Disruptive Impact of Precocene I (juvenile hormone-antagonist) on the Adult Performance and Reproductive Potential of the Egyptian Cotton Leafworm Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae)

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(6)pp: 125(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
The cotton leafworm Spodoptera littoralis has been considered as destructive phytophagous lepidopterous pest of cotton and various vegetable and field crops all over the year (Shonouda and Osman, 2000;El-Khawas and Abd El-Gawad, 2002;Adham et al., 2009).Approximately112 plant species belonging to 44 families are reported as hosts of this pest in tropical and temperate zones of the old world (Magd El-din and El-Gengaihi, 2000).To control the attacks of S. littoralis, several types of conventional insecticides have been used over the past 40 years (Casida and Quistad, 1998).The intensive use of broad-spectrum insecticides against S. littoralis has led the development of resistance to many registered pesticides making their control even more difficult (Miles and Lysandrou, 2002;Aydin and Gurkan, 2006;Davies et al., 2007, Mosallanejad andSmagghe, 2009).As a result of improper and excessive uses, also, these insecticides usually exhibit several adverse impacts on the human health and beneficial animals as well as cause serious toxicological problems to the environmental systems because these chemicals have a long half-life and retention in the environment for long periods (Van Der Gaag, 2000;Costa et al., 2008;Relyea, 2009;Tiryaki and Temur, 2010;Damalas and Eleftherohorinos, 2011;Chowański et al., 2014).Therefore, eco-friendly insecticides have received global attention in recent years as alternative for the conventional insecticides.These alternative compounds are characterized by lower toxicity to non-target organisms than conventional insecticides and they are effective at low concentrations (Attathom, 2002;Gade and Goldsworthy, 2003).Also, they are biodegradable into harmless compounds, which allows for avoiding the problems of environmental pollution (Tiryaki and Temur, 2010;Walkowiak et al., 2015;Li et al., 2017).The potential of juvenile hormone analogues (JHAs) and insect growth regulators (IGRs) for the pest control had been intensively reviewed (Staal, 1982;El-Ibrashy, 1982, 1984;Dhadialla et al., 1998;Gilbert et al., 2000;Tunaz and Uygun, 2004;Chowański et al., 2014).
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 insect growth regulators (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 larval stage (El-Ibrashy, 1982).
The anti-juvenile hormone agents, such as compactin, fluoromevalonate, imidazoles and precocene, are considered as insect growth regulators (IGRs) because they affect either the mevalonate pathway in JH biosynthesis, or the corpora allata (CA) directly, the organ that produces JH (Staal, 1986).
Precocenes are originally plant-derived chromenes (2H-1-benzopyran) with insecticidal activities (Proksch et al., 1983;Isman et al., 1986).On the basis of their functions, precocenes can be described as JH inhibitors or anti-JH agents (Staal, 1986).Precocenes and their synthetic analogues received a great attention by entomologists both doing fundamental and applied work due to their twin advantage; using as a physiological probe in the former avoiding surgical allatectomy and as an effective tool in devising 'fourth generation insecticides' in the latter (Muraleedharan et al., 1986;Sariaslani et al., 1987;Moya et al., 1997;Szczcpanik et al., 2005;Singh and Kumar, 2011).Compounds with anti-JH activity are considered as new representatives of IGRs lacking some disadvantages of juvenoid-type chemicals (Bowers, 1982;Staal, 1982).The chemicals having anti-JH activity are potentially efficacious and superior to JH mimics for control of the major insect pests where most of the damage is caused by larval stage (El-Ibrashy, 1982).
On reproduction in adults of several insect orders, precocenes have been shown to prevent normal vitellogenic development of the oocytes or disturb the embryonic development leading to sterility (Staal, 1986;Kumar and Khan, 2004;Ringo et al., 2005;Amiri et al., 2010).The present study was carried out aiming to assess the effects of Precocene I (PI) on the most important parameters of adult performance and reproductive potential of S. littoralis.

Experimental insect:
A sample of Egyptian cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) pupae was kindly obtained from the culture of susceptible strain maintained for several generations in Plant Protection Research Institute, Agricultural Research Center, Doqqi, Giza, Egypt.A culture was raised under laboratory controlled conditions (27±2 o C, 65±5% R.H., photoperiod 14h L and 10h D).Rearing procedure was carried out according to Ghoneim (1985) and improved by Bakr et al. (2010).Larvae were provided daily with fresh castor bean leaves Ricinus communis.The emerged adults were provided with cotton pieces soaked in 10% honey solution as a food source.Moths were allowed to lay eggs on Oleander branches.The egg patches were collected daily, and transferred into Petri dishes for another generation.

Precocene I administration:
Precocene I (PI)(7-methoxy-2,2-dimethyl chromene) was kindly provided by Dr. Heba Hassan, Prof. at Plant Protection Research Institute, Agricultural Research Center, Doqqi, Giza, Egypt.Molecular formula: C 12 H 14 O 2. PI was diluted in acetone to prepare a wide range of doses.In a preliminary experiment, the sublethal doses of PI were found as 150, 120, 90, 30 and 15µg/larva.Groups of 20 healthy larvae of 1day old 5 th (penultimate) and 1-day old (last) instar larvae were used as replicates for each dose.Each dose was topically applied (once) onto the thoracic sternum of each larva using Hamilton micro applicator (NHN 737).Control larvae had been topically applied only with 1µl acetone.All treated and control larvae were kept individually under the previously mentioned laboratory controlled conditions.All larvae were provided with fresh castor bean leaves every day, during the feeding period.Just after the adult emergence, the most important parameters of adult performance and reproductive potential had been recorded.

Adult performance parameters:
Adult emergence: Number of successfully metamorphosed adults was expressed in % according to Jimenez-Peydro et al. (1995) as follows: [No. of completely emerged adults / No. of pupae] × 100 Adulticidal activity: The adulticidal activity of PI was determined by observing the adult mortality.Morphogenic efficiency: It was determined by the impaired adult morphogenesis as appeared in deformed adult females and recorded in %.It was calculated in percentage as follows: [No. of deformed adults / No. of emerged adults] ×100 Adult longevity: Total longevity of adult females and its major compartments: preoviposition (ovarian maturation) period and oviposition period (reproductive lifetime) were measured in mean days±SD.

Criteria of the reproductive potential:
After pupal stage of control and treated larvae, the emerged adult females of S. littoralis were daily collected and released in plastic jars (3L) provided with sterilized cotton pieces, soaked in 10% honey solution, for feeding, as well as suitable Oleander branches as an oviposition site.The treated adult females were coupled with normal adult males (1:2) of the same age obtained from the main culture.The eggs were collected daily, and carefully transferred to Petri dishes to count eggs.

Oviposition efficiency:
Oviposition efficiency could be detected by the oviposition rate as follows: Number of laid eggs per ♀/reproductive lifetime (in days) x 100 2. Reproductive capacity: Fecundity: The laid eggs were counted for calculating the number of eggs per female.Fertility: The hatchability was usually expressed in hatching percentage of laid eggs.Sterility index: It was calculated according to Toppozada et al. (1966) as follows: Sterility Index = 100 -[(a b / A B) × 100] Where: a: mean number of eggs laid per female in the treatment.b: percentage of hatching in the treatment.A: mean number of eggs laid per female in the controls.B: percentage of hatching in the controls.

Incubation period:
The laid eggs were kept in Petri dishes under the same laboratory controlled conditions, as previously mentioned.Just after the oviposition, eggs were observed until hatching for recording the incubation period (in mean days±SD).

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 difference between means.

Effects of PI on the adult performance of S. littoralis: 1. Adult emergence:
After topical application of PI onto the 5 th instar larvae, data of the most important parameters of adult performance were assorted in Table (1).Depending on these data, PI exerted a blocking action on this crucial physiological process only at the higher three doses (66.6, 58.3 and 50.0%, at 90, 120 and 150 µg/larva, respectively, vs. 100% emergence of control adult females).After topical application of PI onto the 6 th instar larvae, data of adult performance were arranged in Table (2).In the light of these data, PI exerted a similar arresting action on the adult emergence only at the higher two doses (95.5 and 85.7%, at 120 and 150 µg/larva, respectively, vs. 100% emergence of control adult females).

Adult survival:
As clearly shown in Table (1), PI exhibited a slight extended toxic effect on adult females after treatment of 5 th instar larvae only with the higher two doses (28 and 30% mortality, at 150 and 120 µg/larva, respectively, compared to 0% mortality of control adults).With regard to the survival of adult females successfully emerged from pupae after treatment of 6 th instar larvae, PI exerted a slightly extended toxic effect only at the higher two doses (10 and 5% mortality, at 150 and 120 µg/larva, respectively, compared to 0% mortality of control adults, (Table 2).

Adult morphogenesis:
After treatment of 5 th instar larvae, PI failed to exhibit morphogenic efficiency against adults, since no adult deformities were observed (Table 1).In view of data contained in Table (2), topical application of 6 th instar larvae with PI led to the impairment of adult morphogenesis, since some deformities were produced, in no certain trend (21.4,25.0, 10.7 and 3.4% deformed adult females, at 150, 120, 90 and 30 µg/larva, respectively, vs. 0% deformation of control adult females).As obviously seen in Fig.
(1), the most important features of the impaired morphogenesis program appeared in adults with curled wings, adults with non-expanded wings and adults failed to completely get rid of the pupal exuvia, at the posterior extremity.

Adult longevity:
In the present study, S. littoralis is a lepidopteran in which a part of the ovarian maturation has been taken place in the second half of pupal stage and another part has been completed in very few days, or even some hours, after adult emergence.Thus, the pre-oviposition period does not exactly indicate the whole ovarian maturation period.On the other hand, the oviposition period is usually known as 'reproductive life-time'.In addition, the post-oviposition period does not exceed few hours.Therefore, the major compartments of adult longevity are pre-oviposition period and oviposition period.

Table 1. Affected adult performance of S. littoralis after topical application of PI
sublethal doses onto 1-day old penultimate instar larvae.

Effects of PI on the reproductive potential of S. littoralis:
1. Oviposition rate: After topical application of PI onto 5 th instar larvae, data of the most important criteria of reproductive potential were assorted in Table (3).Depending on these data, the oviposition efficiency of adult females was drastically prohibited only at the lower two doses, since the oviposition rate was seriously regressed (75 and 85%, at 30 and 15 µg/larva, respectively, vs. 100% oviposition of control females).Moreover, the suppressing potency of PI on the oviposition rate increased with the increasing dose level, after treatment of 6 th instar larvae (93.8, 91.5, 86.7, 77.7 and 58.3 %, at 15, 30, 90, 120 and 150 µg/larva, respectively, vs. 100% oviposition rate of control females, Table 4).
Another informative parameter of the reproductive capacity is fertility (hatchability= hatching% of laid eggs).Depending on the data distributed in Table (3), treatment of 5 th instar larvae with PI led to complete sterility of adult females, since complete failure of egg hatching was observed.Similar complete sterility had been recorded for eggs laid by those adult females emerged from 6 th instar larvae treated only with doses 150 and 30 µg/larva (see Table 4).At other doses, seriously reduced hatchability was recorded (58.9, 33.1 and 4.2%, at 15, 90 and 120 µg/larva, respectively, vs. 98.3% of control eggs).

Effects of PI on the embryonic development of S. littoralis:
In insects, incubation period can be used as a valuable indicator of the embryonic developmental rate, i.e., longer period usually denotes slower rate and vice versa.In the present study on S. littoralis, no incubation period could be measured for eggs of the adult females after treatment of 5 th instar larvae with PI because no eggs could hatch, regardless of PI dose (see Table 3).
After treatment of 6 th instar larvae with PI, data of

Blocked adult emergence:
Scarce studies have examined the effects of anti-JH compounds on adult emergence in insects.Inhibition of adult emergence in the flesh fly Sarcophaga ruficornis was recorded after larval treatment with PII (Khan and Kumar, 2005).The terpenoid imidazoles, KK-42, was reported to inhibit the adult emergence of the mulberry silk moth Bombyx mori when applied to the newly formed pupae (Kadono-Okuda et al., 1987).In the present study on S. littoralis, the adult emergence was slightly or drastically blocked, especially at the higher three or two doses of PI, depending on the larval instar under treatment, 5 th or 6 th instar.
For interpretation of this result, it important to point out that the adult emergence in insects is a crucial physiological process and regulated by the eclosion hormone.The disturbance of this hormone appeared in partial or complete arresting of adults to emerge.The present result of blocked adult emergence can be interpreted by the disruptive effect of PI on the normal metabolism of insect hormones during the development of the juveniles leading to failure of adult emergence (Trigo et al., 1988).In particular, PI may disturb the adult eclosion hormone release and/or inhibition of the neurosecretion (Al-Sharook et al., 1991;Josephrajkumar et al., 1999).On the molecular basis, anti-JH compounds, like PI, might cause misexpression of certain genes, particularly the brood complex (br-C) transcription factor gene, leading to symptoms of impaired metamorphosis, like blocking of adult emergence (Wilson, 2004;Nandi and Chakravarty, 2011).
In contrast, few studies have examined the adulticidal activities of anti-JH compounds against insects.Different doses of PII were topically applied onto the 3 rd instar larvae of the grey flesh fly Parasarcophaga dux.Toxic effects were recorded on adults, in a dose-dependent course (Nassar et al., 1999).Injection of a single dose (of 50 or 150 µg) of PII into 4-day old adults of the desert locust Schistocerca gregaria led to high mortality of adults (Tawfik et al., 2014).After exposure of the newly moulted 2 nd or 4 th (penultimate) instar nymphs of the grasshopper Euprepocnemis plorans to different doses of PII, various mortality percentages were recorded among the emerged adults (Ghoneim and Basiouny, 2017).
In the present study, PI exhibited a slightly extended toxic effect on the adult females after treatment of 5 th or 6 th instar larvae of S. littoralis, only with the higher two doses.These adult mortalities can be explained by the retention and distribution of PI in the insect body by 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).The adult life in insects depends on healthy immature stages.Digestive disorders, such as starvation, metabolism disturbance, degeneration of peritrophic membranes and accumulation of faecal materials at the hind gut, may be the cause of adult mortality (Soltani, 1984).

Disrupted adult morphogenesis:
Impaired adult morphogenesis, as expressed in the production of deformed adults, was widely reported in the available literature, after treatment of various insects with different JHAs (Kamaruzzaman et al., 2006;Mojaver and Bandani, 2010;Begum and Qamar, 2016).According to the currently available literature, few studies examined the morphogenic effects of anti-JH compounds on the insect adults.Topical application of the highest dose (1 mg) of PII onto 3 rd instar larvae of P. dux led to the production of adult malformation (Nassar et al., 1999).During the study carried out by Khafagi and Hegazi (1999) on the latent effects of PI and PII on the parasitic wasp Microplitis rufiventris reared on its host S. littoralis, they observed various morphologically imperfect moths.Deformed adults of the Eri silk Philosamia ricini were observed after larval treatment with PII (Khan and Kumar, 2000).Formation of deformed S. ruficornis adult flies were recorded after larval treatment with PII (Khan and Kumar, 2005).After topical application of PІ and PIІ onto the 2 nd instar larvae of the Colorado potato beetle Leptinotarsa decemlineata, severe morphological abnormalities were observed (Farazmand and Chaika, 2008).Treatment of the 4 th instar or 5 th instar larvae of the red cotton stainer bug Dysdercus koenigii with PII or the precocenoid compounds 6-hydroxy-DMC and 6-bromo-DMC, the emerged adults appeared with small and pale body and underdeveloped wing pads/wings, at certain doses (Banerjee et al., 2008).Deformed adults of the house fly Musca domestica were produced after treatment of larvae with PIІ (Gaur and Kumar, 2009).In addition, the development of some adult structures and organs, as affected by anti-JH compounds, had been investigated.As for example, treatment of 5 th instar larvae or prepupae of the large fruit tree Tortrix Archips podana with 300, 450, and 600 µg Precocene/insect, morphogenesis of the adult antennae was retarded (Triselyova, 2012).After treatment of 5 th instar larvae of S. littoralis, in the current investigation, PI failed to exhibit morphogenic efficiency against adults, since no deformed adults were produced.On the other hand, treatment of 6 th instar larvae with PI resulted in the impairment of adult morphogenesis, since some adult deformities were produced.
These adult deformities may be explained by an action of PI on the hormonal imbalance during the adult differentiation, in particular the modification of ecdysteroid titer which led to changes in lysosomal enzyme activity causing overt morphological abnormalities (Josephrajkumar et al., 1999).Other suggestions can be conceivable, such as the interference of PI with JH or ecdysteroid metabolism causing a disruption in the chitin metabolic system (Yu and Terriere, 1975), inhibition of chitin synthase by metabolites of PI (Cohen and Casida, 1980a, b), inhibition of DNA synthesis (Mitlin et al., 1977) and/or inhibition of facilitated diffusion and active transport across cell membranes of nucleosides and amino acids (Mayer et al., 1988).

Disturbed adult longevity:
In S. littoralis, the post-oviposition period does not exceed few hours.Therefore, the major compartments of adult longevity can be considered as the pre-oviposition period and oviposition period.

Total adult longevity:
Diverse effects of IGRs on the adult longevity had been reported in a number of insects.The total adult longevity in some insects was shortened after larval treatment of some insects with some IGRs (Pineda et al., 2009;Luna et al., 2011;Hamadah et al., 2015;Aliabadi et al., 2016).On the contrary, adult longevity in other insects was prolonged after larval treatment with some IGRs (Liu and Chen, 2001;Kandil et al., 2012;Sabry and Abdou, 2016).Furthermore, no effect was exhibited by some IGRs on the adult longevity of a number of insects (Saenz-de-Cabezon et al., 2005;Zarate et al., 2011).
Only few results had been reported in the current literature concerning the affected adult longevity by some anti-JH compounds.Depending on the reported results, the adult longevity was shortened after larval treatment with some antijuvenoids.For examples, different doses of PII were topically applied onto the 3 rd instar larvae of P. dux, total adult longevity was significantly shortened (Nassar et al., 1999).After topical application of 25 mg l -1 of the precocenoid compound 6hydroxy-DMC onto the 5 th instar nymphs of D. koenigii, the emerged adults lived shorter longevity than control adults (Banerjee et al., 2008).Khafagi and Hegazi (2004) investigated the effects of PI and PII on the parasitoid wasp M. rufiventris after topical treatment of the host larvae of S. littoralis.Longevity of the parasitoid adult females which developed in hosts treated by Precocenes was shortened.Results of the current investigation on S. littoralis disagreed with those previously reported results, since the major effect of PI was the promotion of adult females to spend longer life-time than control adults.In other words, treatment of 5 th or 6 th instar larvae with PI led to remarkably or slightly prolonged longevity, respectively.An exceptional case was recorded as slightly shortened longevity after treatment of 5 th instar larvae only with the lowest dose (15 µg PI/larva).However, the prevalent prolongation of total adult longevity of S. littoralis, as a response to the action of PI, in the present study, was, to some extent, in agreement with those reported results of extended life span in desert grasshoppers, monarch butterflies and the linden bug Pyrrhocoris apterus, after surgical allatectomy (Herman and Tatar, 2001;Hodkova, 2008).
In the current study, the affected female adult longevity of S. littoralis, after larval treatment with PI, may be attributed to its interference with the hormonal regulation of adult longevity because a close relation between certain hormones and adult longevity was reported in other insects, such as Drosophila melanogaster (Broughton et al., 2005;Carbone et al., 2006;Chamseddin et al., 2012) In insects, the fat body serves many vital functions (Arrese and Soulages 2010) and it is therefore not surprising that longevity mechanisms occur within the fat body (Hwangbo et al. 2004).Also, the prolonged longevity may be due to the preventing physiological trade-offs between survival and egg production (Flatt, 2011;Edward and Chapman, 2011).In addition, the extended longevity was associated with resistance to exogenous stress, like PI, as suggested by Wang et al (2004) for D. melanogaster.
After the attainment of sexual maturity, insects often show degenerative changes in some tissues and organs which can be called 'senility' or 'aging'.In insects, the affected adult longevity can be considered an informative indicator for the adult aging, i.e., prolongation of longevity may denote a delay of aging and vice versa, although the death is usually the destiny of all creatures.As reported by Yamamoto et al. (2013), JH controls aging, to some extent, because it directly affects mechanisms of somatic survival.Therefore, PI may affect the JH level and/or functions leading to prolongation of adult longevity of S. littoralis, in the present study, i.e.PI exerted a delaying action on S. littoralis aging.However, the exact mode of action of PI on the biochemical sites in adults is unknown until now.More information on the adult endocrine system of S. littoralis is required to clarify the mechanism by which PI prolong the adult longevity and delay aging.

Pre-oviposition period:
S. littoralis is a lepidopteran in which the ovarian maturation takes place partially in pharate pupal stage and completes during the pre-oviposition period of the adult females (El-Ibrashy, 1971, 1982).Thus, pre-oviposition period may partially indicate the ovarian maturation rate, i.e. longer period may denotes slower rate and vice versa.The currently available literature contains many reported results of the effects of different IGRs on pre-oviposition period in various insect species (Rashad et al., 2006;Aref et al., 2010;Kandil et al., 2013;Salem, 2015;El-Khayat et al., 2015;Zhou et al., 2016;Hassan et al., 2017;Hamadah et al., 2017).As far as our literature survey could ascertain, no information was available on the effects of anti-JH compounds on the pre-oviposition period in insects.
In the present study on S. littoralis, treatment of 5 th instar larvae with PI led to a slight shortening in the pre-oviposition period of adult females, while treatment of 6 th instar larvae led to a slight or considerable shortening in this period, depending on the dose.This shortened pre-oviposition period may indicate a faster ovarian maturation rate as a response to the action of PI.Depending on this result, PI failed to exhibit anti-gonadotropic activity against S. littoralis but it might exert an enhancing action on this reproductive process.The present result corroborated with Kelly and Fuchs (1978) who reported that precocene is not a specific antigonadotropic agent in adult female Aedes aegypti.Recently, Oliveira et al. (2017) recorded the failure of precocene to affect the ovary development of workers of a highly eusocial wasp.On the contrary, our result was inconsistent with many reported results of inhibitory action of different anti-JH compounds on the ovarian maturation in insects, such as the Mediterranean splendid grasshopper Heteracris littoralis after topical application of PII (20-100 μg/insect) onto 3 rd instar nymphs (Alrubeai, 1986); M. domestica after topical application of 20 μg/fly of PII onto the newly-emerged females (Li et al, 1993); the brown plant hopper Nilaparvata lugens after exposure of 5 th instar nymphs to different doses of PII (Pradeep and Nair, 2000); D. cingulatus after topical application of PII onto eggs of different ages (Gayathri-Elayidam and Muraleedharen, 2001) and the wing-dimorphic cricket Velarifictorus ornatus after injection of PI (dose over 50 μg) into short-winged females (Zhao and Zhu, 2013).However, the exact mode of the enhancing action of PI on the pre-oviposition period and the ovarian maturation rate in S. littoralis is unfortunately available right now!!

Oviposition period:
No reliable information has been obtained regarding the effects of anti-JH compounds on the oviposition period in insects.On the other hand, few reported results of affected oviposition period by IGRs are available.This period was shortened or prolonged, depending on different factors, such as the susceptibility of treated insect, potency of the tested compound, concentration level, time and method of treatment, etc. (For examples, see Kandil et al., 2005;Shahout et al., 2011;Salem, 2015;Hassan et al., 2017).
In the present study on S. littoralis, the oviposition period of adult females was slightly or significantly prolonged, depending on the PI dose and the larval instar under treatment, 5 th or 6 th instar.The shortened oviposition period can be interpreted by the enforcing action of the IGR on the adult females to lay eggs as quickly as possible for avoiding this xenobiotic factor.On the other hand, the prolonged oviposition period in S. littoralis, as a response to the action of PI, in the current investigation, cannot be interpreted right now!!However, PI may interfere with the hormonal control of egg deposition, since every physiological process in insects has been regulated by certain hormone(s).It is important to keep in mind that PI is usually considered anti-JH compound.

Disrupted reproductive potential of S. littoralis by PI:
Reproduction in insects is mainly controlled by the juvenile hormone (JH), which is also responsible for protein metabolism, and is specifically needed for egg maturation (Ghoneim et al., 2014).Effects of IGRs on the insect reproduction can be grouped into: i) reproductive behaviour, ii) oviposition, iii) egg hatchability (ovicidal and embryocidal), and iv) sterilization of adults (Mondal and Parween, 2000).On the other hand, ecdysteroids have essential functions in controlling the processes involved in insect reproduction, i.e., vitellogenesis, ovulation of matured eggs and spermatocyte growth (Wigglesworth, 1984;Hagedorn, 1985).

Inhibited oviposition efficiency of adult females:
In insects, the oviposition rate can be used as an informative indicator for the oviposition efficiency (Ghoneim et al., 2014).Depending on the current literature, oviposition rate of different insect species has been regressed by various IGRs (Al-Dali et al., 2008;Ghoneim et al., 2014;Bakr et al., 2005;Al-Mekhlafi et al., 2011;Hassan et al., 2017;Hamadah et al., 2017).However, very few studies have examined the effects of anti-JH compounds on this important reproductive parameter.Exposure of D. melanogaster females to 0.14 μmol of PI resulted in remarkably regressed oviposition rate (Ringo et al., 2005).Larval treatment of the Sunn pest Eurygaster integriceps with PI led to decreasing egg laying rate (Amiri et. al., 2010).
Results of the present study were well in agreement with those reported findings, since topical application of PI onto 5 th instar larvae of S. littoralis resulted in serious inhibition of the oviposition efficiency of adult females, i.e., oviposition rate was drastically regressed only at the lower two doses.Moreover, treatment of 6 th instar larvae with PI resulted in pronouncedly reduced oviposition efficiency, in a dosedependent course.The prohibited oviposition efficiency of S. littoralis, in the current study, can be explained as a result of the inhibition of ovarian DNA synthesis or the interference of PI with vitellogenesis via certain biochemical processes.However, anti-JH compounds may exert a reverse action to that exerted by the ecdysteroid agonists which stimulate the neurosecretory cells to release a myotropic ovulation hormone (Parween et al., 2001).

Perturbation of the reproductive capacity: 2.1. Prohibited fecundity:
In the present study, topical application of PI onto the 5 th or 6 th instar larvae of S. littoralis resulted in drastic reduction of the adult fecundity, in a dose-dependent manner or in no certain trend, depending on the larval instar under treatment.These results were, to a great extent, in agreement with those reported results of fecundity inhibition in some insects after treatment of immature with some precocenes and other anti-JH compounds.For examples, topical application of PII doses of 0.125 and 0.0625 mg onto 3 rd instar larvae of P. dux caused inhibition of the female natality (Nassar et al., 1999).Exposure of 5 th instar nymphs of N. lugens to different doses of PII resulted in fecundity reduction, in a dose-dependent manner (Pradeep and Nair, 2000).After treatment of E. integriceps nymphs with PI, fecundity of adult females was reduced (Amiri et al., 2010).Repeated daily topical application of PI and PII onto S. littoralis larvae led to reduced fecundity of its parasitic wasp M. rufiventris (Khafagi and Hegazi, 2004).Apart from precocenes, application of the anti-JH compound H17 reduced the fecundity of L. decemlineata (Lehmann et al., 2015).On the other hand, the present findings were inconsistent with those reported results of Precocene failure to affect the fecundity of some insects, such as the bug Panstrongylus megistus (Hemiptera: Reduviidae) of which males were treated with PII and ethoxyprecocene (synthesized PII analogue) but the fecundity did not differ statistically from that of the control groups (Cavalcante and Regis, 1992).
To understand the fecundity inhibition of S. littoralis, in the present study, it is important to point out that the JH is required for post-eclosion development of the vitellogenin-producing adult fat body.In many insects, including S. littoralis, JH modulates fecundity at least in part because JH is necessary to induce yolk proteins uptake into oocytes (Soller et al., 1997), while ecdysone, produced from egg follicles, induces yolk protein mRNA expressed in the fat body (Bownes, 2004;Raikhel et al., 2005;Schwedes and Carny, 2012).In addition, the fecundity inhibition in S. littoralis may be due to the interference of PI with one or more processes from the ovarian follicle development to the egg maturation.(1): PI may cause some disorders in the ovaries, including cell death in the germarium, resorption of oocytes in the pre-vitellarium and vitellarium (Khan et al., 2007;Zhou et al., 2016).( 2): PI may inhibit the synthesis and metabolism of proteinaceous constituents during oogenesis (Salem et al., 1997).(3): PI may exert an inhibitory action against the function of authentic gonadotropic hormone (JH in adults) responsible for the synthesis of vitellogenins and vitellogenesis (Di Ilio et al., 1999).

Reduced fertility:
Another informative parameter of the reproductive capacity is fertility (hatchability).In the present study on S. littoralis, treatment of 5 th instar larvae with PI caused complete sterility, since complete failure of egg hatching was observed, regardless the dose.After treatment of 6 th instar larvae with PI, the egg hatchability was severely reduced.Complete sterility was recorded at the doses 150 and 30 µg/larva.These results were, to a great extent, concomitant to those reported results of sterility and inhibited fertility of some insects after treatment of immature stages with a number of anti-JH compounds.For examples, topical application of PIII onto eggs and 5 th instar nymphs of the grasshopper Aiolopus thalassinus led to sterility of adult females (Osman, 1988).After treatment of E. integriceps nymphs with PI, the hatchability of laid eggs was reduced (Amiri et al., 2010).Apart from precocenes, phenolic chromene and hydroxyethyl chromene (isolated from Ageratum conyzoides) were found to cause sterility in the bug Dysdercus flavidus (Vyas and Mulchandani, 1984;Okunade, 2002).Bowers and Aregullin (1987) isolated an anti-JH compound, polyacetylenic sulfoxide, from Chrysanthemum coronarium which produced sterile adults in the large milkweed bug Oncopeltus fasciatus.
For explicating the fertility reduction and sterility in S. littoralis by PI, in the present study, some suggestions can be provided herein.First: Maturation of the insect eggs depends basically on the vitellogenins, precursor materials of these macromolecules including proteins, lipids and carbohydrates, all of which are necessarily required for the embryonic development (Soltani and Mazouni, 1992;Chapman, 1998).These materials are synthesized primarily by fat body during the immature stages (Telfer, 2009) or by the ovary in situ (Indrasith et al., 1988).Wherever the site of their synthesis, PI may disturb the production of these materials and/or accumulation in adult females of S. littoralis leading to the reduction of fertility.Second: PI may indirectly affect the fertility via its disruptive effect on opening of the intracellular spaces in follicular epithelium or generally inhibited the role of the gonadotropic hormone responsible for the regulation of vitellogenin deposition into oocytes (Davey and Gordon, 1996).Third: The reduction in fertility may be due to the penetration of residual amounts of PI in S. littoralis mothers into their eggs and disturbance of embryonic cuticle synthesis.So, the fully mature embryos had weakened chitinous mouth parts that were insufficiently rigid to perforate the surrounding vitellin membrane and free from the eggs (Sallam, 1999;Sammour et al., 2008).Fourth: The reduced fertility of S. littoralis may be due to serious effect of PI on survival of the developing embryos at certain stages as recorded in decreasing hatching percentage.Fifth: Because some molecular studies revealed the effects of some IGRs on insect reproduction owing to the perturbation of gene expression in the hierarchy cascade of vitellogenesis and/or choriogenesis (Sun et al., 2003), PI may interfere with the gene expression resulting in a reduction of the developed embryos in S. littoralis, in the present study.

Retarded embryonic development of S. littoralis by PI:
In insects, incubation period can be used as an informative indicator of the embryonic developmental rate, i.e., longer period usually denotes slower rate and vice versa.In the present study on S. littoralis, effect of PI on the embryonic developmental rate could not be determine after treatment of 5 th instar larvae because no eggs hatched.After treatment of 6 th instar larvae, PI exerted conspicuously retarding action on the embryonic rate, since the incubation period was remarkably prolonged.
As far as our literature survey could ascertain, no information was available on the effects of anti-JH compounds on the incubation period or the embryonic developmental rate in insects.Therefore, our result can be considered as the first report in the world concerning the effects of precocenes on the incubation period or the embryonic developmental rate in S. littoralis.Unfortunately, a conceivable interpretation of this retardation of embryonic development is not available right now!!

Conclusion:
According to the obtained results in the present study, Precocene I considerably affected the adult emergence, ovarian maturation rate, oviposition period, and adult longevity of S. littoralis.Also, it drastically prohibited the oviposition efficiency, dramatically reduced the reproductive capacity and retarded the embryonic development.Therefore, Precocene I may be a potential control agent being involved in the IPM program against this dangerous polyphagous pest.

Fig. 1 .
Fig. 1.Deformed adult females of S. littoralis after topical application of larvae with the higher four doses of PI. (A) Normal adult.(B & C): Emerged adults with curled wings.(D & E): Moths failed to completely get rid of the pupal exuvia, at the posterior extremity, and the nonexpanded wings.

Table 2 . Affected adult performance of S. littoralis after topical application of PI sublethal doses onto 1-day old last instar larvae.
a, b, c: See footnote of Table(1).

Table 3 . Reproductive potential of S. littoralis as influenced by PI after topical application of sublethal doses onto 1-day old penultimate instar larvae.
Table (4)clearly revealed a retarding action of the tested compound on the embryonic rate in the eggs, since the incubation period was remarkably prolonged (4.7±0.5, 4.2±0.7 and 4.9±0.6 days, at 15, 90 and 120 µg/larva, respectively, vs. 3.5±0.4days of control eggs). considerable

Table 4 . Reproductive potential of S. littoralis as influenced by PI after topical application of sublethal doses onto 1-day old last instar larvae.
See footnote ofTable (1).d: very highly significantly different (P<0.001)