Introduction 1 Increased industrialization, urbanization, population growth and overall man‟s ...

223 Pages·2011·0.92 MB·English

Introduction

Increased industrialization, urbanization, population growth and overall man‟s
greed to overexploit Mother Nature has created a serious threat to all kinds of life in the
form of pollution which has now become a global problem. Among all types of pollution,
aquatic pollution is of greater concern as each and every organism depends on water
(Singh et al., 2010). The pollution of aquatic environment with wide array of xenobiotics
has become a menace to the aquatic flora and fauna and is a problem of immediate
concern. Aquatic pollutants undoubtedly have direct effect on fish health and survival.
Heavy metals and xenobiotics are regarded as the serious pollutants of the aquatic
environment because of their perisistence and tendency to bioaccumulate in aquatic
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Introduction
organisms particularly fishes (Tavares and Carvalho, 1992). Fishes are exposed to such
xenobiotics present in the water by three primary ways viz., (i) skin (ii) gills and (iii)
orally. The resistance of the fish to toxicity also depends on its physiological state (Pal,
2006).
Fish, a key member of many natural food webs is one of the important source of
animal proteins (16-23%) and hence form a valuable commodity for human consumption
(Oshode et al., 2008). Fishes not only meet the food requirements of man within a
country but also earn valuable foreign exchange and so fishing industries do help to
provide employment to millions of people all over the world.
Besides anthropogenically induced changes, natural changes in the aquatic
environments also have an appreciable impact on the fish populations and ultimately on
fisheries. Usually these effects are not possible to quantify because natural changes or
fluctuations in the environment may occur simultaneously with the anthropogenically
induced changes (Richardson, 2001). All these natural (fluctuation of water temperature,
starvation, pH, oxygen concentration etc.) or anthropogenic (xenobiotics) changes in the
water system are bound to continuously disturb the homeostatic mechanism of fishes
besides creating considerable stress to the fishes (Vosyliene and Kazlauskiene, 1999).
Stress reactions involve various physiological changes including alterations in
blood composition and histopathology of various organs particularly haemopoietic and
immune organs (Ololade and Ogini, 2010). Stressors can also become causative of
disease outbreaks, low productivity and mortality in aquaculture. Other toxic end points
include decreased growth, mobility and reproductive effects (Allen, 1994). Changes in
the environmental quality can, therefore, be a major determinant of year-class strength
and eventually the long- term dynamics of fish populations (Rose et al., 1993).
All these stresses may result in significant alteration in such important functions
as feeding, reproduction, growth, haematology etc. in fishes. Among these,
haematological studies on fishes have assumed greater significance due to the increasing
emphasis on pisciculture and greater awareness of the population of natural freshwater
resources. Such studies have generally been used as an effective and sensitive index to
monitor physiological and pathological changes in fishes (Kayode and Shamusideen,
2010).
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Introduction
Haematological evaluation of fish provides valuable facts concerning the
physiological response of fish to changes in the external environment. Study of
haematological parameters on one hand help in establishing the health status of fish and
on other is the cheapest, trusted and well known tool to monitor the ambient aquatic
environment of the fish. Blood is a sensitive indicator of stress and any physiological
dysfunctioing in fish‟s body gets reflected as alterations in its blood constituents.
The count of red blood cells is quite a stable index which, like any other
organisms, fish also tries to maintain within the limits of certain physiological standards
using various physiological mechanisms of compensation. Haematological variables are
also finding considerable usage for clinical diagnosis of fish physiology to determine the
effects of external stressors and toxic substances because there is a close association
between the circulatory system of fishes and the external environment (Wendlaar, 1997).
As an indicator of pollution, blood parameters also help to diagnose and describe the
general health condition of fish species following stress conditions be it pollutants,
diseases, metals, hypoxia etc. (Duthie and Tort, 1985 and Roche and Boge, 1996).
Blood cell responses are important indicators of changes in the external or internal
environment of organisms. In fishes, exposure to chemical pollutants can induce either
increase or decrease in haematological levels. These changes may tend to vary depending
on the fish species, age and cycle of sexual maturity (Luskova, 1997). In corollary to
warm blooded animals, changes/perturbations in the blood parameters of fish can also be
used to determine and confirm the dysfunctioning of various organs or tissues. Thus
haematological analysis can definitely enhance and facilitate fish cultivation through
early detection of stress and diseases that could affect production performance (Kori-
Siakpere et al., 2009).
Blood being the medium of intercellular and intracellular transport, comes in
contact with various organs and tissues of the body and thus can pose a direct threat to
physiological functions of the fish. Xenobiotics (like heavy metals/pesticides) rapidly
bind to the blood proteins and thus may induce haematological changes on one hand and
histopathological on the other (Tyagi and Srivastava, 2005).
Histopathological biomarkers can be best indicators of effects of various
anthropogenic pollutants as well as a reflection of the overall health of the entire
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Introduction
population of an ecosystem (Jordanoska and Kostoski, 2005). These biomarkers are
closely related to other biomarkers of stress since many pollutants have to undergo
metabolic activation in order to be able to provoke cellular changes in the affected
organisms and hence its histology (Mohamed, 2009). For instance the mechanism of
action of several xenobiotics could initiate the formation of a specific enzyme that may
disrupt metabolism leading to cellular intoxication and death at a cellular level. This
usually manifest in the form of necrosis as histopathologcal biomarkers at tissue level.
Histopathology thus can be employed as a sublethal end point or a sort of bioassay to
evaluate the adverse effects of water pollutants in fishes. Such histopathological studies
even can be very useful in establishing water quality criteria (Oliva et al., 2010).
Xenobiotics are also known to have drastic effects on the immune organs in fishes
(Karol, 1998). Study of the cellular components of immune system including their
function and interaction is the central science of immunology. The immune system
generally is of two types viz., innate and acquired or adaptive immune system. The latter
may further have humoral and cellular components. The humoral response is defined as
the interaction between antibodies and antigens. Antibodies are specific proteins released
from a certain class of immune cells (B lymphocytes). Antigens on the other hand are
those that elicit the generation of antibodies. Immunology itself rests on an understanding
of the properties of these two biological entities. Equally important is the cellular
component which not only kills the infected cells in its own way but is also crucial in
controlling the antibody response.
Perturbations of immune functions caused by immunotoxic compounds may lead
to immunosuppression which thereby can result in decreased resistance to the infections.
Presence of contaminants in aquatic environment hence have a definite correlation with
increase in outbreak of infectious diseases in fish populations (Halloran et al., 1998).
Xenobiotics can also result in such adverse immunotoxic effects in tissue as necrosis,
multiple histopathologic effects, chemical induced cellular pathology (abnormal
proliferation and maturation of immunocompetent cells) as well as their functional
alterations. Such structural and functional alterations of the immune system through
immunodepression may ultimately modify host‟s defense mechanisms against infectious
agents (Krzystyniak et al., 1995).
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Introduction
Depending on the nature of the chemicals, duration (i.e. acute or chronic) and
route of exposure, different compartments of the immune system may become
functionally impaired. Alterations in one compartment can often be accompanied by
changes in the other making the interpretation of these changes difficult. Due to its
complex nature, it is difficult to assess the overall responsiveness of the immune system
in the framework of immunotoxicological studies. It is also common to observe, an
enhanced immune response at a low exposure level and suppression at higher exposure
levels (Zelikoff et al., 1995).
Haematological, histopathological and immunological changes hence are among
the first detectable responses of fish to environmental stressors either natural or
anthropogenic (Dethloff et al., 2001). These parameters may respond even after short
term exposure. Exposed fish can compensate for the stressor and these parameters may
even return to pre-stress levels (Wong et al. 1992). If fish cannot compensate for the
stressor‟s effects, an altered physiological state may then be reached to which these
organisms get acclimatized and hence continue to function. In extreme cases,
acclimatization response may even get exhausted and subsequently they may have very
undesirable impacts on their fitness profile (Nagarkatti and Nagarkatti, 1987).Thus,
haematological, histopathological and immunological parameters can act as bioindicators
of not only health status of fish but of the aquatic ecosystem as well.
Presently therefore, an endevour has been made to analyze the effect of seasonal
variations on haematological parameters of two fish species viz., Labeo boga and Labeo
bata. Besides this, the effect of natural (starvation and temperature) and anthropogenic
(heavy metal zinc and carbamate pesticide sevin) stressors on haematological parameters
(Total Erythrocyte Count, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean
Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Total and
Differential Leucocyte Count), histology of haemopoietic organs (liver, head kidney and
spleen) and immune organs (thymus, spleen and head kidney) of fish L. boga has been
evaluated. Additionally, the development of thymus has also been studied in fish L. boga
which is an economically important food fish. Studies have revealed that these
parameters shall find its application not only in assessing the physiological status of fish
but also about the impact of environmental stress on fish health.
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Review of Literature

General Haematology:
First work on haematology of an Indian fish, Channa punctatus was carried out
by Dhar (1948). He conducted preliminary studies on the morphology of corpuscles,
erythrocyte and leucocyte counts and clotting time of this air breathing fish. Since then
different workers have carried out haematological works on various fishes.
Banerjee (1956, 1957) studied the morphology of erythrocytes in an air breathing
fish, Saccobranhus fossilis. Pilay (1958) made routine investigations on the blood of
Hilsa ilisha from river Hooghly. Haematological parameters of three Indian major carps,
Labeo calbasu, Catla catla and Cirrhinus mrigala were studied by Das (1958). Pradhan
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Review of Literature
(1961) reported haemoglobin and haematocrit values of some important fishes like
Tilapia, Channa, Heteropneustes. Banerjee (1966) studied certain haematological
parameters of both sexes of Anabas testudineus. Observations on routine haematological
parameters of carps were made by Qayyum and Naseem (1967 & 1968). Khan and
Qayyum (1969) studied the differential blood cell counts in four species of air breathing
fish. Blaxhall (1972) made a review of some selected literature regarding the use of
haematological techniques in fresh water fish pathology.
Some routine haematological methods for examining fish blood were described
by Blaxall and Daisley (1973). The parameters included haemoglobin estimation,
haematocrit, erythrocyte counts, erythrocyte sedimentation rate and total & differential
leucocyte counts of fish Salmo trutta (L.). Pandey (1974 a, b) studied the blood cell
morphology of erythrocyte and leucocyte of Colisa fasciatus and Garra gotyla.
Mishra et al. (1977) studied some aspects of haematology of an air breathing
Indian mud eel, Amphipnous cuchia. Mahajan and Dheer (1979) made a year long study
based on monthly observations on the haematology of female Channa punctatus with
respect to haemoglobin, haematocrit values, total erythrocyte and leucocyte counts
together with differential enumeration of various leucocytes viz., small and large
lymphocytes, monocytes, neutrophils, eosinophils and basophils. The study showed that
the total erythrocyte count, haemoglobin and haematocrit values decreased during the
breeding time i.e. July to September but thrombocytes number increased significantly and
the neutrophilic number showed a decrease.
Joshi (1980) recorded arneths count in 33 species of freshwater fishes. He also
studied the TEC, TLC and Hb content in the teleost Wallago attu and related them with
the size of fish in year 1981.The effects of repeated weekly blood sampling on blood
parameters in salmonid Salmo gairdneri and cyprinid fish, Leuciscus idus over a period
of 22 days were studied by Hoffmann and Lommel (1984). The procedure was observed
to result in mild anaemia demonstrated by decrease of red blood cell counts, haemoglobin
and PCV. The changes were more marked in L.idus, which showed signs of increased red
cell destruction. No significant trends could be observed in the white blood cell picture.
The results showed that blood tests are possible in individual fish, especially S. gairdneri
over a long period.
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Review of Literature
While working on differential red blood cell counts, Joshi (1986) found
abundance of erythroblasts in normal circulating blood of some freshwater fishes. He also
gave documentation about cytomorphological classification and key to the identification
of blood cells (Joshi, 1987b). Chaudhary et al. (1986) investigated the size and sex
related variations of some blood parameters of an exotic fish, Sartherodon mossambicus.
They found that the TEC, Hb and PCV values were highest in the largest fishes
attributing it to fulfil their increasing energy demands. Parish et al. (1986) identified
seven distinct leucocytes viz., lymphocytes, monocytes and granulocytes from the
peripheral blood of elasmobranch, Scyliorhinus canicula. Rambhaskar and Rao (1987)
observed that active fishes generally exhibit higher values of erythrocyte numbers,
haematocrit and haemoglobin.
Scanning electron microscopic studies on the morphology of blood cells of a
freshwater eel Amphipnous cuchia were made by Ahmad et al. (1990). Ohja et al.
(1991) studies natural destruction of erythrocytes in circulating blood of Rita rita using
scanning electron microscopy. Ohja and Pandey (1997) also evaluated ultrastructural
morphology of blood leucocytes in Clarias batrachus and stated that leucocyte
identification at ultrastructural level is more authentic than conventional histochemical
method.
Age related changes in haematology of hybrid striped bass were studied by
Hrubec et al. (2001). They stated that while the values of PCV and RBC indices were
significantly lower in younger fish, total WBC and lymphocyte count remained
significantly higher in fish at 6 and 9 months of age compared to that of neutrophils and
monocytes which observed higher values at 6, 9 and 15 months of age. Homatowska
et al. (2002) studied haematological indices and cytomorphometry of circulating blood in
the sunbleak Leucaspius delineatus. They also analyzed the mean red blood cell count,
Hb content, Hct, calculated values of RBC indices viz., MCV, MCH & MCHC besides
thrombocyte count, leucocyte count and their composition (lymphoblasts, lymphocytes,
monocytes, neutrophils, basophils and eosinophils).
Daily variations on haematology and blood biochemistry in healthy male and
female Tench (Tinca tinca) during four seasons of the year were studied by De Pedro
et al. (2005). They stated that RBC, Hct and Hb presented a similar daily profile in spring
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Review of Literature
and summer. They further added that total and differential leucocyte counts showed
significant daily fluctuations depending on the season. Gao et al. (2007) compared
diploid, triploid and tetraploid specimens of loach Misgurmus anguillicaudatus with
reference to haematological variations. He observed no significant differences in
haematocrit among polyploids but the erythrocyte count decreased with increased ploidy
level. The haematological profile and description of cell types from the peripheral blood
of six Mediterranean fish species were studied by Pavildis et al. (2007). They reported
that number of differential leucocyte cell types was not influenced by sex or maturity
stage in any species although some variations in the diameter of cells were observed.
Haematological analysis of common carp (Cyprinus carpio), Gold fish (Carassius
auratus), Tilapia (Oreochromis mossambicus) and stinging catfish (Heteropneustes
fossilis) spontaneously infected with Aeromonas hydrophila was carried out by Dhanaraj
et al. (2008). They observed that WBC showed a linear decrease from 0 to 21th day
whereas RBC exhibited a linear decrease from 0 to 2nd day in C carpio, C. auratus and
H. fossilis, but in O. mossambicus, it decreased from day 0 to day 7 and increased from
7th day and reached a high level on day 14th and then decreased again from day 21 when
it again attained the count observed on day 0. Hb content of C. carpio, C. auratus and H.
fossilis were statistically significant but it was not so in O. mossambicus.
Monthly fluctuations in haematology and serum biochemical data in Leuciscus
cephalus were characterized by measuring RBC and WBC counts, Hb, Hct, MCV, MCH
and MCHC by Aras et al. (2008). They obtained highest values during the month of May
and the minimum values during cold months for such parameters like RBC, Hct, MCV
and WBC. The highest and lowest values for Hb, MCH and MCHC were found in
January and in warm months respectively. On the basis of their studies they concluded
that all the studied parameters were affected by many endogenous and exogenous factors
such as reproductive cycle, water temperature and metabolic rate.
Blood parameters such as erythrocyte, leucocyte and thrombocyte counts,
haemoglobin contents, Mean copuscular volume, Mean corpuscular haemoglobin, Mean
corpuscular haemoglobin concentration and haematocrit of African lungfish, Protopterus
annectens were studied by Ikechukwu and Obinnaya (2010) during various
reproductive stages (fingerlings, juveniles, intermediates and adults). Intraspecies
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Review of Literature
haematological relationships in P. annectenes indicated a high positive correlation
between haematocrit and erythrocyte counts and Hb contents respectively in all sizes of
P. annectens with mean r-values of 0.0860 and 0.843 (p<0.05) for Hct/ EC and Hct/ Hb4
respectively.

General Immunology:
The history of fish immunology is much more recent. The techniques employed in
the fish immunology depend upon the species, pathogen and the environment. Many
reviews and papers are now available on the subject of development of immune response
and antibody production in fishes. Phylogeny of immunity, a text edited by Smith et al.
(1966) is a collection of papers by some of the scientists in the field of fish immunology.
Chillar et al. (1969a, b) found that lymphocytes and macrophages of the spleen
and kidney isolated from rainbow trout Salmo gairdneri inoculated with sheep
erythrocytes were able to form the rosette complexes with the specific fish immunogens.
Although these reactions have not been as yet demonstrated with fish pathogens, cellular
reactions must indeed be very important factor in finding off the fish parasites. Most
likely, the cellular activity acts synergistically with other defensive mechanisms. Then
final stages of the engulfment, digestion and elimination of the antigenic materials by the
macrophages and polymorphonuclear leucocytes ultimately rid the fish off the offensive
agents.
Small circulating cells have been indicated to be instrumental in the antibody
production in rainbow trout as stated by Chiller et al. (1969 b) using Jerne plaque
technique. They stated that antibody against fish pathogens is produced by specific cells
which either reside in the anterior kidney, spleen, in small clusters in other areas of the
fish or circulate through out the fish via the humoral system.
The pacific hagfish Eptatretus stoutii was reported to be capable of a variety of
immunologic responses including allograft rejection and serum antibody production to
soluble and cellular antigens by Riviere et al. (1975). Studies revealed that thymus in
young specimens had phagocytic and antigen receptive cell population associated with
the pharyngeal velar muscles. They suggested that a protothymus or precursor of the
thymus of higher vertebrates may be contained within their velar muscle.
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