chapter 6 - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/43324/14/14_chapter6.pdf ·...
TRANSCRIPT
Chapter 6
Black Gill Disease In Tiger Shrimp Penaeus
monodon
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 79
6.1. Introduction
The marine fisheries and aquaculture contribute significantly
towards enhancing the national productivity and socio-economic
development, especially in rural areas. The culture of penaeid shrimps
is now practiced on large scale along coastal waters of India. In
Gujarat, shrimp culture is being practiced in coastal ponds for several
decades, especially in South Gujarat region. Penaeus monodon is an
economically important shrimp, cultured in the coastal ponds of
southern parts of Gujarat and the culture practice is expanding
rapidly. However, the rapid expansion programmes of shrimp culture
have been hindered by diseases affecting production; and outbreaks
of diseases have caused major problems in many countries (Downs et
al., 2001). Shrimp grown in captivity are reported to be susceptible to
viral, bacterial, fungal and protozoan infections and this affect the
production (Da Silva et al., 2011).
The black gill disease in shrimp is reported to be caused by
several factors and it may result in death of infected animals due to
the destruction of gills and by the blockage of gas exchange across the
gill lamellae leading to suffocation (Lightner et al., 1975). This disease
is characterized by the presence of black spots and necrosis in the
gills and eventually gills undergo necrosis and get collapse at the
advanced stages of disease development (Egusa and Udea, 1972). The
black spots in the gills are known to be caused by an inflammatory
reaction involving activation of prophenoloxydase that converts
phenolic compounds to melanin (Bian and Egusa, 1981) leading to
melanization and death of fish. The black gill conditions are known to
produce initially gill discoloration and gradually leading to black gill
conditions; this indicates melanization of gills leading to death (Khoa
et al., 2004; Khoa, 2005). In the black gill disease, black lesions occur
were also reported in cuticular and subcuticular tissues of the
exoskeleton. These are also known to be associated with inflammatory
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 80
responses mainly hemocytic infiltration, hemocytic encapsulation,
melanization and deposition of collagen fibers (Bian and Egusa,
1981).
Lightner et al. (1975) reported that one of the causes of black
gill disease in prawns is an infection caused by a fungus from the
genus Fusarium (Lightner et al., 1975). In marine environment, some
pathogenic anamorphic fungi are known to cause serious diseases to
aquatic animals (Duc et al., 2009). The intensive culture of crustacean
has led to the increase in the number of cases of fungal diseases and
the major diseases are reported to be burn spot disease and black gill
disease (Alderman and Polglase, 1985). The fungal diseases are
reported to be second to bacterial diseases in economic aspects and
these infections are generally associated with chronic, steady losses
(Bruno and Wood, 1994; Ramaiah, 2006).
Fusarium species has been reported to cause black gill disease
in Penaeus japonicus (Khoa et al., 2005), Homarus americanus (Fisher
et al., 1978), Penaeus monodon (Khoa et al., 2004), Austropotamobius
pallipes (Alderman and Polglase, 1985), Penaeus semisulcatus
(Colorni, 1989), P. aztecus (Cook, 1971), P. duorarum (Johnson, 1974),
P. californiensis (Lightner, 1975) and Macrobrachium rosenbergii
(Burns et al., 1979). In the years 2005 and 2006, mantis shrimp,
Oratosqilla oratoria, with black or brown gills were found in Japan
(Duc and Hatai, 2009). In Vietnam, black gill disease has been
reported in shrimps and ornate rock lobster; and Fusarium has been
detected as causative agent for infection (Nha et al., 2009). Apart from
these, other factors such as exposure to nitrite, ascorbic acid
deficiency and infection by infectious hypodermal and hematopoietic
necrosis virus, Flexibacter or fungus Haliphthorus are also known to
produce black gill conditions (Hatai et al., 1992). According to
Maestracci and Vey (1989), Fusarium causes decreases osmolarity of
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 81
hemolymph and concentration of Na+ and Cl−
Fungal infections are common in cultivable species, and can
prove fatal if not treated early; and so in aquaculture, bio-security
programs associated with diseases have become an important focus.
Disease outbreaks have threatened profitability and viable
aquaculture operations throughout the world. During the year 2007-
2008, in the coastal ponds of South Gujarat region, the shrimps
showed gross clinical signs of black gills at the grow-out stage.
Previously, this disease has never been reported in shrimps from
these regions; and not investigated systematically in depth, although
it was known to occur in P. monodon in other part of the world. The
present work deals with the black gill disease in P. monodon cultured
in the coastal farms of South Gujarat region; and describe the gross
and microscopic pathology as well as defense status during this
infection.
in the cryfish
(Maestracci and Vey, 1989). The fungal enzymes are known to be
involved in the destruction of cell membranes and in particular, act
on shrimp carapace (Da Silva et al., 2011).
6.2. Results
6.2.1. Morphological features
During the year 2007 and 2008, Penaeus monodon were
detected with black gill condition in live state from intensive shrimp
culture farms of Olpad area (South Gujarat region) (Fig. 6.1). The
external clinical sign of naturally infected P. monodon was black gills.
The gills were completely black in colour; and the gill lamellae were
observed to be melanized, necrotic, atrophic and collapsed (Fig. 6.1).
The infected shrimps (with black gills) were weak, lethargic, showing
anoxia and had difficulty in respiration, and were swimming near the
pond edges in shallow water. The affected shrimps exhibited
deposition of silt on carapace and body surfaces; and have exhibited
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 82
heavy fouling. The melanization was mainly observed in gills; at the
same time though melanization was not detected in other parts of the
body. However, some shrimps have shown the erosion or necrosis in
cuticular region and appendages.
6.2.2. Wet mount preparation
The wet mount preparation of black gill filaments from infected
shrimps clearly showed the presence of fungal hyphae and
zoothamnium under microscopic observations (Figs. 6.2a, 6.2b). The
heavy fouling by zoothamnium was also detected. The colonies of
zoothamnium with thick stalk myonemes, which were also continuous
with each other and round trophonts, were detected in wet mount
preparation. A close association between fungal hyphae and
zoothamnium was clearly observed in squash preparation of infected
gills (Figs. 6.2a, 6.2b).
6.2.3. Histopathological analysis
The histopathological changes in gills, muscles and
hepatopancreas of naturally infected P. monodon with black gill
disease are shown in Figs. 6.3 to 6.5. The microscopic observation
revealed black spots in gills. The infected gills revealed hypertrophy
and necrosis of gill filaments as well as infiltration of hemocytes as
host inflammatory response with H & E as well as PAS staining
methods (Figs. 6.3c to 6.3f). In the hemocoel of the gill tissue,
amorphous mass of substances caused by coagulation necrosis was
observed, which may be the remnants of hemocytes (Figs. 6.3e, 6.3f)
(Bright red area in gill section stained with PAS). Black deposition
likely to be melanin deposition, which was observed in the hemocoel,
especially in gill sections stained with PAS reaction (Figs. 6.3e, 6.3f).
However, hemocytic and fibrocytic encapsulation of hyphae in gill
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 83
lamellae were not detected clearly. In control gill section, gill filaments
and gill endothelial cells can be clearly observed (Figs. 6.3a, 6.4b).
The muscle sections of uninfected shrimps shows regular
organization of myofibrils without any damage (Figs. 6.4a, 6.4b). The
histological changes in the muscles of infected shrimps consisted of
myofibril necrosis and degeneration of varying extent. In the necrotic
muscles, along with the hemocytic infiltration, fragmentation and
vacuolation have been detected (Figs. 6.4c, 6.4d). The progressive loss
of myofibre parenchyma also led to disorganization of myofibre. At the
same time, myonuclei with hyperchromatism and picnosis were also
observed in the section. The coagulation necrosis as bright red
amorphous mass was also evident along with degenerated muscles in
the sections stained with PAS (Figs. 6.4e, 6.4f).
The hepatopancreas of infected shrimps revealed necrosis in
glandular epithelia, formation of necrotic coagulative mass,
disorganization of secretary cells and infiltration of hemocytes (Figs.
6.5c, 6.5d). In the control sections, granular epithelial cells with clear
nuclei, secretory vesicles and central lumen can be seen (Figs. 6.5a,
6.5b). The nuclei were hypertrophic; and were showing
hyperchromatism and picnosis in some of the cells stained with PAS
(Figs. 6.5e, 6.5f).
6.2.4. Antioxidant enzymes
The activity of hepatopancreatic enzymes associated with free
radical scavenging, mainly catalase, SOD, ALP, SGOT and SGPT, in P.
monodon with black gill disease, possibly infected with pathogenic
fungus, is shown in Table 6.1. Significant changes in antioxidant
enzyme activities were found in shepatopancreas from infected
shrimps as compared to healthy ones. In the hepetopancreatic
homogenate of infected shrimps, significant decrease (p ˂ 0.05) in ALP
SGPT, SGOT and SOD activities have been observed as compared to
Figure 6.1 The naturally infected P. monodon with black gills. (The
indicating black gill region).
Figure 6.2a Squash preparation from black gill region showing the
zoothamnium colony with myonemes and trophants.
Figure 6.2b Photomicrograph showing fungal hyphae in association with
zoothamnium in wet mount preparation from black gill region.
(hy=hyphae; my= myonemes; tr= trophonts)
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 84
Figure 6.3a and 6.3b shows the control gill sections (Fig. 6.3a scale
bare=10 µm and Fig. 6.3b scale bare=50 µm).
Figure 6.3c and 6.3d shows the infected muscle sections with necrosis
(n), myonuclei with hyperchromatism and picnosis (p) (Fig. 6.3c scale
bare=10 µm and Fig. 6.3d scale bare=50 µm).
Figure 6.3e and 6.3f the gill sections statined with PAS shows the
melanin deposition in hemocoel (hemolysis (he) and necrosis (n). (Fig.
6.3e scale bare=10 µm and Fig. 6.3f scale bare=50 µm).
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 85
Figure 6.4a and 6.4b shows the control muscle sections with myofibrils
and myonuclei (Fig. 6.4a scale bare=10 µm and Fig. 6.4b scale bare=50
µm).
Figure 6.4c and 6.4d shows the infected muscle sections with necrosis
(n), myonuclei with hyperchromatism and picnosis (p) (Fig. 6.4c scale
bare=10 µm and Fig. 6.4d scale bare=50 µm).
Figure 6.4e and 6.4f shows the muscle sections with PAS (Fig. 6.4e
scale bare=10 µm and Fig. 6.4f scale bare=50 µm).
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 86
Figure 6.5a and 6.5b shows the control hepatopancreas sections which
are showing secretory lobule (sl); central lumen (cl), secretory epithelial
cells (sc) with clears nuclei (n) and secretory cessicles. (Fig. 6.5a scale
bare=10 µm and Fig. 6.5b scale bare=50 µm).
Figure 6.5c and 6.5d shows the infected hepetopancreas sections with
hemocytic infiltration (hi) with, fragmentation and vacuolation (fr),
hyperchromatism and picnosis (p) in granular epithelial cells in section.
(Fig. 6.5c scale bare=10 µm and Fig. 6.5d scale bare=50 µm).
Figure 6.5e and 6.5f shows PAS stained the infected hepetopancreas
with necrotic coagulative mass, disorganization of secretary cells (sc) and
infiltration of hemocytes (h). (Fig. 6.5e scale bare=10 µm and Fig. 6.5f
scale bare=50 µm).
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 87
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 88
control. However, the catalase activity was found to be significantly
higher in the shrimps with black gill disease when compared to that
in non-infected shrimps.
Table 6.1 Antioxidant enzyme activities in hepatopancreas of infected and healthy shrimps.
Control-
1
Control
2
Infected
1
Infected
2
Catalase (µg
H2O2
7.79
/min/ml) ±0.23
a 9.47
±0.29
b 13.06
±0.35
c 12.10
±0.13
c
ALP
(KA Units)
68.70
±0.54
a 62.73
±0.77
b 58.80
±1.35
b 49.98
±0.82
c
SGPT
(Unit/ml)
29.03
±0.34
a 36.98
±0.92
b 15.58
±0.61
c 12.98
±0.83
c
SGOT
(Unit/ml)
19.14
±0.22
a 23.89
±0.59
b 9.97
±0.90
c 9.63
±0.42
c
SOD (unit
activity/ml)
47.17
±0.44
a 43.99
±0.10
b 36.89
±0.26
c 40.15
±0.27
d
Values are mean ± S.E. (n=6). Values with different superscript are significantly different (p ˂ 0.05).
6.3. Discussion
The shrimp farming has become a highly competitive and
profitable farming practice in India and in many Asian countries
(MPEDA, 1993; Subasinghe and Shariff, 1994). The emphasis is on
enhancing the shrimp production by semi-intensive and intensive
culture techniques which include high density stocking, pond
fertilization and use of nutritionally balanced supplementary feed.
Though the techniques aimed to enhance the production, in many
instances the quality of water deteriorated and the disease occurrence
increased (Tareen, 1982). Diseases have been considered as one of the
major constraints to the development, expansion and intensification
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 89
of shrimp farming; and losses due to diseases are increasing in
shrimp farming throughout the world (Brock and LeaMaster, 1992).
The main factors reported to be involved in development of diseases
are infectious agents like viruses, rickettsia, bacteria, fungi, protozoa,
metazoan as well as feed factors, environmental factors and
husbandry features (Brock and LeaMaster, 1992). It is necessary to
identify the diseases in cultured shrimps in order to develop useful
control measures.
The morphological characteristics like weakness, sluggish
movement, black gills, body fouling and the congregation of shrimps
near pond margins in shallow water were some of the symptoms
detected during present investigation in diseased shrimps; and they
appear quite similar to the symptoms of black gill disease reported in
many shrimp species (Khoa et al., 2004; Khoa and Hatai, 2005; Khoa
et al., 2005). Microscopic examination of wet mount preparation of the
gills and other tissues are known to provide rapid reliable means for
demonstration of fungal and other parasitic diseases in farmed
shrimp (Sparks, 1985). During present investigation, detection of
fungal hyphae along with zoothamnium in fresh mount preparation of
gill (Figs. 6.2a, 6.2b) from P. monodon with black gills suggest that the
disease was caused by fungal infection, possibly Fusarium sp. In
marine environment, fungal infections are commonly observed in
crustaceans. The most common fungi affecting the shrimp are known
to be Fusarium sp.; and several cases of black gill disease caused by
Fusarium sp. have been reported in marine and fresh water shrimps,
cryfish, lobster, mantis shrimp and crabs (Lightner and Fontaine,
1975; Lightner, 1981; Khoa et al., 2004; Khoa and Hatai, 2005; Khoa
et al., 2005; Duc and Hatai, 2009). According to Rhoobunjongde et al.
(1991), in the early stages of black gill disease, the gills show a change
in colour from opaque white to brown, prior to the development of
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 90
black spots. In India, fusariosis and black gill disease caused by
Fusarium has been known to affect penaeid shrimps; and is known to
cause high mortality (Ramaiah, 2006). The Fusarium is reported to
infect shrimp by colonizing the cuticular wound; and they also infect
the gills, walking legs, eye lens and body wall (Brock and LeaMaster,
1992). The high stocking density of shrimp is known to enhance the
spread of the disease (Brock and LeaMaster, 1992). The Fusarium
causes high pathogenicity in Penaeus japonicus, P. monodon, Penaeus
duorarum and Penaeus vannamii (Johnson, 1974; Laramore et al.,
1977; Khoa et al., 2004; Khoa and Hatai, 2005; Khoa et al., 2005).
The freshly mounted infected gills showed the presence of
Zoothamnium (Figs. 6.2a, 6.2b) and the fouling is due to this
infectious agent. The ciliates Zoothamnium, Epistylis, Vorticella and
Suctoreans invade shrimps; their bloom indicates an excessive
nutrient loading and gill fouling; and they cause respiratory and
locomotory difficulties (Brock and LeaMaster, 1992). In the cultured
shrimp, the ciliates are seen to form a mat like structure on the shell
due to the deterioration of water quality. The silt deposition on
animals, high nutrient load, turbidity and low oxygen level are
considered as some of the reasons for ciliate growth and fouling
(Turnbull et al., 1994; Raj, 1995). The lethargy associated stress was
also considered to encourage fouling in animals (Lightner, 1988). The
lethargic movement of shrimp with black gills seems to be the reason
for the presence of Zoothamnium in gills and fouling.
The presently detected histopathological changes in infected
gills- necrosis of gill filament, infiltration of hemocytic cells, formation
of melanized granulomatous mass and hypertrophy indicate the
damage caused by fungal infection in P. monodon (Figs. 6.3c to 6.3d).
The Fusarium infection to shrimps is observed to be associated with
clogging of hemolymph vessels by hyphae and hemocytes, necrosis of
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 91
gills, decrease in oxygen exchanges; and these factors were considered
as the main causes of death (Momoyama, 1987; Souheil et al., 1999).
In penaeid shrimp the black pigment deposit in association with the
site of hemocytic activity has been demonstrated to be melanin; and
the pigment deposition is known to be associated with cellular
inflammatory conditions during infection (Lightner and Redman,
1977). The histopathological response to fungal infection in P.
monodon evident during present investigations appears to be quite
similar to that observed in P. japonicus, P. californiensis and other
shrimp species (Lightner, 1975; Bian and Egusa, 1981;
Rhoobunjongde et al., 1991; Duc et al., 2009). The formation of
melanized lesions as granulomatous nodule has been thought to be
formed by the encapsulation of fungal hyphae by host hemocytes
(Khoa and Hatai, 2004; Duc et al., 2009). This infection has been
known to trigger intense host response which is associated with the
development of melanized lesions; and this is a characteristic of
fungal disease (Brock and LeaMaster, 1992). The presently detected
malanized nodules in black gills (both in H & E and PAS stained
sections) suggest hemocyte accumulation and their lyses in response
to fungal infection; however, the presence of fungal hyphae are not
confirmed in gill sections, though they are observed in wet mount
preparation of infected gills (Figs. 6.2a, 6.2b). According to Soderhall
et al. (1979), phenoloxidase of hemocytes is one of the factors involved
in formation of melanized lesions. The pathogenic fungi in shrimp is
also known to secrete proteins, and involved in adhesion and invasion
in host tissue; and play a significant role in destruction of cell
membrane (Da Silva et al., 2011). The histological study also revealed
the necrosis, hemocytic infiltration and formation of necrotic
hemocytic melanized granulomatous areas in hepatopancreas and
muscles (Figs. 6.4, 6.5). The crustacean hepatopancreas is believed to
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 92
be responsible for major metabolic events; and the open circulatory
system of shrimp in hepatopancreas is known to play a significant
role in pathogenesis (Gibson and Barker, 1979; Rameshthangam and
Ramasamy, 2006). The present study has shown that the
hepatopancreas is also damaged due to fungal infection; and this is
further supported by change in antioxidant enzyme activities in
hepatopancreas observed during present analysis (Table 6.1). The
conidia of fungi are also known to invade the circulatory system (Duc
and Hatai, 2009). The histopathological changes detected in muscles
in infected shrimp during present investigation speculate that
through open circulatory system, fungi may have entered in the
muscles as well as hepatopancreas and caused the damage.
The antioxidants are potential indicators of oxidative stress in
marine organisms. The increase in the activities of detoxifying
enzymes in response to oxidative stress has been suggested to be a
general phenomenon; and oxidative stress in aquatic organisms is
observed to be more profound during change in water quality
parameters (Hwang and Lin, 2002). The fungal infection to P.
monodon, in the present case, resulted into significant decrease (p ˂
0.05) in ALP. SGOT, SGPT and SOD; except catalase, which is found
to have increased significantly (p ˂ 0.05) in hepatopancreas (Table
6.1). The increase in the level of ROS has been known to be associated
with viral infection (Schwarz, 1996; Downs et al., 2001). In the
present study, the decline in the activities of antioxidant enzymes in
shrimps suffering with black gill disease could be due to inactivation
of antioxidant enzymes by oxidative stress as proposed by
Mohankumar and Ramasamy (2006). The decrease in antioxidant
enzyme activities have been observed by Chang et al. (2003) as well as
Mohankumar and Ramasamy (2006) in WSSV infected P. monodon.
The normal cells are observed to possess number of antioxidants that
eliminate toxic metabolites and provide protection against free radical
Chapter 6 Black Gill Disease In Tiger Shrimp Penaeus Monodon
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 93
damage to the organisms under normal conditions (Wayner et al.,
1987). The generation of toxic metabolites has been known to get
increased in pathological conditions (McCord, 1988). The major
antioxidants include superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx), Glutathione S-transferase (GST),
glutathione reductase (GR), reduced glutathione (GSH), Vitamin C,
Vitamin E , ceruloplasmin, alpha lipoic acid, caratenoids, coenzyme
Q10, flavonoids and certain minerals (Asayama et al., 1989). The
present study is believed to be the first report on the effect of black gill
disease on antioxidants enzymes associated with detoxification in
hepatopancreas in P. monodon. The significant changes in antioxidant
enzymes with black gill disease also indicate that the shrimps are
under high oxidative stress probably due to increased production of
ROS. The generation of ROS in crustaceans in response to invading
microorganisms including fungi has been suggested by
Rameshthangam and Ramasamy (2006). The catalase activity is
thought to be important in invertebrates during defense; and increase
in its level indicates need to destroy reactive oxygen species (Arun and
Subramanian, 1989). Presently observed significantly higher level of
catalase in infected shrimp indicates activation of defense response.
This study is believed to be the first report on black gill disease
in cultured P. monodon from the coastal ponds of South Gujarat
region; and hopefully likely to help fish farmers of this region in
proper management of shrimp culture units.