Toxicity and Disruptive Impacts of Novaluron, A Chitin Synthesis Inhibitor, on Development and Metamorphosis of The Olive Leaf Moth Palpita unionalis (Hübner) (Lepidoptera: Pyralidae)

The olive leaf moth Palpita unionalis (Lepidoptera: Pyralidae) is an economic pest of the commercial olive groves in Egypt and different Mediterranean countries. The present study was conducted aiming to assess the effects of Novaluron, a chitin synthesis inhibitor, on survival, growth, development and metamorphosis of this pest. The newly moulted last instar (6th) larvae had been treated with six concentrations (100.0, 10.0, 1.00, 0.10, 0.01 and 0.001 ppm), via the fresh olive leaves, as food. Different degrees of toxicity were recorded on all developmental stages. LC50 was calculated in 0.97 ppm. The somatic weight gain of larvae was drastically reduced and the larval growth rate was severely regressed, regardless the concentration. The larval duration was generally shortened but the pupal duration was remarkably prolonged, in a dosedependent manner. The pupation rate was regressed, especially at the higher four concentrations. The metamorphosis program was impaired, since larvalpupal intermediates had been produced at some concentrations. In addition, the pupal morphogenesis was disrupted, since some pupal deformities had been observed at some concentrations.


I. INTRODUCTION
From the Zoogeographical point of view, the Mediterranean Basin was reported as the original area of the olive leaf moth Palpita unionalis (Hübner)(Lepidoptera: Pyralidae). Now it is an international lepidopterous migratory pest in the tropical and subtropical regions of the Old World [1, 2]. P. unionalis is one of the most dangerous pests of olives in Egypt and other Mediterranean countries [3][4][5][6]. The most important damage of this pest occurs on young trees, nurseries and shoots of old trees [7,8]. The control of P. unionalis on olive trees has relied upon the use of traditional synthetic insecticides [9]. Different pesticides exhibited a good control when applied on the early larval instars [10]. Insecticide residues have been detected in olive oil and in the environment where olives are grown [11]. In addition, the extensive use of conventional insecticides has caused resistant insect strains to emerge [12,13] and serious toxicological problems to humans and the environment [14,15]. Therefore, alternative materials have been initiated recently to minimize the pesticide hazards and introduce of new effective and safer ways and negligible effects on ecosystem.
Over the past four decades, efforts have been made to develop insecticidal compounds with selective properties that act specifically on biochemical sites that are present in particular insect groups but with properties that differ from conventional insecticides [16][17][18]. Insect Growth Regulators (IGRs) belong to a group of compounds which are not directly toxic, but act selectively on normal growth, development metamorphosis and/or reproduction in insects via disrupting the hormonally regulated physiological processes [19][20][21][22][23][24]. Because of their desirable characteristics, such as low toxicity, less environmental pollution, high selectivity, and low impact on natural enemies and people, IGRs are used to control various insect pests [25][26][27]. Several IGRs have been extensively studied for investigating their effects on metamorphosis and reproduction in a number of insect species [28,29]. On the basis of the mode of action, IGRs had been grouped in three categories: (i) Juvenile hormone analogues (JHAs) (also called as Juvenoids), ( inhibitors [30,16,31]. They had been, also, grouped in CSIs and substances that interfere with the action of insect hormones (i.e. juvenile hormone analogues, and ecdysteroids) [32].
CSIs interfere with chitin biosynthesis in insects and thus prevent moulting, or produce an imperfect cuticle [33]. By affecting the hormonal balance, they disrupt several physiological processes in insect body [33]. Also, CSIs are less toxic compounds to the non-target organisms and beneficial biota and have no residual effects [34]. One of the novel benzoylphenyl ureas is the Novaluron. It inhibits the chitin formation on larvae of various insects of different orders [35,36] and exhibits a high toxicity against several dipterous species [37][38][39][40][41][42]. It is, also, a powerful suppressor of lepidopteran larvae [43] and whiteflies [44,45] as well as some species of Hemiptera [46,47] and Coleoptera [48][49][50].  [63]. Larvae were daily provided with fresh olive leaves Olea europaea L, as a food. After the larval stage, the developed pupae were collected and transferred to Petri dishes (5.5×1.4cm). The emerged adults were daily collected and released in plastic jars (3L) provided with cotton pieces, soaked in 10% sugar solution, for feeding, as well as olive twigs ( 20 cm in length) as an oviposition site. After egg deposition, adult males and females were transferred into new plastic jars. The jars of eggs were provided with fresh tender olive twigs fixed in a small bottle containing water, so as to keep the leaves flat and fresh, for feeding of the newly hatched larvae. The fresh tender olive leaves were renewed daily until pupation.

Bioassay of Novaluron.
Novaluron [1-[chloro-4-(1,1,2trifluoromethoxyethoxy) phenyl] -3-(2,6difluorobenzoyl) urea] has the molecular formula: C 17 H 9 ClF 8 N 2 O 4 . It was supplied by Sigma-Aldrich Chemicals. A series of concentration levels of Novaluron was prepared by diluting with distilled water in volumetric flasks as follows: 100.0, 10.0, 1.0, 0.1, 0.01 and 0.001 ppm. Bioassay tests were carried out using the newly moulted last instar (6 th ) larvae. Fresh olive leaves were dipped in each concentration of Novaluron for 5 minutes and air dried before introduction to larvae for feeding. Control larvae were provided with water-treated olive leaves. Ten replicates of treated and control larvae (one larva/replicate) were kept separately in glass vials. The larvae were allowed to feed on treated leaves for 24 hrs. Then, they provided with fresh untreated olive leaves and all biological and physiological parameters were recorded daily. Weight gain: Each individual larva (treated and control) was carefully weighed every day using a digital balance for calculating the growth as follows: Initial weight (before the beginning of experiment) final weight (at the end of experiment).
Growth rate: Growth rate (GR) can be calculated according to Waldbauer [66] as follows: GR = fresh weight gain during feeding period / feeding period X mean fresh body weight of larvae during the feeding period.
Developmental rate: Dempster's equation [67] was applied for calculating the developmental duration, and Richard's equation [68] was used for calculating the developmental rate.
Pupation rate: The pupation rate was expressed in % of the successfully developed pupae.
Deranged metamorphosis: different features of impaired metamorphosis program of P. unionalis were observed as larval-pupal intermediates, pupaladult intermediates or extra moult and calculated in (%). Also, impaired pupal morphogenesis was observed as pupal deformations and calculated in %.
Various features of impaired metamorphosis and morphogenesis were recorded in photos.

Pupal water loss.
Pupal water loss was calculated depending on the data of the initial and final weights of the pupae, as follows: Water loss % = [initial weight -final weight /initial Weight] × 100 4. Statistical analysis of data.
Data obtained were analyzed by the Student's tdistribution, and refined by Bessel correction [69] for the test significance of difference between means.
III. RESULTS

Toxicity and lethal effects.
After treatment of the newly moulted last instar (6th) larvae of P. unionalis with six concentrations of Novaluron (100.0, 10.0, 1.00, 0.10, 0.01 and 0.001 ppm), via the fresh olive leaves, as food, data of toxicity and lethal effect on all developmental stages were distributed in Table ( 14±0.37 and 1.75±0.50 days, at 0.001, 0.01, 0.10, 1.00, 10.00 and 100 ppm, respectively, vs. 3.60±0.69 days of control larvae). Developmental rate of larvae is another parameter indicating an enhancing action of Novaluron, since the treated larvae developed in faster rate than control congeners. As obviously shown in the previously mentioned table, a reversal action of Novaluron was exerted on the developed pupae, since their duration was remarkably prolonged, in a dosedependent manner (9.25±1.98, 9.28±0.75, 9.60±0.54, 10.66±1.52 and 12.50±0.70 days, at 0.001, 0.01, 0.10, 1.00 and 10.0 ppm, respectively, vs. 9.20±0.78 days of control pupae). This prolongation of pupal stage was reflected in a retarded development, i.e., pupae developed in slower rate than that of control pupae (for detail, see Table 2).
Because the pupal death may be due to the desiccation caused by Novaluron, loss of body water was estimated in %. In general, the successfully developed pupae from treated larvae lost more body water than control pupae (28.6, 31.0, 31.0, 31.0 and 38.7%, at 0.001, 0.01, 0.10, 1.0 and 10.0 ppm, respectively, compared to 28.2% of control pupae).
With regard to the effects of Novaluron on metamorphosis and morphogenesis of P. unionalis, data listed in Table (2) exiguously revealed various disruptive effects such as the regressed pupation rate, especially at the higher four concentrations (70,60,80 and 40%, at 0.10, 1.0, 10.0 and 100 ppm, respectively, vs. 100% pupation on control larvae). The metamorphosis program was impaired, since larvalpupal intermediates had been produced at some concentration levels (10, 30 and 10% at 0.10, 1.00 and 10.0 ppm, respectively). Description of these intermediate creatures was provided in Plate (1). Moreover, 10% of pupal-adult intermediates had been produced only at 0.01 ppm (see Plate 2). In addition, the pupal morphogenesis was disrupted, since some pupal deformities had been observed at some concentration levels (12.5 and 20.0%, at 10.0 and 1.0 ppm, respectively). Some malformed pupae had been observed in non-tanned segmented body or segmented body with tanned part and incompletely tanned part, depending on the concentration level of Novaluron (see Plate 3).
As reported in the available literature, LC 50 values of Novaluron and lufenuron against S. litura were determined as 350.45 and 453.78 ppm, respectively [100]; LC 50 of Pyriproxyfen was found to be 0.025% against S. litura larvae [86]; LC 50 of Hexaflumuron against H. armigera was 8.47 mg /L [101]; LD 50 values of RH-5849 and Tebufenozide against E. kuehniella were 0.05 and 0.005 μg/insect, respectively [90]; LC 50 of Methoxyfenozide against Culex pipiens was calculated in 24.54 µg/L [89]; LC 50 of Lufenuron against G. pyloalis was 19 ppm [91] and LC 50 values of Chlorfluazuron, Cyromazine, Lufenuron and Precocene I against C. felis were 0.19, 2.66, 0.20, and 10.97 ppm, respectively [96]. Also, a variation in LC 50 values was reported for Novaluron on S. littoralis, since LC 50 values were 2.71 and 2.65 ppm, after treatment of penultimate instar larvae and last instar larvae, respectively [55]. In the current investigation on P. unionalis, LC 50 of Novaluron was calculated in 0.97 ppm. Thus, LC 50 value depends on several factors, such as susceptibility of the insect and its treated stage or instar, lethal potency of the tested compound and its concentration levels, method and time of treatment, as well as the experimental conditions.
To explicate the recorded toxic effect of Novaluron on larvae, pupae and adults of P. unionalis, in the present study, IGRs exhibit their toxic effects on insects with a mode of action other than that of conventional insecticides. Furthermore, CSIs interfere with the synthesis and/or deposition of chitin on the exoskeleton or other chitinized internal structures, such as the peritrophic matrix, hindering the role of peritrophic membrane in protecting the secreting cells from damage [102,103]. Furthermore, it was suggested that the tested CSI interferes with the transport system of UDP-N-acetyl amine across the membrane [104].
For some detail, the larval deaths of P. unionalis by Novaluron, in the current study, may be attributed to the failure of larvae to moult (lethal moult) owing to the inhibition of chitin formation [105,106], to the inability to shed their exocuticle [107], or to swallow volumes of air for splitting the old cuticle and expand the new one during ecdysis [108]. Also, these larval deaths may be due to the prevented feeding and continuous starvation of the present insect [109].
Although the disturbance of hormonal regulation or the disrupting of normal activity of the endocrine system in insects by IGRs was reported [110,111] and suggested for some mosquito species [35,112], the pupal deaths in P. unionalis, in the present investigation, could not be directly relate to the hormonal activity of Novaluron, but to other causes, such as suffocation, bleeding and desiccation due to imperfect exuvation, failure of vital homeostatic mechanisms, etc. [113]. This suggestion can easily be substantiated since Novaluron exerted a predominant desiccating action on the successfully developed pupae of P. unionalis to lose more body water than control pupae, in the present study. In addition, the adult mortality of P. unionalis after treatment of newly moulted last instar larvae only with 0.01 ppm of Novaluron, in the current study, can be explained by the retention and distribution of this compound in the insect body as a result of rapid transport from the gut of treated larvae into other tissues, by the direct and rapid transport via the haemolymph to other tissues, and/or by lower detoxification capacity of adults against the tested CSI [114].
2. Disturbance of growth and development of P. unionalis by Novaluron.
Depending on the currently available literature, some authors have taking into account the body weight gain by the insect larvae as a valuable indicator for growth [115]. In the present study, both larval weight gain and growth rate had been determined after treatment of newly moulted last (6 th ) instar larvae of P. unionalis with different concentrations of Novaluron. The somatic weight gain of larvae was drastically reduced and the larval growth rate was severely regressed, regardless the concentration. Also, larval duration was generally shortened and the developmental rate of these larvae was enhanced.
On the other hand, the present results of shortened larval duration and enhanced developmental rate of P. unionalis larvae were in agreement with the reported results of shortened larval duration of P. gossypiella after treatment of newly hatched larvae with Methoxyfenozide [99] and other insects, such as Rhynchophorus ferrugineus by Lufenuron and Diofenolan [121], A. ipsilon by Flufenoxuron [122] and Schistocerca gregaria by Lufenuron [123]. On the contrary, the present results disagreed with the reported results of prolonged larval duration of S. littoralis larvae after treatment of penultimate or last instar larvae with by Novaluron [55] and Cyromazine [78]; prolonged larval duration after treatment of 5 th instar larvae of Spodoptera frugiperda with LC 10 and LC 25 of Methoxyfenozide [124] and prolonged larval duration in P. gossypiella after treatment of the first instar larvae with Pyriproxyfen [99].
Lepidoptera belong to the most sensitive groups of insects regarding the growth regulating effects of IGRs. The inhibited growth of P. unionalis by some concentrations of Novaluron, in the current study, may be a result of the blocked release of morphogenic peptides, causing alteration in the ecdysteroid and juvenoid titers [125]. Also, Novaluron may affect the tissues and cells undergoing mitosis [126].
Recently, the developmental duration was prolonged indicating retarded development in some other insects by various IGRs, such as G. pyloalis by Lufenuron [91]; C. pipiens by Methoxyfenozide [89] and N-tertbutylphenyl thenoylhydrazide (ecdysteroid derivative) IJTSRD | May-Jun 2017 Available Online @www.ijtsrd.com [132]; C. cephalonica by Fenoxycarb [94]; P. gossypiella by Lufenuron and Pyriproxyfen [99] and Novaluron [56]; etc. In agreement with those reported results of retarded development, the present study recorded a powerful retarding effect of Novaluron on the development of P. unionalis, since the pupal duration was remarkably prolonged and the developmental rate of pupae was considerably regressed.
In the current study, retarded development of P. unionalis by Novaluron, as expressed in prolonged pupal duration and regressed developmental rate, may be attributed to the indirect interference of this CSI with neuroendocrine organs responsible for the synthesis and release of tropic hormones, like prothoracicotropic hormone (PTTH) [133]. The prolongation of larval or pupal duration may be due to the persistence of juvenile hormone (JH) in the haemolymph where it is only in the absence of JH that ecdysone could be activated and lead to the formation of the next stage [134]. Also, Novaluron may exhibit a delaying effect on the ecdysis and transformation [108]. In particular, the final step of chitin biosynthesis pathway was inhibited by this CSI and the precursor was not converted into chitin leading to a prolongation of developmental duration [112].
The effects exhibited by IGRs on insect metamorphosis may be important from the practical stand-point because they could result in various morphogenic defects as well as mortality [135]. Depending on the available literature, the major symptoms and features of the impaired metamorphosis of an insect after treatment with various IGRs (including CSIs) had been described as reduction of pupation and adult emergence, production of larval-pupal and/or pupal-adult intermediates, deformed larvae and/or pupae and the production of supernumerary larval instars (superlarvae). However, all or some of these features were observed in various insects as responses to the disruptive effects of different IGRs, such as S. littoralis by Chlorfluazuron [136], Triflumuron [72], Lufenuron [105,106], Flufenoxuron [71,72], Methoprene and Fenoxycarb [127]; Novaluron [55] and Cyromazine [78]. Also, some or all of these symptoms of the impaired metamorphosis were recorded after treatment of different insects with several IGRs, such as T. castaneum and T. confusum [137], Liriomyza trifolii [138] and Callosobruchus maculates [139] [140]; Rh. ferrugineus [121] and P. demoleus [79] by Diofenolan; Lobesia botrana by Lufenuron [141]; C. pipiens by Kinoprene [84]; etc.
In the present study on P. unionalis, Novaluron detrimentally prohibited the pupation process, since pupation % considerably decreased, especially at the higher four concentrations. This results was, to a great extent, consistent with those reported results of reduced pupation rate of some insects by various IGRs, such as P. xylostella by Hexaflumuron [142], S. littoralis by Novaluron [55] and Cyromazine [78], G. pyloalis by Lufenuron [91] and Fenoxycarb [93] as well as Encarsia formosa by Pyriproxyfen and Fenoxycarb [24].
In the present study on P. unionalis, the pupal morphogenesis was deranged, since different pupal deformities had been observed, at some concentrations of Novaluron. Some malformed pupae appeared in non-tanned segmented body or segmented body with tanned part and incompletely tanned part, depending on the concentration level of Novaluron. To some extent, similar deranged pupal morphogenesis had been reported for T. castaneum and T. confusum after treatment with Cyromazine [137], Spodoptera frugiperda after feeding of 5 th instar larvae on a diet treated with LC 10 and LC 25 of Methoxyfenozide [124], C. cephalonica after topical application of last instar larvae with Fenoxycarb [94] and P. gossypiella after treatment of the full grown larvae with Novaluron In the current investigation on P. unionalis, Novaluron exhibited a disruptive effect on the metamorphosis program, since larval-pupal intermediates had been produced, after treatment of newly moulted last instar larvae with some concentrations.
This feature of impaired metamorphosis was, also, described as abnormal or lethal pupation [124]. Our result was, to a great extent, in agreement with some of those reported results of disturbed metamorphosis of a number of insect pests by various IGRs, such as H. armigera by Hexaflumuron [101], S. littoralis by Novaluron [55] and Cyromazine [78], C. cephalonica by Fenoxycarb [94] and P. gossypiella by Novaluron [56]. Also, the larval-pupal intermediates were observed after topical treatment of last instar larvae of Spodoptera exempta, Spodoptera exigua, S. littoralis, Mamestra brassicae, Galleria mellonella, Mythimna unipuncta and Spodoptera frugiperda with RH-5849, Tebufenozide or Methoxyfenozide [143,113,116]. Moreover, some pupal-adult intermediates of P. unionalis had been produced only at 0.01 ppm of Novaluron, in the current investigation, as a feature of impaired metamorphosis program. As far as our literature survey could ascertain, no information was available on the production of pupal-adult intermediates.
The production of larval-pupal and pupal-adult intermediates, in the present study on P. unionalis, can be explicated by an inhibitory effect of Novaluron on the DNA synthesis [145] or the chitin biosynthesis and chitin synthase [146]. (4) The molt induction had lethal consequences because the induction of a rapid molt did not provide enough time for the completion of larval-pupal transformation. Thus, the insects molted to nonviable forms between the life stages [147]. Molts induced during the early phase of the last instar produce larval-like individuals, while those formed in the late phase generate pupal-like individuals [148].

CONCLUSION
Depending on results of the present study, it can be concluded that Novaluron exhibited various degrees of toxicity against all developmental stages of P. unionalis, as well as it displayed some disruptive effects on development, metamorphosis and pupal morphogenesis. Therefore, Novaluron may be considered as a promising control agent against this economic pest of the commercial olive groves in Egypt and other olive producing countries as a potential alternative to the conventional pesticides.  [55] K. Ghoneim