Physiological and Ultrastructural Alterations in the Crayfish Procambarus clarkii Treated with Spinosad (Bacterial Derived Insecticide)

Physiological and ultrastructural investigations have been carried out on Procambarus clarkii exposed to sublethal concentrations of Spinosad insecticide. The study showed that the highest mortality percentage was 88% for males and 70% for female occurred at 64 × 103 ppm Spinosad. The lethal toxicity (LC50) was 24.1 g/l and 29.8 g/l for males and females respectively. Exposure to 1/2 LC50 (12.07 g/l). Spinosad for 7 days caused many physiological disorders including decrease the haemolymph glucose levels and elevated uric acid levels.12.07 g/l Spinosad resulted in a significant decrease in the total proteins of haemolymph, hepatopancreas and muscles in both males and females. The total cholesterol and triglycerides levels were significantly increased in both haemolymph and hepatopancreas and decreased in muscles of males. On the other hand, cholesterol and triglycerides levels were significantly decreased in haemolymph, hepatopancreas and muscles of females. TEM examinations of male hepatopancreas exposed to 12.07 g/l Spinosad for 7 days revealed ruptured microvilli of absorptive cells, deformed mitochondria, destructed rough endoplasmic reticulum, vacuolated cytoplasm, pyknotic nuclei and appearance of vesicles containing small dark granules.


Introduction
Procambarus clarkii was introduced in the early 1980's into Egypt for aquaculture [1]. They were left without control and had invaded most of the governorates of Upper and Lower Egypt and its distribution has extended from northern Delta to Assiute governorate [2]. Fresh water crayfish can largely prey on snails and mosquitoes thus can be used as a biological control agent [3,4]. Crayfish utilized by man in many countries including Egypt because they are cheaper than the marine prawns [5]. In California, Hawaii, Spain, Japan and Egypt their burrowing activity has caused considerable agricultural damage to irrigation structures such as dams besides eating the roots and shoots of various aquatic crops [6,7]. P. clarkii was known to cause a lot of damage to the fisheries of the Nile possibly by eating the fry and the young fish and damaging the nets of fishermen [8]. The international introduction of P. clarkii into different countries has almost negative consequences, so considerable effort has been paid to control its dispersal with pesticides [9,10]. Pesticides attract public concern due to their potential transport from one environment to another and their effect on non-target biota [11]. The intensive application of chemical pesticides near an aquatic environment may cause hazards to the organisms inhabiting those areas including non-target animals such as fish and shrimp [12]. Spinosad is a bacterial-derived insecticide that contains two active ingredients, spinosyn A and D which are derivatives of the actinomycete, Saccharopolyspora spinosa that occurs naturally in the soil [13,14]. It Kills through contact and ingestion action. Spinosad causes neuronal excitation in insects and after periods of hyper excitation, lice become paralyzed and die [15]. Due to its safety to the environment, mammals, fishes and beneficial insects, Spinosad was registered under the US Environmental Protection Agency reduced risk program [16]. Little works have been carried out on the use of Spinosad in controlling P. clarkii [17]. Therefore, the present study aimed to spot the light on some physiological and ultrastructural alterations in the freshwater crayfish P. clarkii after being exposed to Spinosad.

Collection and acclimation of specimens
Adult P. clarkii were collected by using 0.7 cm diagonal net size from Bany Helal irrigation canal at Sharkia Governorate during April 2017. The collected specimens were transferred alive to the laboratory, where they maintained in glass aquaria (17.5 h × 38.5 l × 23 w cm). The conditions were maintained at 25°C and a 12:12 h light-dark regime. Water was changed every four days. Animals were fed with carrot.
Determination of Spinosad Lc 50 : Stock solutions of Spinosad were prepared by using distilled water as a solvent to give 4 × 10³, 8 × 10³, 16 × 10³, 32 × 10³ ppm. Three replicates per each concentration were used to determine the LC 50 . Ten full mature animals either males and females (9-12 cm) were placed in each aquarium. An equal number from each sex were left without treatment as a control. Experiments were checked at 24 h intervals up to 96 h. The dead crayfish were counted and reported. Lc 50 was determined according to Finney [18] by the graphic method of the curve dose-effect, using the profit analysis.

Chronic exposure
In the long-term exposure, 1/4 and 1/2 of 96 h LC 50 of the tested pesticides was used and redosed every 4 days in a static renewal manner. Living animals, surviving the effect of the tested pesticide were sacrificed after 7 days of exposure.

Haemolymph and tissue sampling
Haemolymph sampling: Haemolymph was obtained from conscious P. clarkii by direct puncture of the heart using a syringe containing EDTA as anticoagulant for subsequent analysis.
Tissue sampling: After decapitation of P. clarkii, pieces of muscle and hepatopancreas were taken for physiological and ultrastructural studies.

Physiological studies
Haemolymph glucose and uric acid levels were measured according to GOD-PAP method [19]. Haemolymph, hepatopancreatic and muscle total proteins were determined according to the method of the Biuret method [20].
Triglycerides and total cholesterol in haemolymph, hepatopancreas and muscle were measured according to the method of Fernandez et al. [21] (Tables 1-4).

Transmission electron microscopy preparations
The dissected hepatopancreas of crayfish exposed to either clean water or 1/2 LC 50 of Spinosad for 7dayswas fixed in 2.5% glutraldehyde in 0.05 M cocodylate buffer containing 0.15 sucrose at pH 7.2 for 2 h. Ultra-thin sections stained with aqueous urinyl acetate and lead citrate and then examined using a JOEL Transmission Electron Microscopic at the regional center for Mycology and Biotechnology in El-Azhar University, Nasr city, Cairo, Egypt.

Statistical analysis
The statistical analysis was performed using SPSS 14.00. The mean values obtained in the different groups were compared by unpaired student's t-test.

Mortality tests of Spinosad on P. clarkii
Effect of different concentrations of Spinosad on mortality percentages of adult males and females P. clarkii at different exposure periods ( Figure 1).

Lethal toxicities (LC 50 ) of Spinosad pesticide
The different LC 50 values of male and female P. clarkii exposed to Spinosad were illustrated in Table 1.

Analytical studies Transmission
Electron Microscopy examinations of hepatopancreas of P. clarkii: Normal hepatopancreas: Transmission electron microscopy examinations have been revealed the presence of three distinct cell types in the hepatopancreas of P. clarkii. These three types are the absorptive cells (Ac), secretory cells (Sc) and fibrillar cells (Fc). The absorptive cell is large columnar with a centrally located nucleus (N). The apical surface of absorptive cell has numerous microvilli (MV). The cytoplasm contains a number of irregularly shaped mitochondria (M) and parallel tubules of rough endoplasmic reticulum (RER) (plate I: a and b) ( Figure  2). The secretory cell is the largest cell type and is characterized by the presence of a basely located nucleus (N) with small nucleolus (NU). The cytoplasm of this cell contain very thin layer of rough endoplasmic reticulum (RER) found in a perinuclear position and small numbers of mitochondria (plate I: c). the fibrillar cell (FC) has a large nucleus (N) with prominent nucleolus (NU) and massive rough endoplasmic reticulum (RER) (plate I: d).

Effect of spinosad
Plate II (Figures 3a-3d) shows electron micrographs of hepatopancreatic cells of P. clarkii after exposure to 1/2 LC 50 (12.07 g/l) Spinosad for 7 days. Microvilli of the absorptive cells become ruptured    and lost their filaments within the cytoplasm, the rough endoplasmic reticulum had the form of short and thin cisternae and also had the form of concentric whorls (Figure 3a). The cytoplasm of absorptive cell lost its density and became granular and vacuolated, vacuoles contain cellular debris are also noted (Figure 3b). The nucleus become pyknotic (PN), heterochromatin appeared as dense masses scattered in the nucleoplasm and as electron dense aggregates along the inner nuclear envelope (Figure 3c). The rough endoplasmic reticulum become fragmented (Figure 3d).

Discussion and Conclusion
The red swamp crayfish, Procambarus clarkii was introduced worldwide and has become the dominant freshwater crayfish in almost all areas it occupies [22]. P. clarkii greatly spread all over the River Nile [10]. Considerable efforts have been paid to control its dispersal with pesticides [23,24]. Spinosad may be used to control agricultural pests. In the present study, toxicological effects of Spinosad on P. clarkii were determined. This study indicated that there is no mortality in males and females P. clarkii at 8 × 10³ ppm Spinosad. The highest mortality occurs at concentration 64 × 10³ ppm while the lowest mortality at concentration 16 × 10³ ppm for both male and females. It is also clear that adult females are more tolerant to toxicity than adult males of P.
clarkii. Lc 50 of Spinosad for 96 h. for P. clarkii has been determined to be 24.1 and 29.8 g/l for adults male and female, respectively. This result disagree with Abdel-Kader [25] who calculated LC 50 of Spinosad for 5 days to be 858 and 4338 ppm for adults male and female P. clarkii, respectively.
Physiological measurements have been used as indicators of the state of animal health condition and as a biochemical method for assessing the possible mode of action of stressors [26,27]. Analyses of haemolymph constituents have proved to be useful in the detection and diagnosis of metabolic disturbances and disease processes [28]. Blood glucose appeared to be a sensitive and reliable indicator of environmental stress [29]. The present study showed clearly that glucose level in haemolymph of male and female P. clarkii was significantly decreased after treatment with Spinosad. This increase may be due to an increase in haemolymph concentration of catecholamines and corticosetroids [30] as a stress response of animal subjected to environmental alterations. This finding is in agreement with Winkaler et al. [31] reported a high plasma glucose levels in the fish Prochilodus lineatus exposed to neem extract. The present study showed clearly that uric acid in haemolymph of male and female P. clarkii significantly increased after treatment with Spinosad. Low values of uric acid have insignificant meaning but increasing values indicate several disturbances in the kidney [32]. This increase in haemolymph uric acid concentrations may be attributed to the action of pesticides as well as oxygen insufficiency which causes pathological changes of the green glands [33]. Moreover, accumulation of pesticide in the green gland which may cause damage of cells followed by an increase in haemolymph uric acid. This finding is in agreement with Hamdi [34] who showed that there is increase in haemolymph uric acid of crayfish P. clarkii after treatment with Malathion. Proteins are important biochemical components and an assessment of the total protein content in haemolymph, hepatopancreas and muscle could be used as a diagnostic tool for determining the physiological status of an organism [35]. This study shows also that Spinosad cause an increase in haemolymph total protein, but they cause a decrease in hepatopancreatic and muscle total protein .This finding is not in agreement with El-Sheikh [36] who reported a decrease in haemolymph total protein of Spodoptera littoralis exposed to Spinosad and he explained that this decrease may be due to inhibition of DNA and RNA synthesis. This finding is also in agreement with Hamdi [34] who reported a decrease in total protein of both hepatopancreas and muscle of crayfish P. clarkii exposed to Malathion and she explained that this depletion may be attributed to the toxic effect of the pesticide, however. The depletion of the total protein levels in haemolymph and the tissues of hepatopancreas and muscle of test crayfish may be due to the enhanced proteolytic activity in these organs under pesticide stress. Also, this increase in haemolymph total protein may be due increased biosynthesis process occurred by the high enzyme stress to withstand pesticide entrances defensive case. This finding is in agreement with Data are represented as means of three samples ± SE, * Significant difference (p ≤ 0.05) **Highly significant difference (P ≤ 0.01) Exposure of mature male and female P. clarkii to 1/2 LC 50 (12.07 g/l) Spinosad for 7 days caused many physiological disorders such as decreasing the haemolymph glucose levels and elevating uric acid levels. 12.07 g/l Spinosad resulted in also a significant decrease in the total proteins of haemolymph, hepatopancreas and muscles in both males and females The total cholesterol and triglycerides levels were significantly increased in both haemolymph and hepatopancreas while decreased in muscles of males On the other hand, cholesterol and triglyceride levels were significantly decreased in haemolymph, hepatopancreas and muscles of females Senthil et al. [37] and Narra et al. [38] reported decreasing in total protein level in haemolymph, hepatopancreas and muscle of freshwater field crabs Spiralothelphusa hydrodroma and Barytelphusaguerini, respectively exposed to Chlorpyrifos.
Cholesterol is a major concern of diet-conscious persons [39]. The present study showed that Spinosad have induced decreasing in total cholesterol and triglyceride concentrations in haemolymph, hepatopancreas and muscle in both sexes. The decrement in the cholesterol and triglyceride may be due to the increased activity levels of lipase, the enzymes responsible for the breakdown of lipid into free fatty acid and cholesterol. This decline may be due to increase hormonal secretions that enhance metabolic rate which in turn reduce the metabolic reserve of the triglyceride [40]. Also, it may be due to the imposition of high energy demands to counter the toxic stress. A decrease in hepatopancreatic and muscle total cholesterol and triglyceride has been reported in P. clarkii exposed to Malathion [34] and this decrease might be attributed to the alteration of the hepatopancreas which were manifested by vacuolar degeneration of the hepatocytes. On the other hand, the increase in cholesterol and triglyceride in haemolymph and hepatopancreas of males may be due to inhibition of lipase activity and other enzymes of lipid metabolism.
In Crustacea, there is a correlation between the physiological condition of the organism and the structure and the appearance of hepatopancreas [41]. The hepatopancreas represents a corner stone in the body metabolism and considered to be the most sensitive organ to pollutants and toxicants [42] and the main site of accumulation and detoxification in crayfish bodies [43]. The present study showed clearly that there are three main cell types forming the digestive tubules of P. clarkii. These cells were the absorptive, secretory and fibrillar cells. These findings are in agreement with Abdelmonem et al. [5]. The obtained results showed that Spinosad caused ruptured microvilli of the absorptive cells, granular and vacuolated cytoplasm; rough endoplasmic reticulum had the form of concentric whorls and nuclear pyknosis. Heterochromatin condensation and marginalization may be due to progressive inactivation of nuclear component [44]. Electron micrographs showed also the presence of vacuoles containing cytoplasmic debris in the cytoplasm. These vacuoles probably arisen to digest the destructed cellular organelles as a result of treatment with the tested pesticides. Asztalos et al. [45] proposed that the focal development of empty vacuoles might be the starting point of cellular autolysis process. The presences of autophagic vacuole in this study improve the detoxification role of hepatopancreatic cells against pesticides. The fragmentation of RER might be a consequence of final hyperactivity prior to cell necrosis [46].