Biological and histopathological effects of some insecticidal agents against red palm weevil

The red palm weevil Rhynchophorus ferrugineus is the most important insect pest for the date palm trees in the middle east and Gulf states. The objective of this study was to investigate the pathogenicity of different concentrations of a natural plant extract (neem) and a synthetic insect growth regulators (IGRs) (flufenoxuron) on the histology of the ovary and testis, and on some biological parameters of the red palm weevil. Prepupae were grouped and treated with three different doses of neem extract (Neem J, 50, 100, 500 ppm) and a synthetic insect growth regulator (flufenoxuron, 0.05, 0.1, 0.5 ppm). The present study demonstrates that both natural (Neem) and synthetic (flufenoxuron) IGRs are capable of disrupting growth, development and reproduction in Rhynchophorus ferrugineus. Growth and developmental disruptions are resulted from the production of high mortality rate, reduction in body length, sex ratio, and morphological malformations, in a dose-dependent fashion. The study further reveals that IGRs exert effects on histological structure of gonads, in this way, disrupt gamete productions. IGRs disrupt female gamete production through their actions on the accumulation of yolk granules (vitellogenesis) and follicular epithelial cells. Disruption of male gamete production was detected by disorganization of testicular cysts and depopulation of these cysts in addition to degeneration and necrosis in germ cell lineage.


INTRODUCTION
The red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae), is an economically important, tissue-boring pest of date palm in many parts of the world.One of highest destructive pests in palm plantation, the larvae are responsible for damaging the palm, and once they have gained access, the death of the palm generally ensues.The female weevils lay their eggs on palms mostly in cracks, crevices and wounds.Another important site of pest entry into the palm is at the leaf axil and also from where offshoots emerge.Because of the concealed nature of the larvae, effective methods for the management of the red and other palm weevils have been difficult to develop (Murphy and Briscoe, 1999).The pest has caused large economic losses in date palms worldwide for the last 30 yr (Faleiro, 2006;Murphy and Briscoe, 1999), and still there are no effective control measures.The chemical control of this pest is undesirable, since chemicals lead to the pollution of water courses around areas with palm weevil infestation (Moura et al., 1995;Abuzuhairah et al., 1996).Therefore, safer and yet effective control methods should be sought (Nassar and Abdullah, 2001).Isman et al. (1990) reported that the natural biopesticides offer desirable alternatives to using synthetic chemicals in agricultural systems where protection of the environment and preservation of beneficial organisms are important.One such bio-pesticide of interest is the natural insect growth regulator (IGR), azadirachtin, (one of the most active ingredient of neem seed extract) a botanical compound that can be effective, is biodegradable, and rapidly metabolizes in the environment.This compound is a liminoid that accumulates in the seeds of the neem tree (Azadirachta indica), from which it can be extracted efficiently (Schroeder and Nakanishi, 1987).Beside the natural neem extract, flufenoxuron represents another safe category of synthetic insect growth regulators (IGRs), especially chitinsynthesis inhibitors, because of their selectivity and low toxicity to the non-target organisms (Mitsui, 1985).
Therefore, the present study was designed to evaluate some biological, toxicological and histopathological effects of one synthetic IGR (flufenoxuron, a chitin synthesis inhibitor), and another botanical one, neem extract (containing azadirachtin, one of the active ingredients of the neem tree Azadirachta indica), against prepupal larvae of Rhynchophorus ferrugineus.

The experimental insect
Prepupal stage of the Red palm weevil, Rhynchophorus ferrugineus were collected from diseased date palm tree at Al-Manaief Al-Gharbia region, Ismaileya governorate.They were kept in the laboratory in small plastic cages approximately 5x10x13 cm.The insects were incubated at laboratory temperature of (28±2ºC) and relative humidity (80±10% R.H.).Soon after collection, the prepupae were randomly assorted in groups and topically treated individually with different doses of the plant extract (Neem J) or the insect growth regulator (flufenoxuron).

Bioassay and administration of chemicals:
Two insect growth regulators (IGRs) were used in the present study.One of them is a natural plant extract obtained from the neem tree Azadirachta indica (Meliaceae).Azadirachtin is one of the most important active ingredients of neem seed kernel of this tree.
The other IGR used is the chitin synthesis inhibitor (fluefenoxuron), with the formula of Three dose levels were prepared from neem: 500, 100, 50 ppm.On the other hand, three dose levels were prepared from fluefenoxuron: 0.05, 0.1, 0.5 ppm.For each treatment, prepupae were topically treated with 10 µl of the plant extract or the synthetic IGR.These insecticides were topically applied onto the dorsum of the prothorax.Control insects were topically applied with acetone only.All treated and control insects were checked daily until pupation and then adult emergence.Adults were provided daily with portions of fresh sugarcane stem tissue approximately 0.5x2x2cm.
Bioassay parameters and different criteria were observed and recorded just after the prepupal treatment.

Statistical analysis of data:
Biological and histopathological effects of some insecticidal agents against red palm weevil 9 Data obtained were presented as mean ± standard error of mean (SEM).Differences between every 2 groups were analyzed by the unpaired student's t-test.The difference was considered significant if p < 0.05.All graphs and statistical analysis were performed using Microsoft Excel software.

Histopathological studies:
Ovaries and testes were dissected out and fixed with the appropriate fixative to be processed for hematoxylin and eosin staining Treated larvae and the untreated larvae of the red palm weevils were dissected in Ringer's solution.was isolated and fixed in Bouin's solution then embedded in paraffin.Many sections were obtained and stained with haematoxyline and eosin according to method of Drury and Wallington, (1980).

Bioassay studies: Lethal effects
As shown in table (1) the lethal effect of neem and flufenoxuron was dosedependent, increased with the increasing of the dose.The lethal effect of neem extract on RPW was not obvious in a distinct developmental stage while, the lethal effect of flufenoxuron on RPW was more obvious in the pupal stage.Also, topical application with neem extract resulted in reduction in the number of the emerged adults that exhibit many morphological abnormalities.The highest mortality rates attained 26.7% and 13.3% in treated prepupae with the higher concentration of neem (500 ppm) and flufenoxuron (0.5 ppm), respectively (Table 1).Whereas the highest concentration of neem and flufenoxuron caused 22.2 and 14.3% mortality of adult stage respectively compared to 0% of control group.The lower larval mortality was 6.7 and 6.7% while adult mortality was 7.7 and 0% after treatment of both stages with the lower concentration (50 & 0.05 ppm) of neem and flufenoxuron, respectively.Many authors reported toxic effects of neem tree extracts, such as Azt, against various insect species.Using another neem preparation, NeemAzal (with 20% of Azt content), Ghoneim et al. (2000) recorded various mortality percents among larvae, pupae and adults of the Egyptian cotton leafworm S. littoralis.The latter neem preparation exhibited various degrees of lethality on the house fly Musca domestica which decreased if the concentration decreased below 2000 ppm in the artificial diet of larvae (Mohamed et al., 2000).However, so many results had been reported by several authors for Azt or Azt preparations against different species (Osman, 1993;Osman and Bradley, 1993;Linton et al., 1997).
Flufenoxuron is known to be highly effective, acting against many agricultural pests with a relatively low toxicity to mammals and natural enemies (Su and Scheffrahn, 1996).Flufenoxuron has been tested on some insect species, such as Culex quinquefasciatus and Aedes albopictus (Ho et al., 1990); Spodoptera exempta and S. littoralis (Fisk and Wright, 1992); Blattella germanica (Reid et al., 1992);

Biological parameters effects: Body length
In the present study, topical application of neem extract onto prepupae led to pronounced suppression in the maximal body length.Table 2 and Fig. 1&2 summarizes the previous results, males and females of neem-treated red palm weevils groups were generally shorter and smaller than the control.The difference between males and females was similarly significant in both control and neem-treated weevils, independently from the applied dose of neem.However, in comparison to controls, both male and female body lengths were affected similarly and significantly by neem.
However.these reduction was more obvious in females than in males especially at the higher doses.This was reflected on the growth because its smallest index was calculated by the highest dose-level and vice versa.Also, Azt enhanced the development because the prepupal duration was significantly shortened especially at the higher dose-levels.Lastly, Neem-based products have been reported to cause toxicity and growth regulation, reductions in larval survival and weight, effects in other coleopteran insects (Schluter 1985, Schmutterer and Singh 1995, Trisyono and Whalon, 1999;Weathersbee and Tang, 2002).Emerged control female red palm weevils were longer and greater than males.This statistically difference was kept after treatment with flufenoxuron (Tab.2).However, the difference between males and females was not similar in flufenoxurontreated weevils as in controls.The significance increased on increasing the applied dose of flufenoxuron.This may also show that the effect on males was higher than that on females.In the present study R. ferrugineus, prepupal treatment with flufenoxuron resulted in decreasing of body length after emergence.It is similar to, loss of weight by some other chitin inhibitors, in general, had been reported for several insect species, such as M. domestica (Bakr et al., 1991); Spodoptera exempta, S. exigua, Mamestra brassicae and Galleria mellonella (Smagghe and Degheele, 1994), as well as S. littoralis (Ghoneim et al., 1998).Diflubenzuron, the pioneer of benzoylphenyl ureas, affects the development, among other vital criteria, of several insect species (Soltani et al., 1984(Soltani et al., , 1996;;Basiouny, 2000;Chebira et al., 2000).

Sex Ratio:
As clearly shown from the data in Table (2) that neem affected male more than female during their growth.The number of emerged females were more than males, an effect which did not increase with the increase of neem dose.The results of the three applied concentrations were similar.Also It was observed that after flufenoxuron treatment, the number of emerged females were more than the number of male (tab.2).This effect, in contrast to the results of neem, increased with the increase of flufenoxuron dose.The ratio of females increased on increasing the dose, giving an indication that the mortalities were higher among males than females.These data indicated that neem and flufenoxuron was more affected on male than female emergence.Also, treatment with hexaflumuron caused the same results against pectinophora gossypiella (El-Barkey et al. 2009).

Gonadal histopathological effects Ovary:
As shown in table (3) some ovarian follicles in the germarium of RPW treated with lower concentrations of neem and flufenoxuron revealed atrophy and degeneration (Fig. 6).This effect was severe in some RPW treated with higher concentrations (Fig. 7).It was also associated with massive hyperplasia in the nurse epithelial cells.
In higher concentrations treated RPW, some oocytes appeared degenerative with abnormal distribution of yolk granules and vacuolization of cytoplasm was observed (Fig. 8).This effect was accompanied by moderate hyperplasia in the epithelial layer of nurse cells.Some of those cells were flattened instead of cuboidal.
The most outstanding results found in both of the three concentration treatments were the hyperplasia in the surrounding trophic nurse cells and the decrease in the amount of yolk granules of the mature ovarian follicles.(Fig. 9) (Fig. 10).The above mentioned results were summarized in a semi-quantitative manner in table 3. The table clarifies the graduality of the histopathological effect of different concentrations of neem on the ovary of the red palm weevil.
In the present study, ovaries from newly emerged adult of R. ferrugineus prepupally treated with both neem and flufenoxuron exhibited ovarian developmental retardation.The prominent features of retardation involved increases in oocytes resorption, delay of follicle and oocytes development and destruction of follicular epithelium.The delay of oocytes development included abnormal distribution and size and regression in the accumulation of yolk granules.This may result in a decrease in fecundity and egg viability of the females.The destruction of follicular epithelium involved degeneration, hyperplasia and necrosis in the follicular cells.
On the other hand, the maturation of insect eggs is dependent, among other factors, on the materials taken up from the surrounding haemolymph (Telfer et al., 1981), and by materials synthesized by the ovary in situ (Indrasith et al., 1988).These materials include proteins, lipids, and carbohydrates all of which are required for embryogenesis (Kanost et al., 1990).
Since ovarian protein content was decreased in insect growth regulator diflubenzuron-treated female of C. pomonella (Soltani and Mazouni, 1992), also, significant decrease in ovarian protein content in chlorfluazuron-treated female was reported by Perveen and Miyata (2000) and in RH-0345 and pyriproxylen-treated mealworm pupae (Aribi et al., 2001), therefore, the ovarian and oocyte growth development and vitellogenesis retardation showed in the present study may be due to the alteration in ovarian protein content.This possibility is also suggested by Perveen and Miyata (2000) who suggested the interference of chlorfluazuron with vitellogenesis through the lack of protein in ovarioles.

Testis
In general, the testes of the neem-treated RPW were smaller in size than the control.The testicular follicles were also smaller and narrower, even in healthy appeared follicles.
In RPW treated with lower concentrations of neem and flufenoxuron some testes had narrower follicles with reduction in the cellular content (Fig. 13, 14) , with higher concentrations of neem and flufenoxuron the testicular follicles revealed pronounced disorganization characterized by decreasing of the cellular content less than previous (Fig. 16).The cysts of spermatocytes and spermatogonia were severely reduced in number (Fig. 15).The above mentioned results were summarized in a semiquantitative manner in table 4. The table clarifies the graduality of the histopathological effect of different concentrations of neem on the ovary of the red palm weevil.
The present study revealed that topical application of both Azt and flufenoxuron onto prepupae of R. ferrugineus, resulted in testicular architectural defects, which were dose-dependent, increased with the increasing of the dose.Neem extract had the higher effect.The prominent architectural defects were germ cells degeneration, displacement of testicular cysts and depopulation of germ cells, in addition to the decrease in the number of spermatozoa especially in neem treated weevil.
The observed reduction in the spermatozoal number is in agreement with the study of Linton et al. (1997) who observed poisoning and arrest in spermatogenic meiosis at metaphase I in the testis of S. gregaria treated with azadirachtin.The histological destruction detected in the present study are similar to that demonstrated in different insects treated with other IGRs and botanical extracts (Wilson and Hays, 1969;Younes et al., 1994;Mohamed et al., 2000).

Fig. 1 .
Fig.1.Effect of different concentrations of flufenoxuron (ppm) on the difference in body length (cm) between male and female RPW.Values are expressed as mean ± SEM.Statistical analysis: a = p< 0.00001; b = p<0.001;c = p<0.01.

Fig. 5 :
Fig. 5: Photomicrograph of longitudinal section of a vitellarium of control RPW showing heterogeneous cytoplasm and different sphere of yolk granules.Fig. 6: Photomicrograph of longitudinal section of an ovariole of 50 ppm neem treated RPW showing mature ovarian follicle with epithelial cell hyperplasia, mild decrease in yolk granules, degenerative and necrotic cells.

Fig. 8 .
Fig.8.Photomicrograph of longitudinal section of an ovariole of 0.05 ppm flufenoxuron treated RPW showing a vitellarium with epithelial cells hyperplasia, stratified polygonal shaped cells and unequal distribution of yolk granules.

Fig. 9 .
Fig.9.Photomicrograph of longitudinal section of an ovariole of 0.05 ppm flufenoxuron treated RPW showing small mature ovarian follicle with hydropic degenerative, some pyknotic and necrotic epithelial cells (arrow) and displacement of nuclei of epithelial cells.Note the healthy appearance of the nucleus, N.

Fig. 12 .
Fig.12.Photomicrograph of transverse section of testis of control RPW showing cysts with different stages of germ cells development.

Fig. 14 .
Fig. 14.Photomicrograph of longitudinal section of testis of 500 ppm neem treated RPW showing atrophied testicular follicles with few numbers of spermatogonia (g) spermatocytes (c) and spermatozoa (z) within the cysts.

Table 1 :
Lethal effect of different concentrations of neem & flufenoxuron (ppm) applied topically onto the prepupae of the Red Palm Weevil, Rhynchophorus ferrugineus.

Table 2 :
Effect of different concentrations of neem & flufenoxuron (ppm) applied topically onto the prepupae on the pupal duration, sex ratio and body length of the Red Palm Weevil, Rhynchophorus ferrugineus.