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EFFECTS OF 20-HYDROXYECDYSONE AND INSULIN APPLICATION ON REPRODUCTION IN EPHESTIAKUEHNIELLA ZELLER (LEPIDOPTERA: PYRALIDAE).

Byline: H. Delhoum, N. Aribi, S. Kilani-Morakchi and N. Soltani

ABSTRACT

Insulin and 20-hydroxyecdysone (20E) have known roles in insects and mammals, but their interactions are complex. Therefore, the effects of human insulin and 20E have been examined in an insect Ephestiakuehniella. These two molecules, administered separately (5 and 10 u g) on newly emerged pupae, were evaluated on the reproduction; insulin and ecdysone have been given to insects by topical acetone application. The primary effects of human insulin were, firstly, specified. Insulin increased glycogen, carbohydrates and proteins amounts compared to 20E, while it decreased the levels of lipids only with the 5u gdose. Insulin and 20E increased the vitellogenin and vitellin amounts but decreased ecdysteroid content in ovaries. Duration of the oviposition period was not affected by the 20E but is shortened by insulin at the two tested doses. Moreover, the fecundity and fertility (egg hatchability) are affected by the two molecules.

Conclusively, acetone solutions of 20E or human insulin, applied topically, on insects have similar effects on all considered parameters.

Key words: Ephestia kuehniella, Insulin, 20-hydroxyecdysone, Reproduction, Metabolism.

INTRODUCTION

Reproduction and development in insects are based on two main hormones, juvenile hormone (JH) and ecdysteroids (Gade and Hoffman, 2005; Belles and Piulachs, 2015). Ecdysteroidogenesis, widely documented (Brown et al. 2009; Niwa et al. 2010; Quinn et al. 2012; Yamanaka et al. 2013), can also be initiated by insulin or insulin-like peptides (ILPS), a hormone with a major role in the carbohydrate metabolism and which also act as a nutrition-dependant control of insect development (Hua, 2010; Yamanaka et al. 2013; Okamoto and Yamanaka, 2015). Indeed, nutritional signals act in collaboration with other co-factors (rapamycin, protein kinases and nitric oxide), directly on ecdysteroidogenesis (Yamanaka et al. 2013). As in Vertebrates, insect ILPs seem to control others functions like growth, lifespan and reproduction (Antonova et al., 2012; Puig and Mattila, 2011).

Insulin, secreted by insulin-producing cells in the brain, binds to its receptor (insulin receptors) and the complex thus formed acts through the cell signaling cascade via the protein kinases and a transcription factor protein named fork head box class O or FOXO (Antonova et al., 2012). The same factor is also connected with the regulation of ecdysone signaling (Yamanaka et al. 2013).Insulin signaling pathway, conserved in animal evolution, has been studied in insects (Antonova et al., 2012;Hou et al. 2012); in spite of similar signaling pathways, the Mammals and Insects, present structural differences at the ILPS and receptors (Garelli et al. 2012).

Prior studies indicated that bovine insulin induces a biological response in insects but the physiological effects can be nuanced between endogenous ILPS and mammalian insulin (Graf et al., 1997; Riehle and Brown, 1999; Maniere et al., 2004; Brown et al. 2008); the specificity of the connection between the ligand and insulin receptors could explain the nuanced effects which are noted (Brown et al. 2008). The bovine or porcine insulin, compared to endogenous ILPS, show varying effects on vitellogenesis and ecdysteroids synthesis (Brown et al. 2008) in Insects; insulin and 20E act either synergistically (Roy et al. 2007) or antagonistically manner (Colombani et al. 2005, 2006). Moreover, endogenous ILPs are also involved in ovarian steroidogenesis regulation (Riehle and Brown, 1999; Maniere et al., 2004).

Physiological and molecular interactions, between insulin and steroid hormones, have been reported in some species of insects Aedes aegypti (Graf et al. 1997; Roy et al.2007), Diacamma sp(Okada et al. 2010) or Drosophila melanogaster (Mirth and Shingleton, 2012); however, these interactions were not well established in mammals. Colombani et al. (2005) note that this functional interaction, if is conserved in human, could play an important role in cancerous and metabolic pathologies where these signalisation pathways are involved. The factor FOXO is present in Mammals (Human also), and ecdysteroids (20E) can bind to oestrogenic receptors and cause similar effects to those induced by oestrogens (Jian et al. 2013).

Bovine or porcine insulin, tested in insect, show or suggest, in cited studies, an interaction with ecdysteroid hormone but what about the effects of the human insulin to insects and which is its action compared with the 20E? Therefore, the present study aims to examine the impact of human insulin and 20E tested separately in Ephestia kuehniella (Lepidoptera: Pyralidae). This biological model has been the subject of several studies related to reproduction and development (Tefler, 2009), profiles of ecdysteroids (El Ouar et al. 2010) and the impact of molting hormone agonists (Soltani-Mazouni et al. 2012).In lepidopteran species, ILPs have been identified and they are five in Samia cynthia, two in Agruis convolvuli and two in Spodoptera littoralis (Antonova et al., 2012). Still, little is known about the secretion of ILPs, receptor interactions, and their direct effects on specific processes in the target tissues of insects (Antonova et al., 2012).

Then, the objective of this work was to investigate the effects of human insulin and 20E on some indicators of reproduction (vitellogenins, vitellins, ovarian ecdysteroids and reproductive potential) due to the role of these hormones on this process (Riehle and Brown, 1999; Maniere et al., 2004; De Loof, 2008; Belles and Piulachs, 2015). Moreover, in order to highlight the primary effect of human insulin (glucogenesis and associated interconversions), the main biochemical components (carbohydrates, proteins and lipids in fat body) were examined; only two studies have reported that synthetic ILPs directly affect metabolic processes in insects (Antonova et al., 2012). Effects of 20E on metabolic aspects were also evaluated due to possible relationships between ILPs and ecdysteroids in metabolism control (Tatar et al., 2003; Colombani et al., 2005). The expected results will allow us to evaluate the separate action of these hormones to better understand their interactions.

MATERIALS AND METHODS

Insect and breeding: E. kuehniella Zeller (Lepidoptera, Pyralidae) is reared, to the laboratory, in boxes containing flour at a temperature of 25C and a relative humidity of 70% in total obscurity (Soltani-Mazouni et al. 2012). Pupae are dated according to their age (days) from the pupal molting. Under our laboratory conditions the duration of pupal development is above 9 days.

Hormones and treatment of pupae: 20E (Sigma, USA) and insulin or INS (Sigma) were tested separately by topical application (3 u l/ insect), at two doses (5 u g and 10 u g), according to El Ouar et al. (2010) and Roy et al., (2007).The compounds are administrated on the abdomen of newly emerged pupae (less than 6 h and without cocoon). Controls pupae received 3ul of solvent (acetone); acetone allows a better diffusion throughout the cuticle. Insuline is soluble in organics solvents (Brange, 1987) and solubility in acetone was cited in literature (Bergeron et al., 2003). In addition, previous studies, in our laboratory, show that acetone has no significant effect compared with untreated series in E. kuehniella (El Ouar et al., 2006).

Determination of biochemical composition of fat body: The fat body carbohydrates, glycogen, proteins and lipids amounts were determined, at various times during the pupal stage (0, 1, 3, 5 and 7 days), from control and treated series (20E or INS at 5u g and 10u g). Samples drawn from 6-8 individuals/age were conserved at -20C in trichloroacetic acid (20%) until analysis. Extraction and quantification of main biochemical constituents were as previously described (Abdellaoui et al., 2013). Briefly, carbohydrates, lipids and proteins were extracted according to the procedure of Shibko et al. (1966), while glycogen according to Van Handel (1965). The dosage of carbohydrates and glycogen was realized according to the method of Duchateau and Florkin (1959) using anthrone as reagent and glucose or glycogen as standard. The lipids were quantified according to Goldsworthy et al. (1972) using vanillin as reagent and lipids as standard.

Lastly, the proteins were quantified following the method of Bradford (1976) using blue brillant coomassie (G 250) as reagent and bovine serum albumin (BSA) as standard. The different results are expressed in mg.

Extraction and determination of ovarian free ecdysteroid amounts: The ovaries of controls and treated adult females, newly emerged (less than 6 hours), are dissected out and conserved in methanol (500u l); for each treatment, six to eight paired ovaries were analyzed individually. The enzyme immunoassay (EIA), of the free ovarian ecdysteroids, was conducted according to Aribi et al. (1997). Individual samples were diluted in methanol, sonicated and centrifuged. Supernatants were taken out and evaporated. The obtained extracts were dissolved in phosphate buffer (0.1 M, pH 7.4) and stored at -20C. Each sample was analyzed in duplicate using, peroxidase as the enzymatic tracer, a polyclonal antibody (L2) and tetramethyl benzidine as color reagent (L2 antibodies have a six-to eightfold higher affinity for ecdysone than for 20E).

The rabbit polyclonal antibody and the tracer were kindly supplied by Dr J.P. Delbecque (University of Bordeaux I, France) and Dr. C. Blais (UMPC, Paris, France), respectively. Data are expressed in pg equiv. 20E.

Vitellin and vitellogenin quantification: The vitellogenins quantification was made, on fat body samples collected at different ages (0, 1, 3, 5 and 7 days), in female pupae of controls and treated series. Ovaries, collected on newly emerged adult female controls and treated, were used for vitellin quantification. Extraction and quantification were donne as previously described (Boulahbel et al., 2015). The extraction was performed using Tris buffer (0.5 M; pH 7.4) following the procedure of Postlethwait et al. (1980) and Fabre et al. (1992); then, the quantification was made according to Bradford (1976). Assays were conducted with 6-8 repeats by age and for each series.

Evaluation of reproductive potential: The newly emerged adult females, resulting from control and treated (20E or insulin) pupae, are coupled with untreated males. The different couples were maintained under controlled conditions and regular monitoring was allowed to determine the pre-oviposition and oviposition periods, fecundity (number of eggs per female throughout its lifespan), fertility (hatgheg egg by female) and egg hatchability (percentage of neonates that emerged from eggs). Experiments were conducted with 6-8 repeats by age and for each series.

Statistical analysis: Results are presented as means standard deviation (SD). The homogeneity of variances was checked by Levene's test. Data were subjected to Student's t-test and analysis of variance followed by a post-hoc Tukey at level 5%.

RESULTS

Effects of insulin and 20 E on the fat body biochemical components: In the control of E. kuehniella, the carbohydrates amount decreased significantly during the pupal stage at 5 (p = 0.01) and 7 days (p = 0.002) (Fig. 1A). The comparison between controls and treated series shows that insulin increases the carbohydrates amount at all ages tested with 5 and 10 u g (p = 0.0001); however, a dose response relationship with 5u g of insulin shows higher values at days 5 (p = 0.0001) and 7 (p = 0.0001). Treatment with 20E also increases significantly, on day 3, at the lowest dose (p = 0.0001), at day 5 with the two tested doses (p = 0.0001) and day 7, only at the highest dose (p = 0.0001). Insulin induces a marked effects on the amounts of carbohydrates (p= 0.0001), particularly at 1 and 3 days (p = 0.0001) (Fig. 1A). In the control series, the glycogen amounts decreased from day 3 (p = 0.02) of the pupal stage (Fig. 1B).

Treatment with insulin increases glycogen amounts compared to the controls (Pless than 0.01); this rise occurs, at day 5 of pupal stage, with both doses 5 and 10 u g (Pless than 0.001) and also at days 1, 3 and 7 in the high dose (p = 0.02, p = 0.03, p = 0.0002 respectively). The 20E increases glycogen amounts, at day 7 (Pless than 0.01), only with 5 u g of the pupal stage. Insulin, in high dose shows an effect more significant on glycogen amounts at all tested ages (p = 0.006). Proteins, in the control series, decrease during the pupal stage (Fig. 1C). Insulin application, compared to controls, increases protein amounts, at days 3, 5 and 7 with the two tested doses (Pless than 0.001); insulin dose effect is noted, at day 5, with 10 u g (p = 0.0001). Treatment with 20E involves a reduction, on day 1 (Pless than 0.001), with only the low dose (5 u g) but an increase at days 5 (Pless than 0.001) and 7 (Pless than 0.001), respectively.

Insulin has an effect more significant than 20E on protein amounts (p = 0.01), particularly on day 5 of the pupae (Pless than 0.0001). Lipids amount, during the pupal stage, in the control and treated series with 20E and insulin, increases significantly (Pless than 0.01) at days 5 and 7 (Fig. 1D). The comparison, between controls and treated pupae, indicates that only insulin at the low dose (5 u g) causes a decrease in lipid levels at all tested ages (Pless than 0.001). 20E has no effect on lipid amounts.

Effects of insulin and 20E on the ovarian ecdysteroids amounts: In the control series, ovarian hormonal amounts, determined to newly emerged females adults, were 2235220.90 pg equivalent 20E. Insulin and 20E present, compared to controls, a similar and highly significant effect (Pless than 0.0001) in the ecdysteroid amounts. They decrease after treatment by 20E at 5 u g (161.70 23.93; p = 0.0007) and 10 u g (343 7.14; p=0.001). Insulin, also, involves a reduction at 5 u g (335.30 51.97; p=0.001) and 10 u g (552.10 85.59; p = 0.002) (Tab. 1). The high dose of 20E and the two doses of insulin have a comparable effect on hormonal titers, while the 20E at the low dose, causes marked decrease (Pless than 0.0001).

Effects of insulin and 20E on vitellogenin and vitellin amounts: In controls pupae, the vitellogenins show a pic at day 5 of the pupal stage (p = 0.0002); then, the vitellogenins values decrease on the eve of the adult emergence (p = 0.02). The low doses of insulin involves, during the pupal development, an increase in the amounts of vitellogenins at day 1 and 3 (Pless than 0.001), but a reduction at day 5 (Pless than 0.001) (Fig. 2). The 20E, at two doses, increases significantly (Pless than 0.001) the vitellogenin amounts at days 1, 3, 5 and 7 respectively; a dose-response relationship was observed at day 3 (Pless than 0.001). The 20E induces a more significant effect on the vitellogenin amounts (Pless than 0.0001), compared to insulin treatment, particularly, at days 5 and 7 (Pless than 0.0001). The ovarian vitellin, in females of E. kuehniella,has values of 418.01 18.33 u g in controls (Tab. 2).

Treatment with two doses of 20E and the low dose of insulin increase the ovarian vitellin amounts; the high dose of insulin has no significant effect. The 20E has a marked effect (Pless than 0.0001) on the vitellin amounts than insulin (Tab. 2).

Effect of insulin and 20E on reproductive events: Treatments were made topically, on newly ecdysed pupae, and their effects were investigated on the reproductive events of adult females, following treated pupae. The results, presented in table 3, indicate that in control series the duration of pre-oviposition period was 1.40 0.24 days. The treatment with 20E or insulin has no significant effect (p = 0.39)on this parameter. Concerning the duration of the oviposition period, we note a value of 3.80 0.37 days in control. This period is shorter after insulin treatment with 5 u g (2.60 0.24; p = 0.02) and 10 u g(2.50 0.43; p = 0.03). The oviposition period is not affected by 20E. Fecundity is 189 3.2 eggs per female in the control series (Tab. 4). The 20E and insulin, with two tested doses, reduce significantly the fecundity compared to controls (pless than 0.0001).

Indeed, the number of eggs per female fall to 155.7 3.37, after treatment, with 5 u g of 20E; an important drop is recorded after treatment with 10 u g of 20E (123.7 2.26) and with 5 and 10 u g of insulin (118.2 1.28; 127.4 1.50). Insulin and 20E, at high doses, have a drastic effect (Pless than 0.0001).The fertility (egg hatched by female), in control series was 175.20 3.89. This value is reduced after 20E treatment with 5 u g (98.33 4.31; Pless than 0.0001) and 10 u g (98.93 3.06; Pless than 0.0001). Insulin has the same effect as the 20E and shows values of 92.80 1.88 (Pless than 0.0001) and 101.20 0.96 (Pless than 0.0001), for the 5 and 10 u g respectively. So, the lowest fertility is noted with the 20E and the low dose of insulin.

Table 1. Effects of 20-hydroxyecdysone (20E) and insulin (INS), applied topically and separately on newly ecdysed pupae (5 and 10 ug), on total ovarian ecdysteroid amounts (pg equiv 20E) from newly emerged adult females of E. kuehniella. (m SD, n = 6-8). For all series, the values followed by the same lowercase letters are comparable to the 5% threshold.

###Control###20 hydroxyecdysone###Insuline

###5 ug###10 ug###5 ug###10 ug

2235 220.90a###161.70 23.90b###343.70 7.14c###335.30 51.97c###552.10 85.50c

Table 2. Effects of 20-hydroxyecdysone (20E) and insulin (INS), applied topically and separately on newly ecdysed pupae (5 and 10 ug), on total ovarian vitellin amounts (ug) from newly emerged adult females of E. kuehniella. (m SD, n = 6-8). For all series, the values followed by the same lowercase letters are comparable to the 5% threshold.

###Control###20 hydroxyecdysone###Insuline

###5 ug###10 ug###5 ug###10 ug

418.01 18.33a###1327.68 48.00b###508.84 15.84c###447.84 13.34d###413.53 8.82a

Table 3. Effects of 20-hydroxyecdysone (20E) and insulin (INS), applied topically and separately on newly ecdysed pupae (5 and 10 ug), on the duration (days) of pre-oviposition and oviposition in adult females of E. kuehniella. (m SD, n = 6-8). For each parameter and for all series, the values followed by the same lowercase letters are comparable to the 5% threshold.

###Treatments###Pre-oviposition period###Oviposition period

###Control###1.40 0.24a###3.80 0.37a

###5 ug###1.83 0.31a###4.00 0.25a

###20 hydroxyecdysone

###10 ug###2.33 0.33a###3.83 0.47a

###5 ug###1.80 0.37a###2.60 0.37b

###Insuline

###10 ug###1.83 0.31a###2.50 0.43b

Table 4. Effects of 20-hydroxyecdysone (20E) and insulin (INS), applied topically and separately on newly ecdysed pupae (5 and 10 ug), on the fecundity (number of eggs laid/female), hatchability (egg hatchability in %) and fertility (hatched egg/female) in adult females of E. kuehniella. (m SD, n = 6-8). For each parameter and for all series, the values followed by the same lowercase letters are comparable to the 5% threshold.

###Fecundity###Hatchability###Fertility (hatched

###Treatments

###(eggs/female)###(%)###eggs/female)

###Control###189 3.20a###92.70 2.50a###175.20 3.89a

###5 ug###155.70 3.37b###63.10 5.32b###98.33 4.31bc

###20 hydroxyecdysone

###10 ug###123.70 2.26cd###80.00 6.60c###98.83 3.06bc

###5 ug###118.20 1.28c###78.34 2.44c###92.80 1.88b

###Insuline

###10 ug###127.40 1.56d###79.43 3.96c###101.20 0.96c

DISCUSSION

Effects of insulin and 20E on the main biochemical components of fat body: In E. kuehniella, the decreased of carbohydrates, during the pupal stage, could be explained by the energy used in the metamorphosis process. Glycogen, essential form of carbohydrates reserves, is reduced on day 3 of the pupal stage, coinciding with the apolysis and is linked to chitin synthesis (El Ouar et al. 2010). In E.kuehniella,human insulin increases the glycogen amounts, in fat body, similar to synthetic endogenous ILPS (Brown et al. 2008). In E. kuehniella, our results show that the effect of 20E on carbohydrates is comparable to that of insulin, confirming the previous work in D. melanogaster (Hou et al. 2012). The 20E can act as an inhibitor of glycolysis during metamorphosis in Bombyx mori (Tian et al. 2010); indeed, insulin and 20E inhibit the same molecular mechanisms in D. melanogaster (Puig and Mattila, 2011) and Helicoverpa armigera (Hou et al. 2012).

In controls pupae of E. kuehniella, the reduced protein levels recorded could be related to the synthesis of the new cuticle but, also, with the vitellogenesis process (El Ouar et al. 2010); the proteins used for the elaboration of adult tissues may also explained this decrease. The insulin treatment, in E. kuehniella pupae, induces an increase in the protein amounts in accordance with known roles of ILPS in A. aegypti (Graf et al. 1997). The 20E presents, in low doses, a similar action as the insulin; moreover, the absence of effect in the strongest dose can be linked to a regulatory process. The lipids constitute a mobilizable energy quickly during fasting and this can be explained a gradual increase observed during metamorphosis. Insulin, tested at the high dose, has no effect and this is in agreement with the work of D. melanogaster (Ceddia et al. 2003) and A. aegypti (Brown et al. 2008).

Otherwise, the insulin signaling pathway is reported as a potential regulator in the modulation of lipid contents (Baumann et al. 2013); this may explain the observed decrease in the lipid amounts with the low dose (5u g). After application of 20E in E. kuehniella, lipid levels are comparable to those of the control series, confirming the work under taken in other species of Lepidoptera as Bombyx mori (Wang et al. 2010). Thus, the results show effects of human insulin but, also, the role of ecdysteroids in the metabolism on E. kuehniella pupae. Tatar et al. (2003) hypothesized that many effects attributed to ILPs, in insects, may be mediated by ecdysteroids. Moreover, in D. melanogaster, Colombani et al. (2005) noted that ecdysone can exercise its regulation through modulation of the insulin/IGF signalling and the Foxo activity in the fat body.

Effects of insulin and 20E on the reproduction: The endocrine control of reproduction (like the vitellogenesis) is mainly made by juvenile hormone (JH), 20E, neuropeptides and ILPs (Belles and Piulack, 2015). The evolution of vitellogenins amounts in controls pupae of E. kuehniella shows a peak, between 3 and 5 days, which coincides with ecdysteroids increase, cited in various studies like in El Ouar et al. (2010). Separates application of 20E (5 and 10 u g) and insulin (5 u g) involves an increase in vitellogenins and vitellins amounts. This is in agreement with Parthasarathy and Palli (2011) who report a potentialization of the vitellogenins and vitellins synthesis by the 20E; the works of Roy et al. (2007) in Aedes aegypti show that insulin involves, also, a rise of the vitellogenins. In vitro, a direct stimulatory effect of bovine insulin on protein synthesis of ovaries has been demonstrated for A. aegypti (Graf et al., 1997).

Moreover, the literature specifies that these two signaling pathways are essential in the vitellogenesis and oocyte maturation (Parthasarathy and Palli, 2011). Thus, the increase of vitellogenins and vitellins after the insulin treatment seem to be explained. The decrease of vitellogenins amounts observed at day 5 of the pupal stage, after application of insulin, may also be related to the molecular interaction between insulin and JH (Parthasarathy and Palli, 2011). Insulin/20E or insulin/JH interaction, still under review, is based by the complex relations via the factor of transcription FOXO (Antonova et al., 2012; Yamanaka et al., 2013). Insulin may also act in combination with the amino acids and 20E as a third component which regulate vitellogenins (Roy et al. 2007); this regulating effect on vitellogenins could explain the absence of insulin effect at the highest dose (10 u g).

In insects, the ovarian steroidogenesis is a remarkable target of insulin (Riehle and Brown, 1999). Bovine insulin was found to stimulate, in vitro, ecdysteroid production by the ovaries in A. aegypti (Riehle and Brown, 1999) and Phormia regina (Maniere et al., 2004). It was reported that bovine insulin, only to an optimal dose (17 uM), increases ecdysteroids production in Aedes's ovaries (Brown et al. 2008) and porcine insulin also stimulates this parameter in the same species (Graf et al. 1997). However, Mammals insulin, compared to endogenous ILPS seems to constitute bad agonists for the ecdysteroids production (Brown et al. 2008). In E. kuehniella, measurements of ovarian ecdysteroids, before and after enzymatic hydrolysis with porcine liver esterase, show that conjugate ecdysteroids represent only 8.37% of total ecdysteroids (Yezli et al., personal communication).

In our results, application of 20E and insulin shows similar effects corresponding to a decrease in free ovarian ecdysteroids amounts compared to controls. Ecdysteroids decrease may be explained by a same molecular mechanism cited by Puig and Mattila (2011). The most marked effect noted with the low dose of 20E can suggest an endocrine regulation; indeed,in vivo, several factors (hormones, peptides and proteines kinases) can affect the ecdysteroid biosynthesis. In addition, ecdysteroids themselves play a role in their ownbiosynthesis via a feedback regulation of the PTTHaxis in the insect brain, and via direct action on the ecdysteroid-producing (prothoracic glands and/or endocrine organs like ovaries) (Belles and Piulack, 2015). In lepidopteran species, recent studies provide evidence for ILP regulation of ecdysteroid production by the PG, perhaps by signaling nutritional state and via the prothoracicotropic hormone or PTTH (Antonova et al., 2012).

Moreover, the role of ILPs relative to ovary ecdysteroidogenic hormone (OEH) that also stimulates ecdysteroid production is unresolved.

The insulin and 20E treatment reduce fecundity and fertility; furthermore, insulin reduces the oviposition period. Fecundity and fertility reduction can be also explained by the decrease in the ecdysteroid amounts. A reduction of fertility was, also, noted after insulin treatment and may be related to a decrease in egg hatchability (Kuo et al. 2012; Baumann et al. 2013). The insulin signaling pathway can act in the differentiation of the ovary and is essential in the last stages of oogenesis (Okada et al. 2010).Otherwise, the same molecular mechanism controls the adjustment of the functions of endocrine system and the insulin signaling pathway (Puig and Mattila, 2011); so, the regulating role would be thus be possible in the process of reproduction where the 20E and the HJ are also involved. In addition, ecdysteroid actions in certain stages of reproduction like oogenesis or the effects of OEH a being studies (Belles and Piulachs, 2015)

In conclusion, the results, on the metabolic aspects, show that human insulin can act in E. kuehniella. Insulin signaling pathway/insulin receptors, highly conserved and action of the same molecular mechanisms can be explained this action; in addition, studies suggest that multiple ILPs can activate the insulin receptor and stimulate the signal transduction. Moreover, differences of action between human insulin and endogenous ILPs might be explained by a specificity of binding between ligand and insulin receptor. Besides, the 20E can acts also in metabolism control; consequently, the functional interaction between human insulin and 20E seems to be possible. Acetone solutions of 20E or human insulin, applied topically on insects have similar effects on all considered parameters.

The modulations observed, according to doses and age, can be explained by the possible relationships between ILPs and ecdysteroids, cited in the literature; this, in particular, when these hormones are involved in the control of the same functions. In E. kuehniella, our results show that human insulin can acts at the level of fat body but cannot stimulate the ovarian steroidogenese; however, these results seem to be in favour of insulin impact at the level of ovaries. Studies show that ILPs can differ in their ability to activate various physiological processes; indeed, at similar doses, ILPs cannot activate the anabolic processes in the fat body, but activated the ovarian production in vitro (Antonova et al., 2012). Further investigations are needed to understand how do multiple ILPs interact with an insulin receptor and coordinate diverse physiological and behavioral processes in different tissues.

Moreover, the mechanisms of action of insulin and 20E remain to clarify; in addition, a combined treatment of insulin and 20E, at different application time, are needed to precise the interaction between these two hormones.

Acknowledgements: The authors are grateful to Dr. JP Delbecque (University of Bordeaux I, France) and Dr. C. Blais (Paris XI University, France) for providing antibodies and tracer, respectively. This research was supported by the National Fund for Scientific Research of Algeria (Laboratory of Applied Animal Biology to Prof. N. Soltani), by the Ministry of Higher Education and Scientific Research of Algeria (CNEPRU project F 011.2012. 0038 to Prof. N. Aribi).

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