**5. Role of Snail Hemocytes in Innate Immunity**

Circulating blood cells known as hemocytes represent the main cellular component of the molluscan immune system. Hemocytes are composed of a mixture of different subpopula‐ tions of cells, for example, flow cytometric analyses of hemocytes from the freshwater snails *Biomphalaria glabrata* [30] and *Planorbarius corneus* [31] confirmed two types of circulating cells with two distinct functions [31]. Large granular hemocytes of *B. glabrata*, characterized by the absence of the monoclonal antibody BGH1 surface marker [32], are highly phagocytic in nature, while the BGH1+ is nonphagocytic. *Lymnaea stagnalis* also possess two subtypes hav‐ ing specific surface epitopes such as the mature LS1 and nondifferentiated LS1<sup>+</sup> hemocytes [29]. It is presumed that hemocyte subpopulations that differ both chemically and function‐ ally are regulated in their activities or behaviors through specific receptors and the signals conveyed by their interaction with appropriate ligands. It was further concluded [33] that there are five types of cells in the hemolymph of *B glabrata* and *Biomphalaria straminea* which contributes to the knowledge base for studies on hemocytes and their involvement in control‐ ling *Schistosoma mansoni* infection.

If attention is focused on the functional attributes of hemocytes, several reports in this direc‐ tion revealed diverse immunological functions such as phagocytosis [34], cytotoxicity [35], aggregation [36] and pathogen encapsulation [37, 38]. In addition to hemocytes, hemolymph, the humoral component of the molluscan immune system, is reported to exhibit the activities of superoxide dismutase [39], catalase [40] and acid [41] and alkaline phosphatases [42]. Total hemocyte count in mollusc has been considered as an important immune parameter [43]. Elevation of the total hemocyte count indicates augmentation of immunity of invertebrates [44]. Phagocytosis is an established strategy of immune defense in invertebrates including mollusc. It is considered as the major immunological activity evidenced in many molluscan species [45]. Major cytotoxic molecules such as superoxide anion and nitric oxide generated by the circulatory hemocytes of molluscs are functionally associated with the destruction of pathogens [46, 47]. Phenoloxidase is reported to be functionally associated with phagocy‐ tosis, self‐nonself discrimination, cytotoxicity and melanization response [48]. Superoxide dismutase and catalase play a significant antioxidation role in the cellular physiology of mol‐ luscs. In addition, glutathione‐S‐transferase is functionally associated with general detoxifica‐ tion response of xenobiotics and anti oxidation activity [49]. All these enzymes are involved in scavenging and deactivating the toxic oxidative radicals and protect the tissue from oxi‐ dative damage [46]. Acid and alkaline phosphatases are functionally involved in pathogen destruction in phagolysosome which bear immunological significance [50]. Several reports also demonstrated a range of receptors which bind carbohydrates, extracellular matrix pro‐ teins, hormones, growth factors and cytokines resulting specific immunocyte signals not only in vertebrates but also in molluscs [37]. Thus, it can be surmised that signaling systems are evolutionary conserved functions of immunocytes in the animal kingdom.

not sufficient to inhibit bacterial growth [28]. Bacteria in their growth phase appeared to play an important role in the antibacterial activity of achacin. These data illustrate that when snails are infected by pathogens, achacin should bind to the plasma membranes of those that are proliferating. Achacin may attack pathogens during other growth phases too by increasing

are widely distributed in living organisms, appeared to be of import in both vertebrate and

Circulating blood cells known as hemocytes represent the main cellular component of the molluscan immune system. Hemocytes are composed of a mixture of different subpopula‐ tions of cells, for example, flow cytometric analyses of hemocytes from the freshwater snails *Biomphalaria glabrata* [30] and *Planorbarius corneus* [31] confirmed two types of circulating cells with two distinct functions [31]. Large granular hemocytes of *B. glabrata*, characterized by the absence of the monoclonal antibody BGH1 surface marker [32], are highly phagocytic in

[29]. It is presumed that hemocyte subpopulations that differ both chemically and function‐ ally are regulated in their activities or behaviors through specific receptors and the signals conveyed by their interaction with appropriate ligands. It was further concluded [33] that there are five types of cells in the hemolymph of *B glabrata* and *Biomphalaria straminea* which contributes to the knowledge base for studies on hemocytes and their involvement in control‐

If attention is focused on the functional attributes of hemocytes, several reports in this direc‐ tion revealed diverse immunological functions such as phagocytosis [34], cytotoxicity [35], aggregation [36] and pathogen encapsulation [37, 38]. In addition to hemocytes, hemolymph, the humoral component of the molluscan immune system, is reported to exhibit the activities of superoxide dismutase [39], catalase [40] and acid [41] and alkaline phosphatases [42]. Total hemocyte count in mollusc has been considered as an important immune parameter [43]. Elevation of the total hemocyte count indicates augmentation of immunity of invertebrates [44]. Phagocytosis is an established strategy of immune defense in invertebrates including mollusc. It is considered as the major immunological activity evidenced in many molluscan species [45]. Major cytotoxic molecules such as superoxide anion and nitric oxide generated by the circulatory hemocytes of molluscs are functionally associated with the destruction of pathogens [46, 47]. Phenoloxidase is reported to be functionally associated with phagocy‐ tosis, self‐nonself discrimination, cytotoxicity and melanization response [48]. Superoxide dismutase and catalase play a significant antioxidation role in the cellular physiology of mol‐ luscs. In addition, glutathione‐S‐transferase is functionally associated with general detoxifica‐ tion response of xenobiotics and anti oxidation activity [49]. All these enzymes are involved in scavenging and deactivating the toxic oxidative radicals and protect the tissue from oxi‐ dative damage [46]. Acid and alkaline phosphatases are functionally involved in pathogen

ing specific surface epitopes such as the mature LS1 and nondifferentiated LS1<sup>+</sup>

so as not to harm neighboring host cells. Thus, LAOs, which

is nonphagocytic. *Lymnaea stagnalis* also possess two subtypes hav‐

hemocytes

the local concentration of H2

222 Organismal and Molecular Malacology

invertebrate host defenses.

nature, while the BGH1+

ling *Schistosoma mansoni* infection.

O2

**5. Role of Snail Hemocytes in Innate Immunity**

Apart from the above‐mentioned defense mechanisms, snails also undergo starvation and aestivation under any stress condition. Though several reports are available on starvation and aestivation of snails, information on immune‐related parameter of Indian mollusc is scant. In *Helix pomatia*, antioxidant enzymes are stimulated during aestivation [51] and physiologi‐ cal correlation exists between antioxidant defense and metabolic depression [52]. Starvation is reported to compromise the immunological activity of a land snail, *Helix aspersa* [53]. As per these reports, several immunological parameters are shown to be influenced by nutri‐ tion; some of these parameters are hemocyte count, phenol oxidase activity and phagocytosis. One of the elegant reports [54] in this perspective showed modulation of the innate immune parameters during experimental aestivation and starvation in *Parashorea globosa*. The param‐ eters studied by this group included generation of cytotoxic molecules like superoxide anion, nitric oxide and phenoloxidase and the activities of superoxide dismutase, catalase, gluta‐ thione‐S‐transferase, acid phosphatase, alkaline phosphatase and total protein in hemocytes and hemolymph of *P. globosa* during activity, aestivation, arousal and starvation. This finding appears to be important in the field of comparative immunity and physiology for *P. globosa* which is considered as a commercially important mollusc in India.

#### **5.1. C‐reactive protein (CRP), a multifunctional player in Achatina**

C‐reactive protein (CRP) was first discovered in Oswald Avery's laboratory at the Rockefeller Institute for Medical Research [55]. CRP has evolved conservatively, and homologous proteins with similar functional attributes have been found in many other species. The stable preser‐ vation of this protein during evolution implies some biological significance. Thus, CRP is an ancient molecule discovered in humans only about 82 years ago. It belongs to a protein family called pentraxin (from the Greek words "penta" five and "ragos," berries) that constitutes a phylogenetically ancient family of proteins exhibiting a remarkable conservation of structure and binding reactivities. The presence of CRP has been reported from a wide range of differ‐ ent animals such as monkey, dog, goat, rabbit, rat, mice, domestic fowl, fish, shark and lump‐ sucker among vertebrates and horseshoe crab [56] and *A. fulica* [57] among the invertebrates. The finding that CRP is a major blood constituent of primitive animals, for example, horseshoe crab, *L. polyphemus* and dogfish argues strongly for an important role of this protein.

In *A. fulica*, induction of C‐reactive protein (CRP) synthesis was triggered by exogenous admin‐ istration of the steroid 4‐androstenedione (4 AD) [58].Further, it has been suggested that the hepatopancreas is the main site of CRP expression and the CRP gene in the hepatopancreas is acutely responsive to Gram‐negative bacterial infection [59]. Previously, a question had been raised on whether CRP is inducible in *Limulus* [60]. A search of the *Limulus* CRP promoter for the IL‐6 response element and the Drosophila heat shock element in the human CRP promoter [61] revealed an absence of these cis‐elements which led to the conclusion that *Limulus* CRP expression is constitutive [60]. However in mammals, hepatic CRP is soluble in nature which is released into circulation [62] induced by proinflammatory cytokines. Recently, in an interest‐ ing study, the evolutionary significance of TNF, IFNγ and iNOS in immune response has been amply demonstrated in two Indian mollusc species [63]. Besides assessing different toxicologi‐ cal parameters, anti‐bacterial property of the innate immune molecule, namely C‐reactive pro‐ tein (CRP) isolated from *A. fulica*, was also determined. CRP is a prototypic acute phase reactant, which is a phylogenetically conserved protein expressed in invertebrates such as arthropods [56], molluscs [58] and also in all vertebrates [64]. In *Limulus*, an arthropod, CRP acts as a main front‐line innate immune molecule [59] which may be the key to a powerful defense mecha‐ nism of these animals against microbial infections that are potentially lethal in other organisms. Moreover, the presence of high level of endogenous CRP (2–4 mg/ml) in the hemolymph of *A. fulica* [58] might be the sole reason behind their effective survival in the environment.

Several authors reported that CRP can protect mice from infections caused by both Gram‐posi‐ tive *Streptococcus pneumoniae* [65] and Gram‐negative *Neisseria elactamica* [66] and *Haemophilus influenzae* [67] bacteria via direct binding with repetitive phosphorylcholine moieties on the lipo‐ teichoic acid or the lipopolysaccharide (LPS) of these pathogens, respectively. The level of CRP also increases dramatically during periods of immunological challenge and boosts the bacteri‐ cidal activities of monocytes and neutrophils by enhancing the release of reactive oxygen inter‐ mediates [68]. CRP also induces oxidative stress in vitro in endothelial cells, smooth muscle cells and monocyte‐macrophages [69, 70]. Although there are many reports on properties of CRP in a wide range of in vitro and in vivo model systems, clear understanding of the actual biological functions of this phylogenetically ancient and highly conserved molecule remains elusive.

It is also noted that bacterial cells are strongly dependent on metabolic cycles for their sur‐ vival and pathogenicity [71, 72]. Therefore, effect of *Achatina* CRP (ACRP) on these bacterial metabolic cycles comprising key metabolic enzymes such as phosphofructokinase 1(PFK1) in glycolysis, isocitrate dehydrogenase (ICDH) and isocitrate lyase (IL) in TCA cycle and fruc‐ tose‐1,6‐bis phosphatase (FBP1) in gluconeogenesis was also investigated. Various authors have reported the existence of eukaryote‐like programmed cell death and the involvement of caspase‐3‐like proteins in bacteria [73]. Based on this information, it was attempted to delin‐ eate the anti‐bacterial property of ACRP in terms of inhibition of salient metabolic enzymes which decrease bacterial infection accompanied by ROS generation and apoptosis‐like phe‐ notypes during bacterial cell death.

Several authors [74] reported potentiality of human CRP to inhibit superoxide (O2 − ) genera‐ tion and delay apoptosis in neutrophils [64]. Recently, it has been reported that immune‐ potent CRP modulates antioxidant and anti‐inflammatory effects in LPS‐stimulated human macrophages [75]. The anti‐stress property of ACRP was tested in mice which are known to have a very low level of endogenous CRP (∼2 μg/mL) even after an inflammatory stimulus [76]. In order to prove this hypothesis, lead nitrate was administered intraperitoneally at an environmentally relevant dose in mice, and the induced oxidative stress was found to be removed when ACRP was administered prior to treating with Pb*.* Furthermore, in an in vitro study, both native CRP and its subunits were found to accomplish reversal of lead‐induced hepatotoxicity in *A. fulica* [77].

In molluscs, several anti‐microbial peptide (AMP) genes are triggered during onset of a broad range of pathogenic infections. Furthermore, several categories of immune molecules are extracted from snails including glycosaminoglycans, peptides, proteins (glycoproteins) and enzymes which possess diverse biological activities [78, 79]. Interestingly, evolution‐ ary success of *A. fulica* can be associated, in part, with their relatively simple and effective innate immune system comprised of defense molecules present in their hemolymph such as hemocyanins, lectins, C‐reactive proteins and macroglobulins in addition to a large number of granular hemocytes or amoebocytes [78, 79].

It was earlier established that xenobiotics, like heavy metals, are successful in triggering the synthesis of CRP causing inflammatory condition, and in turn, CRP was found to be a very good scavenger in eliminating these heavy metals. In contrast to human and other higher level mammals, the normal fresh water teleost *Channa punctatus* has a high level of CRP [80]. The level of CRP was also found to be significantly high in the snail *A. fulica* [58] during rainy season which is nearer to the level of CRP in the horse‐shoe crab, *Limulus*. It was clearly documented by several authors that level of CRP significantly increases in serum during onset of infection or inflammation and thereby CRP acts as an inducible protein in mam‐ mals. However, in invertebrates, CRP is constitutively expressed, as for example, *Achatina* hemolymph contains a higher level of CRP which is about 2 mg/ml and showed strong cross reaction with *Limulus* CRP antiserum [58].

Snails accumulate heavy metals more in their tissue inducing numerous acute and sublethal effects [81]. Due to this sensitivity, they are considered as excellent bioindicators of heavy metal contamination [82]. The effect of accumulated heavy metals on different molluscan tis‐ sues and possible use of such alterations as biomarkers of exposure to xenobiotics has been investigated [8, 9]. Molluscs have shown considerable promise as biomonitors of metal pollu‐ tion [83], and an extensive literature has appeared concerning mechanisms of uptake, detoxi‐ fication and storage of heavy metals [84]. Few studies on several fresh water and marine species further substantiate the role of molluscs as bioaccumulators [85]. Further, ecological and ecophysiological studies suggest that molluscs react to environmental stress and pollu‐ tion by modifying their behavior [86]. It is reported that terrestrial snails might regulate some metals assimilated from food and xenobiotic exposure [7]. The kinetics of metal accumulation and detoxification are still a subject of discussion, and there is a lack of information regarding metal toxicity in snails [84, 87].
