Efficiency of the chitin synthesis inhibitor lufenuron ( cga-184699 ) on growth , development and morphogenesis of Schistocerca gregaria ( orthoptera : acrididae )

This paper deals with the objective of investigating the effects of Lufenuron (CGA-184699) on the growth, development and morphogenesis of the desert locust Schistocerca gregaria. Five concentration levels (1000, 500, 250, 125 and 62.5 ppm) were given through the fresh plant food to the newly moulted (4 or 5 ) instar or late-aged 5 instar nymphs. All results were obtained 24 h after feeding. The growth of Lufenuron-treated nymphs was profoundly inhibited because their weight gain was drastically reduced. Such reducing action of Lufenuron was dose-dependent after treatment of 4 instar nymphs but at the higher two concentration levels after treatment of newly moulted 5 instar nymphs and at the higher three concentration levels after treatment of late-aged 5 instar nymphs. After treatment of the last instar nymphs, early or late-aged, the developmental rate detrimentally regressed as a response to the prohibiting action of Lufenuron on the development. In contrast, such rate was promoted during significantly shortened duration after treatment of 4 instar nymphs, may be to accelerate the development for avoiding additional adverse effects of Lufenuron. Lufenuron exhibited an inhibitory effect on the adult emergence after treatment of last instar nymphs, regardless of the timing of treatment. On the other hand, Lufenuron exerted no effect on this vital process after treatment of 4 instar nymphs. Moreover, the present compound, at certain concentration levels, induced the adults to emerge in a rate more than that on control congeners. However, the emerged adults suffered a morphogenic action of Lufenuron because different deformed females were produced in increasing % as, at least, the higher three concentration levels were increased. In addition, the adult females spent only shortened longevity and then died. The shortening effect was dose-dependent after treatment of 4 instar nymphs. Kew Words: Schistocerca gregaria, Lufenuron, growth, development, emergence, morphogenesis, longevity. INTRODUCTION Because the use of insecticides for controlling insect pests has several disadvantages to various environmental aspects, including human health and economics, numerous institutions have extensively searched alternatives such as insect growth regulators (IGRs) including juvenile hormone analogues (JHAs), chitin synthesis inhibitors (CSIs) and ecdysteroids (for reviews, see: Post and Vincent, 1973; Slama, 1974; Staal, 1975; El-Ibrashy, 1984; Hoffman and Lorenz, 1998; Tunaz and Uygun, 2004). Retnakaran et al. (1985) identified benzoylphenyl ureas (BPUs), JHAs (or juvenoids), antijuvenoids and miscellaneous IGRs as 4 distinct classes of growth Bakr, R.F. et al. 42 regulators. The BPUs have been subjected to intensive investigations because of their commercial importance and their interference with the moulting and other physiological processes (Soltani et al., 1993; Casida and Quistad, 1998; Ghoneim et al., 2006). The present investigation deals with various growth and developmental impacts of the CSI, Lufenuron (CGA-184699) on the desert locust Schistocerca gregaria. MATERIALS AND METHODS Experimental Insect: A gregarious stock culture of Schistocerca gregaria (Forsk.) was raised by a sample from the established culture of Locust and Grasshopper Res. Division, Agric. Res. Center, Giza, Egypt. The insects were reared under crowded breeding conditions outlined by Hunter-Jones (1961) and Hassanein (1965). Newly hatched hoppers were kept in wooden cages with wire-gauze sides (40x40x60 cm) and small door in the upperside to allow the daily feeding and cleaning routine. The bottom was covered with 20 cm layer of sterilized sand. Each cages was equipped internally with 60 W electric bulb for lightening (17:7 LD) and warming (32±2 C.). The relative humidity varied from 70-80% following the introduction of fresh food plant to 60-70% several hours later. Successive generations were raised before obtaining the nymphs for the present experimental work. Fresh food plant was clover Medicago sativa along the period of study except few weeks every year because of the absence of this plant species. During these weeks, insects were fed on Sesbania eagyptiaca. All experiments were conducted with M. sativa only. Lufenuron Preparation and Administration: Five concentration levels of the chitin biosynthesis inhibitor Lufenuron were prepared using the distilled water: 1000, 500, 250, 125 and 62.5 ppm. A technical concentrate 10% of Lufenuron ( Match, CGA-184699 ) was used. Its chemical formula is: N-{{{ 2,5-dichloro-4-(1,1,2,3,3-hexafluoro-propoxyl)-phenyl} amino}2,6-difluorobenzamide (CA)}}}. The concentration range was chosen depending on some preliminary trials carried out on the present insect species. Feeding technique was applied using fresh clean clover leaves (M. sativa) after dipping for 3 minutes in each concentration level. Feeding on treated food plant was allowed for 24 h for the newly moulted penultimate (4) instar, newly moulted last (5) instar or late-aged (5day old) last instar nymphs. Control insects had been allowed to feed on untreated food plant and kept under the same laboratory conditions. Three replicates (10 nymphs/rep.) were carried out for each treatment. Each individual nymph was kept in a suitable glass vial provided with a thin layer of sterilized sand. The vials were carefully located in a cage supported with a suitable electric bulb for lightening and warming. Criteria Studied and Calculations: Growth, development, metamorphosis and morphogenesis were determined as detailed herein. The fresh body weight was recorded every day using an electric digital balance. The weight gain was calculated as follows: initial weight (before the beginning of experiment) – final weight (at the end of experiment). The growth inhibition was calculated as follows: {aA/A}x 100, where: a: maximal weight of treated nymphs, A: maximal weight of control nymphs. The developmental duration of nymphal instars, treated or control, was estimated using Dempster' equation (1957). The developmental rate was calculated according to the equation: Developmental rate = 100/mean duration (in days). The adult emergence was estimated in % Efficiency of lufenuron (cga-184699) on morphogenesis of Schistocerca gregaria 43 whatever the morphogenesis was perfect or defected. All morphogenic aberrations were counted, calculated in % and recorded in photographic plates. The adult longevity comprised all adult physiological phases: maturation period, reproductive lifetime and post-oviposition period. It was calculated in mean days ±SD. Statistical Analysis : Data obtained were analyzed using the Student t-distribution and were refined by Bessel's correction (Moroney, 1969) for testing the significance of difference between means. RESULTS Treatments of the newly moulted penultimate instar nymphs: a) Growth and Developmental Effects of Lufenuron: Assorted data in Table (1) obviously show the enormous action of Lufenuron on growth and development of S. gregaria. The nymphal growth was prohibited since the weight gain pronouncedly decreased as the concentration level (concentration level) was increased. For some details, while nymphs gained 0.281±0.113 g (not significant) at the lowest concentration level, the reduction of their weight gain increased until 0.120±0.01 g ( p< 0.001) at the highest concentration level . Another parameter may be informative, because the somatic growth change% of control nymphs was recorded as 83.80±9.68% vs 76.70±12.15 and 25.14±12.14% at 62.5 and 1000 ppm, respectively. In addition, the highest growth inhibition% (35.82%) was calculated after treatment with the highest concentration level of Lufenuron. The developmental rate, on the other hand, was undergone to the action of Lufenuron , but in a reverse correlation. As for example, it was 11.76 at 62.5 ppm and 14.49 at 1000 ppm (vs 10.52 of control congeners). Depending on these data, Lufenuron promoted the nymphal rate of development through shortened time intervals (e.g., 6.90±1.03 days at 1000 ppm, vs 9.50±1.15 days of control congeners). Table (1): Growth and developmental effects of Lufenuron on Schistocerca gregaria after treatment of the newly moulted penultimate instar nymphs. Conc. (ppm) Mean weight (g ± S.D.) Weight gain


INTRODUCTION
Because the use of insecticides for controlling insect pests has several disadvantages to various environmental aspects, including human health and economics, numerous institutions have extensively searched alternatives such as insect growth regulators (IGRs) including juvenile hormone analogues (JHAs), chitin synthesis inhibitors (CSIs) and ecdysteroids (for reviews, see: Post and Vincent, 1973;Slama, 1974;Staal, 1975;El-Ibrashy, 1984;Hoffman and Lorenz, 1998;Tunaz and Uygun, 2004).Retnakaran et al. (1985) identified benzoylphenyl ureas (BPUs), JHAs (or juvenoids), antijuvenoids and miscellaneous IGRs as 4 distinct classes of growth regulators.The BPUs have been subjected to intensive investigations because of their commercial importance and their interference with the moulting and other physiological processes (Soltani et al., 1993;Casida and Quistad, 1998;Ghoneim et al., 2006).The present investigation deals with various growth and developmental impacts of the CSI, Lufenuron (CGA-184699) on the desert locust Schistocerca gregaria.

MATERIALS AND METHODS Experimental Insect:
A gregarious stock culture of Schistocerca gregaria (Forsk.)was raised by a sample from the established culture of Locust and Grasshopper Res.Division, Agric.Res.Center, Giza, Egypt.The insects were reared under crowded breeding conditions outlined by Hunter-Jones (1961) and Hassanein (1965).Newly hatched hoppers were kept in wooden cages with wire-gauze sides (40x40x60 cm) and small door in the upperside to allow the daily feeding and cleaning routine.The bottom was covered with 20 cm layer of sterilized sand.Each cages was equipped internally with 60 W electric bulb for lightening (17:7 LD) and warming (32±2 C.).The relative humidity varied from 70-80% following the introduction of fresh food plant to 60-70% several hours later.Successive generations were raised before obtaining the nymphs for the present experimental work.Fresh food plant was clover Medicago sativa along the period of study except few weeks every year because of the absence of this plant species.During these weeks, insects were fed on Sesbania eagyptiaca.All experiments were conducted with M. sativa only.

Lufenuron Preparation and Administration:
Five concentration levels of the chitin biosynthesis inhibitor Lufenuron were prepared using the distilled water: 1000, 500, 250, 125 and 62.5 ppm.A technical concentrate 10% of Lufenuron ( Match, CGA-184699 ) was used.Its chemical formula is: N-{{{ 2,5-dichloro-4-(1,1,2,3,3-hexafluoro-propoxyl)-phenyl} amino}-2,6-difluorobenzamide (CA)}}}.The concentration range was chosen depending on some preliminary trials carried out on the present insect species.Feeding technique was applied using fresh clean clover leaves (M.sativa) after dipping for 3 minutes in each concentration level.Feeding on treated food plant was allowed for 24 h for the newly moulted penultimate (4 th ) instar, newly moulted last (5 th ) instar or late-aged (5day old) last instar nymphs.Control insects had been allowed to feed on untreated food plant and kept under the same laboratory conditions.Three replicates (10 nymphs/rep.) were carried out for each treatment.Each individual nymph was kept in a suitable glass vial provided with a thin layer of sterilized sand.The vials were carefully located in a cage supported with a suitable electric bulb for lightening and warming.

Criteria Studied and Calculations:
Growth, development, metamorphosis and morphogenesis were determined as detailed herein.The fresh body weight was recorded every day using an electric digital balance.The weight gain was calculated as follows: initial weight (before the beginning of experiment) -final weight (at the end of experiment).The growth inhibition was calculated as follows: {a-A/A}x 100, where: a: maximal weight of treated nymphs, A: maximal weight of control nymphs.The developmental duration of nymphal instars, treated or control, was estimated using Dempster' equation (1957).The developmental rate was calculated according to the equation: Developmental rate = 100/mean duration (in days).The adult emergence was estimated in % whatever the morphogenesis was perfect or defected.All morphogenic aberrations were counted, calculated in % and recorded in photographic plates.The adult longevity comprised all adult physiological phases: maturation period, reproductive lifetime and post-oviposition period.It was calculated in mean days ±SD.

Statistical Analysis :
Data obtained were analyzed using the Student t-distribution and were refined by Bessel's correction (Moroney, 1969) for testing the significance of difference between means.

Treatments of the newly moulted penultimate instar nymphs: a) Growth and Developmental Effects of Lufenuron:
Assorted data in Table (1) obviously show the enormous action of Lufenuron on growth and development of S. gregaria.The nymphal growth was prohibited since the weight gain pronouncedly decreased as the concentration level (concentration level) was increased.For some details, while nymphs gained 0.281±0.113g (not significant) at the lowest concentration level, the reduction of their weight gain increased until 0.120±0.01g ( p< 0.001) at the highest concentration level .Another parameter may be informative, because the somatic growth change% of control nymphs was recorded as 83.80±9.68%vs 76.70±12.15and 25.14±12.14%at 62.5 and 1000 ppm, respectively.In addition, the highest growth inhibition% (35.82%) was calculated after treatment with the highest concentration level of Lufenuron.
The developmental rate, on the other hand, was undergone to the action of Lufenuron , but in a reverse correlation.As for example, it was 11.76 at 62.5 ppm and 14.49 at 1000 ppm (vs 10.52 of control congeners).Depending on these data, Lufenuron promoted the nymphal rate of development through shortened time intervals (e.g., 6.90±1.03days at 1000 ppm, vs 9.50±1.15days of control congeners).

b) Metamorphosing and Morphogenic Effects of Lufenuron:
Just a look at the data of Table (2), the eclosion of treated adults had astonishingly exceeded that of control congeners.This metamorphosing event was unexpectedly promoted by Lufenuron and increased by increasing concentration level .Whatever, several grades of affected morphogenesis were recorded.The %s of malformed adults increased as the conc.level was ascended, especially the higher three ones (14.2, 28.5 & 40.0% at 250, 500 % 1000 ppm, respectively).Various symptoms or shapes of deformation are shown in Plate (1), such as: incomplete eclosion with twisted legs and curled wings.It is easy to say all these adult derangements might be due to the morphogenic action of Lufenuron (for more details, see Table 2).Not only the adult morphogenesis and metamorphosis but also the adult longevity was pronouncedly affected by Lufenuron Those successfully emerged adults lasted short longevity and then died.Generally.longevity was found in a reverse correlation with the concentration level of Lufenuron .

Treatments of the newly moulted last instar nymphs: a) Growth and Developmental Effects of Lufenuron:
By the careful examination of the data given in Table (3), it can be concluded that Lufenuron considerably affected the nymphal growth and development.The nymphal weight gain was reversely correlated to the conc.level.It was deduced as 0.515±0.263and 0.497±0.138g at the two lower concentration levels, while as 0.362±0.131and 0.128±0.102g at the two higher concentration levels .
The change% supported the previously mentioned finding (78.86±18.7 and 38.56±15.36% at 62.5 and 1000 ppm, respectively, vs 85.71±16.46% of control congeners).In addition, the highest growth inhibition was found as 23.39 % at the highest concentration level of Lufenuron.Efficiency of lufenuron (cga-184699) on morphogenesis of Schistocerca gregaria 45 Data of the same table clearly provide a dissimilar action of Lufenuron on the nymphal developmental rate, since increased by increasing concentration level (9.90 and 12.82 at 62.5 and 1000 ppm, respectively , vs 9.17 of control congeners ).That is to say, Lufenuron hastened the nymphal development through shortened durations (10.1±1.10 and 7.8±1.95days at the same mentioned concentration levels, respectively , vs 10.9±0.98 days of control congeners ).

b) Metamorphosing and Morphogenic Effects of Lufenuron:
Data assorted in Table ( 4) demonstrate the possible effects of Lufenuron on the adult performance of S. gregaria after treatment of the newly moulted last instar nymphs.Firstly, the adult emergence was partially blocked.This blockage was seen parallely to the concentration level reaching 40% (at 1000 ppm) in comparison to 100% of control adults.Aside with the metamorphic action of Lufenuron, it remarkably affected the adult morphogenesis.The highest % of adult deformity was recorded at the higher two concentration levels .Also, various symptoms of such deformation can be easily observed in Plate (1).
Moreover, the emerged adults survived only a shortened longevity which could be attributed to the death-accelerating action of Lufenuron .The shorter longevities were 37.00±3.96and 37.50±4.60days at 1000 and 500 ppm, respectively (compared to 48.30±2.55days of control adults).

Treatments of the late-aged last instar nymphs: a) Growth and Developmental Effects of Lufenuron:
After treatment of the late-aged last instar nymphs, the obtained results were arranged in Table ( 5).An inhibitory action of Lufenuron can be easily detected because the treated nymphs were prevented to gain normal somatic increments, especially at the higher three concentration levels (0.468±0.137, 0.340±0.131& 0.316±0.116g at 250, 500 & 1000 ppm, respectively , vs 0.598±0.233g of their control correspondings).Moreover, the growth inhibition % expressed and confirmed the previously denoted data since it increased by the increasing concentration level (as for example, 9.90 and 42.23 % at 62.5 and 1000 ppm, respectively ).Also, Lufenuron prohibited the nymphal development because their life duration was prolonged consecutively to the ascending concentration level and vice versa (Table 5).

b) Metamorphosing and Morphogenic Effects of Lufenuron:
Data given in Table ( 6) evidently reveal the affected adult performance, by Lufenuron treatments against the last instar nymphs .
The adult emergence was blocked in different degrees depending on the conc.levels of Lufenuron.These successfully eclosed adults suffered a morphogenic action of Lufenuron because the highest deformity (57.14%) had been recorded at the highest concentration level.However, the adult deformity increased by increasing conc.level.Several malformed adults have been shown in Plate (1).Only short longevity, but statistically insignificant, was lasted by the treated adult females ending in death.

Growth of S. gregaria as influenced by Lufenuron.
Studies on different insects have shown that chitin synthesis inhibitors (CSIs) can affect the production of the peritrophic matrix of the locust Locusta migratoria (Clarke et al., 1977) and the blowfly Calliphora erythrocephala (Becker, 1978).These chemicals also affect hardening of the cuticle of the boll weevil Anthonomus grandis grandis (Haynes and Smith, 1994) and development of the pupal integument of the yellow mealwom Tenebrio molitor (Soltani el al., 1987).
In the present study, five concentration levels of each of the CSI (Lufenuron, CGG-184699) were given to the newly moulted penultimate, newly moulted last, or late-aged last instar nymphs of S. gregaria through the fresh food.Lufenuron obviously deteriorated the growth of nymphs because their weight gain drastically decreased as the concentration level was increased after treatment of the newly moulted 4 th instar nymphs but at the higher two concentration levels after treatment of the last instar (early or late-aged) nymphs.
On the contrary, CGA-184699 and CGA-59205 failed to affect the developmental duration or rate of the immature stages of M. domestica (Ghoneim et al., 2004).Moreover, some CSIs induced the developmental rate along shortened duration, as seen in the present study on S. gregaria after treatment with Lufenuron at certain physiological ages of nymphs.So, many similar findings can be obtained by the literature, as for example, Tanani (2001) recorded shortened developmental duration of Rhynchophorus ferrugineus by CGA-184699 and CGA-59205 and El-Sheikh (2002) observed shortened larval duration of Agrotis ipsilon by Flufenoxuron.Also, Flufenoxuron exhibited a shortening effect on the developmental duration of S. gregaria at certain concentration levels and insignificant lengthening effect at other concentration levels (communication with Tanani, 2007) A variation in the developmental action was clearly observed by the action of Lufenuron, in the present study.Whereas Lufenuron promoted the developmental rate along significantly shortened duration after treatment of the newly moulted nymphs (of 4 th or 5 th instar), it retarded such rate along pronouncedly prolonged duration after treatment of the late-aged last instar nymphs.Anyhow, the considerably shortened nymphal duration usually indicates a fostered developmental rate may be for avoiding a further serious action of Lufenuron during the normal duration.
The presence of great variation in effects of BPUs (or CSIs, in general) on the insect development may be largely due to the large species-variations in respect to relative potency of various BPUs on different insect species.This variation may also be resulted from the different mechanisms of ecdysteroid metabolism existing in different insects (Whisenten et al., 1989).However, the inhibitory action of CSIs on the development (as expressed in prolonged developmental duration) in various insect species may be explained 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 stage (Retnakaran et al., 1985;Sehnal and Bryant, 1993).Adult Performance of S. gregaria as affected Lufenuron.
In regard to the Adult emergence of S. gregaria, in the present study, no inhibitory effect could be detected after treatment of the newly moulted penultimate instar nymphs with Lufenuron.On the contrary, a considerable inhibitory action was exerted on the adult emergence after treatment of the late-aged last instar nymphs.
However, no nymphal deformities, permanent nymphs, nymphal-adult intermediates, supernumerary nymphal instar, extramoult or adultoids of S. gregaria were observed in the present study, but only adult malformations which appeared as twisted legs, coiled antennae, curled wings as well as adult failure to get rid the last nymphal exuvia.Generally, these defected features may provide relevant evidence to a morphogenic action of Lufenuron.Each of them seriously interferes, directly or indirectly, with the programme of adult morphogenesis… In addition, several hypotheses have been introduced to explain the mode of action of CSIs on the morphogenesis including: direct inhibition and/or interference with chitin synthase (Deul et al., 1978), effect on the chitinase levels comprising that chitin is being digested faster than deposited (Soltani et al., 1993), interference with juvenile hormone and ecdysteroid metabolism causing a disruption in the chitin metabolic system (Yu and Terriere, 1975), inhibition of chitin synthase by metabolites of CSIs (Cohen and Casida, 1980), inhibition of protease (s) that activate the chitin synthase zymogen (Leighton et al., 1981), inhibition of DNA synthesis (Mitlin et al., 1977), inhibition of glycosyl transferases that are involved with synthesis of lipidlinked oligosaccharides in cell membranes which possibly provide primer molecules for chitin synthase (Mayer et al., 1980), and/or inhibition of facilitated diffusion and active transport across cell membranes of nucleosides and amino acids (Mayer et al., 1988).Adult longevity of S. gregaria was significantly shortened after the nymphal treatments with Lufenuron, in the present study, but in no certain trend.These results agree with shortening effects of DFB on T. molitor (Soltani et al., 1983), of IKI-7899 and Flufenoxuron on Agrotis ipsilon (Shaurub et al., 1999;El-Skeikh, 2002) , of CGA-184699 and CGA-59205 on M. domestica (Ghoneim et al., 2004) and of Flufenoxuron on S. gregaria (personal observation).Adult longevity depends on healthy immature stages.Digestive disorders such as starvation, disturbance in metabolism, degeneration of peritrophic membrane and accumulation of faecal materials at the hind gut may be the cause of untimely adult mortality as a result of CSIs exposure (Soltani, 1984;Parween, 1997a).Generally, the shortened adult longevity can be explicated by the accumulation of toxic xenobiotics in the insect body which upsets a complicated balance of factors such as absorption, excretion and detoxification (Abdel-Al, 1996;Mustafa, 2002).

Table ( 1): Growth
and developmental effects of Lufenuron on Schistocerca gregaria after treatment of the newly moulted penultimate instar nymphs.

Table ( 2
): Adult performance as affected by Lufenuron after treatment of the newly moulted penultimate instar nymphs of Schistocerca gregaria.

Table ( 3
): Growth and developmental effects of Lufenuron on Schistocerca gregaria after treatment of the newly moulted last instar nymphs.

Table ( 4
): Adult performance as affected by Lufenuron after treatment of the newly moulted last instar nymphs of Schistocerca gregaria.

Table ( 5
): Growth and developmental effects of Lufenuron on Schistocerca gregaria after treatment of the late-aged** last instar nymphs.Develop.Rate and No. of insect used for each replicates of treatment and control: see footnote of Table( 1 ).** the 5-day old nymphs of last instar were treated with Lufenuron.

Table ( 6
): Adult performance as affected by Lufenuron after treatment of the late-agde** last instar nymphs of Schistocerca gregaria.