Assessment of the toxic and disruptive effects of Precocene II on growth and metamorphosis of the grasshopper Euprepocnemis plorans plorans

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(8)pp: 5366(2017) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
As a result of indiscriminate and excessive uses, the conventional insecticides usually exhibit several serious impacts on the human health and beneficial animals as well as cause toxicological problems to the environmental systems (Van Der Gaag, 2000;Costa et al., 2008;Relyea, 2009;Tiryaki and Temur, 2010).Furthermore, the conventional insecticides have a tendency to accumulate in different trophic levels of the food net (Damalas and Eleftherohorinos, 2011;Chowański et al., 2014).In addition, the repeated use of many insecticides results in the development of resistance in the emerging insect strains (Davies et al., 2007, Mosallanejad andSmagghe, 2009).Therefore, eco-friendly insecticides have received global attention in recent decades as an alternative to these conventional insecticides.These alternative compounds should be characterized by a short period of half-life in the environment, lower toxicity to non-target organisms and their efficiency at low concentrations (Attathom, 2002;Gade and Goldsworthy, 2003;Tiryaki and Temur, 2010;Walkowiak et al., 2015;Li et al., 2017).
It should be mentioned that the use of juvenile hormone (JH) or JH-based compounds for pest control was early suggested by some authors (Williams, 1967;Staal, 1976Staal, , 1982) ) as "third generation insecticides".Screening new targets involved in JH-biosynthesis within the corpora allata has been a subject of study in the past four decades (Bede et al., 2001).So, compounds that interact with JH, stimulate JHbiosynthesis, inhibit JH-biosynthesis or interfere with its catabolism can be appreciated as new insecticidal agents against insect pests (Nandi and Chakravarty, 2011).These compounds can be collectively called as 'insect growth regulators' (IGRs).IGRs are not directly toxic, but act selectively on normal growth, development, metamorphosis and/or reproduction in insects via disrupting the hormonally regulated physiological processes (Martins and Silva, 2004;Wang and Liu, 2016).Because of their desirable characteristics, such as low toxicity, less environmental pollution, high selectivity, and low impact on natural enemies and human health, IGRs are usually used to control various insect pests (Cedric, 2005;Wang and Wang, 2007;Resmitha and Meethal, 2016).
As reported by some authors (Staal, 1986;Singh and Bhathal, 1994;Hoffmann and Lorenz, 1998), precocenes either inhibit JH biosynthesis or act as inhibitors of the enzyme action.Therefore, precocenes and other anti-JH agents have been considered prototypes of "fourth-generation pesticides" (Sariaslani et al., 1987;Moya et al., 1997;Szczcpanik et al., 2005;Banerjee et al., 2008;Singh and Kumar, 2011).It has been demonstrated that the design of JH mimics or anti-JH agents is an effective strategy for insecticide discovery (Bede et al., 2001).Compounds with anti-JH activity are considered as new representatives of IGRs lacking some disadvantages of juvenoid-type chemicals (Bowers, 1982;Staal, 1982).These chemicals are potentially efficacious for control of the major insect pests where most of the damage is caused by the larval stage (El-Ibrashy, 1982).
Grasshoppers have been reported as serious pests in Egypt especially in the newly reclaimed area (EL-Garhy et al., 1988).The most adversely economic grasshopper is Euprepocnemis plorans plorans (Charp.)(Orthoptera:Acrididae).Although this grasshopper species caused 95% damage to crops of the Nile Delta, Egypt (Abdel-Fattah, 2002), it received a little attention of research in Egypt (Ghoneim et al., 1994a(Ghoneim et al., ,b, 1995;;El Sayed, 1998a,b;Abdel-Fattah, 2002;Mohamed, 2014).The control strategies against this grasshopper species still depend upon the conventional insecticides.Although these chemicals are often effective, but not always appropriate.The present study was conducted to evaluate the toxicity and anti-hormonal activities of PII on growth and metamorphosis of this grasshopper.

Experimental Insect:
A culture of Euprepocnemis plorans plorans (Charp.)(Orthoptera: Acrididae) was originated by a sample of nymphs and adults from the susceptible culture maintained for several generations along some years in Locust and Grasshopper Department, Plant Protection Research Institute, Giza, Egypt.It was reared under laboratory controlled conditions (32±2°C, 65±5% R.H. and 12h dark: 12h light) at Department of Zoology and Entomology, Faculty of Science, Al-Azhar University, Cairo.Both adults and nymphs were raised in glass fronted cages (30 x 30 x 30 cm in diameter).The top of each cage had a small wire-gauze opening door.The bottom of cages was covered with a layer of sterilized sand (10 cm in depth).All cages were held at the laboratory controlled conditions.All developmental stages of the grasshopper were fed on the maize leaves (Zea mays), and a daily routine feeding and cleaning manipulations were continuously conducted.

Precocene II Treatment:
Precocene II (6,7-dimethoxy-2,2-dimethylchromene, PII) was kindly provided by Dr. Heba Hassan, Prof. at Plant Protection Research Institute, Giza, Egypt.PII was diluted in acetone to prepare a series of doses: 60, 40, 20 and 10 µg/cm 2 .A contact technique, originally described by Unnithan et al. (1980) for PII against Schistocerca gregaria, was applied.Bottom of a Petri dish (15x2 cm) was coated with each dose.After acetone evaporation, groups of 15 newly moulted nymphs of 2 nd or 4 th (penultimate) instar of E. plorans plorans were confined in each dish for 24h (exposure period).By this technique, the precocene fumigant could presumably reach the corpora allata within the insect rapidly via the tracheal system.Groups of 15 newly moulted nymphs of 2 nd or 4 th instar nymphs were confined in only acetonetreated Petri dishes and used as controls.All treated and control nymphs were kept under the previously mentioned laboratory conditions.After the exposure period, treated and control nymphs were transferred into clean Petri dishes and provided with suitable pieces of maize leaves, as fresh food, every day.

Criteria of Study: 3.1. Toxicity:
Initial mortality (%) was recorded within 24 h post-treatment whereas the extended toxic effect of PII was determined according to the recorded mortalities of all developmental stages.Only female nymphs and adults were used in the present study.LD 50 values were calculated by Microsoft Office Excel, 2007, according to Finny (1971).
After exposure of 2 nd instar nymphs to doses of PII, the successfully moulted nymphs of 4 th and 5 th instars were daily weighed (in mg).Also, the treated 4 th instar nymphs and the successfully moulted 5 th instar nymphs were daily weighed.Growth rates (GR) of 4 th and 5 th instar nymphs were calculated according to (Waldbauer, 1968) as follows: GR = G/TA.Where G: fresh weight gain (mg) of nymphs along the instar.T: the instar period (in days).A: mean fresh body weight of nymphs during the feeding period.

Development and Metamorphosis:
After exposure of 2 nd and 4 th instar nymphs to PII, all features of retarded development and impaired metamorphosis were recorded (in %).

Statistical Analysis of Data:
Data obtained were analyzed by the Student's t-distribution, and refined by Bessel correction (Moroney, 1956) for the test significance of the difference between means.

Toxic Effect of PII on E. plorans plorans:
After exposure of the newly moulted 2 nd instar female nymphs to different doses of PII, the toxic effect was detected by mortality (%) of nymphs.Data of mortality had been assorted in Table (1).According to these data, all treated 2 nd instar nymphs completely died at 24 h post-treatment (initial mortality) by the higher two doses (60 and 40 µg/cm 2 ).The lower two doses (20 and 10 µg/cm 2 ) of PII caused only 26.92 and 20.00% mortality, respectively, at 24 h post-treatment.At 11 days post-treatment, the same lower doses caused 28.30 and 23.20% mortality, respectively, compared to 10.67% mortality of control nymphs.Thus, toxicity of PII increased by the time in the same instar.The lethal activity of PII extended to the subsequently moulted nymphal instars and adults.Mortality of 3 rd instar nymphs were 20.10 and 18.12%, at 20 and 10 µg/cm 2 , respectively, vs. 6.0% of control mortality.At the lower two doses, mortality of 4 th instar nymphs were 16.99 and 14.98%, respectively.As obviously seen, the toxic effect of PII appeared in a dosedependent course against 2 nd , 3 rd , and 4 th nymphal instars.PII, only with the lowest dose, affected the survival of 5 th (last) instar nymphs, since 7.98% mortality was recorded.With regard to the emerged adult females, PII exhibited a weak mortal potency after treatment of 2 nd instar nymphs (3.85%),only with 20 µg/cm 2 .LD 50 was calculated as 0.388 µg/cm 2 (Table 1).After exposure of the newly moulted 4 th instar nymphs to different doses of PII, data of the toxicity were summarized in Table (2).Depending on these data, all doses caused various mortalities of the treated 4 th instar nymphs and the moulted 5 th instar nymphs.At 24 h post-exposure, the nymphal mortalities were recorded in 22.22, 40.74, 10.20 and 25.00%, at 60, 40, 20 and 10µg/cm 2 , respectively.Six days later, the toxic potency of PII decreased, since mortalities were 12.12, 30.85, 07.69 and 15.00%, at 60, 40, 20 and 10 µg/cm 2 , respectively, vs. 5.00% mortality of control nymphs.Regardless the dose level, toxic potency of PII decreased by the moulting, since mortalities of 5 th instar nymphs were 10.10, 21.00, 05.18 and 05.00%, at 60, 40, 20 and 10 µg/cm 2 , respectively.In addition, PII displayed its insecticidal activity in no certain trend.In respect of the emerged adult females, only the lower two doses caused 7.69 and 10.00% mortality, respectively.LD 50 was determined as 17.022µg/cm 2 (Table 2).Depending on these data, the 2 nd instar nymphs were more sensitive to PII toxicity than 4 th instar nymphs.

Effect of PII on the Growth of E. plorans plorans:
Table (3) contains data of nymphal growth during only the latter two instars (4 th and 5 th ), as affected by exposure of 2 nd instar female nymphs to PII.In the light of these data, PII exerted a slight inhibitory action on growth of 4 th instar nymphs (0.026±0.003 and 0.025±0.005,at 20 and 10 µg/cm 2 , respectively, vs. 0.030±0.004 of control nymphs).Also, a slight inhibitory action was exerted on the growth of 5 th instar nymphs (0.026±0.002 and 0.027±0.004,at 20 and 10 µg/cm 2 , respectively, vs. 0.029±0.005 of control nymphs).
After exposure of the 4 th instar nymphs to PII, data of nymphal growth were also arranged in the same table.As exiguously shown, PII exerted a weak suppressing action on growth of 4 th instar nymphs after exposure to the highest and lowest doses (0.038±0.009 and 0.039±0.007,at 60 and 10 µg/cm 2 , respectively, vs. 0.043±0.015 of control congeners).On the other hand, the growth rate of 5 th instar nymphs was slightly suppressed at these doses (0.037±0.004 and 0.038±0.005,at 60 and 10 µg/cm 2 , respectively, vs. 0.039±0.001 of control congeners).On the basis of data arranged in the aforementioned table, PII exerted potent suppressing action on the nymphal growth only at the middle two doses.Concerning the 4 th instar nymphs, growth rates were considerably regressed (0.032±0.001 and 0.030±0.005,at 40 and 20 µg/cm 2 , respectively, vs. 0.043±0.015 of control nymphs).Similarly, the growth rate of 5 th instar nymphs was remarkably regressed (0.028±0.005 and 0.029±0.001,at 40 and 20 µg/cm 2 , respectively, vs. 0.039±0.001 of control nymphs).

Effects of PII on Development and Metamorphosis of E. plorans plorans:
Depending on the data distributed in Table (4), two major features of retarded development and deranged metamorphosis could be recorded (in %), as permanent nymphs and precocious metamorphosis.After exposure of 2 nd instar female nymphs to PII, the development was suspended in a feature of permanent nymphs which survived two-fold period of their control congeners and failed to moult into the next instar ending in death.Permanent nymphs were induced in 3.85% in the 2 nd instar (only at dose 20 µg/cm 2 ) and 3.33% in the 4 th instar (only at dose 10 µg/cm 2 ).As clearly seen in the same table, 3.33% of treated 2 nd instar nymphs precociously moulted into 4 th instar, skipping off the 3 rd instar.These precociously developed nymphs survived more than 20 days and eventually perished.
After exposure of 4 th instar female nymphs to PII, no permanent nymphs were observed.On the other hand, the metamorphosis program was impaired; since precocious adultoids (omitting the 5 th instar) had been produced only at the higher two doses (11.11 and 3.70%, at 60 and 40 µg/cm 2 , respectively).These precocious adultoids appeared normally in colour and behaviour but without wings.They survived more than one month and eventually perished with no ability to mate. (2): Precocious development into the 4 th instar (skipping the 3 rd instar). (3): Precocious adultoids (skipping the 5 th instar) had no wings and eventually died without mating.
Results of the present study on E. plorans plorans were in agreement with those reported results of toxicity of some anti-JH compounds, since exposure of 2 nd instar nymphs to the higher two doses (60 and 40 µg/cm 2 ) of PII resulted in complete mortality of nymphs within the first 24h post exposure (initial mortality).At the lower two doses (20 and 10 µg/cm 2 ), PII exhibited a prolong toxicity on the subsequently moulted instars but weak toxicity on adult females.After exposure of the 4 th instar nymphs to PII, various mortalities had been recorded among the treated nymphs and 5 th instar nymphs.Only at the lower two doses, PII affected the adult survival.For the explication of the nymphicidal effect of PII, it may be attributed to the prevention of moulting nymphs to swallow volumes of air for splitting the old cuticle and expand the new one during ecdysis (Linton et al., 1997).Also, these nymphal deaths might be due to the prevented feeding and continuous starvation of the present insect (Ghoneim et al., 2000).The adult mortalities, after exposure of 2 nd or 4 th instar nymphs to PII, 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 nymphs 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 compound (Osman et al., 1984).
It is important to point out that, LC 50 or LD 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 level, method and time of treatment, as well as the experimental conditions.For examples, LD 50 of PII against the red cotton stainer Dysdercus koenigii has been found as 85.46 and 82.37 mgl -1 for 4 th and 5 th instar nymphs, respectively (Banerjee et al., 2008).After treatment of 4 th instar larvae of the Asian tiger mosquito, Aedes albopictus with PI and P II, LC 50 values were estimated in 41.63 and 43.55μg/ml, respectively (Liu and Liu, 2014).LC 50 of PII against the booklice Liposcelis bostrychophila was calculated in 30.4g/cm 2 but LC 50 of PI was found 64.0g/cm 2 (Lu et al., 2014).LC 50 of PI against the cat flea Ctenocephalides felis was estimated in 10.97 ppm (Rust and Hemsarth, 2017).LC 50 values of Pitavastatin against the tobacco hornworm Manduca sexta and the Pacific beetle roach Diploptera punctata were estimated in 5.23, and 395.2 µM, respectively (Li et al., 2017).In the present study on E. plorans plorans, LD 50 values were 0.388 and 17.022 µg/cm 2 , after exposure of 2 nd instar nymphs and 4 th instar nymphs, respectively.Depending on these data, the 2 nd instar nymphs were more sensitive to PII toxicity than 4 th instar nymphs.

Growth Inhibition of E. plorans plorans by PII:
In the current study on E. plorans plorans, PII exerted a slight inhibitory action on the nymphal growth of both 4 th and 5 th instars, after treatment of 2 nd instar nymphs, regardless the dose, but the growth rate was remarkably regressed after treatment of 4 th instar nymphs with 40 and 20 µg/cm 2 .These results were, to some extent, in accordance with some of the reported results of the inhibited growth of various insects by different anti-JH compounds.Several chromene derivatives inhibited the growth of the last instar larvae of the mealworm beetle Tenebrio molitor (Roberto et al., 1998).PI and PII exhibited growth-inhibiting activities against A. aegypti, An. sacharovi and An.stephensi (Saxena et al., 1994;Yasyukevich and Zvantsov, 1999).Larvae of M. sexta were fed on HMG-CoA reductase inhibitors, Fluvastatin, Lovastatin or Pitavastatin-treated food, starting with 1 st instar.The treated larvae grew in significantly slow growth rate (Li et al., 2017).
To understand the growth inhibition of E. plorans plorans, in the current investigation, PII might block the release of morphogenic peptides, causing the alteration in the ecdysteroid and juvenoid titers (Barnby and Klocke, 1990).Also, PII may affect the tissues and cells undergoing mitosis (Nasiruddin and Mordue, 1994).In addition, PII might exert an inhibitory action on the haemolymph and fat body protein contents, as suggested by Lange et al. (1983) for locusts after treatment with precocenes.

Disrupted Development and Metamorphosis of E. plorans plorans by PII: 3.1. Precocious Metamorphosis:
In the current investigation, exposure of 2 nd instar nymphs of E. plorans plorans to PII led to 3.33% precociously moulted nymphs into 4 th instar, skipping off the 3 rd instar (only at the lowest dose).These precocious 4 th instar nymphs survived for more than 20 days and eventually died.After exposure of 4 th instar nymphs to PII, some treated nymphs precociously metamorphosed into adultoids, omitting the 5 th instar, only at doses of 60 and 40 µg/cm 2 .These precocious adultoids appeared without wings.They survived more than one month and eventually perished without mating.These results were, to a great extent, in corroboration with those reported results of precocious metamorphosis in several insects of various orders by different anti-JH compounds.Within Orthoptera, exposure of 4 th instar nymphs of the desert locust Schistocerca gregaria to PII (15 µg/cm 2 ) induced precocious adultoids (Salem et al., 1982 a, b).Different doses of PI or PII (20-100 μg/insect) induced precocious metamorphosis in the Mediterranean splendid grasshopper Heteracris littoralis (Alrubeai, 1986).Among Hemiptera, PII induced precocious metamorphosis in the kissing bugs Rhodnius prolixus and Triatoma dimidiata when applied by either contact exposure or fumigation (Tarrant et al., 1982).Ayoade et al. (1996) observed precocious metamorphosis in the brown plant hopper Nilaparvata lugens after exposure to PII.In Coleoptera, topical application of PI and PIІ onto the 2 nd larval instar of L. decemlineata induced the precocious adultoids (Farazmand and Chaika, 2008).In addition, precocious metamorphosis had been induced by precocenes in several insects of Diptera, such as the flesh fly Neobellieria bullata (Darvas et. al., 1990) and the house fly Musca domestica (Gaur and Kumar, 2009); as well as Lepidoptera, such as the tobacco cutworm Spodoptera litura (Srivastava and Kumar, 1999) and P. ricini (Khan and Kumar, 2000).Moreover, other anti-JH compounds induced such feature of impaired metamorphosis in various insects, such as Fluoromevalonate (FMev) against the fall webworm Hyphantria cunea (Farag and Varjas, 1983) and the lawn armyworm Spodoptera mauritia (Balamani and Nair, 1989); ETB (Kuwano et al., 1988), KK-42 (Kuwano et al., 1985;Akai and Mauchamp, 1989), KK-22 (Asano et al., 1984, 1986) and 3-pyridine derivatives (Yoshida et al., 2000) against B. mori.Treatment of N. bullata larvae with KK-110 and J-2710 resulted in precocious pupation (Darvas et. al., 1990).
For interpretation of the production of precocious 4 th instar nymphs or precocious adultoids, after exposure of of E. plorans plorans nymphs to PII, in the present study, it is well known that the cells of corpora allata, JH-producing organs in insects, are selectively destroyed by precocenes (Bowers et al., 1976;Ohta et al., 1977;Pratt et al., 1980;Brooks and McCaffery, 1990).Thus, precocious metamorphosis in the present grasshopper indicated the prohibition of JH production by PII.On the molecular basis of JH action, Wilson (2004) reported that the effects of JH may be due to interference with the expression or action of certain genes, particularly the broad complex (br-C) transcription factor gene, that direct changes during metamorphosis.In hemimetabolous insects (like the present grasshopper), Erezyilmaz et al. (2006) studied the role of br for inducing the precocious adult molt in O. fasciatus after application of PII to 3 rd instar nymphs, and suggested that a loss of br mRNA was caused at the precocious adult molt.However, a deep discussion on the action mechanisms of anti-JH compounds in insects was clearly shown by many authors (Staal et al., 1981;Hamnett and Pratt, 1983;Brooks and McCaffery, 1990;et al., 1995;Miao et al., 2001;Kumar and Khan, 2004;Chen et al., 2005 a,b;Minakuchi and Riddiford, 2006;Amiri et al., 2010).

Suspended Development:
In insects, a state of suspended development attracts a great attention of some entomologists.The induction of suspended development was recorded in some insect species as a response to some insect growth regulators (IGRs) or botanicals.Among IGRs, some authors (Salem et al., 1982a;El-Gammal and Taha, 1984;Ghoneim, 1988;Abou El-Ela, 1993) observed permanent (over-aged) nymphs of S. gregaria after treatment with certain IGRs.Permanent larvae of the European corn borer Ostrinia nubilalis were induced depending upon the dose of fenoxycarb and the timing of application onto the 5 th instar larvae (Gadenne et al., 1990).Permanent larvae of P. argyrostoma were induced after topical application of last instar larvae with 100 µg/larva of chlorfluazuron (Ghoneim and Ismail, 1995).In addition, some botanicals, plant extracts or isolated plant products, had been reported to induce permanent nymphs in various insects, such as the milkweed bug Oncopeltus fasciatus after injection of the newly moulted last instar nymphs with azadirachtin (Dorn et al., 1986); O. fasciatus and D. peruvianus after topical application of Manilkara subsericea (Sapotaceae) extracts onto 4 th instar nymphs (Fernandes et al., 2013); S. litura after treatment of larvae with acetone leaf extract of Withania somnifera (Solanaceae) (Gaur and Kumar, 2010); and the confused flour beetle Tribolium confusum after treatment of 5 th instar and 6 th instar larvae with 1µg/µl of Andrographolide (a terpenoid isolated from the leaves of Andrographis paniculata, Acanthaceae) (Lingampally et al., 2013).Apart from IGRs and botanicals, El-Gammal et al. (1986) observed permanent nymphs in S. gregaria after exposure of gamma irradiation (dose of 20 gray) against the 3 rd instar nymphs.
In the present work, the deranged development was detected by 'permanent nymphs' that induced in 2 nd instar nymphs (3.85%) after exposure only to 20 µg/cm 2 of PII.Also, similar permanent nymphs were induced during the 4 th instar.No permanent nymphs had been induced after exposure of 4 th instar nymphs to PII.All permanent nymphs survived two-fold period of control congeners and eventually perished as nymphs.As seen in the available literature, no reported permanent nymphs in insects had been induced by precocenes or other anti-JH compounds.Therefore, the present study provides the first report of this feature of suspended development in E. plorans plorans by Precocene in the world.To explicate the induction of permanent nymphs of E. plorans plorans, in the current investigation, PII exerted an inhibitory action on the prothoracic gland (ecdysone-producing gland) and hence the ecdysone could not be synthesized and/or released.It is well known that the absence of ecdysone leads to failure of ecdysis.PII might disrupt the ecdysteroid metabolism or alternatively acted directly to inhibit the release of ecdysis-triggering hormone (Gaur and Kumar, 2010).
In addition, the current investigation obviously revealed that PII exhibited multiple activities against E. plorans plorans: anti-JH activity and anti-ecdysteroid activity.These data have validated the reported anti-ecdysteroid activity of other anti-JH compounds in some insects.The imidazole compound KK-42 was found to delay/inhibit the ecdysteroid production in O. nubilalis and S. gregaria (Gelman et al., 1995;Wang and Schnal, 2001).Another imidazole, SDIII, had been reported to exert strong anti-JH and anti-ecdysteroid actions on B. mori (Tan et al., 1992).Results obtained by Yoshida et al. (2000) revealed that the 3-pyridine derivatives temporarily act as anti-ecdysteroids against B. mori.

Conclusion:
Precocene II exhibited a lethal activity against nymphs and adults of the grasshopper E. plorans plorans, inhibited the nymphal growth, induced precocious moult to last nymphal instar and precocious sterile adultoids.In addition to this anti-JH activity, PII exhibited anti-ecdysteroid activity has appeared in permanent nymphs.In spite of these findings, it may be recommended to use PII for pest control but after study its activity and persistence under the field condition in the foreseeable future

Table 1 :
Mortality (%) of E. plorans after exposure of 2 nd instar female nymphs to PII.

Table 2 :
Mortality (%) of E. plorans after exposure of 4 th instar female nymphs to PII.

Table 3 :
Effects of PII on the growth rate (mean±SD) of the 4 th and 5 th instar female nymphs of E. plorans.

Table 4 :
Effect of P II on development and metamorphosis of E. plorans.