Nurocombi is used as an insecticide in large-scale commercial agricultural applications as well as in consumer products for domestic purposes. The present research purposed to evaluate the effect of sub lethal concentration of nurocombi in hematological parameters of the fresh water crab, Paratelphusa jacquemontii after 0,7,14,21 and 28 days. Total counts of hemocytes showed decreasing trend as the duration of exposure in each concentration of nurocombi increased. Differential hemocytes count showed increasing trend as the duration of exposure in each concentration of nurocombi increased in Agranulocyte and Eosinophilic granulocyte. Differential hemocytes count showed decreasing trend as the duration of exposure in each concentration of nurocombi increased in Hyalinocyte and Basophilic granulocyte. It is concluded that hematological changes provide reliable and discriminatory data to augment pesticide pollution and therefore, long-term monitoring is necessary to assess the eco-health of the mangrove system.

Maharajan A, Narayanaswamy Y, Ganapiriya V (2017) Haematological Changes of Fresh Water Crab, Paratelphusa jacquemontii in Response to the Combination of Chlorpyrifos and Cypermethrin (Nurocombi) Insecticide. Ann Aquac Res 4(3): 1041.

•    Paratelphusa jacquemontii
•    Haemolymph indices
•    Nurocombi

Ecotoxicological risk assessment of environmental chemicals is principally based on the results of laboratory studies where test organisms are exposed to a range of concentrations of single compounds. Haematological characteristics are tools for screening pathological status. The haemotological parameters constitute a good indicator of physiological responses [1]. Crustacean hemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and cell-cell communication. The hemocytes of crustaceans are generally simple [2], that are mainly subdivided into three cell types. Authors do not report all the same information in classifying crustacean hemocytes. Relatively few workers have agreed in common terminology. Parameters used to classify the hemocytes have been based on the ration of cytoplasm to the nucleus, and on size and staining reaction of the cells containing granules. Morphological studies on blood cells of crustacean, however, are scarce, so that it is difficult to identify particular haemocyte types involved in defense reactions.

Crustacean hemocytes play central roles in a host’s immune response; however, there is no uniform classification scheme for crustacean hemocytes [3]. Crustacean hemocytes have been categorized into several types, which circulate in the haemolymph. Their primary functions are coagulation, phagocytosis, detoxification, storage and distribution of nutritive material. There is an inherent variability of hemocytes within a species as well as among closely related species [4]. Crabs are linked to different parts of the food webs in freshwater and terrestrial ecosystems. They are active predators and detritus feeders, occupying an intermediate position in both aquatic and terrestrial food webs. Because of their capacity for bioaccumulation and their relatively elevated resistance to pesticides, crabs could transfer these pesticides to organisms in a higher trophic level, and eventually to humans. There are many requirements that a species must meet to be a good biological indicator of pollution [5]. The species must play a key role in the community, transporting the pollutant from the lower to the upper links of the food webs, or participating in the food chain of a species that is economically important to human.

Crabs constitute a significant portion of the freshwater ecosystem. Very often they become the victim of pesticides used against some other activity or agricultural pest. Therefore their population in this area was found decreasing during the last decade. The toxicity of pesticides depends on many factors such as weight, size, developmental stages, time of exposure and temperature in water content of the medium. The use of native species as sentinel organisms is proposed as a more appropriate way to obtain information about a specific site. P. jacquemontii is a local abundant crab that is widely distributed in I Thiruvarur district, and it is territorial, easy to collect and resistant to pollutants. The nurocombi bioaccumulation capacity of this species contributes to its suitability as a bioindicator for the presence of pollutants in aquatic systems, although more studies are needed. Because the biota may accumulate persistent lipophilic organic pollutants, the transfer of these contaminants in the food web, which eventually may reach humans, must be continually observed.

Animal collection and acclimatization

The experiments were performed in accordance with local/ national guidelines for experimentation in animals and all care was taken to prevent cruelty of any kind. Fresh water crab, P. jacquemontii of carapace size ranging from 5.6 to 6.1 and weight 45–55 g were collected from the paddy field of Muthupettai, Thiruvarur Dist, and Tamil Nadu. They were transported and kept in 100 L tank containing well aerated filtered fresh water maintained at ambient temperature (27 ± 2°C) for a period of one week. Before stocking, the tank was washed with 0.1% KMnO4 for disinfection.

Chemicals

For preparation of stock solution 1 ml of insecticide NUROCOMBI (Chlorpyriphos (CPF) 50% and Cypermethrin (CPM) 5% EC), Cheminova, FMC Corporation, Mumbai, diluted with 1 L of Milli-Q deionised water was purchased.

Test concentration

Crabs were exposed to 0.0187 and 0.0374 ppm sublethal concentration of combined insecticide doses at 10% and 20% respectively of the Maximum Acceptable Toxicant Concentration (MATC), which was 0.187 ppm.

Test procedure

After 2 weeks of acclimatization in a holding tank, ten healthy crabs with carapace size ranging from 5.9 to 6.2 and weight 50– 60 g were transferred to each aquarium. Three replicates were performed for test concentration and control. Crabs were fed twice daily with commercially prepared pellet feed at 10:00 and 16:00 h. Uneaten food was quickly removed from the system. The media were renewed every alternate day. Mortality and behavior were observed everyday in each concentration. Two crabs from each aquarium were sampled at 0, 7,14,21 and 28 days postexposure.

Haematological assay

The haemolymph of the crab P. jacquemontii was drawn from haemocoel through a syringe. The haemolymph was thoroughly mixed with 1% sodium citrate, used as an anticoagulant. Thin hemocyte films were prepared by carefully spreading a drop of unfixed hemolymph. They were then air-dried, fixed in 10% formalin-methanol (1:9) and flooded in Giemsa and haemotoxylin and counterstained with eosin [6]. The identification of hemocyte types were based on cell size, shape, and staining properties [7]. The counting of free hemocytes (THC) was done by using a hemocytometer with improved double Neubauer ruling and a diluting fluid suitable for crustaceans [8]. Whenever cell rupture, agglutination and plasma clot formation appeared either in the dilution pipette or in the counting chamber, the count was not made. Differential haemocyte counts (DHC) were made on freshly stained smears following the method of Mix and Sparks [9]. The different types of haemocytes were counted at random under the microscope (×400) and the number of each cell type expressed in percentage, to which the following formula was applied.

Statistical analyses

To observation of significant differences to appraise, the effect of nurocombi on haemolymph to determine the unity between the concentration and haemolymph parameters. Data were analyzed statistically at p < 0.05 by SPSS software version 16.

Examination of haemocytes obtained from the control P.jacquemontii revealed that the haemolymph contained morphologically distinct types of haemocytes namely, Agranulocytes (AG), Hyalinocytes (HC), Eosinophilic granulocytes (EG) and Basophilic granulocytes (BG). Agranulocytes are larger, irregular cells with elongated and horse shoe shaped nucleus. Thin cytoplasmic layer around the nucleus and the average ratio was 40%. Hyalinocytes are smallest with high nuculeocytoplasmic ratio and with the large nucleus compared with other haemocytes. Eosinophilic granulocytes are small with granules and the nucleus is eccentric. Basophilic granulocytes are larger with granules and fat droplets. Fisheries researchers have adopted different criteria for haemocytes classification although the classification of crustacean hemocytes remains controversial [10]. Classification of the haemocyte type in decapods crustaceans is based mainly on the presence of cytoplasmic granules, in hyaline cells, semigranular cells and granular cells [3]. The same classification was given by Soderhall et al. [11], in freshwater crayfish. In contrast in the Indian spiny lobster, P. homarus, hemocytes were classified into four types – prohyalocytes, hyalocytes, eosinophilic granulocytes, and chromophilic granulocytes [12]. Sanjayan et al. [13], have reported five types of haemocytes in Spilostethus horpes that is prohemocytes, plasmatocytes, granulocytes, spherulocytes and adipohaemocytes. Le Moullac and Haffner [14], reported that the circulating haemocyte number is a stress indicator and haemocyte counts may be expensive tool in monitoring the health status of crustacean species. Studies on the hemocytes of crustaceans contribute to the accumulation of the basic knowledge on hemocytes especially with regard to the physiological condition of the animal.

Exposure to nurocombi, in P. jacquemontii induced enlargement of cell size in all the four types of hemocytes. These cells attain a follicular pattern and clumping of cells was distinguished with degenerated cell types. Sublethal exposure to both concentrations of nurocombi on 7 and 21 days resulted in less phagocytosis relative to that of hemocytes of the control crabs. After 7 days the phagocytic response of haemoctyes of crabs exposed to 0.0374ppm was approximately 40% less than that of controls. In hyalinocytes, the nuclei were pushed towards the periphery and the cytoplasm became more granular. The eosinophilic granulocytes invariably developed granuloplasmic vacuoles, membrane blebs and dense cytoplasmic deposits (Plate 17 g, h & i). Exposure of Macrobrachium rosenbergii to sublethal concentration of carbosulfan has induced morphological aberrations in haemocyte shape and ragged cytoplasmic membrane, vaculation in cytoplasm and nucleus, fragmentation of nucleus, degeneration of cytoplasmic membrane and extrusion of nuclear material [15]. Some of the granulocytes were aberrant in shape with lobate nuclei and highly vacuolized cytoplasm (Plate 1 a-c). Achromophilia of cytoplasm was observed in few hemocytes (Plate 1 d, e & f). Many of the hyalinocytes showed large wavy frills pseudopodia and some of them stretched out into fibroblast like cells. Distruption of haemocytes was evident in higher concentrations of nurocombi after 28 days of exposure (Plate 2 a-f).

In control crabs, the Agranulocytes measured with the diameter of 5-6µm and possess centrally placed large nuclei measuring 4-5µm. Hyalinocytes were observed with variety of shapes and measured 8-13µm. The nucleus occupied more of the cytoplasm as the cell matured. Eosinophilic granulocytes are measuring 9-11µm with eccentric nuclei and the cytoplasm contained coarse granules. The size of all the types of haemocytes in creased in the experimental crabs when compared to control.

In the present study, haemotological variations in terms of the number of cells and also the histopathological changes could be noted for this freshwater crab P.jacquemontii with the toxicity of nurocombi. Exposure of P. jacquemontii to increasing sublethal levels of nurocombi induced significant variation in total and differential haemocyte counts. The total numbers of circulating hemocytes were drastically reduced to over control at higher concentration (0.0374ppm) (1162 ± 12.6 to 577 ± 25.1). According to the shape and size and the structure of nucleus in the cell, the proportion of Agranulocytes were found to be 40% Hyalinocytes 25%, Eosinophilic granulocytes 20% and 7% basophilic granulocytes (Figure 1 & Table 1). Exposure of P. jacquemontii to increasing sublethal levels of nurocombi induced significant variation in total and differential counts. The differential number of circulating haemocytes was drastically reduced to over the control at concentration 0.0374ppm at 28 days. Higher nurocombi stress selectively reduced the percentage occurrence of hyalinocytes (25% to 20%), eosiniphilic granulocytes (20% to 12%) and enhanced the percentage occurrence of agranuocytes (40% to 47%) and basophilic granulocytes (7% to 12%) respectively. The hemocytes particularly the hyalinocytes were found to occur in clumps after its exposure to sub lethal concentration of nurocombi, which would be the possible early sign of agglutination. Highly significant and dose related decrease in total haemocyte counts were observed in the crab P. jacquemontii. This haemocytopenic response may be resulted from reduction in the counts of hyalinocytes and eosinophilic granulocytes. More proliferation of other types of haemocytes clearly demonstrated the neoplastic transformation of haemocytopoietic organ.

The reduction in the number of circulating hemocytes was a common pathogenic response in lobsters and crabs experimentally infused with endotoxins [16]. The formation of hemocyte clumps in the blood sinuses may be affected the decline in the number of circulating hemocytes [17]. Total hemocytes count is an indicator of the individual’s defense in crustacean species, as the population of circulating haemocytes may indicate whether the host defense system was activated or not [18]. Total haemocyte count (THC) of exposed crab was significantly reduced. Four major types of hemocytes were detected. The proportion of granule hemocytes was declined in crabs after exposure to nurocombi. Also, some morphological aberrations in haemocyte cells such as irregularity in their shape and ragged cytoplasmic membrane, vaculation in cytoplasm and nucleus, as well as extrusion of nuclear material were observed in exposed crabs.

A gradual decrease of hyalinocytes was noted as the duration proceeds from 7 days to 28 days. Initially the decrease in the higher concentration after 7 days exposure was 22% and it showed a higher decline of 20% after 28 days of exposure. Hb and Hct values were decreased up to 10th day and after that recovered showing a significant increase in Cyprinus carpio exposed to lindane [19]. This decrease may be due to the migration of hemocytes out of the haemolymph circulation to participate in the encapsulation or phagocytosis [20,21]. The agranulocytes were noted to show the increasing trend when compared to that of control. The increase was ranged from 45% on the exposure after 7 days and 47% after 28 days of exposure (Table 1).

Summarizing, light microscopy and histopathological analyses performed in the haemolymph of P. jacquemontii insight into four different types of haemocytes with morphological alterations. In the present study, haemotological variations in terms of the number of cells and also the histopathological changes could be noted for this freshwater crab P.jacquemontii with the toxicity of nurocombi. Exposure of P. jacquemontii to sublethal concentration of nurocombi has induced morphological aberrations in haemocyte shape and ragged cytoplasmic membrane, vaculation in cytoplasm and nucleus, fragmentation of nucleus, degeneration of cytoplasmic membrane and extrusion of nuclear material. Studies on the haemocytes of crustaceans contribute to the accumulation of the basic knowledge on haemocytes especially with regard to the physiological condition of the animal. The histopathological studies elucidate the variations in haemocytes in response to the toxicity of nurocombi in the fresh water crab P. jacquemontii

Authors would like to acknowledge their gratitude to Science and Engineering Research Board, Department of Science and Technology, New Delhi, India (SB/YS/LS/254/2013) for the financial assistance and Head of the Institution, Khadir Mohideen College, Adirampattinam for the facilities provided.

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