Evaluation of the Potential Side-Effects of Novaluron on the Shrimp Palaemon adspersus: Moulting Hormone Profile, Cuticle Secretion and Chitin Contents

The leaching of a large amount of pollutants derived from agricultural and domestic activities (fertilizers, pesticides, detergents) might contaminate especially the aquatic environments affecting several non-target aquatic organisms such as crustacean species. The current study aimed to evaluate under laboratory conditions the potential side-effects of novaluron (20% Wettable Powder), a potent benzoylurea derivative insecticide on mosquito larvae, against a non-target shrimp, Palaemon adspersus Rathke, 1837 (Decapoda, Palaemonidae). This species is abundant in the lagoon El-Mellah (Northeast Algeria) and a relatively important species for the local fishery industry. The compound was tested at two concentrations (0.91 mg/L and 4.30 mg/L) corresponding respectively to the LC50 and LC90 determined against fourth-instar larvae of Culiseta longiareolata (Diptera, Culicidae). The newly ecdysed adult shrimps were exposed for 15 days, i.e. stage A until D during a moult cycle. Under normal conditions, changes in hemolymphatic ecdysteroid concentrations during the molting cycle presented a peak at stage D, just before the ecdysis while in the treated series, we note an increase in hemolymphatic ecdysteroid concentrations at stages C and D and an absence of the peak as compared to the controls. Histological observations of integuments revealed that novaluron caused a significant reduction in thickness of the new cuticle at its LC50 and an inhibition of the new cuticle secretion at its LC50. The determination of chitin amounts, showed that exposure of shrimps to novaluron resulted in a significant decrease of values at all molting stages with a dose-response manner in comparison to controls. Thus, the overall data confirm the primary mode of action of novaluron on chitin. This insecticide can present secondary effects on this non-target shrimp species commercially important for the local economy.


Introduction
Conventional pesticides are widely used in crop production and very effective against target organisms [1]. So, they are known to make risks and impacts on human health and environment [2]. In this context, several institutions have extensively searched alternatives such as insect growth disruptors (IGDs) with specific mode of action on insect and lower toxicity against non-target organisms than conventional insecticides [3,4]. The IGDs compounds can be grouped according to their mode of action, as follows: substances that interfere with the action of insect hormones (i.e. juvenile hormones, ecdysteroids) and chitin synthesis inhibitors (i.e. of cuticle formation. Among these they are several classes of the chitin synthesis inhibitors, such as pyrimidine-nucleoside peptides, benzoylurea, oxazolines, thiazolidines, tetrazines, thiadiazines, thiophthalimides and certain chromo-and fluorophores [5]. The benzoylurea compounds prevent the formation of chitinous structures and interfere with the molt process which hampers normal development of exoskeleton in many insect orders [6]. During the last decades, an intensive search for more potent benzoylurea derivatives from the prototype compound, diflubenzuron [7], has resulted in synthesis of several Moulting Hormone Profile, Cuticle Secretion and Chitin Contents analogues, such as triflumuron [8], chlorfluazuron [9], teflubenzuron [10], hexaflumuron [11], flufenoxuron [12], lufenuron [13] and more recently, novaluron [14]. Previously, it has been shown that diflubenzuron could affect the cuticle by reducing the thickness and altering their structure [15] due to a decreased amount of chitin in Penaeus kerathurus [16]. An HPLC analysis for residues of diflubenzuron, and has reported that the compound present a low stability in sea water under laboratory conditions [15]. More recently, diflubenzuron affect the levels of different biochemical constituents as proteins, lipids, carbohydrates in hemolymph and muscle during a moulting cycle [17]. Novaluron is a chitin synthesis inhibitor, belonging to the class of benzoylurea insecticide with excellent activity against several important insect pests [18] with a high toxicity level and effectiveness against several mosquito larvae as, Culiseta longiareolata [19] Aedes aegypti [20] and Culex pipiens [21]. It was designated a reducedrisk/organophosphorus alternative as it exhibit low acute mammalian toxicity and no significant subchronic effects in mammals [22,23,24]. So, according to these agencies, novaluron was considered a low risk to the environment and non target organisms. Its use might contaminate rivers which diverse their pollutants into the lakes of El kala (Northeast Algeria) and the Annaba gulf. Therefore, in the present study, we investigate the impact of this compound on a non-target organism, shrimp Palaemon adspersus Rathke, 1837 (Decapoda, Palaemonidae) abundant in the lagoon El-Mellah (Northeast Algeria) and a relatively important species for the local fishery industry. The compound was added to the rearing seawater of newly-ecdysed adult shrimps during a molt cycle. We examine its effects on ecdysteroid profile, cuticle secretion and chitin contents. The data obtained show that this insecticide can present secondary effects on this nontarget shrimp species.

Palaemon adspersus
Rathke, 1837 (Decapoda, Palaemonidae) were collected from the lagoon El-Mellah (Northeast Algeria), in the channel that leads to the Mediterranean Sea ( Figure 1). This site is far from any source of pollution and expected as a relatively clean site away from pollution sources [25,26]. Shrimps were transported to the laboratory alive and reared in laboratory conditions by maintaining them in glass aquaria (100 x 60 x 80 cm) filled with sea water (salinity 37 psu; temperature 22-25°C; photoperiod 14 h of light). Filtration is performed by water filter having a flow rate of 180 1 / h (Rena 225). The animals were daily fed with fresh mussels distributed in the afternoon. Prior to exposure, shrimps were acclimated to laboratory conditions for a week. Shrimps with of similar size (length: 25 mm and weight: 850 mg) were used in the experiment.

Shrimp Datation
The decapod Crustaceans moult cycle is divided into five key stages: A (early postmolt), B (late postmolt), C (intermolt) and D (premolt) and moulting (E). The datation was made according to the method of [28], based on morphogenesis be at the uropod. This method is simple, fast and efficient. Under these conditions, P. adspersus has a molt cycle of 20 days with 20% for A+B, 25% for C, and 65% for D.

Insecticide and Treatment
Novaluron (wettable powder 20% active ingredient), was kindly provided by Pr. G. Smagghe (Ghent University, Belgium) ( Figure 2). The compound was added to the rearing sea water at two final concentrations (0.91 µg/L and 4.30 mg/L) corresponding respectively to the LC 50 and LC 90 obtained with respect to the fourthstage larvae Culiseta longiareolata (Diptera, Culicidae) [19]. Newly-ecdysed adult shrimps (0-8 h old) were exposed continuously to treatment. Control shrimps were reared in sea water only. Samples (hemolymph, cephalothorax and uropod) were collected from each shrimp at different stages of molt cycle (A, B, C, and D) in control and treated series.

Enzyme Immunoassay of Ecdysteroids
Each sample of hemolymph (3 µl) was extracted with methanol by sonication (2-3 min). After centrifugation (5000 g, 10 min), the supernatants were taken and evaporated (60°C). Each sample was resuspended in 500 µl of phosphate buffer (0.1M; pH7.4) and individually analyzed by an enzyme-immunoassay (EIA) according to the method of [29] modified by [30] and previously described [31] using a conjugate of 20-hydroxyecdysone coupled to peroxidase as the enzymatic tracer, tetramethylbenzidine as the colour reagent and a rabbit B polyclonal antibody. Absorbance was read at 630 nm and data was expressed in pg 20E/µl of hemolymph. The tracer and antibodies were kindly provided by Dr. J. P. Delbecque (CNRS, University of Bordeaux I, France) and C. Blaise (Pierre and Marie Curie University, Paris, France), respectively.

Histological Procedure
Uropods were sampled at different stages of moult cycle (A-D), in control and novaluron-exposed series and fixed in formol (10%). After dehydration in serial washes of graded ethanol the samples were passed through three washes in xylene before were embedded in paraffin as according to [32]. Transverse sections of uropod (4 µm) were made using a Leica RM2125T (Leica Microsystems Nussloch GmbH, Wetzlar, Germany) manual rotary microtome and stained with hematoxylin-eosin. Observations were made in a Leica DM500 microscope equipped with a Leica ICC50 HD camera and the thickness of different cuticle was measured with Las EZ Leica software in each series.

Chitin Quantification
Chitin quantification in peripheral integument was performed following the procedure of, previously described [33]. Chitin content was determined at different stages during the molting cycle in control and treated series by quantification of glucosamine derivatives obtained by deacetylation, depolymerisation and deamination of N-acetyl-glucosamine polymer. Briefly, chitin is subjected to an alkaline digestion with KOH (14 M) at 130°C to deacetylate the chitin of each sample, thus forming chitosan. Then a solubilized chitosan solution is depolymerized by NaNO 2 (10%) and KHSO 4 (10%) to liberate the amine residues from the glucosamine, forming a soluble aldehyde. Theses aldehydes generated in a reaction with NH 4 SO 3 NH 2 (12.5%) and with further addition of MBTH and Fe +3 a blue coloration. Absorbance was read at 650 nm and chitin content was expressed as glucosamine equivalents, according to a standard curve made with glucosamine. Weight of cuticle was determined, before chitin quantification to normalize the results.

Statistical Analysis
Statistical analyses were performed using the Prism software version 6.01 for Windows (GraphPad Software Inc., www.graphpad.com). Results are represented as mean ± standard deviation (SD). The homogeneity of variances was checked by Bartlett's test. The linear and non-linear regression was used to establish the reference curves for the determination of chitin and ecdysteroids contents, respectively. Data were subjected to two-way analysis of variance (ANOVA) followed by a post-hoc HSD Tukey test or to a Student's t test at p< 0.05.

Effect of Novaluron on Ecdysteroid Contents
Under normal conditions of P. adspersus, the titers of hemolymphatic ecdysteroids increased during the molt cycle to reach a peak at stage D, just before the ecdysis. The value recorded at the beginning (stage A) and the end (stage D) were 33.48 ± 3.81 and 115.57 ± 2.51 pg/µl, respectively. In treated series by novaluron at the two tested concentrations (LC 50

Effect of Novaluron on Cuticle Secretion
In control series, the thickness of P. adspersus cuticle increased progressively during the three first stages (A, B, C) and decreased at the end of molt cycle (stage D) ( Figure 3A). Cuticle thickness measurement showed that treatment with novaluron at the two tested concentrations (LC 50

Effect of Novaluron on Chitin Contents
The measurement of chitin contents in control series showed a progressive increase from stage A until stage C to reach a maximum of 150.37 ± 6.02 µg /mg and decreased thereafter at stage D (104. 22
In the current study, under normal conditions, hemolymphatic ecdysteroid titers determined by an enzyme immunoassay, vary throughout the molt cycle of P. adspersus. The titers of 20E are low during postmolt (stage AB) and increased progressively in intermolt (stage C). A single peak was recorded in premolt (stage D). It coincides with the apolysis, which results from the destruction of the deep layers of the old cuticle and the beginning of the genesis of the new. In accordance with our results, total ecdysteroid titers in hemolymph vary over the molt cycle in a variety of crustacean species: Orchestia cavimana [40]; Penaeus vannamei [41]; P. kearathurus [31], and Callinectes sapidu [42]. Generally, in crustacean the titers of 20E is low during intermolt and postmolt; during premolt, concentration rise and reach a peak shortly before molting [43,39].
The results obtained after treatment with novaluron, revealed an increase in hemolymphatic ecdysteroid titers with absence of the peak as compared to controls. The increase in hemolymph ecdysteroids is largely due to increased biosynthesis and conversion to active ecdysteroids. Novaluron is known to be very effective against several important insect pests [44,45] and his bioactivity is typically much greater than diflubenzuron and teflubenzuron [14].
Histological study showed a progressive increase of the cuticle thickness during the three first stages (A, B, C) and a decrease at the end of the molt cycle (stage D) in controls. Cuticle thickness measurement showed that novalurontreatment affect the cuticle secretion with a reduction in the thickness of the old cuticle with a dose-response manner as compared to control groups and an inhibition of the new cuticle. The application of chitin synthesis inhibitors typically induces malformations of the cuticle and a significant reduction of chitin amounts [51]. These results showed that the novaluron develop a fragile cuticle unable to support the increased tension during the molting process and the increase in chitin content observed during our experiments may be related to an inhibition of. Indeed, benzoylurea do not directly interfere with catalytic reaction of chitin synthesis, but act on a postcatalytic step [52], blocking the postcatalytic step of chitin synthesis [53]. Our results are consistent with those commonly reported. Indeed, the derivatives of the benzoylurea interfere with the molting process by disrupting cuticle secretion via the chitin synthesis [3,15,54]. Also, ultrastructural analysis revealed abnormal deposition of procuticular layers in response to the treatment with benzoylurea as demonstrated in shrimp P. kerathurus [15], beetles [51].
Chitin a polymer of N-acetyl-b-D-glucosamine, is a major component of the arthropods cuticle. It constitutes up to 40% of the exuvial dry mass depending on the species and varies considerably with the different cuticle types even in a single organism [55]. Chitin is catalyzed by the chitin synthase enzyme from UDP-N-acetylglucosamine precursors [52]. The molting hormone (20E) acts on expression and activity of chitinolytic enzymes, such as chitobiase and chitinase which are involved in exoskeleton degradation and recycling during ecdysis in arthropods [56]. In our experiment, the measurement of chitin contents in controls showed a progressive increase from stage A until stage C and decreased at stage D. These variations were correlated with principal events of cuticle deposition. According to [15], the chitin content varied between 66 and 72% during molting cycle in shrimp P. kerathurus. The same authors reported an incorporation of two precursors, D-[3-3 H (N)]-glucose and N-acétyl-D-[1-3H]-glucosamine (NAGA) in the postmolt (stage A and B) leading to the secretion of endocuticle, followed by a decrease at the intermolt (stage C) (where the secretion of cuticle is complete) and the least content of incorporation of the two precursors is noted in premolt (stage D) where exocuticle secretion is completed. Novalurontreatment increased significantly the chitin content with a dose-response effect probably by inhibit of the incorporation of sugars into the growing chitin chain. This is in accordance with a previous report made with diflubenzuron another chitin synthesis inhibitor on P. kerathurus [19].

Conclusion
In conclusion, the results obtained in this study were the first demonstrating that novaluron exerted negative effects in a shrimp species. It can increase the amounts of ecdysteroids and disrupt the chitin content causing inhibition of cuticular secretion in a non-target organism P. adspersus. These effects could be explained either by a blockage of transport and incorporation of the biosynthetic precursor of chitin, Nacetyl-D-glucosamine (GlcNAc), or directly by inhibition of chitin synthesis. However, these mechanisms of action remained unclear and new experimental approaches are needed. Given the biochemical composition of their cuticle, the crustaceans can be the potential targets of these benzoylurea derivatives.