Biological and behavioral effects of Pyriproxyfen on pheromone production and perception. of Tribolium castaneum (Coleoptera: Tenebrionidae)

Pyriproxyfen (Admiral) is an insect growth regulator (IGR) acts as antijuvenile hormone. The present work aims to investigate the toxicological effect of Pyriproxyfen on both sexes of rust red flour beetle, Tribolium castaneum resulted from treated 4 larval instar with LC50 value (2.4ppm) and its effect on female production and male's perception to pheromone. The results indicated that both treated and untreated sexes could secrete a pheromone that was able to stimulate the opposite sex as well as its own sex. But production and responsiveness of pheromone in untreated groups were significantly higher than treated one. Females secreted a pheromone is a sex pheromone, While the pheromone secreted by males is an aggregation pheromone.


INTRODUCTION
The rust red flour beetle, Tribolium castaneum (Herbst), is one of the serious pests of flour and other cereal products in Egypt and other countries.Mixing chemical protectants with grains is currently one of the main methods for controlling insect pests in such stored products.One promising way to fulfill this need is through the use of insect growth regulators (IGRs).IGR was introduced to describe a new class of biorational compounds.IGRs have very low toxicity to mammals and other non-target organisms and, usually, are rapidly degraded in the environment (Kostyukovsky et al., 2000).These characteristics make IGRs as potential alternatives to conventional insecticides.
Pheromones must be considered a major mode of intraspecific communication in insects that acts to elicit a specific behavioral or developmental response from other organisms of the same species (Nordlund, 1981).
The aim of this study was clarify the possibilities of using IGR (Pyriproxyfen) and sex pheromone in pest control.

MATERIALS AND METHODS Insect colony:
A laboratory colony of the red flourbeetle, T. castaneum was maintained for many generations under constant conditions 30°C and 70% R.H. in the Department of Entomology, Benha University.The rearing medium was wheat flour mixed by weight with Brewer's yeast (95:5, w:w).

Bioassay test:
Fourth larval instar were obtained from the synchronized population reared on flour media, and then transferred to a treated freshly diet for feeding, The feeding technique was used according to Oberlander, (1997).An appropriate stock concentration was prepared in distilled water and mixed with diet.Four replicates were performed for each concentration.A preliminary experiment was carried out to determine the effect of LC 50 of Pyriproxyfen as a Juvenile hormone analogue against 4 th larval instar of T. castaneum.A wide range of concentrations ranging from 0.1 to 10 ppm (0.1, 0.5, 1, 5 and 10 ppm) were used.In addition, a corresponding untreated control was used, Mortality was scored 48 hrs after feeding.A total of 100 beetles were tested per concentration.

Evidance of pheromone production on T. castaneum adult treated as 4 th larval instar by LC 50 of Admiral and untreated one:
The olfactometer used in the present study was a vial type similar to that used by Burkholder (1970).It consisted of a glass vial (15x1.5cm),which had a rubber plug with a movable glass rod.The latter had a broad inner end at which a small piece of masking tape was fixed.The insect tested for pheromone production was held by the masking tape, while that tested for response was placed on the bottom of the vial.The distance between the two insects was 4 cm.
Ten replicates each one contains 10 vials and in each vial two individuals (male and female) were placed separately.The tested males and females were 8-10 days old.
Hexane was the solvent used for extracting pheromone in the following experiment at (0.3) female equivalents (FE) per 10μ of solvent according to Hussien (1982).

Statistical analysis
The results obtained were evaluated using one way analysis of variance "ANOVA" (Snedecor, 1971) and t-test on origin Pro.Lab (version 7.5) statistical program at one level of significance (P< 0.01).
The percentage of adult emergence decreased with the increase of concentrations showing 90, 86, 65, 52 and 46 decrease.The present results indicated that there was gradual inhibition for adult emergence percentages of 10, 14, 35, 48 and 54 with the tested concentrations of 0.1, 0.5, 1, 5 and 10 ppm.Similar effects were obtained by Hitoshi et al. (1989) on cockroach, Miyamoto et al. (1993)  The LC50 value of the tested IGR Pyriproxyfen (Admiral) against the 4 th larval instar of T. castaneum was 2.4ppm.Latent effect of Admiral on larval, pupal and adult malformation of T. castaneum treated as 4 th larval instar.Liu (2003) on Thrips tabaci who thought that reduction of hatchability could be caused by sterilizing eggs, reducing survival of viable eggs, or reducing fecundity of the adults.This reduction may be also due either to an effect on some later steps in the differentiation and function of follicular cells (Gelbic and Sehnal, 1973) or to derangement of humoral control of oviposition (Matalin and Gelbic, 1975).

Morphological abnormalities Larvae affected by Admiral
Failures of larval developmented events as a result of treatment with Admiral are shown in Plate (1) as larva was dark with wing buds on both sides and also larva was dark and swollen in whole body, these abnormalities due to Admiral interfere with JH biosynthesis and consequently prevent larval development to adult stage.

Pupae affected by Admiral after treatment 4 th larval instar
Gradation in morphogenic changes in pupae is shown in Plate (2) such as abnormal pupal appearance with stretched, transparent wings, also, Larval-pupal monstrosity with larval cuticle patches and shrinkage in pupal body.

Adults affected by Admiral after the 4 th larval instar treatment
Symptoms of adult emergence failure induced by feeding of larvae on Admiral are illustrated in Plate (3) such as adult with transparent elytra and with longed, stretched hind wing.Effect of LC 50 (2.4ppm) of Admiral on responsiveness and production of pheromones in male and female adult beetles which resulted from treated 4 th larval instar: Evidence of pheromone production: -Results on the response of treated virgin females and males of T. castaneum, to pheromone produced by either treated sexes (8-10 days old), under constant conditions of 30°C and 70 %R .H. are given in Table (3).

Male response behavior to female:-
The level of response 34.78 % was reached when treated males were tested against treated females.
While in untreated one and used solvent only the response reach 78.26 and 8.00 %, respectively.
The response behavior of treated male beetles to treated female consisted of a sequence of increasing levels of excitation similar to that resulted from adults produced by treated 4 th larval instar by Atabron (Bakr et al. 2010).Male response behavior to male:-Treated males also responded at a level of 21.74 % to treated male beetles.but response of untreated one and used solvent only were 63.04 and 08.00 %, respectively.The response of treated male beetles to their own sex also consisted of a sequence of events which is similar to that resulted from adults produced by treated 4 th larval instar by Atabron (Bakr et al. 2010).

Female response to female:-
Treated females tested against their own sex showed a level of 12.77 % of response.While response of untreated one and used solvent was only 51.06 and 6.00 %, respectively.
The treated females exhibited a sequence of events similar to that resulted from adults produced by treated 4 th larval instar by Atabron (Bakr et al. 2010).

Female response behavior to male:-
The level of response 08.51 % was reached when female beetles were tested against males but response of untreated one and used solvent only were 38.30 and 6.00 %, respectively.In this case, females also exhibited a sequence of events similar to those mentioned in male response behavior to male.
The present study indicates that virgin female adults of T. castaneum produce the sex pheromone.Sex pheromone-producing females have been reported for another untreated related species (Oceallachain and Ryan, 1977 on T. confusum).
The results indicated that both treated and untreated sexes of the rust red flour beetle could secrete a pheromone that was able to stimulate the other sex as well as its own sex.But responsiveness and production of pheromone in untreated groups were significantly higher than treated one.The degree of response varied according to the source of pheromone.Thus, females secreted a pheromone that stimulated and highly excited males more than females.Thus the female pheromone appeared to be a sex pheromone.On the other hand, the pheromone secreted by males seemed to be an aggregation pheromone and both sexes were affected by this pheromone for aggregation.Results obtained in the present study are in agreement with those results obtained by (Narayanan and Nadarajan, 2005 in Antigastra catalaunalis and Ruther et al., 2007

Pheromone extraction by different solvent:-
The obtained results on the response of males of T. castaneum to extracts by different solvents (hexane, diethylether, acetone and chloroform) of virgin females are given in Table (4).
According to the percentage of treated male response to extracts of treated virgin females, the tested solvents could be arranged descendingly in the following manner: hexane 32.61 %, diethylether 30.85 %, acetone 25.00 % and chloroform 18.37 %.While in untreated one, the response reach 76. 09, 72.34, 62.50 and 61.22 %, respectively.Statistical analysis of the data indicated that the difference in response between extracts by either hexane and diethylether, or between acetone and chloroform was not significant and the difference between the two groups of solvents was significantly different at both treated and untreated case.
The present study proved that hexane is the most effective solvent.This was found true by Abdel Kader et al. (1986b) on untreated T. castaneum.

Effect of pheromone concentration on male response:-
The response of males of T. castaneum to different pheromone concentrations or titers of virgin females is given in Table (5).According to the percentage of male response to different concentrations of virgin females extracts, the obtained results showed that the male response increased progressively and significantly with the increase of pheromone titers at both treated and untreated case.
The treated male response started with low level 10.87 % at 0.1 female equivalent and increased with the increase of female equivalents to reach the maximum level of response of 51.11 % at 0.9 female equivalent.While in untreated one the lowest response was 47.83 % and the highest response was 88.89 %.
Fig. (1) declared that larval, pupal and adult malformation percentages had a positive relationship with the different concentrations of Admiral except with percentage of adult malformation at 5 ppm.These data are similar with those obtained by Kostyukovsky et al. (2000), Mojaver and Bandani (2010) on Eurygaster integriceps and Boina et al. (2010) on 5 th larval instar of Diaphorina citri.

Table 2 :
Biological activity of Admiral against Tribolium castaneum, treated as 4 th larval instar.

Table 3 :
Response of virgin Tribolium castaneum males and females (8-10 days old) to adults of both sexes produced by treated 4 th larval instar by LC 50 of Admiral.

Table 4 :
Efficiency of different solvents in sex pheromone extraction of (8-10 days old) virgin Tribolium castaneum females produced by treated 4 th larval instar by Admiral.

Table 5 :
Response of male Tribolium castaneum to pheromone concentrations (female equivalents) of virgin females, (Both sexes were 8-10 days old) produced by treated 4 th larval instar by Admiral.