Immunology deals with the physiological activity of organisms to defend against pathogen and toxin invasion. Invertebrates residing in aquatic ecosystems often face toxicological threat arises from habitat pollution. The aquatic habitat of invertebrates is in the precarious risk of pollution caused by diverse groups of environmental toxins. Immunotoxins have been considered as a special group of pollutants capable of affecting the immunological profile of organisms. Invertebrates residing in water bear ecological, economical, medicinal, industrial, nutritional and biotechnological significance. Global aquatic bioresource is largely composed of invertebrates belonging to multiple Phyla. These organisms, including insects, snails, clams, mussels, crabs and sponges, are physiologically dependent on innate immunological response for defense against pathogen and environmental contaminants. External physicochemical barriers of invertebrates act as primary line of defen against toxin entry. Principal barriers have been identified as shell, tunic, test, carapace, mucus, etc., in diverse species. Toxin-induced morphological damage of specialized immunocytes of invertebrates has been reported. Toxin-induced shift in density, surface adhesion efficacy and aggregation of blood cells or haemocytes have been identified as major xenobiotic stress in invertebrates. Various environmental toxins are capable of initiating alteration in the innate phagocytic response and cytotoxicity of blood cells. Lysosomes of invertebrate haemocytes are functionally involved in intracellular destruction of environmental pathogens. Toxins like arsenic, pyrethroid pesticides, azadirachtin and washing soda were reported to increase the relative fragility of lysosomal membranes of immunocytes. This often leads to impairment in the efficacy of invertebrates to destroy pathogen under the exposure of pollutants. Xenobiotics like pyrethroid pesticides have been recorded to affect apoptosis and necrosis of invertebrate immunocytes. Selected toxin-induced morphological damages of heart, gill, digestive gland, mantle and antennae may result in the overall impairment in homeostatic levels of invertebrates inhabiting the polluted environment. Global environment, in recent times, is under the serious threat of contamination by diverse chemical compounds of unknown or less known toxicity. A thorough ecotoxicological analysis at cellular and molecular levels needs to be carried out in invertebrates occupying the different realms of the planet in future.
- washing soda
- flow cytometry
- respiratory burst
- nitric oxide
Invertebrates constitute more than ninety-five per cent of the existing biodiversity of the world. They present an extreme range of variation in terms of their body architecture, adaptation, food preference, habitat preference, behaviour and physiological response against environmental stressors. Evolutionarily, they are an ancient group of animals which have survived an extreme range of environmental adversities and perturbations from the time of their origin. Invertebrates occupied diverse kinds of habitats including terrestrial, freshwater, marine and estuarine ecosystems. Aquatic invertebrates are usually distributed in the multiple spatial compartments of the water bodies from the surface to the bottom region called the benthic stratum. Aquatic invertebrates are also found in the conspicuous ecological areas of the mud–water interface and subsoil region at the floor of aquifers. Freshwater ponds of India serve as habitat for diverse aquatic organisms including members of Porifera, Mollusca and Arthropoda. Majority of the ecotoxicological research was carried out on
Aquatic invertebrates bear ecological, economical, nutritional and biotechnological significance and demand a special scientific attention. Many of them are the dietary source of nutrition for human and other organisms. Their importance as potential source of bioactive substances and pharmacologically active compounds cannot be denied. Current scientific reports indicate that the rapid and unrestricted contamination of freshwater and marine ecosystem by diverse pollutants poses serious ecotoxicological threat for the existence of invertebrates in their natural habitats. Various anthropogenic activities like habitat destruction and habitat contamination have been identified as the causative factors of dwindling of invertebrate species on earth. Continuous addition of various toxic chemical compounds into the global environment is a major environmental challenge encountered by human and other organisms. Many of these environmental pollutants are of either industrial or agricultural origin and need a thorough toxicological screening in invertebrate models.
The overall evolutionary success and survival efficacy of invertebrates depended on several factors including the development of a highly advanced immunological system. Immunology deals with the typical strategies of biological defence against environmental toxins and pathogens. Components of immune system and their coherent functional attributes enabled the invertebrates to overcome the toxin-induced chemical stresses of the primitive and modern global environment (Figure 1). Many of the environmental pollutants of the current hydrosphere are less researched, with limited toxicological information. Invertebrates over a period of time are assumed to evolve novel and unique modes of immunological reactivities to defend against the toxic insults of environmental pollutants at the cellular and molecular levels. Aquatic invertebrates are relatively a neglected group of organisms with reference to their immunotoxicological status in a biounsafe environment. The principal immunotoxicological responsiveness of invertebrates centre around the reactivities of circulating immunocytes and selected effector organs like gill, digestive gland, intestine, labial palp, etc.
2. Immunotoxins and their impact on aquatic invertebrates
Immunotoxins are chemical compounds that can modulate the immune-related parameters and can adversely affect the biological and physiological functioning of the immune system of organisms including human. The freshwater ecosystem of India is being contaminated by diverse groups of xenobiotics of known and unknown chemistry. Ray et al. (2011) reported mineral acids, alkalis, detergents, metalloids and pesticides as major environmental contaminants of the Indian freshwater ecosystem . Scientists reported  the toxic effects of pyrethroids, cypermethrin and fenvalerate in aquatic molluscan invertebrates and claimed them as immunotoxins for their adverse effect on the immune parameters of host animal. Toxicity of these pyrethroid pesticides on non-target aquatic invertebrates appears to be detrimental and can seriously affect their survival efficacy and reproductive success. Arsenic, another metalloid and a potent immunotoxin, bears toxic effects on aquatic invertebrates and can modulate the histopathology and immune parameters of freshwater mollusc  and estuarine mud crab . Biopesticide azadirachtin has been recently identified as an immunotoxin due to its potential to affect the immunofunctioning of mussels. Moreover, during monsoon and flood, pesticide-laden agricultural runoff often contaminates the freshwater ecosystem and poses serious threat to its inhabitants. Washing soda, chemically known as anhydrous sodium carbonate, is an important aquatoxin that can alter the selected physiological parameters of diverse groups of invertebrates. Mukherjee et al. (2015b) reported the toxicity of washing soda in a freshwater sponge of India with reference to its phagocytic and cytotoxic status . Altered functioning of the immune system may lead to opportunistic invasion of environmental pathogens and parasites into the body of host and increase the vulnerability of these biofilter species in polluted environment.
The nature and magnitude of immunotoxicity depend on multiple parameters including the concentration of toxin, type and span of exposure and route of entry to the host. Immunotoxins are difficult to identify as they can cause a wide magnitude of adversity on the immune status of organisms. Immunotoxicology deals with the assessment of toxicological response in an organism by estimating the responsiveness and reactivity of its immunological parameters. Immunotoxicology of invertebrates, in recent times, has been gaining a special scientific attention for its efficacy in monitoring the health of environment – both aquatic and terrestrial. Accuracy and precision of selected toxicological responses of aquatic invertebrates enabled a few species to function as suitable biomonitoring agents of water pollution . Several effective immunomarkers of aquatic pollution have recently been established in model invertebrates .
3. External physicochemical barriers of invertebrates as first line of immune defence against pollutants
External physicochemical barriers of the invertebrates act as the first line of defence against the invasion of parasite, pathogen and toxin in a polluted aquatic environment. External physicochemical barriers of the invertebrates include shell, tunic, test, cuticle, carapace, pinacoderm and others. In mollusc, hard calcareous shell or valves provide the primary protection against entry of pathogens and toxins. However, in shell-less molluscs, thick external cuticular sheet known as mantle or pallium are assumed to play a significant physiological role against toxin and pathogen entry. Soft body wall of molluscs consists of cuticle, epidermis and muscles, which are thought to take an active part in the innate immune defence against environmental pathogens and toxins. In a polluted environment, where environmental pathogens and toxins are present in sufficient quantity, the two valves of the mussel are kept closed to minimize the entry of undesired agents. In addition to external shell, mucus secreted from the internal viscera provides another line of defence against the invasion of pathogens and toxins. Mucus acts as a protective barrier preventing the direct contact of toxins to epithelia. Secretion of mucus is reported to be an important detoxification and evasive mechanism of invertebrates. Pathogens are trapped within the mucus secreted by the organism leading to elimination. Calcareous shell made up of calcium carbonate and mucus is considered as external physiochemical barrier and plays an important role in the immune defence of aquatic invertebrates. Carapace, the external physiochemical barrier of crab provides the first line of defence in crustaceans. Corrosive toxins including mineral acids, alkalis, pesticides and detergents appear to be the potential threats of the invertebrate. Contamination of water bodies by these pollutants often results in breaches of the physicochemical barriers of external body surface, which leads to facilitation of invasion of toxic microorganisms and parasites into the viscera of target invertebrates. Components of innate immune response comprise physicochemical barrier against external pathogen and parasite entry. Cuticle covered with waxy material serves as a mechanical barrier of crab against parasitic infection. This chitinous exoskeleton or cuticle helps in the process of wound healing by preventing the fatal loss of haemolymph from body, maintenance of tissue architecture and prevention of opportunistic invasion of pathogen. Whenever this cuticle is damaged by injury or infection, the wound is rapidly sealed by clotting of immunocytes, preventing blood loss and pollutant entry. Once the clot is formed, wound is darkened and accumulation of melanin occurs. Melanin is reported to be involved in sealing of wound and synthesis of new cuticle. On the other hand, pinacoderm made up of flattened pinacocytes forms a continuous layer on the external surface of freshwater sponges and acts as a first line of defence against foreign invaders.
An experiment was performed to investigate the possible role of mucus as a primary barrier in removal of external particles. In this context, the shell of freshwater gastropod,
4. Xenobiotic-induced shift in haemocyte density and morphological damages of blood cells
Haemocytes are the immunocompetent cells which are functionally responsive to various xenobiotics present in the aquatic environment. Homeostasis of total haemocyte density of aquatic invertebrates within the permissible physiological limit may be considered as an important immunological parameter  of cell-mediated immune response of molluscs . Chakraborty et al. (2008) reported suppression in the total count of haemocytes of the freshwater edible mollusc,
Mukherjee et al. (2008) reported an increase in relative percentage of pro-haemocytes subpopulation in aquatic mollusc
Morphological alterations of haemocytes under the exposure of environmental toxins play an important role in cell-mediated immune response of molluscs (Figure 3). Ray et al. (2013b) identified cytoplasmic hypervacuolation, rounding up of cell, alteration in cell shape, hypergranulation, increased cytoplasmic spreading, membrane disintegration and membrane blebbing as principal aberrations in the haemocytes of
5. Nonself surface adhesion and aggregation response of haemocytes
Nonself surface adhesion is considered as an important immunological mechanism which is fundamentally related to self–nonself discrimination. Cell adhesion and interaction between cell and the substratum play a pivotal role in the development, maintenance and immune recognition in multicellular animals. Mukherjee et al. (2007) studied the glass surface adhesion efficacy of haemocytes of
In the dynamic freshwater ecosystem, the inhabitants often compete for niche for their better survival and propagation. Overlapping in the niche leads to a state of acute predation and fighting among animals. As a result, the animals may encounter acute competition and subsequent physical damage and loss of body fluid. Cellular aggregation is a functional attribute offered by the haemocytes of invertebrates  to prevent the accidental blood loss by formation of biological plug at the site of wound and resist the entry of pathogenic microorganism. Selected pollutants like sodium arsenite, washing soda, pyrethroid, azadirachtin are reported to affect the aggregation response of many aquatic invertebrates (Figure 4). Hence, cell–cell aggregation is considered as an immunological response for host defence. Aggregation of haemocytes around invaded microorganisms is termed as “encapsulation response” and is considered as an important immunological response . When successful encapsulation occurs, a host animal can restrict the proliferative and invasive property of a pathogen. Encapsulation reaction is mediated by specific population of immunoactive blood cells and is effective in cell-mediated immunity of invertebrates. Mukherjee et al. (2011) reported anticoagulant and carbohydrate-induced interference of cellular aggregation of mussel,
6. Phagocytic response in the face of environmental stressors
Phagocytosis, in general, is considered a classical innate immune response reported in the majority of the invertebrate Phyla. It is an established immunological response and is considered as a biomarker of aquatic pollution . Phagocytic response enables invertebrates to combat against invading pathogens and pollutants of known and unknown chemistry. Haemocyte-mediated phagocytosis of nonself particles provides natural immunity in the bivalves . Chakraborty et al. (2009) reported the inhibitory effect of sodium arsenite on the phagocytic response of
7. Cytotoxicity of blood cells as an effective immune strategy
Cytotoxic molecules generated by the immunocompetent cells of invertebrates are reported to play an important role in the destruction and deactivation of foreign engulfed pathogens . Authors reported superoxide anion, nitric oxide and phenoloxidase as established cytotoxic molecules of freshwater molluscs affected by various environmental pollutants. Contamination of natural habitat by toxic metals, metalloids, pesticides and washing soda results in a significant alteration in the cytotoxic status of invertebrates. Cytotoxic molecules are considered as an effective component of innate immune defence of invertebrates distributed in polluted environment. Nappi and Ottaviani (2000) reported nitric oxide and superoxide anions as potential “killing agents” of invertebrates . Generation of reactive oxygen intermediate and reactive nitrogen intermediate by the phagocytic cells are mediated by NADPH oxidase and nitric oxide synthase (NOS), respectively. According to them, generation of superoxide anions can be correlated with increased respiratory burst activity in phagocytic cells. Mukherjee et al. (2012) reported a dose-dependent increase in the generation of superoxide anion in the cells of freshwater edible bivalve,
8. Lysosomal membrane stability and activity of phosphatases
Lysosome is an important subcellular organelle involved in the process of degradation of foreign engulfed particulate. After phagocytosis, the phagocytic vacuole with engulfed foreign particles fuses with lysosome to form phagolysosome. As lysosome plays an important role in secretion of various digesting enzymes, maintenance of lysosomal membrane integrity has been gaining special scientific attention from the immunological point of view. Chakraborty and Ray (2009) reported impairment of lysosomal membrane integrity by neutral red retention assay in the haemocytes of
9. Pollutant-induced apoptosis and necrosis of invertebrate immunocytes
Apoptosis, the programmed cell death, is functionally involved in developmental and immunological processes of organisms. Moreover, apoptosis helps in the process of removal of damaged cells and thus is considered an important machinery of survival of host animal in polluted environment. It is involved in specific cell signaling mechanism, which is yet to be studied in invertebrates in detail. However, pesticides and other environmental xenobiotics are reported to affect the apoptotic and necrotic pathways of the immunoactive cells of invertebrates (Figure 7). Current scientific reports suggest apoptosis as an effective biomarker of aquatic pollution. Apoptosis presented changes in the morphological characteristics of cells including membrane blebbing, nuclear condensation, cytoplasmic shrinkage and membrane asymmetry. Kiss (2010) reported the translocation of phosphatidylserine from inner leaflet of the plasma membrane to the outer leaftet, which was considered a hallmark of apoptosis . Ray et al. (2013b) reported the apoptotic and necrotic cell deaths of haemocytes of
10. Toxin-induced damage of target organs and tissues
For filter feeding aquatic organisms, the gill, heart, digestive gland, mantle and antennae act as major target organs of common environmental xenobiotics (Figure 8). Mollusca bear a well-developed gill which serves as an organ of gaseous exchange, filter feeding and immunosurveillance. Molluscan gill is a thin membranous and vascularized organ that is in continuous contact with the environmental toxicants distributed in water. During the process of respiration and filter feeding, gill is exposed to various toxins of known and unknown chemistry. Exposure of gill to aquatic sodium arsenite, pyrethroid pesticide and washing soda of pond water yields a structural damage of lamellae. Chakraborty et al. (2010a) reported hyperchromatic anaplastic cells in the gills of
Global environment in recent times is characterised by the presence of various xenobiotics of known, less known and unknown toxicity and chemistry. Information of immunological attributes of chemical compounds in aquatic invertebrates is limited in the current scientific literature. Limited but significant reports indicate a substantial impairment of the immunological status of invertebrates under the exposure of selected ecotoxins (Figure 9). Acute, subchronic and chronic exposure of common toxins like pesticides, arsenic and alkaline washing soda cause severe damage in the morphological and functional profiles of haemocytes, the chief immunoeffector cells of invertebrates and other organs and tissues. Sponges, in general, are devoid of well-developed organ system. A variety of specialized cells of sponges are functionally involved in various immunological activities. Exposure of immunotoxins like washing soda largely affected the density dynamics of sponge cells as well as the cytotoxic and phagocytic status in freshwater sponge. Invertebrates of freshwater ecosystem act as important economical resource for developed and developing countries. A thorough toxicological analysis of the functional performance of target cells and tissues of invertebrates needs to be carried out for the purpose of conservation and culture of this species in their natural environment.
Authors gratefully acknowledge the scientific contributions of Suman Mukherjee, Sudipta Chakraborty and Sanjib Saha – the former members of the Aquatic Toxicology Laboratory. CRNN of University of Calcutta, UGC SAP DRS II and DST INSPIRE are acknowledged for financial and analytical support as well.