Potential hazards of a chitin synthesis inhibitor diflubenzuron in the shrimp Penaeus kerathurus: biochemical composition of the hemolymph and muscle during the molt cycle.
The use of conventional pesticides in agriculture is the most widespread method for pest control. The intensive use of these neurotoxin insecticides becomes environmentally hostile and ecologically unsafe . In this context, the search for new insect-selective insecticides with minimal ecotoxicological risks is relevant. Insect growth regulators (IGRs) seem promising because of their specific mode of action on insects and their lower toxicity against non-target organisms than conventional insecticides [9,8]. Dimilin (25% Wettable Powder), is a trade formulation of diflubenzuron (DFB) an IGR insecticide belonging to the benzoylphenyl urea derivatives (BPU) which is considered as chitin synthesis inhibitors [5,24,25,26,40,41,42,43]. This insecticide is widely used for several years ago in Algeria against the forest pest insects such as Lymantria dispar L. and Thaumetopoea pityocampa Schiff . Moreover, it has been shown that DFB is a potent IGR for mosquito control [24, 37, 44, 51]. Subsequently, these products present potential hazards to non-target aquatic organisms due to its leaching by surface water might contaminate rivers which diverse their pollutants into the Annaba Bay . It is a well known that these BPU insecticides prevent the molting process by inhibiting chitin formation, thereby causing abnormal endocuticular deposition and abortive molting . Although several reports has evaluated the efficacy of these compounds against insect pests, relatively little information is available concerning their toxicity on non-target organisms. The non-target shrimp Penaeus kerathurus (Forskal, 1775) (Crustacea, Decapoda) has been the subject of several studies including histology, biochemistry, endocrinology and toxicology [7, 16, 27, 39, 40] and data obtained provide an experimental basis to study the secondary effects of IGRs on this shrimp species very widespread in East Algeria and especially in the Bay of Annaba. Previously, we have shown that DFB could cause a decrease in cuticle thickness and structural alterations  due to a reduced amount of chitin in treated cuticles . All these data obtained on this biological model provide an experimental basis to study the impacts of IGRs on this non-target organism.
For environmental risk assessment, a comprehensive understanding on adaptation and/or a recovery is important [11,49,51]. Biochemical constituents like glycogen, protein and lipid are considered as sensitive indicators of metabolic activities .Therefore, the main purpose of the present study is to investigate under laboratory conditions the side-effects of diflubenzuron on the changes of main biochemical components (proteins, lipids, carbohydrates) in the hemolymph and muscle during a molt cycle in a non-target organism, P. kerathurus. This shrimp is abundant in the gulf of Annaba and an economically important species for the local fishery industry. The data obtained may provide insight into risks/hazards of diflubenzuron against crustacean species like P. kerathurus and permit us to evaluate the potential use of this shrimp species as biosensor of coastal marine pollution by these chitin synthesis inhibitor insecticides.
MATERIALS AND METHODS
Collection and rearing of animals:
P. kerathurus were captured by fishermen in a natural site situated in the Bay of Annaba (Mafragh river, 36[degrees] 50' N and 7[degrees]56' E) and transported to the laboratory alive. This site is located about 30 km to the East of Annaba and is far from any source of pollution and expected as a relatively clean site away from pollution sources [18, 19]. Based on our previous study , this site was found not contaminated by the tested insecticide. Prior to exposure, shrimps were acclimated to laboratory conditions for a week by maintaining them in glass aquaria (100 x 60 cm x 80 cm; 8 shrimps per aquarium) filled with sea water (salinity 37 psu; temperature 24-27[degrees]C; photoperiod 14 h of light) with continuous aeration as previously described . Shrimps were daily fed with fresh mussels. Exposed and control shrimps with an average length of 12-14 cm and weight of 12-16 g were used in the experiment.
In crustaceans, the molt cycle refers to the modifications that occur between two successive exuviations and has been subdivided into the following major stages: A (early postmolt), B (late postmolt), C (intermolt) and D (premolt). P. kerathrus individuals were molt staged by microscopic examinations of the uropod setae according to . Under our laboratory conditions, P. kerathurus has a molt cycle of 26,5 [+ or -] 4,5 days and the relative uration of each stage is 13% for A+B, 34% for C, and 52% for D .
Insecticide and treatment:
Dimilin[R] (wettable powder 25% active ingredient, a.i.), a trade formulation of diflubenzuron an insecticide belonging to the benzoylphenyl urea derivatives, was kindly provided by Pr. G. Smagghe (Ghent University, Belgium). The compound was added to the rearing seawater at a final concentration of 1 qg active ingredient/L. This sublethal concentration was chosen according to concentrations tested on several shrimp species [17, 46] and particularly on P. kerathurus [27, 40]. Newly-ecdysed adult shrimps (0-8 h old) were exposed continuously kept under treatment. Control shrimps were reared in sea water only. Control shrimps were reared in seawater only.
The shrimps were sampled at different stages during the molting cycle from control and exposed series. From each individual, 50 mg of tissue (abdominal muscle) and 5 ql of hemolymph were removed and analyzed individually for biochemical estimations following standards methods. Carbohydrates, lipids and proteins were extracted from the same sample according to the procedure of  and quantified as previously described . In brief, quantification of total proteins was carried according to  with bleu brilliant of Coomassie (G 250, Sigma Life Science, Germany) as reagent and bovine serum albumin (Sigma) as standard. Total carbohydrates were determined as described by  using anthrone (Merck, Germany) as reagent and glucose as standard. Total lipids were measured by the sulfo-phospho-vanillin method of  using a lipid solution (Lyotrol, BioMerieux, France) as a standard. All biochemical data are expressed as mg/ml hemolymph or mg/g fresh tissue weight.
Results are presented as the mean [+ or -] SD. The numbers of samples tested per series are given with the results. The comparison of mean values was made by the Student's t -test. All statistical analyses were performed using MINITAB Software (Version 16, Penn State College, PA, USA) and p < 0.05 was considered to be a statistically significant difference.
Biochemical composition of the hemolymph:
In control series, the concentration of hemolymph proteins increased during the molt cycle of P. kerathurus (Table 1). The values recorded at the beginning (stage A) and the end (stage D) of the molt cycle were 24.41 [+ or -] 2.07 and 35.2 [+ or -] 3.15 mg/ml, respectively. This increase was found significant (p=) at stages C and D. In treated series, changes in the concentration of hemolymph proteins during the molt cycle presented a similar profile with marked increases at stages C and D as compared with control series. At stage D, there was a significant difference (p= 0.0265) between control and treated series (p= 0.0265) (35.22 [+ or -] 3.15 mg/ml in control vs 45.07 [+ or -] 6.00 mg/ml in treated series).
The measurement of carbohydrate concentrations in the hemolymph from control series showed a progressive increase from stage A until stage C to reach a maximum of 9.46 [+ or -] 1.84 mg/ml, and decreased thereafter at stage D (8.78 [+ or -] 2.90 mg/ml). In treated series, the carbohydrate concentrations in the hemolymph also peaked at stage C (12.37 [+ or -] 0.96 mg/ml). In addition, the values recorded at stages C (p= 0.0032) and D (p= 0.0001) are significantly higher in treated series as compared with controls (Table 2).
In controls series, the hemolymph lipid concentration presents a peak (10.99 [+ or -] 0.74 mg/ml) that occured at stage C during the molt cycle (Table 3). In treated series the lipid concentrations in the hemolymph increased progressively during the molt cycle to reach a maximum of 14.68 [+ or -] 1.27 mg/ml at stage D. Significant differences were recorded at stages B (0.0038) and D (p= 0.0001) between control and treated series
Biochemical composition of the muscle:
The protein content in the flesh varies through the molt cycle. In control series, the protein content increases from stage A (29.20 mg/g) to stage C (76.80 mg/g), and decline in stage D (66.80 mg/g) (Table 4). The changes in protein amounts in treated series show also an increase in stage C (75.35 mg/g) and a fall in stage D (72.80 mg/g). However, there was no significant difference (p> 0.05) in protein contents between control and treated series at all stage during the molting cycle (Table 4).
Concerning the carbohydrates in the muscle, their levels does not change significantly (p> 0.05) in control shrimps during the molt period (Table 5). The values vary between a minimum of 7.72 mg/g at stage A and a maximum of 8.69 mg/g at stage C. After treatment, carbohydrate contents rise slightly, especially in the stages B and C. There was a significant difference (p= 0.0139) between control and treated series only in the value recorded at stage C.
Examination of the data reported in Table 6 reveals that changes in the lipid levels in the flesh from control series presented a peak at stage C (11.04 mg/g). In treated individuals, the peak of lipid levels peaked at stage B.
In addition, the treatment causes an elevation of lipid levels during the molting cycle when compared with control. However, this increase was significant (p< 0.05) only at stage C.
The intensive use of conventional insecticides to control agricultural pests caused secondary effects on the environment . Moreover, these pesticides contaminate through agricultural run-off to the streams, lakes and ponds during rainy season and adversely affect the non-target aquatic organisms. These detrimental effects have prompted the development of alternative control strategies and environmentally softer chemicals such as IGRs which have minimal ecotoxicological risks rendering them important components in IPM programs . On the basis of the mode of action, IGRs are grouped into three categories: juvenile hormones (JHs) and their analogs (JHAs), also called juvenoids; ecdysone agonists (EA); and chitin synthesis inhibitors (CSIs) or molt inhibitors (MIs) . The IGRs such as chitin synthesis inhibitors seem promising because of their specific mode of action on insect and their lower toxicity against non-target organisms than conventional insecticides . However, there is some concern regarding BPUs with respect to their potential effects on crustacean species . In fact, chitin is the second most important natural biopolymer from the shells of crustaceans in the world .
In crustaceans as in other Arthropods, molting is a complex process essential for growth, metamorphosis and reproduction which is affected by a range of environmental cues and regulated by a cascade of hormonal signals . The nature and the levels of ecdysteroids in hemolymph and ovaries by an enzyme immunoassay using two systems of antibodies reveals the presence of two main hormone: 20- hydroxyecdysone and ecdysone, while the changes in hormonal concentration in hemolymph during the molt cycle shows a single peak that occurred at the beginning of the pre-molt (stage D) and coincides with the apolysis [27, 38]. Lastly, the cuticle secretion was examined and correlations with changes in protein and lipid concentration were defined . Moreover, in a degradation study using an HPLC analysis DFB was found to present a low stability in seawater  than in freshwater .
The exposure of an organism to xenobiotic product can modify the synthesis of certain metabolite and disturb its functionality [22, 32, 45]. Similary, in a contaminated ecosystem biochemical biomarkers can be affected. Thus, biochemical biomarkers evaluated in the barnacle Balanus improvisus (Crustacea: Cirripedia) sampled from both polluted and reference sites in the Patos Lagoon Estuary, Southern Brazil showed pollution and seasonal effects . In this current study the main biochemical constituents (carbohydrates, lipids and proteins) were measured in the hemolymph and muscle of P. kerathurus adults during a moulting cycle. In control individuals, level changes of all constituents present a similar profile with a peak at stage C. These variations are correlated with the principal events of cuticle deposition. As in other crustacean species [28, 30, 36, 46, 47], the secretion of the old cuticle continue from stage A until the end of stage C, while during the stage D, we observe the apolysis, the secretion of the new cuticle and the digestion of the old cuticle . Moreover, protein was the major constituent in the hemolymph of P. kerathurus confirming data reported in other penaeid shrimps such as Fenneropenaeus merguiensis and F. penicillatus .
These variations in the levels of the biochemical constituents during the moult cycle were modified by diflubenzuron in P. kerathurus. In fact, after treatment a slight increase in the concentrations of carbohydrates, lipids and proteins in the hemolymph was observed at the end of the molting cycle (stage D). Similarly, diflubenzuron also resulted in an increase in the muscle contents of carbohydrates and lipids. However, this compound had no significant effect on the protein contents. The carbohydrates, as energy elements play a crucial role in the physiology, while the lipids represent the independent source of energy in insects and are transported via the hemolymph from their site of storage towards the user organs, in particular the vitellogenesis and cuticular synthesis [6, 36]. The increase in the concentrations of the biochemical constituents of the hemolymph, particularly carbohydrates and lipids observed at during the pre- ecdysis period, could be explained by the digestion of the old cuticle releasing proteins and carbohydrate in the hemolymph, and an inhibition of secretion of the new cuticle following diflubenzuron treatment . Previously, we have shown that diflubenzuron reduced the cuticle thickness and altered their structure  due to a decreased amount of chitin in treated cuticles of P. kerathurus . The relative accumulation of carbohydrates and lipids in muscle reduction suggests reduction in glycolytic and lipolitic pathways following the alteration of cuticle deposition.
Several conventional insecticides were tested on crustacean species and were reported to affect the biochemical composition. Thus, endosulfan have been known to influence carbohydrate, protein and lipid contents in Macrobrachium malcolmsonii . Monocrotophos applied to crabs Barytelphusa guerini was reported to decrease the glycogen and protein contents, and to increase the lipid contents . Malathion and glyphosate at sublethal concentrations were found to decrease the contents of selected biochemical constituents (total protein, carbohydrate and lipid) when compared with control Streptocephalus dichotomus preadults . Dimethoate was found to inhibit the acetylcholinesterase enzyme, to induce an oxidative stress and to modify total lipid, carbohydrate and protein content in the isopod species Porcellionides pruinosus .
The current study give information on the nutritive of value of P. kerathurus as evidenced by the determination of the main constituents (proteins, lipids, carbohydrates) of the flesh. Moreover, the exposure of this non-target organism to diflubenzuron, a chitin synthesis inhibitor, was found to affect the levels of selected biochemical constituents in the hemolymph and muscle of P. kerathurus adults during a moulting cycle. These biochemical alterations are correlated with cuticle events and suggested that diflubenzuron can pose side-effects on the non-target shrimp P. kerathurus. Finally, this shrimp species could possibly be used as biosensor of coastal marine and estuarine pollution by these chitin synthesis inhibitor insecticides. Such studies are critically important for better assessing the effects of stressors on organisms, populations and communities in a contamination context of ecosystems .
Received 12 November 2014
Received in revised form 31 December 2014
Accepted 22 January 2015
Available online 25 February 2015
The authors wish to thank Pr. G. Smagghe (Ghent University) for donating the insecticide. This study was financed by the Algerian Fund for Scientific Research and by the Ministry of High Education and Scientific Research of Algeria (CNEPRU project to Pr. N. Soltani).
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Seloua Mounira Morsli, Isma Merad, Mohamed El Hedi Khebbeb and Noureddine Soltani
Laboratory of Applied Animal Biology Department of Biology, Faculty of Sciences, Badji Mokhtar University 23000-Annaba, Algeria
Corresponding Author: Noureddine Soltani, Laboratory of Applied Animal Biology Department of Biology, Faculty of Sciences, Badji Mokhtar University 23000-Annaba, Algeria Fax: + 773387633 E-mail: firstname.lastname@example.org
Table 1: Variations in the concentrations (mg/ml) of total proteins in the hemolymph of P. kerathurus during the molt cycle in control and treated series (m [+ or -] SD; n =3-4; for each stage, means followed by the same letter are not significantly different P > 0,05). Stades Control Dimilin (1 [micro]g/L) P A 24.41 [+ or -] 2.07 a 18.14 [+ or -] 5.09 a 0.1187 B 27.48 [+ or -] 2.91 a 31.20 [+ or -] 8.01 a 0.4953 C 56.90 [+ or -] 2.12 a 62.64 [+ or -] 5.20 a 0.0853 D 85.22 [+ or -] 3.15 a 95.07 [+ or -] 6.00 b 0.0265 Table 2: Variations in the concentrations (mg/ml) of total carbohydrates in the hemolymph of P. kerathurus during the molt cycle in control and treated series (m [+ or -] SD; n = 3-10; for each stage, means followed by the same letter are not significantly different P> 0,05). Stades Control Dimilin (1 [micro]g/L) P A 7.27 [+ or -] 1.42 a 5.36 [+ or -] 0.19 a 0.2309 B 8.45 [+ or -] 0.33 a 8.47 [+ or -] 4.73 a 0.8400 C 9.46 [+ or -] 1.84 a 12.37 [+ or -] 0.96 b 0.0032 D 8.78 [+ or -] 1.90 a 11.37 [+ or -] 2.00 b 0.0274 Table 3: Variations in the concentrations (mg/ml) of total lipids in the hemolymph of P. kerathurus during the molt cycle in control and treated series (m [+ or -] SD; n =3-9; for each stage, means followed by the same letter are not significantly different P> 0,05). Stades Control Dimilin (1 [micro]g/L) P A 8.42 [+ or -] 0.92 a 10.71 [+ or -] 3.13 a 0.2911 B 9.55 [+ or -] 0.57 a 12.62 [+ or -] 1.17 b 0.0038 C 10.99 [+ or -] 0.74 a 12.79 [+ or -] 2.23 a 0.1650 D 9.19 [+ or -] 1.49 a 14.68 [+ or -] 1.27 b 0.0001 Table 4: Variations in the contents (mg/g) of total proteins in the muscle of P. kerathurus during the molt cycle in control and treated series (m [+ or -] SD; n =3-7; for each stage, means followed by the same letter are not significantly different P> 0,05). Stades Control Dimilin (1 [micro]g/L) P A 29.20 [+ or -] 8.60 a 29.45 [+ or -] 4.90 a 0.0810 B 45.75 [+ or -] 9.45 a 40.85 [+ or -] 4.22 a 0.3829 C 76.80 [+ or -] 9.20 a 75.35 [+ or -] 6.25 a 0.2231 D 66.80 [+ or -] 6.30 a 72.80 [+ or -] 5.30 a 0.0752 Table 5: Variations in the contents (mg/g) of total carbohydrates in the muscle of P. kerathurus during the molt cycle in control and treated series (m [+ or -] SD; n =3-6; for each stage, means followed by the same letter are not significantly different P> 0,05). Stades Control Dimilin (1 [micro]g/L) P A 7.72 [+ or -] 1.00 a 7.85 [+ or -] 0.85 a 0.3155 B 8.32 [+ or -] 1.80 a 10.05 [+ or -] 1.25 a 0.1638 C 8.69 [+ or -] 0.74 a 10.67 [+ or -] 1.48 b 0.0145 D 7.83 [+ or -] 1.18 a 6.30 [+ or -] 0.59 b 0.0169 Table 6: Variations in the contents (mg/g) of total lipids in the muscle of P. kerathurus during the molt cycle in control and treated series (m [+ or -] SD; n = 3-6; for each stage, means followed by the same letter are not significantly different P> 0,05). Stades Control Dimilin (1 [micro]g/L) P A 8.84 [+ or -] 2.00 a 9.08 [+ or -] 2.00 a 0.1400 B 10.27 [+ or -] 1.38 a 15.91 [+ or -] 1.07 a 0.0011 C 11.04 [+ or -] 2.99 a 15.01 [+ or -] 3.00 b 0.0428 D 10.88 [+ or -] 3.14 13.02 [+ or -] 2.13 a 0.1548
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|Author:||Morsli, Seloua Mounira; Merad, Isma; Khebbeb, Mohamed El Hedi; Soltani, Noureddine|
|Publication:||Advances in Environmental Biology|
|Date:||Feb 1, 2015|
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