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J. Aqua. 23 (2015)
HAEMATOLOGICAL AND INNATE IMMUNOLOGICAL REFERENCE
INTERVALS FOR FARMED YELLOW CATFISH HORABAGRUS
BRACHYSOMA AND THEIR SEASONAL VARIATIONS
P. K. Sahoo, S. K. Sahoo, B. R. Mohanty, A. Das, S. S. Giri and M. Paramanik
ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751 002, Odisha, India
*Corresponding author: [email protected]
The reference intervals for haematological and innate immune response variables of healthy adult
yellow catfish Horabagrus brachysoma raised in captivity were determined. Reference ranges
were established for all parameters and significant (P < 0.05) seasonal variations in most of the
haematological and innate immune parameters were observed. Total erythrocyte count, packed
cell volume, serum lysozyme and myeloperoxidase activities were found to be higher during
winter season whereas most of the red blood cell indices, superoxide production by phagocytes,
serum ceruloplasmin and anti-protease activities were marked to be significantly higher during
rainy season of the year. Except high total leucocyte count and mean corpuscular haemoglobin,
other parameters were found to be significantly lower during summer, possibly indicating a higher
disease risk in summer months for this species. However, no significant variations in these
variables were obtained between male and female catfish during breeding season. The information
generated would be indirectly helpful for determining health status of this endangered species.
INTRODUCTION
Hematological evaluation is gradually becoming a routine practice for determining
health status, disease or stress conditions of intensively cultured farmed fish (Bowden et al., 2004;
De Pedro et al., 2005; Sahoo et al., 2005; Swain et al., 2007; Tavares-Dias and Moraes, 2007a,
b). Haematological and immunological variables vary substantially between species (Hine, 1992;
Sahoo et al., 2005; Swain et al., 2007) and even within a major group of fish species cultured in
the similar environment (Sahoo et al., 2005). The major biotic and abiotic factors such as
temperature, season, sex, species, age, strain, photoperiod, nutritional status and environmental
factors influence blood parameters in fish (De Pedro et al., 2005; Tavares-Dias and Moraes,
2007a, b). Thus, the establishment of species-specific reliable reference values under standard
environmental conditions is a prerequisite before haematology or immunological parameters
being used for determining biomarkers of health status of fish (Handy and Depledge, 1999) or
exposed to pollutants, stress or infections (Sahoo et al., 2005).
Red blood cell indices are used to diagnose anaemia and to indicate systemic responses
to external stimulus (Tavares-Dias and Moraes, 2007b). Total leucocyte count may reveal
leucopenia or leucocytosis, suggesting possible immune function alterations (Huffman et al.,
1997). The total leucocyte count (TLC) level increases in infected fish (Harikrishanan et al.,
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J. Aqua. 23 (2015)
2003). Previous studies have indicated a reduction in total erythrocyte count (TEC), haematocrit
and haemoglobin (Hb) in infected fish (Rehulka, 2002; Harikrishanan et al., 2003) or fish exposed
to toxic chemicals (Svobodova et al., 2003). Reduction in blood glucose and total protein has
been recorded in fish exposed to Aeromonas spp. (Rehulka, 2002; Harikrishanan et al., 2003).
Immunity is an important physiological defence mechanism to protect against infection
and maintain internal homeostasis (Ingram, 1980). Many cells (leucocytes, nonspecific cytotoxic
cells, eosinophilic granular cells, macrophages and other cells) and their products
[myeloperoxidases (MPO), superoxides, acute-phase proteins, lysozyme, interferon,
complement, properdin, lysins and agglutinins] contribute to the general immunological defence
mechanism. Seasonal influence dominates the life cycle of fish and is also believed to co-ordinate
their immune response (Bromage et al., 2001). Fish appear to exhibit seasonal fluctuations in
their susceptibility to different infectious diseases (Lillehaug et al., 2003; Kumari et al., 2006).
For example, Karvonen et al. (2010) showed higher disease incidences during summer with
prolong high water temperature in farms. However, the pattern was opposite or there was no
pattern. Reference ranges for each parameter are needed to be known in order to access the health
status of fish. Reference intervals for each variable are defined by upper and lower limits that
cover the majority of the values obtained for healthy individuals in the reference population (i.e.,
a set of individuals meeting certain criteria, particularly absence of any disease) (Rehulka et al.,
2004; Tavares-Dias and Moraes, 2007a). Horabagrus brachysoma or Asian sun catfish or yellow
catfish is an endangered species endemic to few southern states of India (Bhat, 2001; Kurup et
al., 2004), and also found in few Asian countries. The positive attributes in this species viz.,
adaptability in varied environment conditions, maturity in captivity, and acceptance to wide range
of food and good growth within short time span in culture conditions enable it as a perspective
species for aquaculture. However, there is lack of information on haematological and
immunological indices in H. brachysoma.
MATERIALS AND METHODS
Fish and experimental design
H. brachysoma juveniles were collected from their natural habitat and transported to the
Institute farm. They were reared in cement tanks of 20 m2 size for a period of two years under
brood stock raising programme. The cement tank covered with linen shed was provided with 2-3
cm soil base and water depth of 45 cm was maintained. The tanks were provided with plastic
pipes for hiding of fish to simulate natural conditions. Water exchange was carried out
periodically to maintain optimum water quality. Fish were provided with pellet feed (30% crude
protein) once daily at 2% of their body weight. The broods raised in cement tanks were collected
during the month of July for induced breeding. The larvae thus obtained were reared for a period
of two years till they mature. The mature fish were collected and equal size (50-55 g) fish were
segregated before releasing those to experimental tanks for further study. Three cement tanks of
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J. Aqua. 23 (2015)
16 m2 size were prepared as described previously and each tank was stocked 48 fish (three
fish/m2) for a period of one year under continuous supply of freshwater under natural photoperiod
and temperature. The aerated ground water was stored in overhead tank before supply to
experimental tanks. Other managemental protocols were similar as mentioned earlier for brood
stock raising. The blood samplings were undertaken in last week of December, April and July
representing winter, summer and rainy seasons, respectively. The size ranges of fish used were
72-85 g, 75-95 g and 85-105 g during December, April and July, respectively. The water
temperature ranges recorded during three major seasons varied from 31.0-34.0 ºC (average 33.0
ºC) in summer (March-June), 29-31 ºC (average 30.5 ºC) in rainy (July-September) and 18.7-20.2
ºC (average 19.4 ºC) in winter (November-January) seasons. The mean of water quality
parameters measured during entire period of study were dissolved oxygen 5.65 ± 0.70 ppm, pH
7.2 ± 0.6, total ammonia 0.109 ± 0.024 ppm, nitrites 0.015 ± 0.009 ppm and hardness 92.0 ± 8.2
ppm. During the study period, fish were observed at quarterly interval for any clinical signs of
diseases and found to be free from any gross signs of disease.
Blood was collected in the morning (10:00-11:00 hours) after 24 h fasting from caudal
vein of rapidly caught anaesthetized (0.1 ml/l 2-phenoxy ethanol) fish through plastic syringe.
Heparinized syringe was used to collect blood from 15-20 fish (randomly collected from three
tanks) to measure haematological indices, blood (plasma) glucose level and nitroblue tetrazolium
assay. Similarly, sera were obtained from blood collected with non-heparinized syringe from 17
to 32 fish (representing equal numbers approx. from each tank) to measure immune parameters
during each season. The sample size details vary in each season and are given in results section
against each test. The blood collected during July (when morphological sex differentiation was
prominent) was processed separately sex-wise. Two of the innate immune parameters such as
natural haemolysin titre and bacterial agglutination titre were not recorded during rainy season
and hence, not incorporated in seasonal or sex impact analysis study. All the assays were carried
out in triplicate.
Haematology and immunology
TEC and TLC were carried out manually after dilution (200 × and 50 ×, respectively)
using modified Dacie’s fluid (Blaxhall and Daisley, 1973). Values were expressed as number of
cells/mm3 of blood. The haematocrit was measured by microhaematocrit method and Hb
concentration using the cyanomethaemoglobin method with Drabkin’s reagent (Blaxhall and
Daisley 1973). Secondary Wintrobe indices, such as mean corpuscular volume (MCV), mean
corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)
were derived from the primary indices. Plasma glucose content was quantified by enzymatic
colorimetric method with GLUCOSE FL kit (Chema Diagnostica, Italy).
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J. Aqua. 23 (2015)
The oxygen radical production by blood phagocytes during respiratory burst activity was
measured through nitroblue tetrazolium (NBT) assay as described previously by Anderson and
Siwicki (1995). The total myeloperoxidase content present in serum was measured according to
Quade and Roth (1997) and a partial modified technique (Sahoo et al., 2005). A turbidometric assay
utilizing lyophilized Micrococcus lysodeikticus cells (Sigma) was used to determine lysozyme
activity in serum following Kumari et al. (2006). Lyophilized hen egg white lysozyme, HEWL
(Sigma) was used to develop a standard curve. Serum lysozyme values were expressed as g/ml
equivalent of hen egg white lysozyme activity. Alternative complement activity was assayed
following a previously described technique (Matsuyama et al., 1988; Yano, 1992) with partial
modifications (Kumari and Sahoo, 2005) by using rabbit red blood cells (RaRBC). The results are
expressed as ACH50 (U/ml), the reciprocal serum dilution giving 50% haemolysis. The total protein
content in serum was measured following Bradford (1976) method, using bovine serum albumin as
a standard protein. The natural haemolysin titre was performed as per Sahoo et al. (2005) using
rabbit RBC. The titre was defined as the last dilution of serum showing complete lysis of RBC.
Values are expressed as reciprocal of haemolysin titre. Natural agglutinin levels in the serum of
individual fish were determined by plate agglutination technique using formalin-killed Aeromonas
hydrophila (Sahoo et al., 2005). The bacterial agglutination titre was defined as the last dilution of
serum showing minimal positive agglutinin. Values were expressed as reciprocal of the
agglutination titre. Total serum antiprotease in fish serum was determined according to Zuo and
Woo (1997) with partial modification. Serum (10 l) was mixed with 100 l of trypsin (bovine
pancreas type I, Sigma; 200 g ml of PBS) and incubated at 25o C for 30 min. It was further
incubated with 1 ml of casein dissolved in PBS (2.5 mg/ml) for 15 min at 25o C. The reaction was
terminated with the addition of 500 l of 10% trichloroacetic acid (TCA). The sample was
centrifuged at 10,000 × g for 5 min to remove protein precipitates. The OD of the supernatant was
measured at 280 nm and the percentage trypsin inhibition was calculated. Ceruloplasmin activity in
serum sample was measured as p-phenylene diamine (PPD) oxidase activity (Sigma) according to
methods of Pelgrom et al. (1995) with slight modification (Sahoo et al., 2008).
Statistical analysis
The data expressed as Mean ± SE and had a non-Gaussian distribution (except for TEC,
Hb, haematocrit, lysozyme and total protein). Thus, reference intervals (25th and 75th percentiles)
were established using non-parametric methods. The normality of the data was assessed using
Kolmogorov-Smirnov test. Difference between means were assessed by Student’s T-test (for
difference between male and female, and also for ACH50 activity in seasonal study) or by one-
way ANOVA (for seasonal study) followed by Duncan’s multiple range tests using SPSS 13.0
package (SPSS Inc., Chicago, USA). A probability level of P < 0.05 was considered statistically
significant. Data from males and females of rainy season were pooled when statistical differences
between sexes were absent.
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J. Aqua. 23 (2015)
RESULTS
The mean values, lower (25th) and upper (75th) percentiles and range of each parameter
obtained from samples collected during all the seasons over one year duration are presented in
Table 1. No difference in haematological and immunological indices was found between male
and female catfish sampled during rainy season (Table 2). However, a marked influence of season
on most of the parameters studied was observed (Table 3).
TEC, Hb and haematocrit were found to be significantly (P<0.05) lowest during summer
season compared to other seasons whereas TLC showed a higher value during summer followed
by rainy and winter seasons. The MCH level was significantly higher during summer season
when compared with winter and rainy seasons. On the other hand, MCV and MCHC showed no
significant fluctuation over the year. Similarly, the blood glucose level was consistent over
different seasons. Superoxide production, serum antiprotease, total protein and ceruloplasmin
were significantly higher during rainy season; whereas lysozyme and myeloperoxidase activities
in the serum of catfish were shown to be higher during winter season. This was not the case for
ACH50 level, as no clear trend with regard to seasonal variations was observed (Table 3).
Table 1 : Haematological and innate immune parameters reference intervals for farmed
yellow catfish obtained from observations made over one year.
Parameter N Mean ± SE 25th-75th
percentile Range
TEC (x 106/ mm3 of blood) 52 1.88 ± 0.07 1.53-2.18 0.68-3.28
TLC (x 103/ mm3 of blood) 53 16.78 ± 1.16 8.73-22.5 4.43-34.45
Hb (g/ dL-1) 54 7.21 ± 0.25 6.0-8.2 3.6-11.4
Haematocrit (%) 59 27.44 ± 0.91 22.5-33.0 12.0-40.0
MCV (fL) 52 157.36 ± 10.82 117.97-171.99 59.07-492.75
MCH (pg) 52 40.12 ± 1.62 31.92-46.51 21.71-68.24
MCHC (%) 54 27.71 ± 1.24 23.13-30.99 12.67-51.82
Glucose (mg/ dL) 56 75.21 ± 3.29 57.06-89.69 37.02-133.95
NBT activity (OD at 540 nm) 58 0.34 ± 0.01 0.29-0.40 0.21-0.53
ACH50 activity (units/ mL) 47 40.27 ± 2.23 30.70-48.25 12.47-68.18
Lysozyme activity (µg/ mL) 53 7.39 ± 0.39 4.67-9.88 2.22-12.33
Myeloperoxidase activity
(OD at 450 nm)
86 0.71 ±0.04 0.46-0.90 0.14-1.78
Ceruloplasmin (units 25 µL of
serum)
82 0.21 ± 0.01 0.12-0.26 0.08-0.63
Anti-protease (% inhibition) 89 52.89 ± 1.63 41.39-62.19 24.84-85.07
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J. Aqua. 23 (2015)
Parameter N Mean ± SE 25th-75th
percentile Range
Total protein (g/ dL) 75 7.14 ± 0.34 4.80-8.61 2.26-13.92
Haemolysin titre 27 1.26 ± 0.13 1.0-2.0 1.0-4.0
Bacterial agglutination titre 24 4.25 ± 0.61 2.0-4.0 2.0-16.0
TEC, total erythrocyte count; TLC, total leucocyte count; Hb, haemoglobin; MCV, mean
corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular
haemoglobin concentration; NBT, nitroblue tetrazolium activity; N, number of fish sampled
Table 2 : Effect of sex on various haematological and innate immune parameters measured
in farmed yellow catfish during rainy season. Data are presented as Mean ± SE with number
of fish sampled in the parenthesis.
Parameter Male Female
TEC (x 106/mm3 of blood) 2.07 ± 0.05 (13) 2.07 ± 0.18 (11)
TLC (x 103/mm3 of blood) 17.60 ± 0.89 (13) 19.82 ± 1.44 (10)
Hb (g/dL) 8.48 ± 0.43 (13) 8.29 ± 0.36 (11)
Haematocrit (%) 31.21 ± 1.33 (14) 30.73 ± 1.71 (11)
MCV (fL) 153.26 ± 6.26 (13) 154.66 ± 10.10 (11)
MCH (pg) 41.19 ± 2.30 (13) 41.94 ± 2.40 (11)
MCHC (%) 27.44 ± 2.23 (13) 27.40 ± 1.09 (11)
Glucose (mg/dL) 85.22 ± 7.98 (11) 85.92 ± 7.48 (14)
NBT activity (OD at 540 nm) 0.41 ± 0.02 (14) 0.38 ± 0.02 (11)
ACH50 activity (units/mL) 42.37 ± 4.42 (16) 35.65 ± 3.16 (18)
Lysozyme activity (µg/mL) 5.26 ± 0.74 (11) 5.44 ± 0.45 (14)
Myeloperoxidase activity (OD at 450 nm) 0.49 ± 0.05 (15) 0.61 ± 0.05 (13)
Ceruloplasmin (units/25 µL of serum) 0.25 ± 0.03 (22) 0.25 ± 0.03 (19)
Anti-protease (% inhibition) 61.75 ± 3.65 (23) 59.93 ± 3.47 (21)
Total protein (g/dL) 9.36 ± 0.61 (20) 9.89 ± 0.74 (11)
TEC, total erythrocyte count; TLC, total leucocyte count; Hb, haemoglobin; MCV, mean
corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular
haemoglobin concentration; NBT, nitroblue tetrazolium activity
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Table 3 : Effect of season on various haematological and innate immune parameters
measured in farmed yellow catfish. Data are presented as Mean ± SE with number of fish
sampled in the parenthesis. Means bearing common superscript in a row are not
significantly (P < 0.05) different.
Parameter Winter season Summer season Rainy season
TEC (x 106/mm3 of blood) 2.10 ± 0.11b (15) 1.47 ± 0.11a (18) 2.09 ± 0.11b (19)
TLC (x 103/mm3 of blood) 12.84 ± 0.38a (17) 23.98 ± 1.57c (18) 18.96 ± 0.88b (18)
Hb (g/dL) 7.24 ± 0.20ab (16) 6.21 ± 0.55a (19) 8.18 ± 0.31b (19)
Haematocrit (%) 28.60 ± 0.96b (20) 22.89 ± 1.91a (19) 30.60 ± 1.28b (20)
MCV (fL) 136.36 ± 9.22a (15) 161.05 ± 9.07a (18) 147.13 ± 6.51a (18)
MCH (pg) 36.42 ± 2.18a (15) 43.32 ± 2.41b (18) 40.37 ± 2.07ab (19)
MCHC (%) 26.60 ± 1.03a (16) 27.14 ± 0.59a (19) 27.23 ± 1.61a (19)
Glucose (mg/dL) 71.27 ± 4.82a (17) 71.82 ± 5.81a (19) 81.77 ± 6.08a (20)
NBT activity
(OD at 540 nm) 0.31 ± 0.01a (19) 0.35 ± 0.02ab (19) 0.37 ± 0.02b (20)
ACH50 activity (units/mL) ND 37.05 ± 3.12a (26) 44.26 ± 3.03a (21)
Lysozyme activity (µg/L) 9.43 ± 0.35c (27) 3.86 ± 0.49a (19) 6.02 ± 0.46b (17)
Myeloperoxidase activity
(OD at 450 nm) 0.93 ± 0.07b (26) 0.69 ± 0.06a (32) 0.55 ± 0.04a (28)
Ceruloplasmin (units/25 µL
of serum) 0.16 ± 0.01a (25) 0.19 ± 0.02a (27) 0.27 ± 0.02b (30)
Anti-protease (% inhibition) 45.62 ± 1.56a (27) 43.24 ± 1.68a (31) 68.88 ± 2.12b (31)
Total protein (g/L) 6.18 ± 0.30a (26) 5.55 ± 0.35a (32) 11.60 ± 0.23b (17)
TEC, total erythrocyte count; TLC, total leucocyte count; Hb, haemoglobin; MCV, mean
corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular
haemoglobin concentration; NBT, nitroblue tetrazolium activity; ND, not done
DISCUSSION
The haematological and immunological assessment of intensively farmed fish can be
considered as an integral part of the evaluation of their health status. Any deviation in the
proportion of the parameters leads to diagnostic significance. Hence, the establishment of
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J. Aqua. 23 (2015)
reference intervals for these variables are important and the same has been defined for many
species viz., hybrid striped bass Morone saxatilis (Walbaum) × Morone chrysops (Rafinesque)
(Hrubec et al., 2001), channel catfish Ictalurus punctatus (Tavares-Dias and Moraes, 2007b),
Indian major carps Labeo rohita, Catla catla, Cirrhinus mrigala (Sahoo and Mukherjee, 1999;
Sahoo et al., 2005), common carp Cyprinus carpio (Tripathi et al., 2003), hybrid tilapia
Oreochromis niloticus (L.) × Oreochromis mossambicus (Peters) (Hrubec et al., 2000) and
southern bluefin tuna Thunnus maccoyii (Rough et al., 2005).
The reference intervals obtained for the parameters studied in yellow catfish were more
or less similar to those defined for other species. A minimal difference that was observed in all
the variables might be due to species difference, age and environment.
No variation was observed in any of the immune parameters studied between male and
female fish that were bled during rainy or breeding season. Similar observations were also made
for few other species viz., L. rohita (Swain et al., 2007) for innate immune parameters, and wild
yellowfish (Barbus holubi), C. carpio, and two mudfish species (Labeo umbratus and L. capensis)
(Van Vuren and Hattingh, 1978) for haematological parameters suggesting that both the sexes
equally immuno-competent.
The present results indicate the existence of seasonal variations in haematological and
innate immunological parameters in the blood of the yellow catfish. This is the first time a normal
range for all these variables being established for this endangered species, which has both food
and ornamental value. Thus, season must be considered as a key factor when blood parameters
are used as biomarkers for prediction of pollution, stress, disease problems or environmental
alterations.
Erythrocyte profiles (red blood cells count, haemoglobin content and haematocrit value)
exhibited almost similar levels in rainy and winter seasons whereas significantly lower levels
during summer. The rise in water temperature for a prolonged period during summer season might
be playing detrimental role on the physiology of fish leading to unnoticed reduced feed intake,
thereby reducing red blood cells and its related parameters. The effect of temperature on number
of physiological and immune parameters of fish has already been described (Hernandez and Tort,
2003). TLC is an important defence activity of fish (De Pedro et al., 2005). The variation in TLC
of yellow catfish clearly depicts the variation in immune response with respect to season. Similar
to our study, a higher TLC was marked in tench (Tinca tinca) during summer and autumn
compared to winter and spring suggesting shortened day length as possible cause inducing
changes in immune system (Collazos et al., 1998). Secondary Wintrobe indices viz., MCV, MCH
and MCHC are indicative of types of anaemia. There was no influence of season on MCV and
MCHC values whereas MCH value was higher during summer.
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J. Aqua. 23 (2015)
The blood glucose level is indicative of stress and in our study, it was observed that the
plasma glucose level remained stable in different seasons. As the fish were deprived of food for
24 h before sampling, the effect of change in feed intake due to seasonal differences in
temperature is minimized. Further, these results indicate absence of any external stress during the
period of study. Hence, blood glucose can actively be considered as a valuable test for evaluating
the general physiological state of fish.
Many researchers have studied the changes in non-specific immune parameters of fish
with relation to infection, toxicity, diet, stressors, temperature fluctuations or pollution (Ingram,
1980; Studnicka et al., 1986; Anderson et al., 1992; Dalmo et al., 1997; Ellis, 2001; Kumari and
Sahoo, 2006). Nevertheless, just a few of these studies are related to catfish species i.e. in Clarias
batrachus (Kumari et al., 2006; Kumari and Sahoo, 2006) and Ictalurus punctatus (Plumb and
Areechon, 1990). Thus, the present work was undertaken to find the normal physiological
presence and ranges for some of the important non-specific immune parameters of this
endangered yellow catfish species.
Natural haemolysins are considered to be important in innate immunity in vertebrates.
Potent haemolytic activity was also observed in the sera of yellow catfish. Other investigators
have also reported natural haemolytic activity in normal serum of different fish species against a
diverse array of cellular antigens (Ingram, 1980; Sakai, 1983a, b; Sahoo et al., 2005; Swain et al.,
2007). Natural factors found in normal, healthy fish such as lysins, agglutinins and precipitins
may help to overcome various diseases much earlier than that required to produce specific
immunity. These natural agglutinins are structurally different from known immunoglobulins.
These natural agglutinins also react with a wide variety of bacteria causing agglutination. The
observed bacterial agglutination titre against a common contaminant/pathogen is clearly
indicative of the presence of agglutinins in yellow catfish sera.
Phagocytes produce large quantities of superoxide anion during phagocytosis or upon
stimulation. The NBT reduction product obtained after reaction with superoxides is a very good
indicator of the health status or the immunization effectiveness in fish (Anderson et al., 1992). A
higher NBT activity was noticed in yellow catfish in rainy season. However, earlier studies have
indicated higher superoxide production at low water temperature (Le Morvan et al., 1998) and
during winter in C. batrachus (Kumari et al., 2006).
MPO is an important enzyme having antimicrobial activity. It utilizes hydrogen peroxide
during respiratory burst to produce hypochlorous acid (Dalmo et al., 1997). Reduced activity may
indicate the presence of contaminants or stress (Anderson and Siwicki, 1995). The highest MPO
activity in yellow catfish was noticed in winter as compared to the lowest activity in C. batrachus
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J. Aqua. 23 (2015)
during the same time (Kumari et al., 2006). The trend obtained for MPO and NBT activities in
yellow catfish thus reverse as compared to Asian catfish C. batrachus.
Lysozyme is an important enzyme in blood that actively lyses bacteria; an increased
level has been considered to be a natural protective mechanism in fish (Ingram, 1980).
Neutrophils are thought to be the source of lysozyme, and the enzyme appears to be much more
bactericidal in fish than that of higher vertebrates (Ellis, 2001). The lysozyme level varies widely
among the fish species (Anderson and Siwicki, 1995), as was also observed in our study.
Lysozyme activity was found to be higher in winter in yellow catfish, which is well correlated
with high MPO in this species at the same period, thus indicating the release of these enzymes by
similar cell population like neutrophils. Similar to our previous study in C. batrachus (Kumari et
al., 2006), a lower lysozyme activity was noticed during summer in yellow catfish. On the other
hand, a higher lysozyme activity was noticed in broods of L. rohita during summer (Swain et al.,
2007) and in dab (Limanda limanda) (Hutchinson and Manning, 1996).
The ACH50 activity is very active in fish serum when compared with mammals (Yano,
1996), suggesting that this pathway is very important in the defence mechanisms of fish (Ellis,
2001; Holland and Lambris, 2002). ACH50 values of fish serum were extremely high when
compared with those of mammalian sera. The ACH50 values of C. carpio, yellow tail and eel
displayed 68, 142, and 134 units/ml of serum, respectively (Matsuyama et al., 1988). Similarly,
we also recorded high ACH50 value (40.27 units/ml) in yellow catfish. Saha et al. (1993) marked
ACH50 values of 26 and 16 units/ml of serum in C. batrachus and Heteropneustes fossilis,
respectively, which are comparatively lower than that of yellow catfish. In this study, the
measured ACH50 in summer and rainy seasons did not vary significantly. Similarly, any change
in alternative complement activity in snapper (Pagrus auratus) with relation to variable
temperatures (12 or 24 °C) was not observed in an earlier study (Cook et al., 2003). However,
variable effects have been noticed in different fish species with relation to temperature variations
or seasons in ACH50 activity (Collazos et al., 1994; Le Morvan et al., 1998; Kumari et al., 2006).
A higher activity of two important innate defence molecules viz., serum ceruloplasmin
and anti-protease activities was observed in yellow catfish during rainy season. Thus, the higher
values obtained in most of the haematological and innate immune parameters in catfish evident
during rainy season i.e., breeding season of the year possibly indicates a natural physiological
phenomenon for making a brood fish healthy from defence point of view to avoid post-breeding
immune suppression and/or for maternal transfer of immunity to eggs.
CONCLUSION
In the present study on yellow catfish the normal baseline values for several
haematological and innate immunological parameters have been established, even for different
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J. Aqua. 23 (2015)
sex. This study highlights the relevance of seasonal variations, when monitoring health or
immune status of fish. Although a clear seasonal variation was marked in haematological and
innate immune parameters of this species, the fluctuations are not consistent to any season, except
summer season showing less value in most of the parameters. The probable compensatory
mechanism among the various defence factors might be playing role to protect from diseases
during different seasons. The data generated will help for subsequent studies with relation to
immune-modulation or stress response in this species.
REFERENCES
Anderson, D. P. and A. K. Siwicki, 1995. Basic haematology and serology for fish health programs.
In: Proceedings of the Diseases in Asian aquaculture, Asian Fisheries Society, 185 pp.
Anderson, D. P., T. Moritomo and R. de Grooth, 1992. Neutrophil, glass adherent, nitroblue
tetrazolium assay gives early indication of immunization effectiveness in rainbow trout.
Vet. Immunol. Immunopathol., 30: 419-429.
Bhat, A., 2001. New report of the species, Horabagrus brachysoma in the Uttarakhanda
district of Karnataka. J. Bombay Nat. Hist. Soc., 98: 294-296.
Blaxhall P. C. and K. W. Daisley, 1973. Routine haematological methods for use with fish
blood. J. Fish Biol., 5: 771-781.
Bowden, T. J., R. Butler and I. R. Bricknell, 2004. Seasonal variation of serum lysozyme level
in Atlantic halibut (Hipoglossus hipoglossus L.). Fish Shellfish Immunol., 17: 129-135.
Bradford, M. M., 1976. A rapid and sensitive method for the quantification of microgram
quantities of protein. Anal. Biochem., 72: 248-254.
Bromage, N., M. Porter and C. Randall, 2001. The environmental regulation of maturation in
farmed finfish with special reference to the role of photoperiod and melatonin.
Aquaculture, 197: 1-4.
Collazos, M. E., C. Barriga and E. Ortega, 1994. Optimum conditions for the activation of the
alternative complement pathway of a cyprinid fish (Tinca tinca, L.): Seasonal variations
in the titres. Fish Shellfish Immunol., 4: 499-506.
Collazos, M. E., E. Ortega, C. Barriga and A. B. Rodriguez, 1998. Seasonal variation in
haematological parameters in male and female Tinca tinca. Mol. Cell. Biochem., 183: 165-168.
Cook, M. T., P. J. Hayball, W. Hutchinson, B. F. Nowak and J. D. Hayball, 2003. Administration
of a commercial immunostimulant preparation, EcoActiva as a feed supplement enhances
macrophage respiratory burst and the growth rate of snapper (Pagrus auratus, Sparidae
(Bloch and Schneider)) in winter. Fish Shellfish Immunol., 14: 333-345.
11
J. Aqua. 23 (2015)
Dalmo, R. A., K. Ingebrightsen and J. Bogwald, 1997. Non-specific defense mechanisms in fish,
with particular reference to the reticuloendothelial system (RES). J. Fish Dis., 20: 241-273.
De Pedro, N., A. I. Guijarro, M. A. Lopez-Patino, R. Martinez-Alvarez, M.J Delgado, 2005.
Daily and seasonal variations in haematological and blood biochemical parameters in
the tench, Tinca tinca Linnaeus, 1758. Aquacult. Res., 36: 1185-1196.
Ellis, A. E., 2001. Innate host defense mechanisms of fish against viruses and bacteria. Dev.
Comp. Immunol., 25: 827-839.
Handy, R. D. and M. H. Depledge, 1999. Physiological responses: their measurement and
use as environmental biomarkers in ecotoxicology. Ecotoxicol., 8: 329-349.
Harikrishnan, R., M. N. Rani and C. Balasundaram, 2003. Hematological and biochemical
parameters in common carp, Cyprinus carpio, following herbal treatment for Aeromonas
hydrophila infection. Aquaculture, 221: 41-50.
Hernandez, A. and L. Tort, 2003. Annual variation of complement, lysozyme and
haemagglutinin levels in serum of the gilthead seabream Sparus aurata. Fish Shellfish
Immunol., 15: 479-481.
Hine, P. M., 1992. The granulocytes of fish: a review. Fish Shellfish Immunol., 2: 79-98.
Holland, M. C. H. and J. D. Lambris, 2002. The complement system in teleosts. Fish Shellfish
Immunol., 12: 399-420.
Hrubec, T. C., J. L. Cardinale and S. A. Smith, 2000. Haematology and plasma chemistry reference
intervals for cultured tilapia (Oreochromis hybrid). Vet. Clin. Pathol., 29: 7-12.
Hrubec, T. C., S. A. Smith and J. L. Robertson, 2001. Age-related changes in hematology and
plasma chemistry values of hybrid striped bass (Morone chrysops x Morone saxatilis).
Vet. Clin. Pathol., 30: 8-15.
Huffman, P. A., M. R. Arkoosh and E. Casillas, 1997. Characteristics of peripheral blood cells
from rainbow trout evaluated by particle counter, image analysis and hemocytometric
techniques. J. Aquat. Anim. Health, 9: 239-248.
Hutchinson, T. H. and M. J. Manning, 1996. Seasonal trends in serum lysozyme activity and
total protein concentration in dab (Limanda limanda L.) sampled from Lyme Bay, U.K.
Fish Shellfish Immunol., 6: 473-482.
Ingram, G. A., 1980. Substances involved in the natural resistance of fish to infection-A
review. J. Fish Biol., 16: 23-60.
12
J. Aqua. 23 (2015)
Karvonen, A., P. Rintamaki, J. Jokela and E. Tellervo Valtonen, 2010. Increasing water
temperature and disease risks in aquatic systems: Climate change increases the risk of
some, but not all, diseases. Int. J. Parasitol., 40: 1483-1488.
Kumari, J. and P. K. Sahoo, 2005. Effects of cyclophosphamide on the immune system and disease
resistance of Asian catfish Clarias batrachus. Fish Shellfish Immunol., 19: 307-316.
Kumari, J. and P. K. Sahoo, 2006. Non-specific immune response of healthy and
immunocompromised Asian catfish (Clarias batrachus) to several immunostimulants.
Aquaculture, 255: 133-141.
Kumari, J., P. K. Sahoo, T. Swain, S. K. Sahoo, A. K. Sahu and B. R. Mohanty, 2006.
Seasonal variation in the innate immune parameters of the Asian catfish Clarias
batrachus. Aquaculture, 252: 121-127.
Kurup, B. M., K. V. Radhakrishnan and T. G. Manoj Kumar, 2004. Biodiversity status of fish
inhabiting rivers of Kerala (South India) with special reference to endemism, threats and
conservation measures. In: Proceedings of the Second International Symposium on the
Management of Large Rivers for Fisheries, Phnom Penh, Cambodia, 163-182.
Le Morvan, C., D. Troutand and P. Deschaux, 1998. Differential effects of temperature on
specific and non-specific immune defences in fish. J. Exp. Biol., 201: 165-168.
Lillehaug, A., B. T. Lunestad and K. Grave, 2003. Epidemiology of bacterial diseases in
Norwegian aquaculture- a description based on antibiotic prescription data for the ten-
year period 1991-2000. Dis. Aquat. Org., 53: 115-125.
Matsuyama, H., K. Tanaka, M. Nakao and T. Yano, 1988. Characterization of the alternative
complement pathway of carp. Dev. Comp. Immunol., 12: 403-408.
Pelgrom, S. M. G. J., R. A. C. Lock, P. H. M. Balm and S. E. W. Bonga, 1995. Integrated
physiological response of tilapia, Oreochromis mossambicus, to sublethal copper
exposure. Aquat. Toxicol., 32: 303-320.
Plumb, J. A. and N. Areechon, 1990. Effect of malathion on humoral immune response of
channel catfish. Dev. Comp. Immunol., 14: 355-358.
Quade, M. J. and J. A. Roth, 1997. A rapid, direct assay to measure degranulation of bovine
neutrophil primary granules. Vet. Immunol. Immunopathol., 58: 239-248.
Rehulka, J., 2002. Aeromonas causes severe skin lesions in rainbow trout (Oncorhynchus
mykiss): clinical pathology, haematology and biochemistry. Acta Vet. Brno., 71: 351-360.
Rehulka, J., B. Minarik and E. Rehulkova, 2004. Red blood cell indices of rainbow trout
Oncorhynchus mykiss (Walbaum) in aquaculture. Aquacult. Res., 35: 529-546.
13
J. Aqua. 23 (2015)
Rough, K. M., B. F. Nowak and R. E. Reuter, 2005. Haematology and leucocyte morphology
of wild caught Thunnus maccoyii. J. Fish Biol., 66: 1649-1659.
Saha, K., K. Dash and A. Sahu, 1993. Antibody dependent haemolysin, complement and
opsonin in sera of a major carp, Cirrhina mrigala and catfish, Clarias batrachus and
Heteropneustes fossilis. Comp. Immunol. Microbiol. Inf. Dis., 16: 323-330.
Sahoo, P. K. and S. C. Mukherjee, 1999. Normal ranges for diagnostically important
haematological parameters of laboratory-reared rohu (Labeo rohita) fingerlings.
Geobios, 26: 31-36.
Sahoo, P. K., J. Kumari and B. K. Mishra, 2005. Non-specific immune responses in juveniles
of Indian major carp. J. Appl. Ichthyol., 21: 151-155.
Sahoo, P. K., K. Das Mahapatra, J. N. Saha, A. Barat, M. Sahoo, B. R. Mohanty, B. Gjerde,
J. Ødegård, M. Rye and R. Salte, 2008. Family association between immune parameters
and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo
rohita. Fish Shellfish Immunol., 25: 163-169.
Sakai, D. K., 1983a. The assessment of the health condition of the salmonids by non-specific
haemolytic activity of serum. Bull. Jpn. Soc. Sci. Fish., 49: 1487-1491.
Sakai, D. K., 1983b. Lytic and bactericidal properties of salmonid sera. J. Fish Biol., 23: 457-466.
Studnicka, M., A. Siwicki and B. Ryka, 1986. Lysozyme level in carp (Cyprinus carpio L.).
Israel J. Aqua., 38: 22-25.
Svobodova, Z., V. Luskova, J. Drastichova, M. Svoboda and V. Zlabek, 2003. Effect of
deltamethrin on haematological indices of common carp (Cyprinus carpio L.). Acta Vet.
Brno., 72: 79-85.
Swain, P., S. Dash, P. K. Sahoo, P. Routray, S. K. Sahoo, S. D. Gupta, P. K. Meher and N.
Sarangi, 2007. Non-specific immune parameters of brood Indian major carp Labeo
rohita and their seasonal variations. Fish Shellfish Immunol., 22: 38-43.
Tavares-Dias, M. and F. R. Moraes, 2007a. Leukocyte and thrombocyte reference values for
channel catfish (Ictalurus punctatus Raf.) with an assessment of morphological,
cytochemical, and ultrastructural features. Vet. Clin. Pathol., 36: 49-54.
Tavares-Dias, M. and F. R. Moraes, 2007b. Haematological and biochemical reference
intervals for farmed channel catfish. J. Fish Biol., 71: 383-388.
Tripathi, N. K., K. S. Latimer and V. V. Burnley, 2003. Biochemical reference intervals for
koi (Cyprinus carpio). Comp. Clin. Pathol., 12: 160-165.
14
J. Aqua. 23 (2015)
Van Vuren, J. H. J. and J. Hattingh, 1978. A seasonal study of the haematology of wild
freshwater fish. J. Fish Biol., 13: 305-313.
Yano, T., 1992. Assays of haemolytic complement activity. In: Techniques in Fish
Immunology, Vol. 2. (Eds. J.S. Stolen, T.C. Fletcher, D.P. Anderson, S.L. Kaatari, A.F.
Rowley), SOS, USA, pp. 131-141.
Yano, T., 1996. The non-specific immune system. In: The Fish Immune System: organism,
pathogen and environment (Eds. G. Iwama and T. Nakanishi), San Diego, USA,
Academic Press, pp. 105-157.
Zuo, X. and P. T. K. Woo, 1997. Natural anti-proteases in rainbow trout, Oncorhynchus
mykiss and brook charr, Salvelinus fontinalis and the in vitro neutralization of fish 2-
macroglobulin by the metalloprotease from the pathogenic haemoflagellate, Cryptobia
salmositica. Parasitology, 114: 375-381.
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GENETIC VARIATIONS AMONG FAMILIES OF SELECTIVELY BRED
MACROBRACHIUM ROSENBERGII (DE MAN) BY RAPD-PCR ANALYSIS
G. Patra1*, J. Mohanty, S. K. Garnayak, P. K. Sahoo and Bindu R. Pillai
ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751002, Odisha, India 1Present address: Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture and
Farmers’ Welfare, Government of India, Krishi Bhawan, New Delhi-110114
*Corresponding author: [email protected]
Giant freshwater prawn, Macrobrachium rosenbergii is an important freshwater crustacean widely
cultured in several countries including India. Of late, its production has come down due to slow
growth rate and disease occurrences. The ICAR-Central Institute of Freshwater Aquaculture
(ICAR-CIFA), Bhubaneswar in collaboration with the WorldFish, Malaysia has initiated a
selective breeding programme for growth improvement of this species. In the present study, two
groups of families (I. six numbers of families for growth and II. six numbers of families for disease
resistance) were selected for experimentation from the families produced in the fourth generation
of selection programme. Each group consisted of two extreme sub-groups of three families in each
with higher and lower growth (based on weight) under group I and, susceptible and resistant
families (based on larval survival following challenge with Vibrio harveyi) under group II. RAPD-
PCR was used to evaluate the genetic variations between and within groups separately. Twelve
selected decamer primers were used to amplify DNA fragments of three individuals of each family
and data were analyzed by POPGENE version 1.31 software. In group I, a total of 102 bands were
scored by the primers out of which 41 bands (40.19%) found to be polymorphic. Genetic diversity
within the group varied from 0.0272 0.0965 to 0.0463 0.1316. UPGMA dendrogram of this
group based on Nei’s genetic distance showed that families 5 (low growth family 2) and 6 (low
growth family 3) are distantly related to high growth families. In the second group of disease
resistance, 35 bands (36.46%) were found to be polymorphic out of 96 bands scored. Genetic
diversity varied between 0.0301 ± 0.0957 to 0.0438 ± 0.1381 within this group. UPGMA
dendrogram showed that families 1 (susceptible group 1) and 2 (susceptible group 2) are distantly
related to three resistant families. Thus, the present results showed the existence of genetic
variations in both growth and disease resistance traits that could be utilized in the selective
breeding programme in M. rosenbergii.
INTRODUCTION
The genetic structure of a population is changeable. The degree of change depends on
intensities of interventions. Wild populations are less prone to changes in their gene pool than
hatchery populations as interventions in wild populations are negligible or very less. Therefore,
wide genetic variations are found among the wild populations. Whereas, reduction in genetic
variation through inbreeding, negative selection and genetic drift are very common in a hatchery
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population (Alam and Islam, 2005). Loss of genetic variation is considered to be the loss of
genetic potential for stock improvement and adaptation to environmental changes. It is therefore,
essential to maintain the genetic variations in the hatchery populations through a systematic
selective breeding programme to avoid inbreeding. The effect of any selection programme will
be to change allele frequencies at loci influencing targeted phenotypes because certain alleles will
be favoured and less favourable alleles will be reduced in frequency or eliminated (Gjedrem and
Thodesen, 2005). Thus the long-term success of any breeding program will depend to a significant
extent on the amount of genetic variations available in the parental population (Falconer and
Mackay, 1996). Several studies have proved that breeding programs on culture stocks with low
amount of genetic variation in the parental population are unsuccessful (Moav and Wohlfarth,
1976; Hulata et al., 1986; Huang and Liao, 1990). Therefore, quantifying the levels of genetic
diversity for every generation among produced families is important for the target traits in any
selective breeding programme.
Molecular markers offer the realistic method to assess the genetic status of a population,
and are powerful tools to detect genetic uniqueness of individuals, populations or species
(Chauhan and Rajiv, 2010). Application of molecular markers has allowed rapid progress in
investigations of genetic variability and inbreeding, parentage assignments, species and strain
identification, and in the construction of high-resolution genetic linkage maps for aquaculture
species (Liu and Cordes, 2004). Several molecular markers like mitochondrial DNA (mtDNA)
random fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD),
amplified fragment length polymorphism (AFLP), microsatellites and single nucleotide
polymorphism (SNP) have been used widely to assess the genetic variations among populations.
These markers in general have been categorized into different classes by various authors.
Danzmann and Gharbi (2001) classified the genetic markers largely into two groups, i.e.,
sequence specific (e.g. microsatellite simple sequence repeat SSR) and sequence-independent
markers (e.g. AFLP and RAPD).
RAPD, a multi-locus dominant marker system, is quite popular amongst researchers as
it provides multiple markers without any prior knowledge of the DNA sequences. These
oligonucleotides serve as both forward and reverse primers and usually are able to amplify
fragments from 3 to 10 genomic sites simultaneously. The variable lengths of DNA are inherited
as classical Mendelian traits (Williams et al., 1990) and thus can be used for genetic analysis
(Horn et al., 1996). RAPDs have gained considerable attention particularly in population
genetics, species and subspecies identification, phylogenetic study, linkage group identification,
chromosome and genome mapping, analysis of interspecific gene flow and hybrid speciation and
as a potential source for single-locus genetic fingerprints (Brown and Epifanio, 2003). These
markers have been used for species identification or interspecies genetic relationship in fishes
(Naish et al., 1995; Partis and Wells, 1996; Dahle et al., 1997; Das et al., 2005; Lakra et al.,
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J. Aqua. 23 (2015)
2007), molluscs (Klinbunga et al., 2000), Argulus parasites (Sahoo et al., 2013) and shrimp (Shi
et al., 1999; Song et al., 1999; Zhuang et al., 2001), and analysis of population structure in shrimp
(Tassanakajon et al., 1997; 1998; Klinbunga et al., 2001; Mishra et al., 2009), catfish (Liu et al.,
1999) and scampi (See et al., 2008; Islam et al., 2014; Mohanty et al., 2014). Sun et al. (2000)
applied the techniques of RAPD and AFLP to analyze the relationships among four species of
Artemia species and strains, and reported that RAPD markers successfully detected diversity and
genetic differentiation among them. This technique has also been employed to study the genetic
variations among the wild populations in selective breeding programme for establishment of base
population of Penaeus monodon (Garcia and Benzie, 1995) and Macrobrachium rosenbergii
(Mohanty et al., 2011), and to identify heritability for growth in Fenneropenaeus indicus
(Rezvani Gilkolaei et al., 2011). RAPD markers having genetically linked to a trait of interest
could be used for individual and pedigree identification, and trait improvement in genetics and
breeding programme (Yoon and Kim, 2001; Shikano and Taniguchi, 2002). Mohanty et al. (2011)
evaluated the genetic variation among the three Indian state (Odisha, Kerala and Gujarat)
populations of M. rosenbergii by RAPD and reported substantial genetic variation within and
between the three populations.
M. rosenbergii is one of the most important cultured species in India and many other
Asian countries. The farmed production of M. rosenbergii in India has shown phenomenal
increase from less than 178 tonnes in 1996 to 42,780 tonnes in 2005 (FAO, 2008). However, the
production has been declining steadily since 2006. Poor quality seed and low survival have been
found to be main reasons of decrease in production. Besides, the scampi culture industry in India
relies mostly on wild or undomesticated lines, which are not pathogen free and often provide
inconsistent quality compared with genetically improved lines. Implementation of stock
improvement programs for scampi in India was necessary to allow this industry to develop in a
sustainable way as it has been demonstrated in other aquatic species, namely Atlantic salmon
(Thodesen and Gjedrem, 2006), common carp (Bakos et al., 2006), GIFT tilapia (Eknath et al.,
2007) and rohu carp (Mahapatra et al., 2006). Therefore, ICAR-Central Institute of Freshwater
Aquaculture (CIFA), Bhubaneswar in collaboration with the WorldFish, Malaysia initiated a
systematic selective breeding programme for improving growth rate of M. rosenbergii in 2007
(Pillai et al., 2011, 2015). In the present study an attempt was made to assess the genetic variations
among the produced families of fourth generation (between high growth and low growth groups)
and between disease resistant and susceptible groups) through RAPD-PCR method.
MATERIALS AND METHODS
Sample collection
The samples were collected from the ongoing selective breeding programme for
M. rosenbergii at the Institute. From the families produced in the fourth generation of selection,
three higher (HG1-3) and three lower (LG1-3) growth families were identified based on the
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weight at final harvest out of 45 families generated (Pillai et al., 2013). In addition, the natural
resistance of larvae of generation 4 (G4) to Vibrio harveyi was assessed by immersion challenge
experiment and a wide variation in survival (0-93.3%) was noticed among the families after
bacterial challenge (Patra et al., 2014). The families were grouped into susceptible or resistant
based on the challenge test. Three extreme susceptible (<20% survival) (S1-3) and three most
resistant (R1-3) families (>80% survival) were selected for present experiments. Pleopods from
prawns (weight 30-40 g) belonging to above twelve selected families were collected for DNA
extraction.
Isolation of genomic DNA
The genomic DNA was isolated from pleopods of prawns by phenol-chloroform
extraction method of Sambrook and Russel (2001) with minor modifications. DNA from three
individuals of each family of each group (total 36 individuals of 12 families) was utilized in the
current study. The pleopod sample was homogenized using a sterile mortar and pestle in the
presence of 700 µl TEN buffer (50 mM Tris-HCL, pH 8.0, 10 mM EDTA, 100 mM NaCl) and
transferred to a 2.0 ml eppendorf tube. Proteinase K and SDS were added at a final concentration
of 500 µg ml-1 and 1%, respectively. The mixture was mixed thoroughly and incubated overnight
at 37 0C. DNA was extracted from the aqueous phase (after centrifugation at 10,000 rpm for 10
min at 4 0C) of phenol, phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl
alcohol (24:1) and was precipitated by adding 0.3 M sodium acetate and absolute alcohol. DNA
pellet was obtained by centrifugation at 10,000 rpm for 10 min at 4 0C. Further, DNA pellet was
washed with 70% ethanol. The pellet was air dried and dissolved in TE buffer (10 mM Tris and
1 mM EDTA, pH 8.0). Extracted DNA was checked for its purity and quantity using NanoDrop
(ND1000, Thermo Scientific, Wilmington, DE, USA) and diluted with distilled water for a
working concentration of 50 µg ml-1.
RAPD-PCR
The PCR reaction was carried out using 25 ng of genomic DNA as template in a total
volume of 25 µl reaction mixture. The reaction conditions optimized for amplification of random
fragments were 0.75 U Taq polymerase (Genei, India), 1x Taq buffer A, 100 mM dNTPs and 20
picomole RAPD primer per reaction. The amplification was carried out in a thermal cycler (MJ
research, Waltham, MA, USA) as per the following programme: initial denaturation at 94 ºC for
5 min, 40 cycles of 94 ºC for 1 min, 36 ºC for 1 min and 72 ºC for 2 min followed by final
extension at 72 ºC for 7 min. Twelve random decamer primers {(OPA02, OPA04, OPA06,
OPA07 OPA08, OPA09, OPA10, OPA11, OPA12, OPA14, OPA15 and OPA17 (IDT Milpitas,
CA, USA)} were selected depending upon repeatability and reproducibility of amplified fragment
patterns and were used in experiments.
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Agarose gel electrophoresis
Amplified products were separated by electrophoresis on 1.5% agarose gel containing
ethidium bromide in 1x TBE buffer at 100 v for 2 h. To determine the molecular size, 100 bp
DNA ladder was run alongside RAPD products. The gels were visualized in a UV gel
documentation system (ALPHA INNOTECH, San Leandro, CA, USA).
Analysis of Data
The bands observed in each lane were compared with all other lanes on the same gel.
The reproducible bands were scored visually as either presence (1) or absence (0). Scores with
respect to all the primers were used for constructing a single data matrix for growth and disease
resistance separately. High growth families (HG1-3) followed by low growth families (LG1-3)
were sequentially considered as population 1-6. Similarly, disease susceptible families (S1-3)
followed by resistant families (R1-3) were considered sequentially as population 7-12. The data
were analyzed using Pop-Gene version 1.31 software (Yeh et al., 1999) for estimation of
polymorphic loci, genetic diversity within populations, interpopulation genetic diversity and for
construction of dendrogram among populations based on genetic distances (Nei, 1972).
RESULTS
The representative RAPD profiles of 18 individuals of M. rosenbergii from growth
group generated by primers OPA2 and OPA4 are depicted in Fig.1. RAPD profiles of M.
rosenbergii obtained by twelve random primers are summarized in Table 1. From all the twelve
random primers 102 bands were scored, out of which 41 bands (40.19%) were polymorphic.
Number of the scored fragments varied from 2 to 13 with a size range of 125 to 2042 bp. Gene
diversity within populations varied from 0.02720.0965 to 0.04630.1316 (Table 2). Population
3 (HG-3) was found to have maximum gene diversity with 11.76% polymorphic loci and
population 1 (HG-1) having the minimum with 7.84% polymorphic loci (Table 2). Genetic
similarity among families ranged from 0.8176 to 0.9320. The highest genetic distance was 0.2013
between populations 3 (HG-3) and 6 (LG-3) while the lowest was 0.0705 between populations 3
(HG-3) and 4 (LG-1) (Table 3). UPGMA dendrogram based on Nei’s genetic distance for 6
populations of M. rosenbergii (Fig. 2) showed that populations 5 (LG-2) and 6 (LG-3) form a
different clade and are distantly related to other families.
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Fig. 1. RAPD profile of 18 individuals of M. rosenbergii of growth group with OPA 2 (A) and
OPA4 (B) primers. Lane M: 100 bp DNA ladder; lanes 1-18: three samples from each family
(lanes -1-3: LG1; 4-6: LG2; 7-9: LG3; 10-12: HG1; 13-15: HG2; 16-18: HG3).
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Fig. 2. UPGMA dendrogram of growth group for six populations (families) of M. rosenbergii
based on Nei’s genetic distance (Pop1- HG1; Pop2- HG2; Pop3- HG3; Pop4- LG1; Pop5- LG2;
Pop6- LG3)
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Table 1 : RAPD profiles of M. rosenbergii families from growth group obtained by twelve
random primers.
Primer Sequence
(5’ to 3’)
No. of
bands
scored
Size of
fragments
(bp)
Total
no. of
bands
No. of
polymorphic
bands
Polymorphic
bands (%)
OPA2 TGCCGAGCTG 6-9 207-1504 10 4 40
OPA4 AATCGGGCTG 7-10 160-1018 10 3 30
OPA6 GGTCCCTGAC 10-13 198-1969 13 3 23.07
OPA7 GAAACGGGTG 4-7 233-1625 7 3 42.85
OPA8 GTGACGTAGG 3-7 143-1204 8 6 75
OPA09 GGGTAACGCC 5-7 125-961 7 2 28.57
OPA10 GTGATCGCAG 5-7 179-1200 9 5 44.44
OPA11 CAATCGCCGT 8-11 137-2042 11 3 27.27
OPA12 TCGGCGATAG 3-6 181-1051 7 5 57.14
OPA14 TCTGTGCTGG 7-9 161-1056 9 3 22.22
OPA15 TTCCGAACCC 5-7 378-1563 7 2 28.57
OPA17 GACCGCTTGT 2-4 295-730 4 2 50
Total 102 41 40.19
Table 2 : Number and percentage of polymorphic loci and gene diversity values within
M. rosenbergii families from growth group.
Population Polymorphic loci Gene diversity
(Mean ± SD) No. %
Population 1 (HG1) 8 7.84 0.0272 0.0965
Population 2 (HG2) 11 10.78 0.0416 0.1240
Population 3 (HG3) 12 11.76 0.0463 0.1316
Population 4 (LG1) 10 9.80 0.0331 0.1036
Population 5 (LG2) 11 10.78 0.0397 0.1186
Population 6 (LG3) 11 10.78 0.0416 0.1240
HG- Higher Growth, LG- Lower Growth
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Table 3 : Nei’s unbiased genetic identity (above diagonal) and genetic distance (below
diagonal) values between M. rosenbergii families from growth group.
Population 1 (HG1) 2 (HG2) 3 (HG3) 4 (LG1) 5 (LG2) 6 (LG3)
1 (HG1) **** 0.8924 0.8681 0.9011 0.8630 0.8265
2 (HG2) 0.1138 **** 0.8871 0.9195 0.8553 0.8606
3 (HG3) 0.1414 0.1198 **** 0.9320 0.8986 0.8176
4 (LG1) 0.1042 0.0839 0.0705 **** 0.8998 0.8762
5 (LG2) 0.1473 0.1564 0.1069 0.1056 **** 0.8933
6 (LG3) 0.1906 0.1502 0.2013 0.1322 0.1129 ****
The representative RAPD profiles of 18 individuals of M. rosenbergii samples from
disease resistance group generated by primers OPA2 and OPA4 are depicted in Fig. 3 and RAPD
profiles of M. rosenbergii obtained by twelve random primers are summarized in Table 4. From
all the twelve random primers 96 bands were scored, out of which 35 bands (36.46%) were
polymorphic. Number of the scored fragments varied from 2 to 13 with size ranges of 125 to 2042
bp. Gene diversity within populations varied from 0.0301±0.0957 to 0.0438±0.1381 (Table 5).
Population 7 was found to have maximum genetic diversity with 11.46% polymorphic loci and
population 11 having the minimum with 9.38% polymorphic loci (Table 5). Genetic similarity
among families ranged from 0.8706 to 0.9371. The highest genetic distance was 0.1386 between
populations 7 (S1) and 9 (S3) while the lowest was 0.0650 between populations 7 (S1) and 8 (S2)
(Table 6). UPGMA dendrogram based on Nei’s genetic distance for 6 families of M. rosenbergii
(Fig. 4) showed that families 1 (S1) and 2 (S2) form a different clade, and are distantly related to
other families.
25
J. Aqua. 23 (2015)
Fig. 3. RAPD profile of 18 individuals of M. rosenbergii from disease resistance group with
OPA2 (A) and OPA 4 (B) primers. Lane M: 100 bp DNA ladder; lanes 1-18: three samples from
each family (lanes 1-3: S1; 4-6: S2; 7-9: S3; 10-12: R1; 13-15: R2; 16-18: R3).
26
J. Aqua. 23 (2015)
Fig. 4. UPGMA dendrogram of disease resistance group for six populations (families) of
M. rosenbergii based on Nei’s genetic distance (Pop1- S1; Pop2- S2; Pop3- S3; Pop4- R1; Pop5-
R2; Pop6- R3).
27
J. Aqua. 23 (2015)
Table 4 : RAPD profiles of M. rosenbergii families from disease resistance group obtained
by twelve random primers.
Primer Sequence
(5’ to 3’)
No. of
bands
scored
Size of
fragments
(bp)
Total
no. of
bands
No. of
polymorphic
bands
Polymorphic
bands (%)
OPA2 TGCCGAGCTG 6-9 207-1504 10 3 30.0
OPA4 AATCGGGCTG 5-10 160-1018 10 5 50.0
OPA6 GGTCCCTGAC 10-13 198-1969 13 4 23.07
OPA7 GAAACGGGTG 4-6 233-1625 6 3 33.33
OPA8 GTGACGTAGG 3-4 143-1204 6 3 50.0
OPA09 GGGTAACGCC 5-7 125-961 7 2 28.57
OPA10 GTGATCGCAG 5-8 235-1200 8 3 37.5
OPA11 CAATCGCCGT 8-10 137-2042 10 2 20.0
OPA12 TCGGCGATAG 3-5 234-616 6 3 50.0
OPA14 TCTGTGCTGG 5-8 161-1197 9 3 33.33
OPA15 TTCCGAACCC 4-6 280-1563 7 2 28.57
OPA17 GACCGCTTGT 2-4 295-730 4 2 50.0
Total 96 35 36.46
Table 5 : Number and percentage of polymorphic loci and gene diversity values within
families of M. rosenbergii from disease resistance group.
Population Polymorphic loci Gene diversity
(Mean ± SD) No. %
Population 7 (S1) 11 11.46 0.0402 ± 0.1160
Population 8 (S2) 9 9.38 0.0438 ± 0.1381
Population 9 (S3) 9 9.38 0.0320 ± 0.1029
Population 10 (R1) 9 9.38 0.0399 ± 0.1276
Population 11 (R2) 9 9.38 0.0301 ± 0.0957
Population 12 (R3) 10 10.42 0.0410 ± 0.1247
S-Susceptible, R-Resistant
28
J. Aqua. 23 (2015)
Table 6 : Nei’s unbiased genetic identity (above diagonal) and genetic distance (below
diagonal) values between families of M. rosenbergii from disease resistance group.
Population 7 (S1) 8 (S2) 9 (S3) 10 (R1) 11 (R2) 12 (R3)
7 (S1) **** 0.9371 0.8706 0.8934 0.9017 0.8873
8 (S2) 0.0650 **** 0.8977 0.9011 0.8890 0.9021
9 (S3) 0.1386 0.1080 **** 0.9288 0.9221 0.9308
10 (R1) 0.1127 0.1041 0.0738 **** 0.9233 0.8993
11 (R2) 0.1035 0.1177 0.0811 0.0798 **** 0.9239
12 (R3) 0.1196 0.1030 0.0717 0.1061 0.0792 ****
DISCUSSION
The present study was conducted to assess the genetic variability within and between
families of selectively bred M. rosenbergii by RAPD analysis. RAPD has long been used in
studying genetic variability in various species owing to its capabilities to run without known
genetic sequence and to generate high polymorphic loci (Vaseeharan et al., 2013). High number
of polymorphic bands in RAPD reflects a high level of polymorphism in the populations. In our
study, RAPD generated a moderate level of polymorphism both in growth (40.19%) and disease
resistance (36.46%) groups. Mohanty et al. (2011) reported a higher level (~76%) of polymorphic
bands with eight RAPD primers, while comparing 3 populations (Odisha, Kerala and Gujarat)
from India which were used as base populations for the selective breeding program leading to
development of populations used in present study. Islam et al. (2014) observed a similar level of
polymorphism (41%) through RAPD with five primers while studying post larvae of M. rosenbergii
broods stocked under different male: female ratio in Bangladesh. The reduced level of
polymorphism noticed in this study (36.46 and 40.19%) as compared to base population (~76%)
might be obvious and due to selection pressure or genetic interventions as the population under
study are from fourth generation of selection.
However, the genetic diversity was low at the population level in both growth
(percentage of polymorphic loci varying from 7.84 to 11.76; Nei’s gene diversity varying from
0.0272 to 0.0463) and disease resistance (percentage of polymorphic loci varying from 9.38 to
11.46; Nei’s gene diversity varying from 0.0301 to 0.0438) groups. These observed intra-
population diversity in selectively bred populations were lower, when compared with the studies
conducted earlier on its base populations (Mohanty et al., 2011). It was reported that percentage
polymorphic loci to be 45 to 52.5% and gene diversity to be 0.1330 to 0.1921. See et al. (2008)
however, detected a very high level of polymorphism (94.3 to 100%) with all five primers while
comparing 11 populations of M. rosenbergii in Malaysia. Hence, selection of base populations
play crucial role in a selection programme.
29
J. Aqua. 23 (2015)
The result of the present study showed presence of genetic variations among the
produced families of the selection programme. The genetic distance between populations varied
from 0.0705 to 0.2013 for growth group and 0.0650 to 0.1386 for disease resistance group. A
comparison with the earlier study in their base populations (genetic distance varied from 0.1161
to 0.2076) (Mohanty et al., 2011) indicated a reduced genetic distance between some populations.
However, Mohanty et al. (2014) reported the genetic distance varying from 0.175 to 0.856 while
studying genetic diversity of 5 Indian populations of M. rosenbergii by microsatellite markers. In
UPGMA dendrogram, out of three families of lower growth groups two families were genetically
distant and one family closely related from higher growth families. Similarly, out of three
susceptible families, two were genetically distant from resistant group whereas one susceptible
family genetically close with the resistant families. The results thus indicate that the genetic
differentiation between families has not stabilized as per breeding traits after four generations of
selection and the populations may require further directional breeding activities to reach the
genetic stability. However, data generated from more number of families of successive
generations would confirm the robustness of these findings.
Hence, it may be concluded that the genetic diversity within populations (families) has
reduced, whereas, the genetic difference between families are still maintained. Hence, further
selective breeding program may help to harness the full potential of traits. Though genetic
diversity studies on selectively bred populations have not been conducted in freshwater prawn
species, there are some reports on marine shrimps and fish. Cruz et al. (2004) found selected
strains through breeding programs tended to lose genetic diversity compared with wild
populations in Pacific white shrimp (Litopenaeus vannamei). A similar study was conducted by
Li et al. (2006) using amplified fragment length polymorphism (AFLP) markers to investigate the
genetic structure of a wild base population and three generations of marine shrimp,
Fenneropenaeus chinensis, selected for fast growth (F5–F7). As time under selection increased,
the genetic diversity tended to reduce, the differentiation between generations became less, and
the variation of genetic structure of the populations became smaller. Luo et al. (2015) studied the
genetic diversity and structure of 5 consecutive selected populations of golden mandarin fish
(Siniperca scherzeri Steindachner) with microsatellite markers and observed reduced genetic
diversity over generations and there was increased genetic distance between adjacent generations.
They opined that the generation populations of breeding had not fully adopted to the existing
selection pressure and environment, and thus the population genetic structure had not yet
stabilized. Additionally, the populations may require further breeding activities to reach a stable
genetic structure in order to ensure the genetic stability of breeding traits.
The reported polymorphism level found in the present investigation indicates that RAPD
markers could be useful to assess genetic variations in selectively bred populations in freshwater
prawn. Further, the results of genetic diversity among different families of M. rosenbergii based
30
J. Aqua. 23 (2015)
on RAPD markers can contribute significantly to the development and implementation of further
genetic improvement programs. However, using more powerful markers with large sample size
may reveal better results which can help to establish genetic relationships among the families in a
particular generation of any selective breeding programme.
ACKNOWLEDGMENTS
This study was carried out at ICAR-Central Institute of Freshwater Aquaculture (ICAR-
CIFA), Bhubaneswar, Odisha, India under a bilateral collaborative research project between
Indian Council of Agricultural Research (ICAR), New Delhi, India, and WorldFish, Malaysia.
The authors are thankful to the Director, ICAR-CIFA, for encouragement and for providing
necessary facilities.
REFERENCES
Alam Md. S. and Md. S. Islam, 2005. Population structure of Catla catla (Hamilton) revealed by
microsatellite DNA markers. Aquaculture, 246: 151-160.
Bakos, J., L. Varadi, S. Gorda, and Z. Jeney, 2006. Lessons from the breeding program on
common carp in Hungary. In: Development of aquatic animal genetic improvement and
dissemination programs: current status and action plans. (Eds. R. W. Ponzoni, B. O.
Acosta and A. G. Ponniah) WorldFish Center Conferences Proceedings 73: 27-33.
Brown, B. and J. Epifanio, 2003. Nuclear DNA. In: Population Genetics: Principles and
Applications for Fisheries Scientist, (Ed. Hallerman, E. M.) American Fisheries Society,
Bethesda, pp. 458-472.
Chauhan, T. and K. Rajiv, 2010. Molecular markers and their applications in fisheries and
aquaculture. Adv. Biosci. Biotechnol., 1: 281-291.
Cruz, P., A. M. Ibarra, H. Mejia-Ruiz, P. M. Gaffney, and R. Perez-Enriquez, 2004. Genetic
variability assessed by microsatellites in a breeding program of Pacific white shrimp
(Litopenaeus vannamei). Mar. Biotechnol., 6: 157–164.
Dahle G., M. Rahman and A.G. Eriksen, 1997. RAPD fingerprinting used for discriminating
among three populations of Hilsa shad (Tenualosa ilisha). Fish. Res., 32: 263-269.
Danzmann, R. G. and K. Ghabri, 2001. Gene mapping in fishes: a means to an end. Genetica,
111: 3-23.
Das, P., H. Prasad, P. K. Meher, A. Barat and R. K. Jana, 2005. Evaluation of genetic relationship
among six Labeo species using randomly amplified polymorphic DNA (RAPD).
Aquacult. Res., 36: 564-569.
31
J. Aqua. 23 (2015)
Eknath, A. E., H. B. Bentsen, R. W. Ponzoni, M. Rye, N. H. Nguyen, J. Thodesen and B. Gjerde,
2007. Genetic improvement of farmed tilapias: composition and genetic parameters of
a synthetic base population of Oreochromis niloticus for selective breeding.
Aquaculture, 273: 1-14.
Falconer, D. S. and T. F. C. Mackay, 1996. Introduction to quantitative genetics. Pearson, Harlow, 464 pp.
FAO, 2008. FAO yearbook 2006. Fisheries and Aquaculture Statistics. FAO, Rome, Italy, 57 pp.
Garcia, D. K. and J. A. H. Benzie, 1995. RAPD markers of potential use in penaeid prawn
(Penaeus monodon) breeding programs. Aquaculture, 130: 137–144.
Gjedrem, T. and J. Thodesen, 2005. Selection. In: Selection and Breeding Programs in
Aquaculture. (Ed. T. Gjedrem) Springer, the Netherlands, pp. 89-111.
Horn, P. L., J. A. Rafalski and P. J. Whitehead, 1996. Molecular genetics (RAPD) analysis of
breeding mapping genes. The Auk, 113: 552-557.
Huang, C. M. and I. C. Liao, 1990. Response to mass selection for growth rate in Oreochromis
niloticus. Aquaculture, 85: 199-205.
Hulata, G., G. W. Wohlfarth and A. Halevy, 1986. Mass selection for growth rate in the Nile
tilapia (Oreochromis niloticus). Aquaculture, 57: 177-184.
Islam S. S., Md. S. Shah and Md. L. Rahi, 2014. Assessment of genetic variability of prawn
(Macrobrachium rosenbergii) post larvae (PL) from brood stocks under different sex
ratios. Int. J. Aquacult., 4: 55-63.
Klinbunga, S., D. Siludjai, W. Wudthijinda, A. Tassanakajon, P. Jarayabhand and P. Menasveta,
2001. Genetic heterogeneity of the giant tiger shrimp (Penaeus monodon) in Thailand
revealed by RAPD and mitochondrial DNA RFLP analyses. Mar. Biotechnol., 3: 428-438.
Klinbunga, S., P. Ampayup, A. Tassanakajon, P. Jarayabhand and W. Yoosukh, 2000.
Development of species-specific markers of the tropical oyster (Crassostrea belcheri)
in Thailand. Mar. Biotechnol., 2: 476-484.
Lakra, W. S., M. Goswami, V. Mohindra, K. K. Lal and P. Punia, 2007. Molecular identification
of five Indian sciaenids (Pisces: Perciformes, Sciaenidae) using RAPD markers.
Hydrobiologia, 583: 359-363.
Li, Z., J. Li, Q. Wang, Y. He and P. Liu, 2006. The effects of selective breeding on the genetic
structure of shrimp Fenneropenaeus chinensis populations. Aquaculture, 258: 278-282.
Liu, Z. J. and J. F. Cordes, 2004. DNA marker technologies and their applications in aquaculture
genetics. Aquaculture, 238: 1-37.
32
J. Aqua. 23 (2015)
Liu, Z. J., P. Li, B. J. Argue and R. A. Dunham, 1999. Random amplified polymorphic DNA
markers usefulness for gene mapping and analysis of genetic variation of catfish.
Aquaculture, 174: 59-68.
Luo, X. N., M. Yang, X. F. Liang, K. Jin, L. Y. Lv, C. X. Tian, Y. C. Yuan and J. Sun, 2015.
Genetic diversity and genetic structure of consecutive breeding generations of golden
mandarin fish (Siniperca scherzeri Steindachner) using microsatellite markers. Genet.
Mol. Res., 14: 11348-11355.
Mahapatra, K. D., R. K. Jana, J. N. Saha, B. Gjerde, and N. Sarangi, 2006. Lessons from the
breeding program of Rohu. In: Development of aquatic animal genetic improvement and
dissemination programs: current status and action plans (Eds. R. W. Ponzoni, B. O.
Acosta and A. G. Ponniah). WorldFish Center Conferences Proceedings, 73: 34-40.
Mishra P. S., A. Chaudhuri, G. Krishna, D. Kumar and W. S. Lakra, 2009. Genetic diversity in
Metapenaeus donovani using RAPD. Biochem. Genet., 47: 421-426.
Moav, R. and G. Wohlfarth, 1976. Two-way selection for growth rate in the common carp
(Cyprinus carpio L.). Genetics, 82: 83-101.
Mohanty, J., L. Sahoo, S. Sahu, B. R. Pillai and K. Dasmahapatra, P. K. Sahoo and P. L.
Lalrinsanga, 2011. Evaluation of genetic variation in Macrobrachium rosenbergii by
RAPD-PCR. In: Book of Abstracts, Asian Pacific Aquaculture, Kochi, 105 pp.
Mohanty, P., L. Sahoo, B. R. Pillai, P. Jayasankar and P. Das, 2014. Genetic divergence in Indian
populations of M. rosenbergii using microsatellite markers. Aquacult. Res., 47: 472-481.
Naish, K. A., M. Warren, F. Bardakci, D. O. F. Skibinski, G. R. Carvalho and G. C. Mair, 1995.
Multilocus DNA fingerprinting and RAPD reveal similar genetic relationships between
strains of Oreochromis niloticus (Pisces: Cichlidae). Mol. Ecol., 4: 271-274.
Nei, M., 1972. Genetic distance between populations. Am. Nat., 106: 283-292.
Partis, L. and R. J. Wells, 1996. Identification of fish species using random amplified
polymorphic DNA (RAPD). Mol. Cell. Probes, 10: 435-441.
Patra, G., B. R. Pillai, P. K. Sahoo, P. L. Lalrinsanga, A. Das, S. Mohanty, S. Sahu and N. Naik,
2014. Immune responses and susceptibility patterns in different families of selectively
bred Macrobrachium rosenbergii (de Man) in response to Vibrio harveyi infection. In:
Book of Abstracts, 10th Indian Fisheries and Aquaculture Forum, held at National Bureau
of Fish Genetic Resources, Lucknow, 12-15 November 2014, AAH- 48, pp: 372.
33
J. Aqua. 23 (2015)
Pillai, B. R., K. D. Mahapatra, R. W. Ponzoni, L. Sahoo, P. L. Lalrinsanga, W. Mekkawy, H. L.
Khaw, N. H. Nguyen, S. Mohanty, S. Sahu and G. Patra, 2015. Survival, male
morphotypes, female and male proportion, female reproductive status and tag loss in
crosses among three populations of freshwater prawn Macrobrachium rosenbergii (de
Man) in India. Aquacult. Res., 46: 2644-2655.
Pillai, B. R., K. D. Mahapatra, R. W. Ponzoni, P. L. Lalrinsanga, H. L. Khaw, S. Mohanty, S.
Sahu, G. Patra and N. Naik, 2013. Genetic improvement of Macrobrachium rosenbergii
(de man) in India through selective breeding, In: Souvenir; Final workshop of the
collaborative project between Central Institute of Freshwater Aquaculture (CIFA),
Bhubaneswar and WorldFish Centre (WFC), Malaysia on ‘Genetic Improvement of
Freshwater Prawn, Macrobrachium rosenbergii, (de Man) in India-Phase-II’ 20-21
December, 2013.
Pillai, B. R., K. D. Mahapatra, R.W. Ponzoni, L. Sahoo, P. L. Lalrinsanga, N. H. Nguyen, S.
Mohanty, S. Sahu, K. Vijay, S. Sahu, H. L. Khaw, G. Patra, S. Patnaik and S. C. Rath,
2011. Genetic evaluation of a complete diallel cross involving three populations of
freshwater prawn (Macrobrachium rosenbergii) from different geographical regions of
India. Aquaculture, 319: 347-354.
Rezvani Gilkolaei, S., R. Safari, F. Laloei, J. Taqavi and A. Matinfar, 2011. Using RAPD markers
potential to identify heritability for growth in Fenneropenaeus indicus. Iran. J. Fish.
Sci., 10: 123-134.
Sahoo, P. K., J. Mohanty, S. K. Garnayak, B. R. Mohanty, B. Kar, J. K. Jena and H. Prasanth,
2013. Genetic diversity and species identification of Argulus parasites collected from
major aquaculture regions of India using RAPD-PCR. Aquacult. Res., 44: 220-230.
Sambrook J. and D. W. Russel, 2001. Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York, NY, USA.
See, L. M., R. Hassan, S. G. Tan and S. Bhassu, 2008. Genetic characterization of wild stocks of
prawns, M. rosenbergii using random amplified polymorphic DNA markers.
Biotechnology, 7: 338-42.
Shi, T., J. Kong, P. Liu, L. L. Hart, Z. M. Zhuang and J. Y. Deng, 1999. RAPD analysis of genetic
diversities in Penaeus chinensis: in the western coast of Korean Peninsula. Oceanol.
Limnol. Sin., 30: 609-614.
Shikano, T. and N. Taniguchi, 2002. Using microsatellite and RAPD markers to estimate the
amount of heterosis in various strain combinations in the guppy, Poecilia reticulate as a
fish model. Aquaculture, 204: 271-281.
34
J. Aqua. 23 (2015)
Song L. S., J. H. Xiang, C. X. Li, B. Z. Liu and R. Y. Liu, 1999. Analysis of RAPD markers of
the genetic structures in the natural population and hatchery stock of Penaeus japonicus.
Oceanol. Limnol. Sin., 30: 261-264.
Sun, Y., W. Q. Song, Y. C. Zhong, R. S. Zhang and R.Y. Chen, 2000. Phylogenetic study of
Artemia from China using RAPD and AFLP markers. Yi Chuan Xue Bao, 27: 210-218.
Tassanakajon, A., S. Pongsomboon, V. Rimphanitchayakit, P. Jarayabhand and V. Boonsaeng,
1997. Random amplified polymorphic DNA (RAPD) markers for determination of
genetic variation in wild populations of the black tiger prawn (Penaeus monodon) in
Thailand. J. Mar. Biotechnol., 6:110-115.
Tassanakajon, A., S. Pongsomboon, V. Rimphanitchayakit, P. Jarayabhand and V. Boonsaeng,
1998. Genetic structure in wild populations of black tiger shrimp (Penaeus monodon)
using randomly amplified polymorphic DNA analysis. J. Mar. Biotechnol., 6: 249-254.
Thodesen, J. and T. Gjedrem, 2006. Breeding programs on Atlantic salmon in Norway-lessons
learned. In: Development of aquatic animal genetic improvement and dissemination
programs: current status and action plans (Eds. R. W. Ponzoni, B. O. Acosta and A. G.
Ponniah). WorldFish Center Conferences Proceedings, 73: 22-26.
Vaseeharan, B., P. Rajakamaran, D. Jayaseelan and A. Y. Vincent, 2013. Molecular markers and
their application in genetic diversity of penaeid shrimp. Aquacult. Int., 21: 219-241.
Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski and S. V. Tingey, 1990. DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
Acids Res., 18: 6531-6535.
Yeh F. C., R. C. Yang and T. Boyle, 1999. Popgene 32-version 1.31: Population Genetics
Software. University of Alberta, Edmonton, AB Canada T6G 2H1.
Yoon, J. M. and G. W. Kim, 2001. Randomly amplified polymorphic DNA-polymerase chain
reaction analysis of two different populations of cultured Korean catfish Silurus asotus.
J. Biosci., 26: 641-647.
Zhuang, Z., T. Shi, J. Kong, P. Liu, Z. Liu, X. Meng and J. Deng, 2001. Genetic diversity in
Penaeus chinensis shrimp as revealed by RAPD technique. Prog. Nat. Sci., 11: 332-338.
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REPRODUCTIVE CHARACTERISTICS AND GERM CELL STATUS OF
INDIAN MAJOR CARP, LABEO ROHITA REARED IN ELEVATED WATER
TEMPERATURE REGIME
S. Patra a, G. Mishraa, B. Pandaa, D. K. Verma a, S. K. Dashb, S. Nandic and P. Routraya*
aCryobiology Laboratory, Aquaculture Production and Environment Division,
ICAR- Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751002, Odisha, India bBerhampur University, Berhampur, Odisha
cFish Genetics and Biotechnology Division, ICAR- Central Institute of Freshwater Aquaculture,
Kausalyaganga, Bhubaneswar-751002, Odisha, India
*Corresponding author : [email protected]
Rearing water temperature and fluctuations in it has a profound effect on survival and gonadal
development of fish. Reproduction in fish, compared with other physiological processes, only
occurs in a bounded temperature range; therefore, small changes in water temperature could
significantly affect this process. Here we analysed the effects of different rearing water
temperatures (28 °C, 30 °C, 32 °C, 34 °C and 36 °C) and a cytotoxic drug (busulfan) on germ cell
status and maturation in Indian major carp, Labeo rohita. The effectiveness of the treatment was
assessed by gonadosomatic index, histology and dye uptake of GC. Thermo-chemical treatments
were given either: as elevated water temperature alone (0.69±0.09) or in combination with
busulfan that showed a low GSI value (0.49±0.26) as compared to control (0.88±0.009). Gonadal
morphology visibly shrunk after the thermochemical treatments. Similarly, the gonadal histology
confirmed that the GC depletion took place when the rohu were reared at elevated temperature
along with the application of a cytotoxic drug busulfan (40 mg/kg). According to the deteriorating
reproductive responses of the fish by temperature fluctuations, it is plausible that changes may
affect aquaculture production and affecting future populations of fish, so new strategies for
amelioration should be anticipated.
INTRODUCTION
Reproduction in fish is influenced by different abiotic and biotic factors. Compared with
other physiological processes, reproduction occurs in a specific temperature range (Pörtner and
Farrell, 2008), thus small changes in water temperature could significantly affect this process
(Van der Kraak and Pankhurst, 1997; Zięba et al., 2010; Zucchetta et al., 2012). Temperature is
a critical physical factor in the lives of fish that is directly related to the control of all fish
reproductive processes from gamete development, maturation, spawning to larval and juvenile
development and survival (Sponaugle and Cowen, 1996; Pauly and Pullin, 1988; Ito et al., 2008;
Pankhurst and Munday, 2011). Temperature plays a crucial role in regulating reproductive cycle
in many fish, particularly in carps (Davies et al., 1986). However, these optimal temperature
regimes vary from species to species. Teleost fish like carps prefer a temperature range of 24 ºC
37
J. Aqua. 23 (2015)
to 30 ºC for their growth and reproduction (FAO, 1989). In many parts of the Indian sub-
continent, maximum surface water temperature in summer months (April to July) rises above 38
ºC. This has been a usual scenario for the last decade in the eastern state of Odisha, India. Rapid
and high fluctuating temperature influence fish reproduction as maturation process of gonad of
carps commences during February-March when the temperature gradually increases and
completes prior to onset of monsoon in May-June. Under these compelling temperature regimes,
what happens to the gonadal status of cultured carp is neither known clearly nor reported by other
researchers.
Moreover, there is scanty literature available about the gonadal growth, maturation and
reproduction under elevated temperature for Indian major carps (Dash et al., 2009). Elevated
water temperature has been found to cause gonadal degeneration in fish, including the partial or
complete loss of germinal elements that might impair fertility and reproductive performance
(Strüssmann et al., 1998; Ito et al., 2008). Fish being cold blooded animal is affected by the
temperature of the surrounding water which influences the body temperature, growth rate, food
consumption, reproduction and other body functions. Germ cells are the building blocks of future
gametes which proliferate under optimal conditions of environment. Strüssmann et al., (1998)
reported the occurrence of GC-deficient fish among groups exposed to high temperatures during
gonadal sex differentiation. Germ cell depletion is believed to be one of the major factors that
are responsible for gonadal sterility and infertility in fish. It has been reported that maturation of
carp broodstock is affected by elevated temperature and also this has been a continued observation
by the authors who state this phenomena (germ cell depletion/ non-attainment of maturity in
carps) occurs when water temperature rises beyond 34 °C.
This study was conducted to ascertain our hypothesis that the gonadal development and
maturity of carps is affected by thermo-chemical parameters. Here, an attempt has been made to
establish how the elevated water temperature and a cytotoxic drug affects the proliferation/depletion
of germ cells in Indian major carp rohu, Labeo rohita. A cytotoxic drug is used in this study to
compare the effect of elevated water temperature on germ cells, as many reports on different fish
species showed that busulfan suppress spermatogenesis and gonad sterilization, such as the Nile
tilapia Oreochromis niloticus (Lacerda, et al., 2010), the Patagonian pejerrey Odontesthes hatcheri
(Majhi, et al., 2009a), the zebrafish Danio rerio (Nóbrega, et al., 2010).
Indian major carps (IMCs) are a group of tropical fish that belong to the family
cyprinidae, which contributes most to the aquaculture production in India and widely found and
cultured in the Indian sub-continent that includes three major species viz. catla, Catla catla, rohu,
Labeo rohita and mrigal, Cirrhinus mrigala. Hence, L. rohita was taken for this study as a
representative of Indian major carps. This is widely cultured in the freshwater systems of the
Indian sub-continent due to its high economic value and consumer preference.
38
J. Aqua. 23 (2015)
MATERIALS AND METHODS
Tank setting and experimental fish rearing
Adult fish Labeo rohita (mean body weight of males 400.6 ± 1.44 g and 400.2 ± 0.86 g
of females) were collected from 0.2 ha brood rearing earthen ponds and kept for acclimatization
for two weeks in a cemented tank of 5100 L capacity (3.4 m L × 1.5 m B × 1.0 m H) at 28°C
water temperature prior to the thermo-chemical treatments. The stocking density was maintained
at the rate of 1.0 kg/m3 in each tank for the entire experimental period of 28 days.
At every one week of interval, samples were taken from each tank for gonadosomatic
index, histology, germ cell localization using marker dyes and confocal microscopy. To avoid
experimental error, the dimensions of all the tanks (nine numbers in each group) were kept same,
covered with polyethylene sheets and fitted with 45 W fluorescence lamps with electronic timers
for regulating the duration of illumination in different tanks. Fish were reared at 28 °C, 30 °C, 32
°C, 34 °C and 36 °C under a 14-hour light and 10-hour dark photoperiod. The temperature of the
water was modulated using two electric heaters (capacity 300 W) (RS Electrical, Zhongshan
RISHENG Electrical Product Co. Ltd., China) with thermostat control and filters were placed in
each tank along with aerators to maintain the water quality. The physico-chemical parameters of
rearing water were tested at weekly intervals following standard methods described in APHA,
1998 (Table 1) (Clesceri, 1998). Fish were fed twice a day till satiation using a commercial
pelleted diet (Abis Exports India Pvt. Ltd., Rajnandgaon, India).
Table 1 : Physico-chemical parameters (mean values and SE) in Labeo rohita rearing tanks
during the experimental period. Values are represented for 1st, 3rd and 7th week showing
the status of rearing water. (DO - Dissolved oxygen )
Parameters pH DO CO2 Alkalinity NH3
1st Week
Tank 1 7 ± 0.11 4.5 ± 0.04 NIL 178±0.09 0.006±0.004 Tank 2 7.2 ± 0.04 4.4 ± 0.04 NIL 159 ± 0.14 0.003±0.001 Tank 3 7 ± 0.12 4.8 ± 0.04 NIL 169 ± 0.07 0.008±0.002 Tank 4 7.2 ± 0.04 4.8 ± 0.2 NIL 158±0.9 0.008±0.001 Tank 5 7 ± 0.04 5.7 ± 0.04 NIL 162±0.16 0.004±0.002 Tank 6 7 ± 0.05 4.4 ± 0.004 NIL 165±0.04 0.005±0.003 Tank 7 7.2 ± 0.03 4.5 ± 0.007 NIL 170±0.09 0.004±0.002 Tank 8 7.2 ± 0.07 4.5 ± 0.007 NIL 172±0.07 0.007±0.004 Tank 9 7.1 ± 0.03 4.7 ± 0.009 NIL 160±0.09 0.008±0.005
39
J. Aqua. 23 (2015)
Parameters pH DO CO2 Alkalinity NH3
3rd Week
Tank 1 7.3±0.09 4.5±0.04 NIL 170±0.03 0.006±0.001 Tank 2 7±0.02 4.8±0.02 NIL 180 ±0.07 0.008±0.0002 Tank 3 7.5±0.04 4.8±0.07 NIL 176±0.09 0.007±0.001 Tank 4 7.4±0.09 5.8±0.02 NIL 156±0.09 0.004±0.002 Tank 5 7.2±0.09 5.6±0.07 NIL 149±0.02 0.0043±0.00 Tank 6 7±0.05 4.6±0.007 NIL 168±0.07 0.005±0.003 Tank 7 7.2±0.007 4.7±0.09 NIL 181±0.15 0.0043±0.001 Tank 8 7.3±0.007 5.1±0.09 NIL 179±0.07 0.004±0.001 Tank 9 7±0.03 5.5±0.01 NIL 175±0.04 0.005±0.001
7th Week
Tank 1 7 ± 0.02 4.5 ± 0.07 NIL 162 ± 0.1 0.007 ± 0.004 Tank 2 7.1 ± 0.04 4.6 ± 0.05 NIL 180 ± 0.04 0.005 ± 0.003 Tank 3 7 ± 0.07 5.2 ± 0.10 NIL 170 ± 0.07 0.006 ± 0.04 Tank 4 7.2 ± 0.05 5.0 ± 0.02 NIL 183 ± 0.07 0.005 ± 0.01 Tank 5 7.1 ± 0.04 5.6 ± 0.02 NIL 180 ± 0.05 0.008 ± 0.001 Tank 6 7 ± 03 4.8 ± 0.004 NIL 165 ± 0.04 0.006 ± 0.004 Tank 7 7.1 ± 0.02 4.7 ± 0.004 NIL 179 ± 0.09 0.005 ± 0.003 Tank 8 7 ± 0.03 5.1 ± 0.02 NIL 180 ± 0.08 0.005 ± 0.003 Tank 9 7.2 ± 0.02 5.27 ± 0.04 NIL 182 ± 0.07 0.043 ± 0.001
Thermo-chemical treatments
First group of male and female fish were reared in water temperature regimes of 28 °C,
30 °C, 32 °C, 34 °C, and 36 °C only, the second group received busulfan dosage of 40 mg/kg and
reared at 28 °C temperature and third group of fish received a combination of busulfan (40 mg/
kg) and elevated water temperature (34 °C). Ten numbers of male and female fish were used in
each of the experimental groups. Each treatment was performed in replicate tanks except the
controls. Busulfan dose was prepared by dissolving it in dimethyl sulfoxide (DMSO) and further
diluting it with freshwater fish Ringer solution to avoid precipitation and maintained at 30 °C
following the methods described by Wenzhi et al., 2011. Busulfan was intra-peritoneally
administrated in two doses (1st week 20 mg/kg and then 40 mg/kg) to fish that were anesthetized
using 200 ppm 2-phenoxyethanol (MP Biomedicals, Inc. Ohio 44139). Control group reared at
28 °C received the vehicle DMSO (Merck Limited, Mumbai) only.
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J. Aqua. 23 (2015)
GSI and histological analysis
For GSI and histological observation, each time two fish were humanely sacrificed at 0,
7, 14, 21 and 28 days. GSI was calculated using the formula (GSI =Gonad weight
Bodyweight×100). For
histology, middle portion of the right and left lobes of the gonads from the sampled fish were
taken after dissection. The gonad samples (1-1.5 mm thickness) were immersed in Bouin’s
fixative for 24 hour and 5µm thick sections were cut using a mechanical microtome (WESWOX
Optik Rotary Microtome, Ambala Cantt, India) and stained using haematoxylin and eosin (Merck,
India Ltd). Gonads were processed for examination with light microscopy using routine
histological procedures (Luna, 1968).
Isolation and gradient separation of testicular germ cells
Testis tissue were collected under sterile conditions, cut in small pieces (~ 2 mm3), rinsed
in phosphate buffered saline (PBS), kept in Leibovitz (L-15) medium (Sigma Aldrich, St.Louis,
MO, USA) and enzymatically digested with trypsin (Sigma Aldrich,St.Louis MO,USA).
Thereafter, germ cell isolation was done by percoll (MP Biomedicals, LLC, France) gradient
centrifugation. This involved centrifuging testicular cells for 10 min (800 g) at 25 °C, resulting
in three bands. The phase containing the largest cells (germ cells) was harvested, rinsed and
subjected to a cell viability test by trypan blue (0.4 %) dye exclusion assay. The protocol
described by Lacerda et al., 2006 was followed to obtain rohu germ cells.
Enumeration and labelling of germ cells
To detect the germ cell population before and after treatment, fluorescent cell linker mini
kit of PKH 26 and PKH 67 (Sigma- Aldrich Inc. CA, USA) were used. Approximately 10 million
cells were suspended in 0.4 mL of diluent C (an iso-osmotic aqueous solution provided with the
dye) in which PKH was diluted to a ratio of 4 µL of dye: 0.4 mL of diluent C. The diluted dye
was then incubated with the cells (final concentration, 10 µmol /L) for 5 min. The cells were
centrifuged at 100×g for 5 min, washed two times, suspended again in L-15 and stored in ice until
use. The stained and unstained germ cells were tagged with the fluorescent membrane dye PKH
26 and PKH 67 and observed under a fluorescent microscope at an excitation wavelength of 551
nm and 490 nm.
Statistical analysis
All qualitative data are presented descriptively, whereas quantitative data were tested
statistically using ANOVA (analysis of variance). Student’s t-test was used to determine the
significant differences between the treatments. Statistical analysis was performed using SPSS 18.0
for Windows 7. Differences between groups were considered as statistically significant at P < 0.05.
RESULTS
The tolerance limit of rohu to elevated water temperature was recorded at different
temperature regimes and shown in Fig. 1. None of the fish held in experimental tanks in control group
41
J. Aqua. 23 (2015)
(28 °C) died or showed symptoms of stress during the experiment but highest mortality (100 %) was
recorded at 36 °C. It was noticed that temperature tolerance capacity of fish decreased with increasing
temperature beyond 34 °C and a significantly low survival was noticed at 34 °C and beyond this water
temperature. After 14 days of rearing marked differences were clearly evident in the survival pattern.
After 28 days of thermo-chemical exposure it was seen morphologically that the gonad
size of rohu shrunk (Fig. 2B) significantly, compared to the control (Fig. 2A) which was further
ascertained by lower GSI (0.49 ± 0.26 and 1.78 ± 0.99). The GSI value decreased steadily with
busulfan administration (40 mg/kg) also. GSI of rohu male (0.88 ± 0.009) and female (3.30 ±
0.11) in the control group indicated healthy and well developed gonad. GSI of male and females
in the treated group-I (elevated temperature only) were (0.69 ± 0.09 and 2.50 ± 0.32) and
similarly, in the treated group-II (only busulfan administration) it was 0.52 ± 0.15 and 1.81 ± 0.84
for males and females respectively. Significantly lower GSI of males and females (0.49 ± 0.26
and 1.78 ± 0.99) were recorded from treated group-III that received a combination of elevated
temperature with busulfan administration. The gonadosomatic index (GSI) of all groups
decreased steadily especially the group-III that received a combination treatment of elevated
temperature and busulfan administration at 28 days in females (Fig. 3A) and males (Fig. 3B).
Fig. 1. Effect of temperature on survival of adult rohu at different rearing water temperatures for
a period of 28 days experimental period. Data shown as mean ± S.E.M (vertical bars) (n=10 of
each sex). Asterisks indicate significant values.
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J. Aqua. 23 (2015)
Fig. 2. Changes in the testis morphology of Labeo rohita before (A) and after (B) busulfan
treatment in combination with that were elevated ambient water temperature (> 34 °C).
Fig. 3. Effect of elevated rearing water temperature 34˚C, busulfan treatment 40 mg/kg and a
combination of both 34˚C water temperature and busulfan treatment 40 mg/kg on the GSI of
Labeo rohita, (A: female and B: male). Data shown as mean ± SEM (vertical bars), n= 10 of each
sex. Asterisks indicate significant values between treatments.
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J. Aqua. 23 (2015)
Gonadal histology
The histological analysis of treated and control fish gonads after 7 days of treatment
showed active oogenesis with different stages of oocytes, perinuclear oocytes, cortical alveolus
oocytes observed. After 14 days of treatment there were prominent cysts of oogonia with decrease
of primary oocytes. At the end of 28 days of treatment it was observed that the number of atretic
oocytes increased with concomitant decrease of cortical alveoli and vitellogenic oocytes in treated
females (Fig. 4). In treated males, initially there was active spermatogenesis within the lobules,
as the days of treatment progressed, gradual decreases of spermatogenic cysts were observed.
After the completion of 28 days of treatment, reduced number of spermatogonia cells was visible
that seemed to lack the capacity for initiation of spermatogenesis (Fig. 5).
Fig. 4. Histological changes in the ovary subjected to (I) Elevated water temperature (34 °C), (II)
Intraperitoneal busulfan administration (40 mg/kg) and (III) Combination of elevated temperature
with 40 mg/kg busulfan dose. A, E, I: Ovary occupies mostly with primary oocutes of various
classes at the start of the treatment experiment (0 days); B, F, J: showing absence of prominent
cysts of oogonia after 14 days of treatment represented by arrow heads; C, G, K: showing absence
of oogonia and other types of GCs after 21 days; D, H, L: degeneration of oogonial cells with
atretic oocytes after 28 days indicated by arrow head; perinuclear oocytes (pns) or immature
oocyte, cortical alveolus oocytes (cas) nucleolus (nu).
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J. Aqua. 23 (2015)
Fig. 5. Histological changes in the testes of males subjected to (I) elevated water temperature
(34 °C), (II) Intraperitoneal busulfan administration (40 mg/kg) and (III) combination of elevated
temperature with 40 mg/kg busulfan dose. A, E, I; active spermatogenesis within the lobules at
the start of experiment (0 days). B, F, J; absence of spermatogenic cysts after 14 days of
treatments indicated by arrowhead; C, G, K; absence of spermatogonia after 21 days indicated by
arrow head; D, H, L; absence of GCs after 28 days indicated by arrow head.
Germ cell labelling
The study implies that the GCs can be dyed with florescent dye without compromising
cell viability. After 2 h staining, it was observed that most (90%) of the germ cells have taken-up
both the dyes (PKH 26 and PKH 67). Similar uptake and retention of PKH 26 and PKH 67 dye
was observed after one week time (Fig. 6). However, some cells (nearly 5%) showed less
fluorescent intensity. This may be due to the fact that many of the cells are in dividing stage. The
fish reared in elevated temperature and in combination with busulfan showed less number of GCs
as evident from dye uptake studies.
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J. Aqua. 23 (2015)
Fig. 6. Proliferative and depletion status of isolated germ cells observed under confocal
microscope A: red color showing florescent PKH 26 labelled germ cells in the control; B: green
color showing PKH 67 labelled germ cells at different stages of development (represented by
arrow head) in control; C and D: poor expression of PKH 26 and PKH 67 in the treated fish
(reared in elevated water and administered with busulfan).
DISCUSSION
Temperature is one of the most pervasive environmental factors that influence
physiology and ecology of aquatic organisms including fish. Fish reproduction is likely to be
affected by increasing and decreasing water temperatures arising from climate change, which has
the capacity to affect endocrine function that may either advance or retard gametogenesis and
maturation. The present study revealed that elevated water temperature and a cytotoxic drug
46
J. Aqua. 23 (2015)
(busulfan) affected the reproductive characteristics and germ cell proliferation of rohu, Labeo
rohita. Elevated rearing water temperature (>34 °C) beyond their thermal threshold resulted in
impairment in germ cell proliferation as seen from GSI and histology. The thermal threshold of
adult rohu has been reported by Das et al., 2004; 2005; 2006. This study is relevant in the present
context of global warming that predicts the water temperature to rise (Wohlschlag et al., 1968;
Franklin et al., 1995; Schmidt- Nielsen, 1997) that may affect the aquatic fauna in terms of their
physiology and reproduction. Here, it was observed that elevated rearing water temperature (>34
°C) has a fatal effect on their survival. The thermal limit of rohu has been reported to be 35 °C
(Chatterjee et al., 2004). Our studies are also in agreement with their findings and further give
more insight into the reproductive status of carps reared at elevated water temperature that were
not reported before in carps. These findings are important because water temperatures above 34°C
impair fish physiology (Das et al., 2004). In temperate teleosts such as pejerrey, the increase of
water temperature during summer signals the end of reproductive episodes (Pankhurst and Porter,
2003). Here experimental data showed that rohu has a temperature tolerance limit of 36 °C as no
fish survived beyond this temperature. It was observed that rohu has certain thermal tolerance
range beyond which it has adverse effects on physiological, metabolic and reproductive activities.
Further, it was investigated whether this rearing water temperature rise is affecting the gonadal
status and reproductive ability of carps. This becomes more important when the problem of global
warming (temperature rise) is believed to affect the food production sector including aquaculture.
Germ cell depletion is believed to be one of the major factors that are responsible for gonadal
sterility and infertility in fish. Here, GC status of rohu was assessed when they were reared in elevated
water temperature along with a cytotoxic drug busulfan that is known to destroy endogenous germ
cells (Brinster and Zimmermann, 1994; Lacerda et al., 2006). These thermo-chemical treatments
showed that depletion of endogenous germ cells of rohu took place as evident from shrunken gonad
and lower GSI. Fish reared in elevated water temperature alone (GSI 0.69±0.09) and in combination
with busulfan showed low GSI value (0.49±0.26) as compared to control (GSI 0.88±0.009). Similar
results of germ cell depletion by warm water temperature and busulfan have been reported in other
teleost species such as Pattagonian pejerrey (Odontesthes hatcheri) (Majhi et al., 2009 a, b).
Treated fish exposed to thermo-chemical treatments showed increased number of atretic
oocytes with concomitant decrease of cortical alveoli and vitellogenic oocytes in females and
absence of spermatogenic cysts within the lobules with complete depletion of GCs in males.
These observations indicates that somatic cells (such as sertoli and Leydig cells in males and
follicular cell in females) that support the proliferation and development of germ cells, were not
critically affected by the thermo-chemical treatments at this doses and duration. Similar results
were reported in Odentesthes bonariensis reared in higher water temperature (Ito et al., 2008;
Soria et al., 2008). It is worth to mention that pejerrey requires an optimum range of water
temperature of 5 °C to 25 °C for its growth and propagation (Majhi et al., 2009b). In other species,
47
J. Aqua. 23 (2015)
study on exposure to higher temperature resulted in degeneracy of sertoli and germ cells in the
seminiferous tubules of rat (Strüssmann et al., 1998) and in Table 2 some studies on other animals
have been discussed for reference. Here in rohu, the effect of elevated temperature was shown to
be negatively affecting germ cell proliferation but sertoli cells remained unaffected. Similarly,
busulfan, a known cytotoxic drug was used to deplete the GC content and compare the same with
the temperature treatments. Moreover, it is not known at what dosage it can make the fish
completely GC deficient. The toxicity and sensitivity of busulfan is reported to be varying from
species to species and requires a specific dose for each animals viz. pigs and goats (7.5 mg/ kg),
mice (10-50 mg/ kg), pejerrey (30-40 mg/ kg) (Honaramooz et al., 2005; Majhi et al., 2009a;
Wang et al., 2010) which resulted in complete removal of germ cells without any lethal effect.
Here, fish administered with 40 mg/ kg busulfan showed no mortality but high mortality was
recorded when applied in combination with elevated rearing water temperature beyond 34 °C.
Table. 2 : A comparative account of rohu (Labeo rohita) germ cell depletion studies with
other vertebrates with special focus on endogenous GC depletion and spermatogenesis.
Species Treatment Effects Year Reference
Mice (Male) Busulfan Depletion of
spermatogonial germ cell
1994 Brinster et al.
Swiss nude Mice Busulfan Depletion of endogenous
germ cell in testes
1999 Ogawa et al.
Tilapia 6-n-Propyl-2-
Thiouracil
(PTU)
Loss of germ cells with
increase of dose
2002 Matta et al.
Mice Ionizing
radiation
Depletion of seminiferous
epithelium of host mice
2002 Creemers et
al.
Pig Busulfan Suppression of
endogenous
spermatogenesis
2005 Honaramooz
et al.
Goat Radiation Testicular irradiation
results in reduction of
endogenous germ cell
population
2005 Honaramooz
et al.
Dog Irradiation Depletion of endogenous
spermatogenesis
2008 Kim et al .
Nile-tilapia
(Oreochromis
niloticus)
Busulfan with
Elevated
temperature
Depletion of endogenous
germ cell
2006 Lacerda et al.
48
J. Aqua. 23 (2015)
Species Treatment Effects Year Reference
Patagonia
pejerrey
(Odontesthes
hatcheri)
Busulfan and
high temperature
Incipient gonadal
degeneration and germ
cell loss
2009 Majhi et al.
Chicken Busulfan Reduction of number of
endogenous primordial
germ cells (PGCs) in
embryonic gonads and
hatchability.
2010 Nakamura et
al.
Cat Busulfan with
local X-ray
radiation
Depleting endogenous
spermatogenesis
2012 Silva et al.
Chicken Busulfan Depletion of endogenous
germ cells in embryonic
gonads
2013 Lee et al.
Rohu (Labeo
rohita)
Busulfan and
elevated
temperature
Depletion of endogenous
germ cells
2015 Present study
In this study the increase in rearing water temperature consistent with climate change
predictions shown to affect the gonadosomatic index of rohu, due to depletion of germ cells. To our
knowledge, the present study provides the first hand evidence that germ cells proliferation in carps
is temperature sensitive. As can be inferred from this study for Labeo rohita, germ cell depletion
occurs when exposed to elevated water temperature. To ascertain the germ cell status in the treated
fish fluorescent dye (PKH 26 and 67) was used and the effectiveness of thermo-chemical treatment
in the gonad assessed. The proliferative cell were dyed and shown positive fluorescence under a
confocal microscope. It was verified from this study that elevated rearing water temperature beyond
their thermal limit (>36 °C) grossly affected the gonadal maturation process as evident from the
depleted GC content. In conclusion, the results of this study indicate that elevated water temperature
affects germ cell proliferation and gonadal status in carps as temperature fluctuation (>34 °C)
adversely affected reproductive characteristics of carps that suggests that climate change is an
additional stressor to fish populations (brood stock). The potential importance of water temperature-
induced reproductive dysfunctions must not be underestimated as freshwater fish constitute the
largest harvestable natural food resource and production of freshwater fishes has been dominated
by carps including Indian major carps (71.9%, 24.2 million tonnes, in 2010) (FAO, 2012).
According to the deteriorating reproductive responses of the fish to temperature fluctuations, it is
plausible that changes may affect aquaculture production and affecting future populations of fish,
so new strategies for amelioration should be anticipated.
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J. Aqua. 23 (2015)
ACKNOWLEDGEMENTS
We thank ICAR-CIFA, Bhubaneswar for logistics to carry out the study. The present
work was conducted under ICAR funded project (NICRA) and DST INSPIRE fellowship to S.P.
(IF 110666) and G.M. (IF120375). The funding support received from DST, New Delhi and
ICAR-CIFA is thankfully acknowledged.
REFERENCES
Brinster, R. L and J. W. Zimmermann, 1994. Spermatogenesis following male germ cell
transplantation. Proc. National Acad. Sci., USA, 91: 11298–11302.
Chatterjee, N., A. K. Pal, S. M. Manush, T. Das and S. C. Mukherjee, 2004. Thermal tolerance
and oxygen consumption of Labeo rohita and Cyprinus carpio early fingerlings
acclimated to three different temperatures. J. Therm. Biol., 29: 265-270.
Clesceri, L. S. 1998. In: Standard Methods for the analysis of water and wastewater (20th Edn.)
American Public Health Association (APHA), Alexandria: VA.
Creemers, L. B., X. Meng, K. D. Ouden, A. M. V. Pelt, F. Izadyar, M. Santoro, H. Sariola and D.
G.D. Rooij, 2002. Transplantation of germ cells from glial cell line-derived neurotrophic
factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by
fractionated irradiation. Biol. Reprod., 66: 1579-1584.
Das, T., A. K. Pal, S. K. Chakraborty, S. M. Manush, N. Chatterjee and S. C. Mukherjee, 2004.
Thermal tolerance and oxygen consumption of Indian major carps acclimated to four
temperatures. J. Therm. Biol., 29: 157-163.
Das, T., A. K. Pal, S. K. Chakraborty, S. M. Manush, N. P. Sahu and S. C. Mukherjee, 2005.
Thermal tolerance, growth and oxygen consumption of Labeo rohita fry (Hamilton,
1822) acclimated to four temperatures. J. Therm. Biol., 30: 378-383.
Das, T., A. K. Pal, S. K. Chakraborty, S. M. Manush, R. S. Dalvi, K. Sarma and S. C. Mukherjee,
2006. Thermal dependence of embryonic development and hatching rate in Labeo rohita
(Hamilton, 1822). Aquaculture, 255: 536-541.
Dash, C., P. Routray, S. N. Dash, P. Swain, D. K. Verma and P. K. Nanda, 2009. Localization of
primordial germ cells (PGCs) in different developmental stages of Labeo rohita (Ham.).
Indian J. Anim. Sci., 79: 111-114.
Davies, P. R., I. Hanyu, K. Furukawa and M. Nomura, 1986. Effect of temperature and
photoperiod on sexual maturation and spawning of the common carp: III. Induction of
spawning by manipulating photoperiod and temperature. Aquaculture, 52: 137-144.
FAO, 1989. Selected documents of warm water fish culture. Culture of the Indian major carps.
FAO Corporate Document Repository, 181.
50
J. Aqua. 23 (2015)
FAO, 2012. The state of world fisheries and aquaculture, Rome: 209.
Franklin, C.E., I. A. Johnson, T. Crockford and C. Kamunde, 1995. Scaling of oxygen
consumption of lake magadi tilapia, a fish living at 37 °C. J. Fish Biol., 46: 829-834.
Honaramooz, A., E. Behboodi, C. L. Hausler, S. Blash. S. Ayres, C. Azuma, Y. Echelard and I.
Dobrinski, 2005. Depletion of endogenous germ cells in male pigs and goats in
preparation for germ cell transplantation. J. Androl., 26: 698-705.
Ito, L. S., C. Takahashi, M. Yamashita and C. A. Strüssmann, 2008. Warm water induces
apoptosis, gonadal degeneration, and germ cell loss in sub-adult Pejerrey Odontesthes
bonariensis (Pisces, Atheriniformes). Physiol. Biochem. Zool., 81: 762-774.
Kim,Y., D. Turner, J. Nelson, I. Dobrinski, M. McEntee and A. J. Travis, 2008. Production of
donor-derived sperm after spermatogonial stem cell transplantation in the dog.
Reproduction, 136: 823-831.
Lacerda, S. M. S. N., S. R. Batlouni, G. M. Costa, T. M. Segatelli, B. R. Quirino, B. M. Queiroz,
E. Kalapothakis and L. R. França, 2010. A new and fast technique to generate offspring
after germ cells transplantation in adult fish: the Nile tilapia (Oreochromis niloticus)
model. PLoS One, 5: e10740.
Lacerda, S. M. S. N., S. R. Batlouni, S. B. G. Silva, C. S. P. Homem and L. R. França 2006. Germ
cells transplantation in fish: the Nile-tilapia model. Anim. Reprod., 3: 146-159.
Lee, H., S. Kim, T. Park, D. Rengaraj, K. Park, H. Lee, S. B. Park, S. Kim, S. B. Choi and J. Han,
2013. Compensatory proliferation of endogenous chicken primordial germ cells after
elimination by busulfan treatment. Stem Cell Res. Ther., 4: 136.
Luna, L.G., 1968. In: Manual of Histologic Staining Methods of the Armed Forces Institute of
Pathology (Ed. L. G. Luna) (3rd Edn.). Blakiston Division, McGraw-Hill, New York,
pp.1-32.
Majhi, S. K., R. S. Hattori, S. M. Rahman, T. Suzuki and C. A. Strüssmann, 2009a.
Experimentally induced depletion of germ cells in sub-adult Patagonian pejerrey
(Odontesthes hatcheri). Theriogenology, 71: 1162-1172.
Majhi, S. K., R. S. Hattori, M. Yokota, S. Watanabe and C. A. Strüssmann, 2009b. Germ cell
transplantation using sexually competent fish: An approach for rapid propagation of
endangered and valuable germlines. PLoS One, 4: e6132.
Matta, S. L. P., D. A. R. Vilela, H. P. Godinho and L. R. França, 2002. The goitrogen 6-n-propyl-
2-thiouracil (PTU) given during testis development increases sertoli and germ cell
numbers per cyst in fish: The tilapia (Oreochromis niloticus) model. Endocrinology,
143: 970-978.
51
J. Aqua. 23 (2015)
Nakamura, Y., F. Usui, T. Ono, K. Takeda, K. Nirasawa, H. Kagami and T. Tagami, 2010.
Germline replacement by transfer of primordial germ cells into partially sterilized
embryos in the chicken. Biol. Reprod., 83: 130-137.
Nóbrega, R. H., C. D. Greebe, H. Van de Kant, J. Bogerd, L. R. França and R. W. Schulz, 2010.
Spermatogonial stem cell niche and spermatogonial stem cell transplantation in
zebrafish. PLoS One, 5: e12808.
Ogawa, T., I. Dobrinski, M. R. Avarbock and R. L. Brinster, 1999. Xenogeneic spermatogenesis
following transplantation of hamster germ cells to mouse testes. Biol. Reprod., 60: 515-521.
Pankhurst, N. W and M. J. R. Porter, 2003. Cold and dark warm and light: variations on the theme
of environmental control of reproduction. Fish Physiol. Biochem., 28: 385-389.
Pankhurst, N. W and P. L. Munday, 2011. Effects of climate change on fish reproduction and
early life history stages. Mar. Freshwat. Res., 62: 1015-1026.
Pauly, D and R. S. V. Pullin, 1998. Hatching time in spherical, pelagic, marine fish eggs in
response to temperature and egg size. Environmen. Biol. Fish., 22: 261-271.
Pӧrtner, H. P and A. P. Farrell, 2008. Physiology and climate change. Science, 322: 690-692.
Schmidt-Nielsen, K, 1997. In: Animal physiology: adaptation and environment. (5th Edn.)
University of Cambridge Press; Cambridge: UK.
Silva, R. C., G. M. Costa, S. M. Lacerda, S. R. Batlouni, J. M. Soares, G. F. Avelar, K. B. Böttger,
Jr. S. F. Silva, M. S. Nogueira, L. M. Andrade and L. R. França, 2012. Germ cell
transplantation in felids: A potential approach to preserving endangered species. J.
Androl., 33: 264-276.
Soria, F. N., C. A. Strüsmann and L. A. Miranda, 2008. High water temperatures impair the
reproductive ability of the pejerrey fish Odontesthes bonariensis: Effects on the
hypophyseal-gonadal axis. Physiol. Biochem. Zool., 81: 898-905.
Sponaugle, S and R. K. Cowen, 1996. Larval supply and patterns of recruitment for two caribbean
reef fishes Stegastes partitus and Acanthurus bahianus. Mar. Freshwat. Res., 47: 433-447.
Strüssmann, C. A., T. Saito and F. Takashima, 1998. Heat-induced germ cell deficiency in the
teleosts Odontesthes bonariensis and Patagonina hatcheri. Comp. Biochem. Physiol.
Part A: Mol. Integ. Physiol., 119: 637-644.
Van Der Kraak, G and N. W. Pankhurst, 1997. Temperature effects on the reproductive
performance of fish. In: Global warming: implications for freshwater and marine
fish. Cambridge: Cambridge University Press; pp 159-176.
52
J. Aqua. 23 (2015)
Wang, D. Z., X. H. Zhou, Y. L. Yuan and X. M. Zheng, 2010. Optimal dose of busulfan for
depleting testicular germ cells of recipient mice before spermatogonial transplantation.
Asian J. Androl., 12: 263-270.
Wenzhi, M., A. Lei, W. Zhonghong, W. Xiaoying, G. Min, M. Kai, M. Wei and T. Jianhui, 2011.
Efficient and safe recipient preparation for transplantation of mouse spermatogonial
stem cells: pretreating testes with heat shock. Biol. Reprod., 85: 670-677.
Wohlschlag, D. E., J. N. Cameron and J. J. Jr. Cech, 1968. Seasonal changes in the respiratory
metabolism of the pinfish (Lagodon rhomboides). Contrib Mar. Sci., 13: 89-104.
Zięba, G., M. G. Fox and G. H. Coop, 2010. The effect of elevated temperature on spawning of
introduced pumpkinseed Lepomis gibbosus in Europe. J. Fish Biol., 77: 1850-1855.
Zucchetta, M., G. Cipolato, F. Pranovi, P. Antonetti, P. Torricelli, P. Franzoi and S. Malavasi,
2012. The relationships between temperature changes and reproductive investment in a
Mediterranean goby: insights for the assessment of climate change effects. Est. Coast.
Shellfish Sci., 101: 15-23.
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EXPRESSION PATTERN OF IMMUNE-RELATED GENES IN THE LIVER OF
ROHU, LABEO ROHITA (HAMILTON) EXPOSED TO CARBON
TETRACHLORIDE TOXICITY
Amruta Mohapatra, Barsa Nayak and Pramoda Kumar Sahoo*
ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, Odisha, India
*Corresponding author : [email protected]
Immune responses in the liver play a crucial role in the detoxification process and rendering
protection against different diseases. The present study was targeted to analyze the expression
kinetics of few immune genes (pro-inflammatory, immune receptors, anti-microbial and innate
immunity-related) using qPCR in liver tissue of carbon tetrachloride (CCl4) treated rohu (Labeo
rohita). Rohu juveniles were injected with single intraperitoneal toxic dose of 30% CCl4 to induce
liver damage. Histopathological findings showed the presence of focal necrosis and vaccuolation
of hepatocytes. Increase in the level of pro-inflammatory cytokine (interleukin-6, IL-8 and IL-15)
transcripts was observed immediately after CCl4 post-exposure (from 6 h to 120 h), thus indicating
the role of enhanced level of cytokines inducing liver damage. The degree of liver damage was
also reflected by reduction in expression of immune related molecules (viz., TLR 22, transferrin
and hepcidin), which are mostly synthesized in liver tissue. No effect was noticed in synthesis of
mitochondrial antiviral-signaling protein (MAVS) transcripts with the current level of CCl4
exposure. These results suggest that there is a critical balance between immune molecules that
may play essential roles in the orchestration of immune defense in fish during liver dysfunction.
INTRODUCTION
Liver plays a crucial role in metabolism of the toxic chemicals in both fish and mammals
because of its portal location within the circulation, its anatomic and physiologic structure (Allis
et al., 1996). Liver which is a site of xenobiotics metabolism and transformation, possesses high
risk of toxic damage (Lattuca et al. 2009). Xenobiotics induce production of reactive oxygen
species (ROS) which is detoxified by the antioxidative system of liver. When the antioxidant
system is unable to cope with the excessive production of ROS, it results in oxidative stress
leading to tissue damage. Carbon tetrachloride (CCl4) is a model solvent for classical
hepatotoxicity, commonly found in freshwater that contaminates both the aquatic environment
and accumulate toxic substance in aquatic organisms (Statham et al., 1978). CCl4 causes
multifactorial damage ranging from lipid peroxidation and inflammation to apoptotic or necrosis
reaction via oxidative stress pathway. Endoplasmic cytochrome P450 induces CCl4 activation
producing trichloromethyl radical. These free radicals cause lipid peroxidation and over
production of inflammatory cytokines leading to damage of hepatic tissues (Boelsterli, 2003).
CCl4 has been shown to increase the levels of glutathione pyruvate transaminase (GPT),
glutamate oxalate transaminase (GOT), malondialdehyde (MDA) enzymes and reduce the levels
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of antioxidant enzymes viz., superoxide dismutase (SOD), and glutathione peroxidase (GPx),
catalase, glutathione (GSH) and total antioxidant capacity (T-AOC) in common carp (Cyprinus
carpio L.) intraperitoneally injected with 30% CCl4 in arachis oil (0.5 ml/kg body weight) at 72
h post-injection (Jia et al., 2014). Upregulations in the gene expressions of toll-like receptor 4
(TLR - 4), cytochrome P450 2E1 (CYP2E1), nuclear factor-ƙB (NF-ƙB), inducible nitric oxide
synthase (iNOS), and inflammatory cytokines like interleukin-1β (IL-1β), IL-6 and IL-12 after
CCl4 exposure are also seen (Jia et al., 2014). Different fish species respond differently to CCl4
exposures. Only 8 mM concentration of CCl4 dose causes severe damage to carp hepatocytes and
liver tissue got impaired at a dose of 0.15 ml/kg body weight of carp (Jia et al., 2012). The dose
related damage by CCl4 to the liver in various fish species, English sole (Casillas et al., 1983),
Nile tilapia (Chen et al., 2004), rainbow trout (Statham et al., 1978), brown trout (Krasnov et al.,
2007), rosy barbs and amphioxus (Bhattacharya et al., 2008) has already been reported. The
toxicity of CCl4 has mostly been investigated in various fish species by means of measuring blood
biochemical changes and histopathological examination. However, the detail cellular and
molecular immune-related events by CCl4 toxicity particularly in Indian major carps have not
been investigated earlier, and remain unclear. Hence, the present study was undertaken to study
the pattern of expression of several immune related genes in liver tissue of rohu for understanding
immunological changes at molecular level.
MATERIALS AND METHODS
Fish
Juveniles of rohu (Labeo rohita) weighing 30.00 ± 10.20 g were collected from the farm
of the ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India.
Fish were reared in 700-L ferro-cement tanks and acclimated to the experimental conditions
before two weeks of initiation of the experiment. A commercial pellet diet at 3% of their body
weight was given to fish twice a day prior to the experiment. One–tenth of the tank water was
exchanged with freshwater daily in order to remove waste feed and faecal materials from bottom
of the tanks. Appropriate aeration was provided in the tanks to maintain optimal oxygen level in
water. The basic physico-chemical parameters were maintained at their optimal level and the
water temperature during the experiment varied between 25-28 °C.
Exposure of fish to carbon tetrachloride
After acclimation, 28 numbers of fish were divided randomly into two groups, control
group containing 4 numbers of fish and experimental group containing 24 fish. Four fish were
kept in 200-L FRP tank for each treatment with similar management conditions. Control group
fish were intraperitoneally injected with olive oil, whereas, the treatment group was
intraperitoneally injected with 30% (v/v) CCl4 in olive oil at a volume of 1 µl/g body weight. The
fish were sacrificed with overdose of anaesthesia (MS222, Sigma) at different time periods (6,
12, 24, 36, 48 and 120 h) post-exposure to CCl4 and liver samples were collected, and one portion
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(~ 100 mg) was kept in RNAlater (Sigma, USA). Rest part of the liver tissue was preserved in
10% neutral buffered formalin for histology following routine protocol of haematoxylin and eosin
staining. Except for histological assay, all tissue samples were stored at 4 °C for 24 h and then
transferred to -20 °C until RNA extraction to study the gene expression pattern.
RNA extraction and cDNA synthesis
RNA extraction was done using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA)
according to the manufacturer’s instruction, followed by DNase I (Fermentas, Canada) treatment
to remove genomic DNA contamination in addition to 1 µl (1 U/µl) of RNase inhibitor
(Fermentas, Canada) to inhibit degradation of RNA. Subsequently, cDNA synthesis was carried
out using M-MLV reverse transcriptase (Sigma-Aldrich, St. Louis, MO, USA) following the
manufacturer’s instructions. The quality and quantity of RNA samples were checked using
NanoDrop ND 1000 (NanoDrop Technologies Inc., USA).
PCR amplification
The semi-quantitative PCR was performed for different immune relevant genes and
β-actin as house-keeping gene (Table 1) to know the specificity of primers and products. All
amplification reactions consisted of an initial denaturation at 95 °C for 2 min prior to 35 cycles
of 95 °C denaturation for 30 s with different annealing temperatures for 45 sec and 72 °C
extension for 1 min 30 sec, followed by a final 72 °C extension for 10 min using 1.5 units of Taq
DNA polymerase and finally by cooling at 4 °C (Genie, India). The generated PCR products
(8 µl) were then analyzed by electrophoresis on 1.0% agarose gel.
Real-time PCR
Real-time PCR was carried out with different primers using Light Cycler 96 SW 1.1
(Roche, Germany). Briefly, 1 µl of cDNA was used as template in a total reaction mixture of 10
µl containing 5 µl of 2X Fast Start Essential DNA Green Master (Roche, Germany), 0.5 µl (5
pmole) of each forward/revese primers (Table 1) and 3 µl of PCR grade H2O provided in the kit.
The qPCR program included a pre-denaturation at 95 ºC for 10 min and 40 cycles of amplification
at 95 ºC for 10 s, at respective annealing temperatures for 10 s, and 72 ºC for 20 s followed by
melt curve analysis at 95 ºC for 10 s, 65 ºC for 60 s and 97 ºC for 1 s and cooling at 37 ºC for 30
s. β-actin was used as a reference gene for the study (Robinson et al., 2012). No template controls
were run each time. Tm analysis was done to check primer specificity. The quantification cycle
(Cq) values were imported in an excel file.
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Table 1 : Primer details used for gene expression analysis
Primer Id Primer Sequence (5’-3’) Amplicon
size (bp)
Annealing
temp. Reference
β-actin F-TGGCAATGAGAGGTTCAGGT
R-TGGCATACAGGTCCTTACGG
139 56 °C Robinson et
al. (2012)
ApoAI –RT F-TGGAGGCTGTGCGTGTA
R- GCTCGCCCAGTTCATTC
164
59 °C Mohapatra et
al. (2014)
IL-1β F-GTGACACTGACTGGAGGAA
R-AGTTTGGGCAAGGAAGA
164 51 °C Self-designed
IL-6 F-GGACCGCTTTGAAACTCT
R-5’-GCTCCCTGTAACGCTTGT-3’
212 54 °C Dash et al.
(2014)
IL-8 F-AAAGGGTTCTTACTGG
R-TTTAGACATCTCGGACT
170 57 °C Self-designed
IL-15 F-ACCAACAATCTCGCTTTCG
R-GTTCAACGGGCATTCCAT
160 56 °C Das et al.
(2015)
Hep F-TACAGTACATCAGCTCCTC
R-GATCAGAATTTGCAGCAGTA
330 50 °C Mohapatra et
al. (2011)
MAVS F-CACCTCCTGTCATAAATAGC
R-AAGCCAAGAAAGACACCT
160 52 °C Self-designed
Transferrin F-GGACTACCAGCTGTTGTGCAT
R-GCCACCATCGACTGCAAT
487 48 °C Sahoo et al.
(2009)
TLR 22 F-TCCTACAATGCCAAAGATGAG
R-CAGGAACACCAGAATCAGTACATCC
273 54 °C Panda et al.
(2014)
Relative expression analysis
The quantification cycle values (Cq) were calculated using Light Cycler 96 SW 1.1 and
the data were exported. n-fold differential expression was calculated using the comparative Cq
method (Livak and Schmittgen, 2001) by calculating the average of each Cq for the triplicate
samples. Cq value for the sample of each cDNA was deducted from its respective Cq value of β-
actin to get ΔCq value. The mean of each sample was done, as the samples were taken in
triplicates. Further, ΔΔCq as obtained by subtracting ΔCq value of sample from ΔCq of the
control. Fold difference was calculated as 2-ΔΔCq.
Statistical analysis
The average fold expression for replicate samples for each time period was calculated
and presented as mean ± SE. The average fold expression of four control group fish was
considered as 0 h value. Further, differences between the mean values were analyzed using one-
way ANOVA followed by Duncan's multiple range tests, with values P <0.05 as significantly
different. All values of n-fold differential expression were plotted in a graph.
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RESULTS
Quality check of isolated RNA from samples
The quality of the RNA from the samples isolated as examined by taking OD260
nm/OD280 nm was found to be in the range of 1.8 to 2.0. Further integrity of the RNA samples
were checked by RT-PCR using β-actin primer pairs and strong amplification products for β-actin
gene (139 bp) were found for all the samples.
Amplification of various gene products in liver cDNA samples using RT-PCR and agarose
gel electrophoresis
All the primer sets for various gene products were checked by running RT-PCR using
control liver RNA samples. The expected product sizes of 139 bp, 164 bp, 164 bp, 212 bp, 170
bp, 160 bp, 330 bp, 160 bp, 487 bp and 273 bp were obtained for β-actin, apolipoprotein A-I
(ApoA I), interleukin (IL)-1β, IL-6, IL-8, IL-15, hepcidin, mitochondrial antiviral-signaling
protein (MAVS), transferrin, and toll-like receptor 22 (TLR 22) genes, respectively.
Expression of inflammatory molecules in rohu liver following CCl4 exposure
The expression of IL-6 transcripts increased immediately from 6 h of exposure of CCl4 in
liver tissue of rohu and its highest level was observed at 120 h among all time points taken here
(Fig. 1a). Similarly, the expression of IL-8 gene showed an increase in liver tissue samples of
experimental fish immediately after 6 h with a peak level of expression being detected at 48 h in
comparison to control fish (Fig. 1b). However, the expression of IL-15 was slightly high at 120 h
post-exposure of CCl4 in rohu liver tissue in comparison to all other periods of exposure or control
fish liver sample (Fig. 1c). On the other hand, the expression of IL-1β didn’t vary up to 48 h of post-
exposure of CCl4 in comparison to control, except a mild increase at 120 h post-exposure (Fig. 1d).
Expression of innate immune molecules in rohu liver following CCl4 exposure
The expression of transferrin gene showed a significant decline immediately after CCl4
exposure till the completion of the experiment, except a transient rise at 12 h post-exposure in
comparison to control (Fig. 1e). Similarly, the expression of TLR 22 declined significantly post
CCl4 exposure from 6 h onwards in comparison to control except a mild transient rise at 24 h
post-exposure (Fig. 1f).
Expression of anti-bacterial molecules in rohu liver following CCl4 exposure
The expression of anti-bacterial molecules, apolipoprotein A1 showed a transient rise at
24 h post-exposure (Fig. 1g). In sharp contrast the expression of hepcidin was below detectable
limit at 6 h post-exposure of CCl4. However, its expression gradually increased 48 h of post-
exposure of CCl4 till 120 h (Fig. 1h).
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Expression of anti-viral molecules in rohu liver following CCl4 exposure
The mRNA levels of MAVS showed a variable levels of expression in the CCl4 exposed
fish in comparison to control (Fig. 1i), although the levels remained higher than the control fish
throughout the period of study.
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Fig. 1. Expression analysis of various innate immune genes; IL-6 (a), IL-8 (b), IL-15 (c), IL-1β
(d), Transferrin (e), TLR 22 (f), ApoA I (g), Hepcidin (h) and MAVS (i) in the liver of rohu at
different time periods of post-exposure to CCl4. Data are presented as mean ± S.E. of three
samples at each time period. Bars bearing the different letter(s) are significantly different
(P<0.05). The fold difference was calculated as 2-ΔΔCq, where ΔΔCq = (ΔCq sample - ΔCq
calibrator) and ΔCq = (Cq value of gene of interest - Cq value of β-actin). The control group (0 h
of post-exposure) was taken as calibrator in the analysis.
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Histopathology
The injected fish liver tissue revealed massive focal areas of necrosis without
inflammatory reaction after 24 h – 36 h of CCl4 exposure (Fig. 2). The hepatocytes in general
revealed massive cytoplasmic vacuolation followed by nucleolysis in the affected areas.
Fig. 2. Liver showing massive focal necrosis and degeneration of hepatocytes. CCl4 - 36 h post-
exposure.
DISCUSSION
The aim of the study was to investigate the possible effect of CCl4 toxicity on immune
genes in liver of rohu. CCl4 hepatotoxicity was confirmed by the histopathological changes in the
liver tissue of rohu. CCl4 induced hepatotoxicity has also been studied in other fish species earlier
(Jia et al., 2014; Cao et al., 2015; Liu et al., 2015). In fish, lesions commonly observed in acute
toxicity by CCl4 include areas of diffuse focal necrosis, laminar or subcapsular necrosis (Chen et
al., 2004), cytoplasmic vacuolations and nucleolysis (Jia et al., 2014). The histopathological
findings noticed in this study generally support earlier findings in other fish species.
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It is generally believed that CCl4 is metabolised by the liver tissue using the cytochrome
P450 system (Yin et al., 2011). CCl4 causes oxidative stress and lipid peroxidation to induce liver
damage. CCl4 induced reactive oxygen species (ROS) production causes tissue damage and also
initiates inflammation. The inflammatory responses were mostly mediated by non-parenchymal
cells of the liver tissue; following activation by CCl4 they release large amounts of inflammatory
associated cytokines (Domitrovic et al., 2011).
TLR 22 specific to teleosts is recently shown to recognize multiple pathogens associated
molecular patterns (PAMP) and other products of damage tissue. It plays an important role in the
regulation of innate immune system in the infectious and inflammatory diseases. Liver is one of
the important sites of TLR 22 synthesis (Panda et al., 2014). The down-regulation of TLR 22
noticed in our study could be due to damage to the hepatic tissue.
Geier et al. (2002) reported that CCl4 rapidly induces the production of pro-
inflammatory cytokines in Kuffer cells leading to liver damage. Further studies have also
demonstrated that cytokine inhibition effectively reduces liver damage in mice. The elevated
levels of IL-6, IL-15, and IL-8 transcripts in the liver of CCl4 treated rohu was noticed in our
study. These data indicated and further confirmed the role of cytokine-related up-regulation being
an important mechanism for hepatocellular damage.
Liver is also an important site of synthesis of antimicrobial proteins viz., apolipoprotein
A I and hepcidin; and iron binding proteins like transferrin. In our study, the level of transferrin
transcripts was significantly low at all the time period except at 12 h post-exposure, thus
indicating the degree of damage to the liver tissue. Similarly, the level of antimicrobial peptide
hepcidin transcript was too low till 36 h post exposure followed by a marked rise thereafter. The
increasing level of transcripts of hepcidin after 2 days of CCl4 exposure in rohu probably indicates
the stabilization of hepatocytes to CCl4 induced damage or regeneration of new hepatocytes
leading to increase in synthesis that needs further in depth study. On the contrary, the level of
apolipoprotein A I transcripts did not show a major change after CCl4 induced liver damage. It
might be due to transport or migration of this transcripts from other sites of synthesis, for example
intestinal tissue that might not have been damaged by CCl4 and may be serving as a site of
synthesis of ApoA I (Glickman and Green, 1977) or their slow breakdown from the system.
Mitochondrial antiviral-signaling protein (MAVS) has been identified from many fish
species and it plays a major role in innate immune response against viruses (Kasthuri et al., 2014).
In this study an increase in level of transcripts in MAVS in CCl4 induced liver damaged tissue at
most of the time periods indicates poor effect of CCl4 on MAVS synthesis, but this conjecture is
speculative and needs to be elucidated by various experiments in teleosts.
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The present study demonstrated that CCl4 could induce hepatotoxicity in rohu within 6
h of post-exposure. An array of indication of synthesis of pro-inflammatory cytokines (increased
level of IL-8, IL-6, IL-15) might be playing major role causing damage to the liver, thus leading
to reduction in synthesis of liver specific innate immune molecules viz, transferrin, TLR 22 and
hepcidin. The findings of the study suggest that the presence of CCl4 in the aquatic environment
might be playing important role in suppression of the immune system of fish through focal/limited
damage without causing significant mortality thus increasing their susceptibility to various
infections.
ACKNOWLEDGEMENT
The authors express sincere gratitude to the Director, ICAR-CIFA, Bhubaneswar for
encouragement and providing necessary facilities during this study.
REFERENCES
Allis, J. W., B. L. Brown, J. E. Simmons, G. E Hatch, A. McDonald and D. E. House, 1996.
Methanol potentiation of carbon tetrachloride hepatotoxicity: the central role of
cytochrome P450. Toxicology, 112: 131–140.
Bhattacharya, H., S. Zhang and Q. Xiao, 2008. Comparison of histopathological alterations due
to sublethal CCl4 on rosy barb (Puntius conchonius) and amphioxus (Branchiostoma
belcheri) with implications of liver ontogeny. Toxicol. Mech. Methods, 18: 627–633.
Boelsterli, U. A., 2003. Mechanistic Toxicology. Taylor & Francis, New York, NY, USA.
Cao, L., W. Ding, J. Du, R. Jia, Y. Liu, C. Zhao, Y. Shen and G. Yin, 2015. Effects of curcumin
on oxidative activities and cytokine production in Jian carp (Cyprinus carpio var. Jian)
with CCl4-induced liver damage. Fish Shellfish Immunol., 43: 150-157.
Casillas, E., M. Myers and W. E. Ames, 1983. Relationship of serum chemistry values to liver
and kidney histopathology in English sole (Parophrys vetulus) after acute exposure to
carbon tetrachloride. Aquat. Toxicol., 3: 61–78.
Chen, C. Y., G. A. Wooster and P. R Bowser, 2004. Comparative blood chemistry and
histopathology of tilapia infected with Vibrio vulnificus or Streptococcus iniae or exposed
to carbon tetrachloride, gentamicin, or copper sulfate. Aquaculture, 239: 421–443.
Das, S., A. Mohapatra, B. Kar and P. K. Sahoo, 2015. Molecular characterization of interleukin
15 mRNA from rohu, Labeo rohita (Hamilton): its prominent role during parasitic
infection as indicated from infection studies. Fish Shellfish Immunol., 43: 25-35.
67
J. Aqua. 23 (2015)
Dash, P., P. K. Sahoo, P. K. Gupta, L. C. Garg and A. Dixit, 2014. Immune responses and
protective efficacy of recombinant outer membrane protein R (rOmpR)-based vaccine
of Aeromonas hydrophila with a modified adjuvant formulation in rohu (Labeo rohita).
Fish Shellfish Immunol., 39: 512-523.
Domitrovic, R., H. Jakovac and G. Blagojevic, 2011. Hepatoprotective activity of berberine is
mediated by inhibition of TNF-α, COX-2 and iNOS expression in CCl4 intoxicated mice.
Toxicology, 280: 33-43.
Geier, A., S. K. Kim, T. Geloff, C. G. Dietrich, F. Lammer and S. J. Karpen, 2002. Hepatobiliary
organic anion transporters are differentially regulated in acute toxic liver injury induced
by carbon tetrachloride. J. Hepatol., 37:198-205.
Glickman, R. M. and P. H. Green, 1977. The intestine as a source of apolipoprotein A1. Proc.
Natl. Acad. Sci. U. S. A., 74: 2569-2573.
Jia, R., L. Cao, J. Du, J. Wang, Y. Liu, G. Jeney, P. Xu and G. Yin, 2014. Effects of carbon
tetrachloride on oxidative stress, inflammatory response and hepatocyte apoptosis in
common carp (Cyprinus carpio). Aquat. Toxicol., 152: 11-19.
Jia, R., L. Cao, P. Xu, G. Jeney and G. Yin, 2012. In vitro and in vivo hepatoprotective and
antioxidant effects of Astragalus polysaccharides against carbon tetrachloride-induced
hepatocyte damage in common carp (Cyprinus carpio). Fish Physiol. Biochem., 38:
871–881.
Kasthuri, S. R., Q. Wan, I. Whanq, B. S. Lin, S. Y. Yeo, C. Y. Choi and J. Lee, 2014. Functional
characterization of the evolutionary preserved mitochondrial antiviral signaling protein
(MAVS) from rock bream, Oplegnathus fasciatus. Fish Shellfish Immunol., 40: 399-406.
Krasnov, A., S. Afanasyev and A. Oikari, 2007. Hepatic responses of gene expression in juvenile
brown trout (Salmo trutta lacustris) exposed to three model contaminants applied singly
and in combination. Environ. Toxicol. Chem., 26: 100–109.
Lattuca, M., G. Malanga, C. A. Hurtado, A. Pe´rez, J. Calvo and S. Puntarulo, 2009. Main features
of the oxidative metabolism in gills and liver of Odontesthes nigricans Richardson
(Pisces, Atherinopsidae). Comp. Biochem. Physiol. B., 154: 406–411.
Liu, Y., J. Du, L. Cao, R. Jia, Y. Shen, C. Zhao, P. Xu and G. Yin, 2015. Anti-inflammatory and
hepatoprotective effects of Ganoderma lucidum polysaccharides on carbon
tetrachloride-induced hepatocyte damage in common carp (Cyprinus Carpio L.). Intl.
Immunopharmacol., 25: 112-120.
Livak, K. J. and T. D. Schmittgen, 2001. Analysis of relative gene expression data using realtime
quantitative PCR and the 2-AACT method. Methods, 25: 402-408.
68
J. Aqua. 23 (2015)
Mohapatra, A., B. Kar, A. Dixit, L. C. Garg and P. K. Sahoo, 2014. Cloning, sequencing and
characterization of apolipoprotein A1 in Labeo rohita. In: 10th Indian Fisheries and
Aquaculture Forum held at NBFGR, Lucknow during 12-15 November 2014; pp. 383.
Mohapatra, A., M. Mishra, S. Das, A. Das, M. Biswas and P. K. Sahoo, 2011. Cloning,
sequencing and expression analysis of hepcidin gene of Labeo rohita to Aeromonas
hydrophila infection. In: Eight Symposium on Diseases in Asian Aquaculture held at
Mangalore, India, during November 21-25, pp. 205.
Panda, R. P., V. Chakrapani, S. K. Patra, J. N. Saha, P. Jayasankar, B. Kar, P. K. Sahoo and H.
K. Barman, 2014. First evidence of comparative responses of Toll-like receptor 22
(TLR-22) to relatively resistant and susceptible Indian farmed carps to Argulus
siamensis infection. Dev. Comp. Immunol., 47: 25-35.
Robinson, N., P. K. Sahoo, M. Baranski, K. D. Mahapatra, J. N. Saha, S. Das, Y. Mishra, P. Das,
H. K. Barman and A. E. Eknath, 2012. Expressed sequences and polymorphisms in rohu
carp (Labeo rohita, Hamilton) revealed by mRNA-seq. Mar. Biotechnol., 14: 620-633.
Sahoo, P. K., B. R. Mohanty, J. Kumari, A. Barat and N. Sarangi, 2009. Cloning, nucleotide
sequence and phylogenetic analyses, and tissue-specific expression of the transferrin
gene in Cirrhinus mrigala infected with Aeromonas hydrophila. Comp. Immunol.
Microbiol. Infect. Dis., 32: 527-537.
Statham, C. N., W. A. Croft and J. J. Lech, 1978. Uptake, distribution, and effects of carbon
tetrachloride in rainbow trout (Salmo gairdneri). Toxicol. Appl. Pharmacol., 45: 131–140.
Yin, G. J., L. P. Cao, P. Xu, G. Jeney, M. Nakao and C. P. Lu, 2011. Hepatoprotective and
antioxidant effects of Glycyrrhiza glabra extract against carbon tetrachloride (CCl4)-
induced hepatocyte damage in common carp (Cyprinus carpio). Fish Physiol. Biochem.,
37: 209-216.
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DIVERSIFIED SUSTAINABLE INCOME GENERATION OPTIONS FOR
RURAL WOMEN IN ODISHA
P. Jayasankar*, H. K. De, B. B. Sahu, Nirupama Panda, U. L. Mohanty, S. Behera
and D. P. Rath
ICAR- Central Institute of Freshwater Aquaculture
Kausalyaganga, Bhubaneswar-751002, Odisha, India
*Corresponding author: [email protected], [email protected]
Rural prosperity and women empowerment are the two sides of a coin. Promoting aquaculture and
related enterprises among women holds immense potential for ensuring their livelihood and socio-
economic empowerment. A study was undertaken in three villages namely Jaipur, Fakirpada and
Paribasudeipur of Puri and Khordha districts of Odisha during the period 2012-13 to 2015-16. A
random sample of 161 women from 10 Women Self-Help Groups (WSHGs) were selected as
beneficiaries for disseminating ICAR-CIFA’s aquaculture technologies like carp seed rearing,
carp culture (grow out), integrated fish farming, post-harvest technologies with value addition of
fish and fish hydrolysate. The women were empowered with skill development in aquaculture and
related enterprises. Due to frequent occurrence of natural calamities and environmental hazards,
the women are forced to diversify their group activities in order to sustain their livelihoods. In the
study area, women SHGs were encouraged to diversify their activities to mushroom cultivation,
rice and vegetable cultivation, floriculture, mini-dairy, backyard poultry, mid-day meal
preparation, coir work, etc. in order to strengthen their livelihood in a sustainable way. On the
whole, the present paper explores different pathways for rural women to avail various
opportunities in terms of multiple diversifications as Sustainable Livelihood (SL) options based
on available natural resources, financial and human capital. Socio-economic impact of such
changes on the beneficiaries was assessed and emerging issues were identified.
INTRODUCTION
Concept of Sustainable Livelihood (SL) attempts to go beyond the conventional
definitions and approaches to poverty eradication, rural development and environmental
management. Structural transformation and development of traditional theories raises, when the
positive impact on rural society is required for non-traditional, non-agricultural and often multiple
livelihood options. Diversification is a strategic approach to an expanded horizon for new
economic activities, which provide better opportunities than the existed before. Several studies
have emphasised ‘multiple livelihoods’ (Bryceson, 2000; Francis, 2000) or ‘occupational
multiplicity’ (Breman, 1996). Diversification can either refer to an increasing multiplicity of
activities (regardless of the sector), or it can refer to a shift away from traditional rural sectors to
non-traditional activities in either rural or urban space (Anon, 2004). An individual has a
diversified livelihood, where he/she has multiple jobs or incomes, but a household can have
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multiple livelihoods, even though each member is in fact specialising in one activity (Ellis, 2000).
It is now recognized that more attention must be paid to the various factors and processes, which
either constrain or enhance poor people’s ability to make a living in an economically,
ecologically, and socially sustainable manner. The SL concept offers the prospects of a more
coherent and integrated approach to poverty (Krantz, 2001).
The objective of the study is to make an attempt to demonstrate different pathways for
rural women to avail various opportunities in terms of multiple diversifications as SL approaches
based on physical, natural resources and human capital. Besides the impact of such changes on
their social and economic development, emerging issues and its solutions are discussed in the
present paper.
MATERIAL AND METHODS
A total of 161 women from 10 Women Self-Help Groups (WSHGs) were selected in a
Simple Random Sampling method from Jaipur village in Puri district and Paribasudeipur and
Fakirpada villages from Khordha districts of Odisha. ICAR-CIFA disseminated different
aquaculture technologies like carp seed rearing, carp culture (grow out), post-harvest technology
i.e. fish processing, packaging, preparation of fish pickle, fish papad and fish hydrolysate to these
women. The women have eight ponds with total water area of five acres in three villages.
RESULTS AND DISCUSSION
Equitable access to more and better jobs in rural areas has enabled rural women to
become effective economic actors and engines of growth; as well as to produce or acquire food,
water, fuel and social services their families need (Anon, 2010). To be able to leverage agriculture
as an engine of inclusive growth, there was a need to expand the horizon into many diversified
areas such as horticulture (mushroom cultivation, nursery management and floriculture), animal
husbandry (backyard poultry and mini dairy) and other allied sectors like coir making (door mate
and rope making) and plastic flower for cycle decoration, etc. Due to ICAR-CIFA’s intervention,
the women could learn carp seed rearing, carp culture and value addition of fish. The women
were sincerely involved in all aquaculture activities throughout the year starting from pond
management, manuring, fertilization, feeding, fish health management, harvesting and marketing.
They expressed their keen interest to adopt aquaculture as their group activity and continue it in
scientific manner. Due to some natural calamities and adverse situations, the members of the
WSHGs face various constraints in doing aquaculture and therefore they add up some other
income generating activities along with aquaculture. Thus they can earn steady income
throughout the year and sustain their livelihoods.
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A) Jaipur village
Due to non-availability of pond on lease, the women of the self-help groups could not
do aquaculture and hence, they started rice cultivation during 2014-15. Some of the group
members were involved in other group activities like coir work (making rope and door mat) and
mushroom cultivation.
1. Rice cultivation
Ten members of a WSHG named ‘Maa Kalijai’ do rice (‘Sarala’ and ‘Pooja’ variety)
cultivation in one acre land area, which is leased to them every year. In Khariff season only they
do rice cultivation. They sow the seeds in July and transplant in August and harvest rice in
December. All these work are carried out by them without hiring any labour from outside. An
amount of Rs. 1,300/- is spent, which includes the cost of 20 kg of seeds, fertilizers and pesticides.
A total of 3.50 quintal of rice is harvested and half of which is given to the lessor. Remaining
1.75 kg of rice is distributed equally among the 10 members of the group of which the sale price
would be approximately Rs. 2,500/-.
Fig. 1 & 2. Paddy culture by the women in Jaipur village, Satyabadi block, Puri district
2. Coir work
The members of two WSHGs comprising 23 women make ropes and door mats from
the coir they purchase from the local vendor. The cost of coir for 50 kg is Rs. 3,200/- including
transportation charge and out of which 2,500 ropes of 40 ft each are prepared. Labour charge for
one rope making is Rs. 1/- and hence total Rs. 2500/- is spent for making 2,500 ropes. One door
mat is made of three ropes and one door mat is sold for Rs. 50/- thus making profit of Rs. 20/-
per mat. In total, profit of Rs. 18,000 is earned from 50 kg of coir. The profit is equally distributed
(approx. Rs. 783/-) among the members.
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J. Aqua. 23 (2015)
Fig. 3 & 4. Coir work by the WSHGs in Jaipur village, Satyabadi Block, Puri district
3. Flower making for cycle decoration
In the leisure time, some women make plastic flowers for cycle decoration and earn
Rs. 1,000-2,000 annually. They distribute the money among themselves (Rs 100-200 per person)
which is used for their family expenses.
Fig. 5 & 6. Plastic flower making by the members of one WSHG for cycle decoration
4. Backyard poultry
In the lean period, the women of 8 WSHGs in Jaipur village add poultry with their group
activities. They rear the indigenous and improved varieties of poultry bird (Vanaraja) on an
average of five birds per WSHG in scientific and hygienic manner. They spend Rs. 189/- per
Vanaraja bird to grow 3.8 kg of body weight and sell the meat at Rs. 90/- per kg. In case of
indigenous species, in total, Rs. 80/- was spent per bird to grow up to 1.5 kg of body weight and
the selling price was Rs. 90/- per kg. Out of meat sale, Rs. 55/- and Rs. 153/- per bird was the net
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J. Aqua. 23 (2015)
return from these two species respectively. Thus, in total, 8 WSHGs earn Rs. 4160/- in a year. By
selling eggs, they also earn an extra income of Rs. 2,700/- per year.
Fig. 7 & 8. Backyard poultry by the WSHGs in Jaipur village, Puri district
B) Fakirpada village
As seasonal routine work, the women are engaged in transplanting of paddy and
harvesting in Kharif and Rabi season and earn Rs. 250/ as daily wages. This is an additional
livelihood option along with regular aquaculture activities. Scarcity of water in ponds during
summer is one of the major constraints, which inhibit the group for doing fish farming. Hence, in
the lean period, the WSHGs do dairy and poultry as their alternative livelihood options.
1. Mid-day meal (MDM) preparation
Two members of the Maa Mangala WSHG in Fakirpada village are involved in mid-day
meal preparation i.e. cooking and supply of cooked meal in the adjacent government primary
school. They work with a spirit of social welfare and not merely for income generation. They are
paid Rs. 4.80 each per student with a total of 26 students available in the school. Thus they
generate additional family income of Rs 125/- per day and 3,750/- per month.
2. Mini-dairy
Ninety per cent of three self-help groups have reared jersey and indigenous varieties of
cows. The women buy cows and buffaloes by taking loan from the group savings. They sell milk
at Rs 25 per litre and get an average monthly income in a range of Rs. 3,000/- to 5,000/- per
household. Out of this amount, they pay the loan partially and rest they spend for their family
expenses.
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J. Aqua. 23 (2015)
Fig. 9 & 10. Dairy development by the group members in Fakirpada village
3. Nursery management
The women SHG members comprising 27 women of Fakirpada have initiated nursery
rearing of various decorative plants (ornamental plants); flowering plants viz., rose, China rose,
Chrysanthemum, Marigold, white tulip, Moon beam, Ashoka, etc.; vegetable saplings like
papaya, capsicum, tomato, chilly, etc. They buy seeds/planting material and grow them upto
standard size. Then they sell the plants in Bhubaneswar, Odisha market at a good price to the tune
of Rs. 2,000-3,000/- per month.
Fig. 11. Nursery management
4. Horticulture and floriculture
Members of one WSHG grow horticultural crops like lady’s finger and sweet gourd on
pond embankment. They sell lady’s finger at Rs. 20/- per kg and Rs 10/- per sweet guard. On an
average they get Rs. 200/- per month. They also sell chrysanthemum and marigold to nearby
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J. Aqua. 23 (2015)
market and earn a net income of Rs. 500/- per month during winter. They do sun flower
cultivation around their pond and sell these in nearby market and fetch about 1,500/- in a year.
Fig. 12. Sunflower cultivation Fig. 13. Lady’s finger cultivation
5. Mushroom cultivation
Mushroom (straw mushroom) (Volvariella sp.) cultivation is one of the most profitable
income generating activities of the self-help groups in Fakirpada village. The women spend Rs.
40/- in total per bed which includes mushroom spawn, straw, rice bran. They sell at Rs.110/- per
kg of mushroom, thereby making profit of 50/- per kg in one cycle. Thus, they cultivate 400
mushroom beds in one season. In total, an amount of Rs. 20,000/-is earned by the group annually.
Fig. 14 & 15. Mushroom cultivation
6. Fish pickle preparation
The ICAR-CIFA imparted training to the women beneficiaries of Jaipur, Fakirpada and
Paribasudeipur on value addition of fish i.e., fish pickle preparation. The women acquired the
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J. Aqua. 23 (2015)
skill and started fish pickle preparation by themselves. For 1 kg of rohu fish pickle they spend
Rs.200/- which includes the cost of fish, spices and bottle for packaging. They sell the pickle @
Rs.400/- per kg in the nearby market and earn good profit to the tune of Rs.200/-. In total, 10 kg
of fish pickle is prepared by the women of three villages and earn Rs.2,000/- in a year
Fig. 16 & 17. Fish pickle preparation by the group members
C) Paribasudeipur village
The ponds in this village are seasonal and dry up by the end of January. Many ponds are
having weak embankments that allow predatory animals like snake etc. to enter and as a
consequence the productivity suffers. They have to completely harvest the ponds by December
and engage in floriculture activities. They grow flowers like marigold in leased land and earn
approximately Rs. 500/- per month.
Fig. 18. Marigold saplings planted in Paribasudeipur village
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J. Aqua. 23 (2015)
Major issues
Rural women encounter several socio-economic constraints in adopting any income
generating activity. Absence of ownership title in favour of women; poor access to other
production resources; no role in decision making etc. prevent women from participating in those
activities. Mainstreaming gender concerns in agriculture in general and aquaculture in particular
has not happened to the desired extent. In the process of adoption of diversified and sustainable
livelihood options, following issues emerged:
1. Poor adoption of scientific technologies
2. Non-availability of quality seeds
3. No supplementary feeding in aquaculture
4. Absence of marketing outlets
5. Non-cohesiveness in the group
6. Inadequate capacity building (training, extension support and exposure visit)
7. Disorganized way of functioning WSHGs
Addressing the issues through intervention of ICAR-CIFA
1. Promotion of scientific agricultural practices of ICAR and particularly of ICAR-CIFA for
aquaculture technologies – carp seed production, composite carp culture and integrated fish
farming. Technologies were demonstrated in SHG pond and skill training provided to the
women at the pond site. Training was also imparted at ICAR-CIFA, Bhubaneswar to selected
members and exposure visits organised.
2. Demonstration of fish hydrolysate preparation was conducted in the village level and
imparted skill development training to women of the WSHGs for use in their fields.
3. Motivated and trained the women for preparing fish pickle, fish cutlet and fish papad as small
scale enterprises.
4. Liasoning made with the state government line departments for disseminating the
technologies and proper monitoring of the progress in respective fields.
5. Stakeholder’s meet was conducted at the village level for convergence for integrated
development.
6. Facilitated marketing of the produce and fish products in nearby markets and different fairs
and exhibitions.
7. In order to have dynamic functioning of the women SHGs, the ICAR-CIFA motivated the
women for regular conduct of meeting of the groups with updating of pass books and ledgers.
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CONCLUSION
In order to utilize spare time and add to their annual income, the women involve in
various diversified income generating activities. These activities not only improve their socio-
economic conditions but also empower them economically and socially which inspires other
women and even their counterparts to follow up. Several approaches i.e. institutional
mechanisms such as technology transfer through Agricultural Technology Management Agency
(ATMA), Krishi Vigyan Kendras (KVK) and Government’s incentive schemes, innovative e-
models of many institutions, tele-marketing and e-marketing (future’s marketing), initiatives of
nongovernmental organizations (NGOs), public-private partnership in agriculture and
NABARD’s Rural Infrastructure Development Fund (RIDF) can enhance various diversified
activities of the farm women and thereby sustain and strengthen their livelihood and food
security.
REFERENCES
Anon, 2004. Working Paper 233. Livelihood Options? The Political Economy of Access,
Opportunity and Diversification. Daniel Start and Craig Johnson. Overseas
Development Institute, 111 Westminster Bridge Road, London, SE1 7JD, UK, 62p.
Anon, 2010. Gender dimensions of agricultural and rural employment: Differentiated pathways
out of poverty. Status, trends and gaps. Published by the Food and Agricultural
Organization of the United Nations, the International Fund for Agricultural
Development and the International Labour Office. Rome, 210 pp.
Breman, J., 1996. Footloose Labour: Working in India’s Informal Economy, Cambridge :
Cambridge University Press.
Bryceson, D. F., 2000. Disappearing Peasantries? Rural Labour Redundancy in the Neo-liberal
Era and Beyond. In: Disappearing Peasantries? Rural Labour in Africa, Asia and Latin
America, Bryceson, (Eds D. F. C. Kay and J. Mooij). Intermediate Technology
Publications, London.
Ellis, F., 2000. Rural Livelihoods and Diversity in Developing Countries. Oxford: Oxford
University Press.
Francis, E., 2000. ‘Rural Livelihoods, Institutions and Vulnerability in South Africa’, Paper
presented at the DESTIN conference on ‘New Institutional Theory, Institutional Reform
and Poverty Reduction’, London School of Economics 7–8 September 2000.
Krantz, L., 2001. The Sustainable Livelihood Approach to Poverty Reduction: An Introduction.
Swedish international development cooperation agency. Division for policy and socio-
economic analysis. 44pp.
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STATEMENT ABOUT OWNERSHIP AND OTHER PARTICULARS ABOUT
‘JOURNAL OF AQUACULTURE’ (From IV; See Rule 8)
1 Place of publication : Bhubaneswar, Odisha, India
2 Periodicity of publication : Annual
3 Printer’s name :
Whether citizen of India : Yes
Address
:
4 Publisher’s name : J. K. Sundaray, Ph. D.
Whether citizen of India : Yes
Address
: ICAR-Central Institute of Freshwater Aquaculture,
Kausalyaganga, Bhubaneswar - 751 002, Odisha,
India
5 Chief Editor’s name : J. K. Sundaray, Ph. D.
Whether citizen of India : Yes
Address
: ICAR-Central Institute of Freshwater Aquaculture,
Kausalyaganga, Bhubaneswar - 751 002, Odisha,
India
6 Name and address of
individuals own the newspaper
and partners or shareholders
holding more than one per cent
of the total capital
: Association of Aquaculturists,
C/O-ICAR-Central Institute of Freshwater Aquaculture
Kausalyaganga, Bhubaneswar - 751 002, Odisha,
India
I, J. K. Sundaray, hereby declare that the particulars given above are true to the best of
my knowledge and belief.
Dated : 29th September, 2017 (sd/-)
J. K. Sundaray
President
Association of Aquaculturists
81
J. Aqua. 23 (2015)
ASSOCIATION OF AQUACULTURISTS
Correspondence address:
Association of Aquaculturists
ICAR-Central Institute of Freshwater Aquaculture
PO: Kausalyaganga, Bhubaneswar-751002, Odisha, India.
Tel:+91-674-2465446, 2465421
Fax:+91-674-2465407
E mail: [email protected]
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