Effects of Three Essential Oils and / or Gamma Irradiation on the Greater Wax Moth , Galleria Mellonella

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INTRODUCTION
Wax moths are serious pests of bees wax worldwide.Greater wax moth (GWM), Galleria mellonella L., and lesser wax moth, Acheroia gresilla L., are known to be harmful to deposited and stored beeswax.GWM causes the greatest damage in apiaries which lead to financial losses every year, beside damaging wax combs by larval feeding, and destroying frames and wooden parts in the hive.Adult wax moths and larvae can also transfer pathogens of serious bee diseases, e.g.foulbrood (Charrière andImdorf, 1997 andOwayss andAbd-Elgayed, 2007).
Possibilities for controlling wax moth include some manipulations in the hive and other treatments to stored combs i.e. technical, physical, biological and chemical methods e.g.sulphur fumigation, acetic and formic acids evaporation and applying paradichlorobenzene (Owayss and Abd-Elgayed, 2007).
The use of chemical agents to prevent or control insect infestation has been the main method of protection against GWM, since it is the simplest and most costeffective means of dealing with stored product pest (Hidalgo et al., 1998).
However, insecticides have serious drawbacks such as pest resurgence and resistance, lethal effects on non-target organisms, the risk of user´s contamination, food residues, and environmental pollution (Tapondjou et al.., 2002).In addition, the precautions necessary to work with traditional chemical insecticides (Fields et al., 2001), and the poor storage facilities of traditional farmers in developing countries, make conventional chemical control unsuiTable (Tapondjou et al., 2002).Thus, there is an urgent need to develop safe alternatives for the protection against this insect.Considerable efforts by many researchers have been focused on plant derived materials, potentially useful as commercial insecticides (Padin et al., 2013, Wondafrash et al., 2012, Sreekanth 2013and Sharma et al., 2014).Since these plant materials with insecticidal properties are often active against a limited number of species , are often biodegradable to non-toxic products and are potentially suiTable for use in integrated pest management , they could lead to the development of new classes of safer insect control agents (Kim et al ., 2003).
The use of sub sterilizing doses of radiation to produce sexually competitive moths has been suggested by many researchers.Furthermore, by using this method, the F 1 offspring of irradiated lepidopterous species are often partially or completely sterile.So, reproduction is suppressed in at least two generations with only one release.This phenomenon is known as F 1 sterility or inherited sterility.(Abd El-Hamid, 2004).The combined effects of gamma irradiation and bioinsecticides on Lepidopterous insects have been studied by several authors (Sallam et al., 1991;Mohamed, 2004;Mohamed et al., 2004;El-Nagar et al., 2004;El-Shall & Mohamed, 2005;Mohamed, 2006 andEl-Naggar et al., 2012).
The present study aimed to clarify the possibility of using certain essential oils, namely, Mentha piperita, Pelargonium graveolens and Ocimum basilicum alone or combined with gamma radiation in the control of this serious pest.
Emerged adults were collected and kept in similar empty glass containers (egg laying cages) provided with paper lids (egg laying substrate) to the glass container.Paper carrying the eggs were removed for collection and replaced by new one.Eggs were gently removed and transferred to other rearing jar containing 250gm media, closed tightly with double muslin layer to prevent the escape of neonatal larvae, and incubated.Fresh food was add frequently (1-2) times per week.

Source of irradiation
The source of gamma radiation used during the present study was from a Cobalt 60 ( 60 Co) irradiator installed in the cyclotron project, Nuclear Research Center, Abu Zaabal, Egypt; the dose rate of irradiation source was 1 Gray/ second.

Volatile oils
Three commercially available volatile oils were tested in this study.
Peppermint oil, Mentha piperita; geranium oil, Pelargonium graveolens; and basil oil, Ocimum basilicum.All tested oils were purchased as pure oils (Branded in Egypt) from kateo aromatic Company of medicinal and aromatic oils.The oils were extracted from the dried plants by steam distillation.

Experimental technique Contact toxicity
Four different concentrations (0.625%, 1.25%, 2.500% and 5.00 %) of each of the tested oils were prepared from the stock solution by dilution with acetone in volumetric flasks to give the necessary concentrations.The contact toxicity was tested in Petri dishes (10 cm), each of them containing 10 larvae.Filter paper disks (Whatman No. 1) were cut (10 cm) and impregnated with series of concentrations of each essential oil.Ten replicates were run for each concentration and control (untreated and acetone).After 24, 48, 72 and 96 h from the beginning of exposure, numbers of dead and alive larvae and the percentage total larval mortality at the end of larval period were recorded.The (LC 10 , LC 50 and LC 90 ) values were assessed by Probit analysis (Finney, 1971).The effect of sub lethal concentrations (LC 50 ) on some biological aspects (pupation, emergence, survival and sex ratio) of the treated stage and its subsequent developmental stages were determined.

Irradiation process
Full-grown pupae of GWM (Parental male ; P 1 ) were irradiated 24-48 h before adult emergence (male line and female line) with 3 sub-sterilizing doses 100, 150, and 200 Gy and two sterilizing doses 250 and 300 Gy, in a 100 cc plastic cup.The fecundity and fertility were determined.Four crossing combination(5 replicates for each) were set up as follows for each dose: treated ♂♂ Χ untreated ♀♀, untreated ♂♂ Χ treated ♀♀, treated ♂♂ Χ treated ♀♀, untreated ♂♂ Χ untreated ♀♀.The later combination was used as control.The daily-deposited eggs of irradiated and non-irradiated females were collected, counted, recorded and kept for calculating the percentage of eggs hatched.

Combined effect of gamma irradiation and essential oils on certain biological aspects of G. mellonella
One dose level of gamma irradiation from the previous doses was chosen (100Gy) to study the combined effect of gamma irradiation with the (LC 50 ) of the two tested essential oils M. piperita and P. graveolens on some biological aspects of the greater wax moth, Galleria mellonella (larval mortality, pupation, emergence, sex ratio and the survival) among F 1 generation.
To continue, the F 1 generation for the male line, newly hatched larvae resulting from irradiated P 1 males were kept in groups, in glass jars, and provided with a semi-synthetic diet.The contact toxicity of three acetone concentrations (LC 50 ) of the two volatile oils has been tested as previously described.Rearing was continued in the same way until larvae reached the last larval instar, then larvae were transferred individually to small plastic vials (10 c) for pupation.For each treatment, the newly emerged males of the first generation (F 1 ) descendants of irradiated parental males were paired with newly emerged untreated females using all possible crosses between them in order to obtain the F 2 generation.The daily-deposited eggs of irradiated and non-irradiated females were collected, counted, recorded and kept for calculating fertility.

Statistical analysis
Data were statistically analyzed using the Analysis of Variance (ANOVA) technique and the means were separated using Duncan s multiple range test (P> 0.05) (Steel and Torrie, 1980).

RESULTS AND DISCUSSION
Data obtained in The lowest percentage of total larval mortality, 18%, was recorded at 0.625 concentrations of the same oil.Many authors recorded the mortality of Lepidopteran insects as a result of treatment with plant oils; as Sharma et al.., (2001) ;Moawad, (2001) and Pavela & Chermenskaya (2004).Our results runs parallel to what have been found by Mohamed (2012), who declared that, the effect of the plant extract (Neem) on the percentage of larval mortality of G. mellonella increased by increasing the period horizontally and the concentration vertically.The activity of Pelargonium graveolens against another insect pest was reported by Kabera et al., (2011).The authors stated that this oil had high contact and oral toxicity against the maize weevil (Sitophilus zeamais).
Data summarized in Table (2) illustrate the biological effect of the selected oils on 4 th larval instar and its subsequent developmental stages of the Greater wax moth, G. mellonella.Percentages pupation was reduced by increasing the concentrations of the applied oils.A complete inhibition of pupation was observed at 2.5 and 5% of O. basilicum followed by 21% at 5 % of M. piperita.
The data presented in the same Table also show that the percentage of adult emergence reduced at all tested concentrations of plant oils compared to the control.A complete inhibition of adult emergence resulted from the treatment of larvae with 2.5 % of O. basillicum.5% of P. graveolens caused 97.50% reduction in adult emergence.Whereas, a dose of 5% of M. piperita resulted in 73.33% % reduction in adult emergence.
Treatments with the oils obviously decreased the survival percentages.All insects failed to survive when treated with 2.5 % of O. basilicum.The percentages of survival were 17% at 5 % of Mentha piperita while the highest percentage of survival recorded was 78% at 0.625 % Pelargonium graveolens.The sex ratio was extremely affected at all concentrations of the tested oils; it was in favor of male at highest concentrations.A significant variation was found between means of larval-pupal period at all treatments.All treatments prolonged the larvalpupal period especially at 5 % Mentha piperita where it reached 44.34 days compared to 35.28 day in the control.Decrease in survival of insects treated with essential oils was also reported by many authors as Padin et al., (2013) on Tribolium castaneum and Kazem & El-Shereif (2010) on some piercing sucking cotton pests, Sreekanth (2013) on mussel scale (Lepidosaphes piperis).Our results are in agreement with Mohamed (2012) who found that the treatment of Galleria mellonella with Neem extract decreased the percentage of pupation, adult emergence and survival.
The average number of eggs laid by females was significantly decreased by increasing the irradiation doses at all treatments.The highest decrease in the average number of eggs was at the mating (Normal Male x Treated Female) among the three doses 200 Gy, 250 Gy and 300 Gy, it was 377.2, 225.6 and 177.2 respectively compared with the control treatment.While, in the treatment (Treated Males x Treated Females), the insects failed to lay any eggs.This reduction in the fecundity of treated insects may be due to the reduction in longevity, the number of oocytes per ovary and oviposition period (Soltani and Mazouni 1992).Also, fertility of the treated insects was significantly decreased by increasing the dose at all treatments when compared with the control.Fertility was sharply decreased to 3.93% at 250 Gy TM x NF, 3.00 % at 200 Gy NM x TF, and 1.04% at 250 Gy NM x TF.No eggs were hatched at 200 or 250 Gy at the mating TM x TF and at all mating combinations of the dose 300 Gy.This apparent decrease in fertility may be due to sterilization of both eggs and sperms or may be due to inability of the sperms to be transferred to females during copulation.The decrease in the fecundity and fertility of irradiated insects was noticed by many other investigators as Hofmeyr et al., (2004);Tate et al. (2007); Aye et al. (2008); Mohamed (2006); El-Kholy and Abd-El-Aziz (2010); Mohamed (2012).In all treatments tested, irradiation and/or oils significantly increased larval mortality.The recorded mortalities were 30.50% in case of 100 Gy radiation and 51 & 55% in case of P. graveolens and M. piperita, respectively and it were 68 & 70% in case of the combined treatment with P. graveolens and M. piperita, respectively.No significant difference was found between the activities of both oils in case of separate or combined treatments.
In all treatments tested, irradiation and/or oils significantly decreased the percentage of pupation at the F 1 generation.Pupation percentages were 69.5% in case of radiation and 45 & 49% in case of M. piperita and P. graveolens, respectively and it were 29 & 32% in case of the combined treatment with M. piperita and P. graveolens, respectively.No significant difference was also found between the activities of both oils on pupation or adult emergence in case of separate or combined treatments.The sex ratio of F 1 generation skewed toward males in the control and at all treatments.
The data in the same Table (Table  4) show that the percentage of F 1 larvae survived to the adult stage was significantly decreased among all single or combined treatments.Percentages of survival were 62.0% in case of radiation and 42 & 47% in case of M. piperita and P. graveolens, respectively and it were 26 & 28% in case of the combined treatment with M. piperita and P. graveolens, respectively as compared to 91% survival in control.No significant difference was also found between the activities of both oils on survival in case of separate or combined treatments.Mortalities of many Lepidopteran insects due to treatments with plant extracts and/or gamma irradiation was reported by many investigators.El-Shall et al, (2005) in their study on Spodoptera littoralis, found that ethanol, petroleum either and chloroform extracts of Eucalyptus camaldulensis induced serious chronic effect on larvae, pupae and adult emergence when used alone or combined with gamma radiation.As well, El-Naggar et al., (1992) reported that the effect of gamma irradiation and extracts from Tafla leaves Nereum oleander and nabk Flacourtia indica on Spodoptera littoralis either used alone or combined with gamma radiation reduced the development of larvae or pupae and inhibited adult emergence.In addition, Sileem (2004) declared that, the effect of gamma irradiation and extracts from Malissa azedrach fruits or Schintis terebinthifdies leaves on Agrotis ipsilon, used alone or combined, reduced the development of larvae or pupae and inhibited adult emergence.Sharma and Seth (2005) in their study of Malia azedrach and its combination with gamma radiation on S. litura found that the growth and the development of larvae were reduced.
Generally, joint treatment of irradiation plus oils tested in the present work was more efficient than oil or irradiation separately.Combination of irradiation and plant oils increased larval mortality and reduced pupation as well as adult emergence and survival.These deleterious effects of combined treatments may be due to an increase in the susceptibility of irradiated insects to oils (El-Naggar et al., 1992).Seth and Sehagel (1993) stated that the growth index in S. littura of F 1 progeny resulting from irradiated male parents decreased by increasing gamma irradiation dose.El-Naggar et al. (1999) stated that joint treatment of irradiation plus plant extracts was more toxicant than the plant toxicity or irradiation separately.Hazaa (2005), also stated that the growth index in S. littura of F 1 progeny resulting from irradiated male parents and treated with red gum plant extract, Eucalyptus camaldulensis was obviously reduced more than control in most treatments.Also, Mikhaiel (2011) stated that the adverse effect on growth and reproduction of the progeny of Sitotroga cerealella moth, descending from irradiated male pupae with 175 Gy and treated with four plants extracts was increased by increasing the concentrations applied.Similar explanations were also made by El-Shall andMohamed (2005) on A. ipsilon, Fadel et al. (2003) on Ceratitis capitata, Mohamed (2012) on Galleria mellonella and Mohamed (2013) on Agrotis ipsilon (Hufnagel).

CONCLUSION
It may be concluded that LC 50 of Mentha piperieta combined with the dose 100 Gy can be efficient for the effective control of Galleria mellonella in particular and lepidopterous pests in general.Moreover, Ocimum basillicum is more toxic against Galleria mellonella if used alone without any combination treatment.Also, we can conclude that the combined treatment with gamma irradiation and plant oils is more efficient in control than single treatments.

Table 1 :
Table (1)show the effect of four different concentrations of the tested oils on 4 th larval instar of G. mellonella at different time intervals.Results show that all concentrations tested have toxic effect on the treated larvae compared to the control.The percentage of larval mortality increased vertically by increasing the concentration and horizontally by the time of exposure.The highest larval mortality (100%) was obtained with 2.5 % of Ocimum basilicum.Effect of some volatile oils on the percentage larval mortality (Mean ± SE) of the Greater wax moth, galleria mellonella Means followed by the same letter in each column are not significantly different at (p> 0.05) *

Table 2 :
Effect of some volatile oils on some biological aspects of the greater wax moth, Galleria mellonella aMeans followed by the same letter in each column are not significantly different at (p > 0.05)

Table 3 :
Effect of gamma irradiation on fecundity and fertility of the greater wax moth, Galleria mellonella parents Means followed by the same letter in each column are not significantly different at (p > 0.05)

Table 4 :
Combined effect of 100 Gy of Gamma irradiation (irradiated parental male) and LC 50 of some volatile oils (treated F 1 descending of irradiated parental male) on some biological aspects of the greater wax moth, Galleria mellonella Means followed by the same letter in each column are not significantly different at (p > 0.05)