Disinfestation of Stored-Grains with Tribolium castaneum by Using Joint Action of Gamma Radiation and some Essential Oils

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INTRODUCTION
The storage pest Tribolium castaneum(Herbst) (Coleoptera: Tenebrionidae), can be a major pest in stored grains.This species has been found associated with a wide range of commodities including grain, flour, peas, beans, cacao, nuts, dried fruits, and spices, but milled grain products such as flour appear to be their preferred food Campbell and Runnion( 2003).Members of genus Tribolium are known to produce toxic quinones, which contaminate flour and flour products (Gorham 1987).
Both larvae and adults with a reduction in quality and quantity of stored products (BagheriZenouz, 1997) inflict damage.Gamma radiations are used to produce mortality or sterility in the insects.This technique can be used by irradiating the insects at doses sufficiently high to produce the desired effects (Shishir et al.,2009).Gamma radiation has greater penetrating power for killing insects infesting commodities Brower and Tilton ( 1970).Gamma radiation has been combined with other means in attempts to reduce the effective radiation dose required to achieve control.In order to control of red flour beetle Tribolium castaneum (Herbst), fumigants are mostly used against stored-grain insect pests, not only because of their broad activity spectrum, but also because of their penetrating power resulting in minimal or no residues on the treated products.Although effective fumigants (e.g.methyl bromide and phosphine) are available, there is global concern about their negative effects, such as ozone depletion, environmental pollution, toxicity to nontarget organisms, pest resistance, and pesticide residues (Benhalimaet et al., 2004).The wide use of these fumigants has raised residue levels and has led to the development of resistance in certain species (Rossi et al., 2010).Due to environmental unfavorable effects of the fumigants, their use has recently been banned in many countries (Carpenter et al., 2000).Thus, there is an urgent need to develop a safe alternative fumigants for stored-grain pest management.Essential oils Fumigation as natural pesticides and gamma irradiation are two ecologically safe methods that could be used in the management of stored-grain insects Ahmadi and Moharramipour (2016).The red flour beetle Tribolium castaneum (Herbst) is one of the main stored-product pests that were reported to be sensitive to essential oils.
According to (Clemente et al.2003), the toxic effect of the extracts of R .officinalisL. can be used to control T. castaneum.As well as (Ashwin, et al., 2017)using essential oils likes Cinnamon oil (Cinnamom umverum), Clove oil (Syzygium aromaticum), Rosemary oil (Rosmarinus officinalis), Bergamot oil (Citrus Bergamia) and Japanese Mint oil (Mentha arvensis).These oils showed potential which can be developed as possible natural fumigants or repellents for control of the pulse beetle.There are several reports on the sensitivity of T. castaneum to radiation (Tuncbileket et al., 2003).(Ayvazet et al., 2002) found that a dose of 200 Gy gamma radiations was sufficient for acceptable mortality of T. castaneum adult in 30 days.On the other hand, the use of gamma radiation and essential oils alone at high doses is very expensive and time-consuming; therefore to reduce costs, it is important to look for a strategy that uses low doses without reducing the efficacy.Also used of low irradiation doses combined with other treatments is a way to minimize the negative effects of irradiation on food quality (Dionisio et al.,2009).The use of gamma radiation and plant essential oils are the most promising new approaches for controlling this insect.In this study, we explored the combined effect of low doses of gamma radiation and lower concentrations of two essential oils, rosemary and cinnamon, aiming to reduce the cost of effective management of T. castaneum.

Insect Rearing:
The red flour beetle T. castaneum was reared in the glass container (250ml) containing wheat flour covered with a fine mesh cloth for ventilation.Adults were obtained from laboratory stock cultures maintained at the Plant Protection Dept.Faculty of Agric., Benha University, Egypt.Adult insects (1-3 days old) were used.The cultures were maintained in the dark in an incubator at 28 ± 1 ºC and 65±5% RH.All experiments were carried out under the same environmental conditions.

Irradiation:
The source of gamma radiation used during the present study was from a Cobalt 60 (Co 60 ) irradiator installed in the cyclotron project, Nuclear Research Center, Abu Zaabal, Egypt; the dose rate of irradiation source was 1 Gray/ second.To study the effect of gamma irradiation, healthy and active adult insects (1-3 day old) were irradiated with five doses (150;200;250;300 and 350 Gy), five replicates were prepared, after irradiations, the insects were kept in the dark in the growth room, and the mortality was measured for 7 days post-treatment.

Essential Oils Used:
Two essential oils Rosemary oil(Rosmarinus officinalis L.) and Cinnamon oil (Cinnamomum aromaticum Nees)belonging to families; Lamiaceae and Lauraceae were used during these investigations.Both essential oils were bought from Al-Gomhuria Company of drugs, chemicals and medical supplies in Egypt.The fumigant toxicity of these oils was tested to the adults of T. castaneum.

Fumigation Test:
In this experiment, 200 ml glass jars with tight covers were used as fumigation chambers for the plant oil.A series of essential oil concentrations in the air was used 62.5, 125, 250, 500 and 1000 μl/l.air for both essential oils to evaluate the mortality of insects after the initial concentration setting experiment.Five replicate were made for treated and untreated (control) adults.Inside every jar, one filter paper was inserted at the bottom.Then one ml from each oil concentration was taken and added to every glass jar on a filter paper for achieving the mentioned oil dosages inside the well-closed jars.Thirty adults were put inside each jar in cotton bags (2×1 cm) with a few amounts of crushed wheat.The jars well-closed and incubated at 28±1 °C and 65±5% R.H.The same steps were followed in the control treatment.Mortality for each dose was investigated independently, the numbers of dead (immobile) and live insects in each bottle were determined until 7 days post-treatment.The 25%, 50% and 90% lethal concentrations (LC25, LC50 and LC90) values were assessed by Probit analysis (Finney, 1971).The effects of sub lethal concentrations (LC50) for the two oils on some biological aspects of an adult's stage of T. castaneum were calculated.

The Combined Effect of Gamma Radiation and Two Essential Oils on Certain Biological Aspects of T. castaneum:
In this experiment, we combined the various doses of gamma irradiation with the LC50 from each essential oil to investigate the toxicity effects on adults of T. castaneum, after 24h from irradiation, the irradiated adults were exposed to the LC50 of both essential oils R. officinalis and C. aromaticum oils; mortality was measured during 7 days post-treatment.Insects were considered dead if no leg or antennal movements were observed.Each experiment was conducted in five replicates with thirty insects in each replicate.For evaluating the joint action of Gamma radiation and essential oils in various mixtures the following equation adopted by (Mansour et al., 1966)was used: Co-toxicity factor = % Observed mortality -% Expected mortality X 100 % Expected mortality This factor was used to classify the results into three categories: A positive factor of 20 or more meant potentiation (synergistic effect), a negative factor of 20 or more meant antagonism, and any intermediate value i.e. between +20 and -20 was considered only an additive effect.TheLD50of gamma irradiation was calculated from the results of different gamma ray doses and combined with LC50 of Rosemary oil & Cinnamon oils and its effect on certain biological aspects of T. castaneum adults.

Statistical analysis:
The dosage mortality response was determined by probit analysis (Finney 1971) using a computer program of Noack and Reichmuth(1978).

RESULTS
Gamma radiation, two essential oils and their combination were investigated against the adults' stage of T .castaneum.The mortality of T. castaneum increased markedly with an increase of the radiation dose and oil concentration, that's shown in Table 1 and Fig 1.As irradiation doses of 150;200;250;300 and 350 GY caused 6.6,13.3,19.9,23.3 and 38.8% mortality after one day post-irradiation, respectively.These mortality percentages were increased with the increasing irradiation doses and exposure period to reach 35.5, 53.3, 63.3 76.6 and 81.1% mortality after one-week post-irradiation.The results showed that the doses need to kill 50% of the population(LD50) value at 7 days post-treatment was 191.95 GY Table2, Also the time required to obtain 50% mortality for the adult of T. castaneum was 1.54 days, as will show in Table 2 and3.In the case of the fumigation toxicity of essential oils, the results showed that the mortality was increased by increasing the plant oils concentrations and exposed period.At 1000 μl/lair adult mortalities were 23.3 and 16.6% after 1-day exposure and increased after 7 days post-treatment to 88.8 and 71.1 % for rosemary and cinnamon essential oils, respectively, while at 62.5 μl/l.air adult mortalities values were 2.2 and 0.0 % after 1-day exposure and increased at 7 days post-treatment to 31.1 and 24.4% for rosemary and cinnamon essential oils respectively.
Table (1): The effect of gamma radiation and two essential oils on the mortality percentage of T. castaneum adult after exposure periods.The lethal concentrations (LC50) of rosemary and cinnamon essential oils to adult stage of T. castaneum are shown in Table (2).The results showed that at 7 days post-treatment the LC50 value was 162.56and 296.30μl/l.air.For rosemary and cinnamon essential oils, respectively, the results showed that Rosemary was more toxic to T. castaneum than cinnamon essential oils.As well, the LT50 for the adults of T. castaneum were 2.18 and 4.05 days, for rosemary and Cinnamon essential oils, respectively Table (3).Results showed that mortality increased in insects with the increase in the concentration and exposure time of essential oils.A significant difference was observed in mortality among the essential oils.In all experiments, the mortality rate of insects treated with either gamma radiation or any essential oils separately was much lower than the mortality of the combined treatment.When we combined various doses of Gamma radiation 150; 200; 250; 300 and 350 Gy with LC50of rosemary oils (162.56 μl/l.air)mortality percentages of53.3,46.6, 30, 25.5 and 18.8% after one day post-treatment have occurred, these percentages were increased with increasing doses and exposed period to reach 100, 100, 86.6, 73.3 and 24.4% after one week post-treatment.Furthermore, a combination of these radiation doses with the LC50 of C. aromaticum( 296.30μl/L.air)increased the mortality from 36.6, 31.1, 25.5, 17.7 and 12.2% after one day.4) and Fig (1).Also, the times needed to obtain 50% mortalities for the adult of T. castaneum were 1.12 and 1.54 days, with the combination of gamma and rosemary and cinnamon oils, respectively.This result clearly indicates that the combination of gamma radiation and rosemary oils was the most toxic effect to the adult stage of T. castaneum, while the least effective one was using cinnamon oil alone.Although it was shown that the combination of gamma radiation and two essential oils could be provided a significant increase in mortality of T. castaneum, according to the Co-toxicity formula, the additive effect was observed when combined most gamma radiation doses andLC50 of two essential oils.Those are shown in Table 5 and Fig 2. that, combinations of gamma radiation and essential oils could be used as an effective alternative to chemical insecticides in grain against stored product insects.This method has the advantages of enhancing the efficacy of each treatment alone and reducing tile exposure period required for complete kill especially for the most tolerant insect stages.As well as, when we studied the effect of sublethal dose or concentration of gamma radiation, rosemary oil, cinnamon oil and their combination on certain biological aspects of T. castaneum in the laboratory at 28±1° C, the results indicated that the treatment of T. castaneum adults with all treatment caused significantly decreased.In the number of eggs laid per female, hatchability rate of eggs, total larval duration, the duration of pupal stage and total developmental period of immature stages while the pre-oviposition period of eggs and incubation period were obviously increased.Meanwhile, the larval mortality in the treatment was higher than control.On the other hand, no significant differences were found between the treatments and the control for the incubation period and pupal stage duration.These results are given in Tables (6).
Table4.The effect of the combined action of gamma radiation and two essential oils on the percentage mortality of adult T. castaneum .afterexposed period.

Conc.
Accumulative adult mortality % after indicated days

DISCUSSION
Irradiation and fumigation by essential oil are two main methods that could be used in IPM.Gamma radiations are used to produce mortality or sterility in the insects.This technique can be used by irradiating the insects at doses sufficiently high to produce the desired effects.The mortality of T.castaneum increased markedly by increasing the radiation dose and oil concentration.These data were agreed with Rejesus and Lapis (1973)who irradiated the insects to 200 Gy of gamma radiation killed all T. castaneum within 28 days after the irradiation, which suggests that the mortality observed in our shorter-term experiments could be higher at longer observations.After radiation treatments, the larval feeding activity was sharply reduced.Bothaina et al.,(2004)reported that feeding and damage caused by navel orange worm larvae could be reduced by as much as 78% after 300 Gy treatments .Irradiation can also extend the shelf life of various products and maintain the quality of the product over a longer period.The study also agreed with (Abbas 2010)as stated that the effect of gamma radiation on Oryzae philuss urinamensis by doses between (60,200,350 and 700 Gy) the required dose of radiation to prevent adult emergence from irradiated 5-days-old pupa was 700 Gy.The dose of 200 Gy caused 100% mortality of irradiated adults 28 days after treatment.Hassan et al., (2013)reported that the lethal effect of gamma radiation doses 0.75 and 1.0 KGy on the adults Callosobruchus maculates reared on cowpeas and chickpeas were slow during the first and third days post treatments.By increasing the dose to 1.5 KGy, the percentage of adult's mortality in both seeds was 53.0 and 40.0, respectively after 24h of treatment.On the other hand the dose 2KGy caused acute mortality for adults post treatment for cowpeas.Saad, and Kabbashi(2014),who irradiated the insects to doses between (0, 3.5, 4, 5, 6 and 8 KGY) of Gamma rays.The obtained results show that all the used doses of Gamma rays effect a mortality of 80 -100% and 93 -100% adults of the red flour beetle Triboluim castaneum Herbest, respectively.The fumigant treatment of essential oils has a great effect on adult mortality percentage of T. castaneum.These results are confirmed with (Choi et al., 2004), these oils are safe to humans and other mammals, and they are environmentally non-persistent.This study has shown that fumigation with the essential oils from R. officinalis and P. atriplicifolia has a toxic effect on T. castaneum.The essential oil from R. officinalis is an effective antioxidant in foodstuffs.The mode of action of oils may involve elements of acetylcholinesterase inhibition and octopaminergic effects toward insects.Isman (2006) stated that the rapid effect of essential oils is because of their neurotoxic mode of action.According to the results obtained from the present study insects mortality increased with increasing concentration levels and exposure time.Also,Papachristos andStampoulos ( 2004) reported an insecticidal effect of R. officinalis on Acanthosce-lidesobtectus (Say) adults when the concentration of the oil in the air was in the range from 0.8 mg/L to 47.1 mg/L.Shakarami(2013)stated that the effects of four plant essential oils, Menthaaquatica, Thymus daenensis, Myrtuscommunis and Artemisia hausskenechtii against two adult flour weevils, T. castaneum and T. confusum.Similar results were observed by (Azab et al. 2017).The data indicated clearly that the mortality of tested insects increased with increase in concentration, exposure period and temperature.The complete mortality of S. oryzae adults was obtained at conc.1.5 % (v/w) for the three tested oils, after 14 days exposure period at 30°C.In addition to toxicity action against adult insects..The combination of several independent techniques for the control of pest as integrated pest management (IPM) is one of the main strategies.Combined treatments with gamma radiation and essential oils from R. officinalis and C. aromaticum caused a higher mortality of T. castaneum than did each of the two treatments alone.The data in agreement with Ahmadi and Moharramipour (2011) indicated that the combination of gamma radiation and R. officinalis essential oil could reduce the feeding of T. castaneum larvae.They showed that the relative growth rate had significantly decreased (P < 0.05) by combining the gamma radiation and R. officinalis and that the severity of the reduction rate increased by increasing doses.Also, relative food consumption rate decreased when gamma radiation and R. officinalis were combined and its reduction severity had a relative convert relative with increasing of doses.Ahmadi and Moharramipour (2016) found that .The mortality rate was assessed 72 h after the last treatment.Integration of gamma radiation and R. officinalis oil enhanced the mortality of C. maculates compared with control treatments of either irradiation or fumigation alone.Synergistic effects on mortality of 1-2 days old adults of C. maculates were observed when exposed to the combination of gamma radiation and essential oil.In addition to toxicity action against adult insects, plant oils generally have an ovicidal action; most of the tested oils caused a high reduction in eggs deposition and larval penetration.Egg mortality is caused by the physical properties of the oil coating, blocking respiration, rather than by a specific chemical effect.Hassan (2013) obtained that the latent effects of different plant oils on adult stage when beetles of C. maculates were fed on seeds treated with the lowest two concentrations (0.0312, 0.0625%) of different oils, the number of eggs laid per female was decreased in all different oils especially in case of cowpea treated with sweet basil and lemongrass.Most of the different oils caused a high reduction in larval penetration in both types of seeds.The use of different plant oils caused the reduction in the progeny comparing to the control.Mbata and Payton (2013) on C. maculate.The data have shown that the rate of development beetle was slower in F1 progeny resulting from treated both seeds with the tested oils than those resulting with the control (33.33 and 38.33 days, respectively).On the other hand the concentration 0.0625% of sweet basil or geranium was found to be highly effective in decreasing the percentage of emergence (30.0and 40.0) in cowpeas, respectively.The highest percentage of emerged adults (76.5 and 64.0) was obtained when treated cowpeas and chickpeas with concentration 0.0312 % of petitgrain oil as compared to 91.0 and 85.0% in control, respectively.

CONCLUSION
In this work, we attempted to control T. castaneum with certain essential plant oils which seem to be safer and less contaminant to the environment.Also, these materials are cheap, available, and could be used to control other stored-product pests.Also, we conclude that the combined treatment of gamma radiation and two essential oils were established results which have a more significant effect than both gamma radiation and plant essential oil each of them alone.

Fig. ( 1 ):
Fig. (1): The toxicity effect of gamma radiation, two essential oils and their combination on the percentage mortality of adult T. castaneum, after the exposed period at the highest dose

Table 2 .
Lethal concentrations values and parameters of probit regression line estimates of gamma radiation and the two essential oils against T. castaneum adults after 7 days post-treatment.

Table 3 .
Lethal time values and parameters of probit regression line estimate for the adult of T. castaneum exposed to gamma radiation and the two essential oils after 7 days post-treatment.

Table 5 .
The combined effect of radiation and two essential oils on mortality of T. castaneumadult after 7 days post-treatments.

Table ( 6
): Effect of sublethal dose (LD50) of gamma radiation, Rosemary oil, Cinnamon oil and their combination on certain biological aspects of T. castaneum