Ultrastructural changes in integument of Tribolium castaneum (Coleoptera: tenebrionidae) induced by chitin synthesis inhibitor (IGR) chlorfluazuron

The present study was planned to investigate the histopathological effects of the chitin synthesis inhibitor chlorfluazuron against the 4 larval instar of Tribolium castaneum (Coleoptera: Tenebrionidae) under electron microscope level. Toxicity of chlorfluazuron was evaluated under three different temperatures. The sublethal concentrations LC40 were 2.118, 39.76 and 1.207 ppm, at 20, 29 and 38 C respectively. Toxicity of this compound increased at relatively high temperature. Ultrastructural studies were carried out on the integument of the prepupae treated as larvae with LC40 of chlorfluazuron. The treated samples revealed that exuviae of the old cuticle were existed above the new formed integument, some densely–stained material was deposited in endocuticle, procuticle lamellae were disorganized and partially degenerated. Disappearance of moulting fluid. The hypodermis was destructed and detached from endocuticle. Mitochondria were swollen and fusion. Lysis of lysosomelike bodies.


INTRODUCTION
Tribolium castaneum is common pest found in indoor food storage facilities.Many research studies in which IGRs have been tested against this species and other indoor stored-product pests have involved mixing the chemical with insect diet (Oberlander et al., 1997).With this technique, the IGR is often effective because the earlier an insect is exposed to IGRs in the larval life cycle; the less likely it is to reach the adult stage.
Insect cuticles form an exoskeleton that exhibits only a limited capacity to keep space with body growth because it is a more or less rigid structure due to the presence of chitin and sclerotized proteins.To allow growth and development, insects are therefore periodically forced to replace their old cuticle with a new and looser one during moulting (ecdysis).Ecdysis is initiated by apolysis, the process that separates epidermal cells from the old cuticle by moulting fluid secretion and ecdysial membrane formation.The moulting fluid contains proteases and chitinases enzymes that digest the main constituents of the old endocuticle (Reynolds and Samueles, 1996).Shortly before ecdysis, the moulting fluid, which has accumulated in the apolysial space, is reabsorbed, allowing the recycling of old cuticle components.
Insect growth regulators (IGRs) are third-generation insecticides less toxic and compatible with insect pest management that were developed to reduce the pollution of food and environment.These compounds have a specific mode of action on insects and a lower toxicity against vertebrates than conventional insecticides (Grenier and Grenier,1993).IGR's include compounds that affect moulting and metamorphosis by mimicking juvenile hormone (JH agonists) or usually antagonizing JH activity, ecdysteroid agonists, or by interfering with cuticle formation ,chitin synthesis inhibitors, (Smet et al., 1990;Oberlander et al., 1991 andOberlander et al., 1997).
The chitin synthesis inhibitor benzoylphenylurea (BPU) insecticides prevent the moulting process by inhibiting chitin synthesis, thereby causing abnormal endocuticular deposition and abortive moulting, (Ishaaya, 1990 andDhadialla et al., 2005).Chlorfluazuron is an insect growth regulator inhibiting chitin synthesis and acts as an anti-moulting agent, inhibits biosynthesis of chitin of an important constituent in insect cuticle, loses cuticle elasticity and results in abortive moulting.
The objective of this study were to determine the efficacy, through laboratory bioassays of chitin synthesis inhibitor chlorfluazuron against the fourth larval instar of T. castaneum under different temperatures, and determine some ultrastructural changes induced in the prepupal integument treated as larvae with sublethal dose at 29 • C MATERIALS AND METHODS

Insect colony:
A laboratory colony of the red flour beetle, T. castaneum was maintained for many generations under constant conditions 29 • C ±1 and 65-75% R.H. in the Department of Entomology, Ain Shams University.The rearing medium was wheat flour mixed by weight with Brewer's yeast (19:1, w: w).

Bioassay test:
A preliminary experiment was carried out to determine the sublethal concentrations of chlorfluazuron as a chitin synthesis inhibitor against T. castaneum at different temperatures of 20, 29 and 38 • C. The feeding technique used was according to Oberlander et al., (1997).Fourth instar larvae were fed on treated flour till pupation.A control experiment was also performed using flour alone.Four replicates each containing 25 larva / jar was used for each concentration and for the control.The effect of temperatures on the inhibition of adult emergence by chlorfluazuron at the LC 40 was determined.

Histopathological studies and ultrastructural techniques:
The effect of a sublethal dose LC 40 at temperature 29 • C on the integument of normal and treated prepupae was examined by the use of electron microscope.Samples were prepared one day after pupation.The control and treated prepupae were fixed in 2.5% glutaraldehyde (0.1 M Sorensen phosphate buffer, pH7.2) for 2 hrs at 4 • C.They were rinsed overnight in 0.1 M phosphate buffer: 12.5% sucrose (1:1) and post fixed in 1% osmium tetroxide (prepared in 0.1 M phosphate buffer, pH7.2).Samples were dehydrated through ethanol series, and twice in propylene oxide and then embedded in ERL (Spurr, 1969), as generally described by Smagghe et al. (1996).Ultrathin sections were prepared with an ultramicrotome and stained with uranyl acetate and lead citrate in LKB automatic stainer prior to examination by JEOL electron microscope.Electron microscope studies were conducted at the electron Microscopy Unit, Ain Shams University, Cairo, Egypt.

Statistical analysis
The mortality in susceptibility bioassay was corrected by Abbott's formula, Abbott (1925).F Probit analysis was used to determine the sublethal concentrations as described by Finney (1971).

Susceptibility test:
Larvae of T. castaneum were susceptible to chlorfluazuron at all the tested temperatures (Table 1).However, the Lc40 was 39.76 ppm at 29 • C, lower and higher temperature increase the sensitivity of insects to the (IGR).While the sensitivity of insect much higher at high temperature, LC 40's were 2.118 and 1.207 at 20 • C and 38 • C, respectively.A significant difference (P< 0.05) were found among the sublethal concentrations at each tested temperatures.According to the obtained results, it is clear that chlorfluazuron can be used to control Tribolium castaneum at relatively high temperature.The same conclusion was achieved by El Shazly and Refaie, (2002) in case of (IGR) pyriproxyfen on Culex pipiens.Also, this finding is in agreement with Arthur (2003) who reported that the (IGR) hydroprene may effectively control T. castaneum and T. confusum in confined spaces inside food storage facilities.However, environmental conditions may affect control of these insects.More larvae of both species were arrested in the larval stage and more adults died after they emerged in exposure times conducted at 32 • C and 75% R.H.He also reported that as temperature increase, larvae would be expected to complete development in a shorter time, thereby decreasing the time in which they are exposed to the chemical.Data on emergence inhibition of adult T. castaneum by chlorfluazuron are presented in Table (2).It is noticed that the emergence inhibition from the treatment of survivors with Lc40 generally increased at low temperature.Similarly, decreased adult emergence rate in T. castaneum due to larval treatment with chlorfluazuron was also reported by Degheele, (1990) ;Mostafa, (2002) and Abdel Fattah and Khaled, (2008) as a result of blocking of the imaginal discs or due to deformation of adult chitin.Histology and Ultrastructure of the integument of untreated prepupae.
The integument of untreated prepupae consists of, cuticle, hypodermis(or epidermis), and basement membrane from outside to inside.The cuticle is differentiated into an outer epicuticle and an inner procuticle (exocuticle and endocuticle) with clear lamellae.The hypodermis consists of a single layer of epidermal cells with large nuclei and mitochondria that are scattered through the cytoplasm.In the epidermal cells, there is also, some scattered lysosomelike bodies.The epidermal cells are held together near their apices.At greater distances from the cuticle, were no tightly to each other, and the spaces between them called lateral lymph spaces, Locke, (1991).Gap junctions between epidermal cells probably provide a pathway for the movement of low molecular weight substances, such as hormones.This enhances coordination between cells, reducing any minor differences that might occur due to slight differences in the timing or amounts of hormonal signals carried by the hemolymph (Chapman, 2002).Because the samples examined were in prepupal stage during moulting time.The moulting fluid appeared in the apolysial space between the cuticle and the epidermal cells.Figs.(1, 3, 4 and 5).Histology and Ultrastructure of the integument of treated prepupae.
Histopathological examination of prepupal instar of T. castaneum, resulting from 4 th larval instar fed on Lc40 of chlorfluazuron was illustrated in Figs.(2, 6, 7 and 8).Treated specimen, revealed detachment of the epidermal cells from the endocuticle (Fig. 2).Also, the epidermal cells are not arranged in the form of a single layer epithelium, but rather undistinguishable and almost completely destroyed (Figs. 2 & 8).Ultrastructural examination of the treated specimen also showed absence of moulting fluid in the apolysial space and adherence of the old larval cuticle (exuviae) to the newly formed cuticle, Fig. ( 6).In the endocuticle, some densely -stained material were deposited, and adjacent lamellae were partially digested.Fig. ( 7).On the other hand, chlorfluazuron caused drastic effect on the epidermal cells where the nuclei shrank; vacuoles appeared in the cytoplasm; the mitochondria completely lose their normal form and being swollen and fused and lysis of lysosomelike bodies, Fig.
(8).Liu, (1989) suggested that juvenile hormone III activated the lysosome system on the hypopharyngeal glands of Apis mellifera, which caused cytolysis.Cytolysis could eventually lead to degeneration of the epidermal layer.
In general, the IGR chlorfluazuron interferes with the normal development and disrupts the structure of the different cuticular components in treated prepupae of T. castaneum.The undistinguishable and destroyed epidermal cells may be prevent the pathway of hormonal secretion and thus prevent the coordination between cells.The effect of chlorfluazuron on the treated samples of T. castaneum , may be also explicated by causing an imbalance in the hormone titers at critical times of moulting because the proper balance in the hormone titers is necessary for normal growth and transformation into the pupal and adult stage, and this may be led to failure of epidermal cells to secrete the moulting fluid.In addition, this may induce a great disturbance in the moulting process to produce the new cuticle and shed out of the old one.Similar results have been reported by Abdel Fattal and Khaled (2008).In addition the observed damage of the mitochondria in epidermal cells may lead to a great lack of energy production in the treated samples.All these factors may give some interpretations for the failure of the IGR treated insects to complete their life cycles.These histopathological changes by chlorfluazuron to great extent, agree with the results recorded for the same IGR on Musca domestica Bakr and Hussein (1988).In agreement with our obtained results, for some other IGRs on some insect species as Schistocerca gregaria Bakr et al., (2008), Lucilia cuprina Binnington et al., (1987).
Finally, two conclusions can be reached from this investigation.First, comparison of my results with those obtained with chitin synthesis inhibitors indicated that treatment with chlorfluazuron results at least partly, in similar symptoms.Second, the chlorfluazuron can be used as a larvicidal agent against Tribolium castaneum at relatively high temperature.

Table ( 1
) Toxicity of chlorfluazuron against last instar larvae of T. castaneum at three constant