and a characteristic feature of viruses of the genus
Nuclear Polyhedrosis virus ŽNPV. belonging to the family Baculoviridae ŽFrancki et al.,
1991.. Occlusion bodies appear to stabilize virions and maintain their viability over
prolonged adverse conditions. In the shrimp, infection probably occurs through a process
similar to that of NPV of insects ŽRohrmann, 1992..
Our observations on the morphometry of MBV virions with single- and double-layered
envelopes revealed size differences in agreement with earlier reports on MBV virions by
Fegan et al. Ž1991. and Chen et al. Ž1995..
MBV infections in post-larval P. monodon can be divided into three phases. The
early eclipse phase is difficult to detect suggesting that major cellular functions had not
been altered. In the final degenerative phase, lysis of the nuclei and cytoplasm of the
hepatopancreatic cells occurs with occlusion bodies and virions released into inter, or
intracellular spaces, or into the lumen of the hepatopancreatic tubules. In adult P.
monodon, this does not occur, either because they are resistant to infection or they are
perhaps able to replace the cells at a faster rate than they are being destroyed, and so,
may be considered to act as carriers of MBV. Vogt Ž1992. also described the MBV
infection in R, F and B cells of the hepatopancreas in the anterior midgut cells of P.
monodon but, MBV has not yet been detected in the generative E cells of the
hepatopancreas ŽFegan et al., 1991; Vogt, 1992..
The fungus Lagenidinum callinectus together with bacterial cells have been found in
MBV-infected hepatopancreatic cells. They probably occur as secondary invaders
subsequent to a viral infection and may promote degeneration of the tissues leading to
rapid death of the host. Similar observations were reported by Baticados et al. Ž1991.,
Ramasamy Ž1995; 1996. and Ramasamy et al. Ž1995.. Hatchery studies have shown that
the MBV infected larvae harboured 10 times more bacteria than uninfected larvae
reflecting the role of secondary invaders in the mortality of shrimp larvae.
The persistent occurrence of MBV-infections was observed in two of the four
hatcheries investigated in Tamil Nadu, India. These infections may be due to a number

60

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

of factors such as, contaminated rearing tanks, inadequate UV-treatment or chlorination
of the source water, or the use of infected brood stocks for spawning. The current study
also revealed that from a total of 42 collections of P. monodon post-larvae, MBV
infection was present in nine samples Ž21%.. In contrast, the prevalence of MBV
infections in hatcheries in Taiwan and Thailand were reported at 98% when females
spawned in large numbers Žup to 40 femalesrtank.. However, it was found that rinsing
brood stocks, eggs and nauplii with clean sea water containing iodophor significantly
reduced infections ŽFegan et al., 1991.. In the present study, MBV infections in brood
stocks from wild-caught P. monodon were estimated at 20%, however, Fegan et al.
Ž1991. reported a 5.7% MBV infection rate in Thailand. The prevalence of MBV peaked
in the post-larval stage and decreased with increasing age through to adults. This may be
due to a number of factors, Ži. brood stocks with light infections may have become
MBV resistant, Žii. the virus may exist in a latent form with the host acting as a vector to
transmit the infection to the larvae, and Žiii. the presence of contaminated faecal matter.
Alternatively, MBV may be non-virulent in the juveniles, sub-adults and brood stocks.
According to Fegan et al. Ž1991., if MBV is carried from the brood stocks to the nauplii,
mysis and post-larvae through the oocytes as latent viruses, they would have to be in the
form of virions without occlusion bodies, or as free viral DNA. Vogt et al. Ž1986. and
Fegan et al. Ž1991. reported that MBV infected the hepatopancreatic cells of poorly
nourished, weak or stressed P. monodon, however, when the post-larvae are well fed the
infection is unable to establish itself and the post-larvae continue to develop. Further
research on the role and nature of the nutrients in the diet may help to improve the
health and fitness of the larvae.
The mortality rates in the post-larval stage of P. monodon were greater than in either
the zoea or mysis, possibly due to the effect of acute MBV infection in the hepatopancreatic epithelial cells over a period of several days Ž10–15 days. or rapidly rising virion
production. However, in the case of the zoea and mysis, the infection is only in the early
phase and because the time period associated with this developmental stage is short the
mortality rate is low. Though mortalities were observed in nauplii, histopathological and
electron microscope studies were unable to confirm the occurrence of MBV virions or
occlusion bodies. Mortalities observed in the zoea and mysis might possibly be

Fig. 15. Enlarged image of a type 1 crystalline occlusion body ŽOB. in which MBV virions are absent.
Bacterium Žb.. Scale bar s 0.5 mm.
Fig. 16. Transmission electron micrograph of a type 2 non-crystalline, granulin-like MBV occlusion body
ŽOB. within the nucleus of an hepatopancreatic cell of post-larval P. monodon, showing non-occluded virions
at the periphery ŽVi.. Scale bar s1 mm.
Fig. 17. An enlarged image of Fig. 16, showing the details of the type 2 non-crystalline, granulin-like MBV
occlusion body ŽOB. and the associated virions ŽVi.. Nucleocapsid Žnc., empty capsule Žec.. Scale bar s 0.5
mm.
Fig. 18. Transmission electron micrograph of cytolysed hepatopancreatic cells of post-larval P. monodon
showing a number of type 2 MBV occlusion bodies ŽOB. and bacteria Žb.. The boundaries of the cells are
maintained by a thin cell membrane Žcm.. Scale bar s 3 mm.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

61

62

Table 3
Prevalence and mortality of MBV-infected hatchery reared post-larval P. monodon
Date of collection

Hatchery

Post-Larval
stages

Total no. of
post-larvae
Examined

1

May 1996

2

June 1996

3

July 1996

4

August 1996

5

March 1997

6

May 1997

A
A
A
B
B
A
A
A
B
A
A
B
C
C
A
A
A
B
B
C
A
D
A
A
B

PL 11
PL 15
PL 17
PL 13
PL 14
PL 8
PL 15
PL 18
PL 15
PL 18
PL 20
PL 20
PL 16
PL 20
PL 15
PL 17
PL 20
PL 14
PL 18
PL 18
PL 10
PL 16
PL 8
PL 10
PL 12

100
100
100
100
125
100
100
125
100
100
100
100
100
100
100
100
125
100
125
100
100
100
100
200
200

No. of MBVInfected
post-larvae
0
0
0
0
0
0
0
0
0
0
0
0
82
83
0
0
0
0
0
83
0
80
0
0
0

Percentage
Infection

Percentage of
mortality
within 48 h

Percentage of
survival in post-larvae

0
0
0
0
0
0
0
0
0
0
0
0
82
83
0
0
0
0
0
83
0
80
0
0
0

0
0
1.0
0
2.0
0
2.0
4.0
0
0
0
0
54.0 c
76.0 c
0
0
0
0
0
47.0 c
0
38.0 c
0
2.0
2.5

100.0 a
100.0 a
99.0 a
100.0 a
98.0 a
100.0 a
98.0 a
96.0 a
100.0 a
100.0 a
100.0 a
100.0 a
46.0 b
24.0 b
100.0 a
100.0 a
100.0 a
100.0 a
100.0 a
53.0 b
100.0 a
62.0 b
100.0 a
98.0 a
97.5a

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

Sl.
no.

June 1997

8

July 1997

A
A
A
B
B
B
C
A
B
B
B
B
B
C
C
C
D

PL 9
PL 10
PL 11
PL 13
PL 15
PL 20
PL 7
PL 13
PL 9
PL 10
PL 11
PL 14
PL 16
PL 9
PL 10
PL 16
PL 13

150
350
100
200
150
50
150
100
300
450
150
125
150
100
175
100
150

0
0
0
0
0
0
125
0
0
0
0
0
0
76
155
92
138

0
0
0
0
0
0
83.3
0
0
0
0
0
0
76
88.6
92
92

4.0
2.6
2.0
3.0
2.0
0
30.0
3.0
3.3
1.0
3.0
1.6
1.0
40.0
56.0
76.0
74.0

84"2

55"6

Mean percentage"SE
a

96.0 a
97.4 a
98.0 a
97.0 a
98.0 a
100.0 a
70.0 b
97.0 a
96.7 a
99.0 a
97.0 a
98.4 a
99.0 a
60.0 b
44.0 b
24.0 b
25.4 b
99"0 a
45"6 b

Percentage of survival in MBV-uninfected larvae.
Percentage of survival in MBV-infected larvae.
c
Note: Ž1. The rate of mortality was significantly higher in MBV-infected post-larvae than in MBV-free post-larval P. monodon. Ž2. Hatcheries C and D exhibited
MBV infection while hatcheries A and B were free of MBV infection.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

7

b

63

64

Table 4
Ža. Mortality of MBV-infected nauplii, zoea, mysis and post-larvae of P. monodon. Percentage mortality Ž%.
Tank
no.

Total no. of
nauplii stockeda

Total no. of zoea
surviving after 3
days of stocking

Total no. of mysis
surviving after 8 days
of stocking

Total no. of post-larvae
surviving after 25 days
of stocking

801,700 Ž2.88.
712,900 Ž3.10.
578,600 Ž3.32.
675,900 Ž3.24.
523,900 Ž2.98.
753,400 Ž3.48.
3"0.08

624,100 Ž24.4.
645,000 Ž13.42.
406,800 Ž31.46.
493,400 Ž29.36.
438,900 Ž18.72.
610,500 Ž21.80.
23"3

321,000 Ž61.11.
395,100 Ž46.97.
310,200 Ž47.73.
347,800 Ž50.21.
2,900,100 Ž46.28.
450,900 Ž42.24.
49"2

150,800 Ž81.7.
140,000 Ž81.20.
120,700 Ž79.83.
130,500 Ž81.32.
105,000 Ž80.55.
150,500 Ž80.71.
81"0

Nil

28"2

57"3

91"1

Žb. Mortality of uninfected controls, nauplii, zoea, mysis and post-larvae of P. monodon. Percentage mortality Ž%.
Tank
no.

Total no. of
nauplii stockeda

1
680,900
2
895,800
3
750,500
4
788,400
5
543,200
Mean percentage mortality"SE
a
b

Total no. of nauplii
surviving before moulting
into zoea Ž18 h.

Total no. of zoea
surviving after 3
days of stocking

Total no. of mysis
surviving after 8 days
of stocking

Total no. of post-larvae
surviving after 25 days
of stocking

660,500 Ž2.99.
870,500 Ž2.82.
728,800 Ž2.89.
761,600 Ž3.39.
521,800 Ž3.94.
3"0.19

640,700 Ž5.9.
860,900 Ž3.89.
708,300 Ž5.62.
748,700 Ž5.04.
506,400 Ž6.77.
5"0.4

625,500 Ž8.14.
838,800 Ž6.36.
688,300 Ž8.28.
724,100 Ž8.16.
491,800 Ž9.46.
8"0.4

605,900 Ž11.01.
814,100 Ž9.12.
665,700 Ž11.29.
706,900 Ž10.34.
480,200 Ž11.59.
11"0.4

Initial stock of napulii which metamorphose into zoea, mysis and post larvae, examined in the study.
Initial stock of napulii, which metamorphose into zoea, mysis and post larvae, examined in the study.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

1
825,500
2
745,000
3
598,500
4
698,500
5
540,000
6
780,600
Mean percentage
mortality"SE
Mean percentage of
infection"SE

Total no. of nauplii
surviving before moulting
into zoea Ž18 h.

P. Ramasamy et al.r Aquaculture 184 (2000) 45–66

65

associated with stress in combination with a viral infection and, or other unknown
factors.
This study has established the occurrence of two types of MBV infections in the
Indian penaeid shrimp P. monodon. MBV-infected hepatopancreatic cells exhibited a
300% volume increase in the hypertrophied nuclei. Cytolysis and bacterial invasion was
observed. Studies in the hatcheries showed that MBV infected larvae were 10 times
more susceptible to bacterial infection.

Acknowledgements
This work was funded by The Department of Biotechnology, Government of India,
New Delhi.

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