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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



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



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



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



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)

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).

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).

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).



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



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



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



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



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



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.

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