Synergistic Effects of Nanocomposite Films Containing Essential Oil Nanoemulsions in Combination with Ionizing Radiation for Control of Rice Weevil Sitophilus oryzae in Stored Grains.

The fumigant toxicity of eight individual essential oils (EOs; basil, cinnamon, eucalyptus, mandarin, oregano, peppermint, tea tree, and thyme) and one binary combination (thyme and oregano) for control of the rice weevil, Sitophilus oryzae, were investigated. In bioassays, all individual and combined EOs were toxic to the rice weevil. Eucalyptus EO exhibited the highest toxicity among the individual EO treatments, causing 100% mortality at a minimum concentration of 0.8 µL/mL after 24 hr of exposure. The combination treatment of oregano and thyme EO displayed higher fumigant activity than the individual oregano or thyme treatments. A stable oil-in-water nanoemulsion was evaluated using high-pressure homogenization (microfluidization [MF]) and varying the pressure and number of cycles. The droplet size of the emulsions was found to decrease from 217 to 71 nm and encapsulation efficiency increased from 37% to 84% with increasing MF pressure and number of cycles. The optimum conditions for preparing the mixture of oregano and thyme EO nanoemulsions were evaluated to be homogenization pressure of 103 MPa and three cycles. Incorporating an oregano:thyme nanoemulsion (0.75%) into cellulose nanocrystal (CNC) containing chitosan (CH/CNC), methyl cellulose (MC/CNC), and polylactic acid (PLA/CNC) composite films resulted in extended diffusion matrices causing 32% to 51% rice weevil mortality after 14 days exposure. Irradiation at 200 Gray alone caused 79% mortality and increased to 100% when combined with the bioactive chitosan film containing the oregano:thyme nanoemulsion. PRACTICAL APPLICATION: A binary combination of oregano:thyme has potential as a biopesticide against stored product pests. The encapsulation of EO nanoemulsions into biopolymeric support could be used for bioactive packaging to prevent food spoilage and extend shelf life. Combining bioactive films with irradiation can provide complete control of rice weevil in packaged rice. The system developed in this research may also be extended to explore other food-packaging films with various food models to control different types of stored pests.

Effects of Irradiation Dose on Sperm Production, Insemination, and Male Mating Possible Period in the Sweetpotato Weevil (Coleoptera: Brentidae).

The sterile insect technique (SIT) has been used for the control or eradication of target insect pests. To successfully apply SIT, it is very important to clarify the effect of irradiation on male reproduction in the target pest, because their mating and spermiogenesis abilities affect the success of eradication program. The sweetpotato weevil, Cylas formicarius (Fabricius) (Coleoptera: Brentidae) is a notorious and worldwide pest of sweet potato. We investigated the effect of irradiation at five doses ranging from 0 (control) to 150 Gy on 9-d-old males. Survival rate of the control (no treatment) remained high from day 10 to 20 of adult life, whereas higher doses of irradiation reduced it, maximally by approximately 70%. Mating rates showed a similar tendency. Radiation dose neither affected sperm production nor sperm transfer at any dose, although spermiogenesis is active during the adult stage. However, radiation dose affected the lifetime total of ejaculated sperm number, likely because of fewer matings by irradiated males. These results suggest that use of a dose of 150 Gy or higher is appropriate for the final step of eradication of this weevil. At least, lower dose of irradiation may arise the inadequate sterilization, resulting in a failure of eradication program. We conclude that weekly release of sweetpotato weevil sterilized with high dose, achieving complete sterilization, could be useful for eradication program after reducing the population by male annihilation method.

Comparative toxicity and micronuclei formation in Tribolium castaneum, Callosobruchus maculatus and Sitophilus oryzae exposed to high doses of gamma radiation.

The effects of gamma radiation on mortality and micronucleus formation in Tribolium castaneum Herbst, Callosobruchus maculatus (F.) and Sitophilus oryzae (L.) genital cells were evaluated. Two groups of healthy and active adult insects 1-3 and 8-10 days old were irradiated with various doses (50-200 Gy) gamma ray. Seven days post-irradiation; mortality rates and micronucleus formation were assessed in genital cells of the irradiated insects. The results show that with increasing gamma doses, the mortality rate of each species increased and T. castaneum and S. oryzae showed the low and high sensitivity respectively. It was shown that the micronucleus appearance in the tested insects had correlation with amount and intensity of radiation doses. Moreover our results indicate different levels in the genotoxicity of gamma radiation among the insects' genital cells under study. The frequency of micronuclei in genital cells of 1-3 days old insects exposed to 50 and 200 Gy were 12.6 and 38.8 Mn/1000 cells in T. castaneum, 20.8 and 46.8 Mn/1000 cells in C. maculatus and 16.8 and 57.2 Mn/1000 cells in S. oryzae respectively. A high sensitivity of the genital cells to irradiation exposure was seen in S. oryzae correlated with its high mortality rate compared with the other two species. These results might be indicative of inflicting chromosomal damage expressed as micronucleus in high mortality rates observed in the pest population; an indication of genotoxic effects of radiation on the studied species.

Fitness and field performance of a mass-reared biological control agent, Rhinoncomimus latipes (Coleoptera: Curculionidae).

Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae), a biological control agent of mile-a-minute weed, Persicaria perfoliata (L.) H. Gross, has been mass reared with no infusion of new genetic material for 8-9 yr (at least 24-36 generations), while insects from the same genetic stock have been subject to field conditions in North America for that same period of time. Our main objective was to compare the laboratory population with the field population (and in 1 yr with a Chinese field population) to determine whether genetic changes had occurred, especially ones that may reduce the effectiveness of the laboratory population when released in the field. The laboratory insects laid more eggs and had reduced survival compared with field weevils in several comparisons, and had reduced responsiveness to cues that induce reproductive diapause. Exposure to older plants had the greatest effect on induction of reproductive diapause in both laboratory and field weevils, with effects of daylength and temperature less pronounced. At least a portion of the laboratory weevil population overwintered successfully. Results suggest that it is not necessary to add wild-type genetic material to the rearing colony at this time.

Green light synergistally enhances male sweetpotato weevil response to sex pheromone.

Sweetpotato, commercially grown in over 100 countries, is one of the ten most important staple crops in the world. Sweetpotato weevil is a major pest of sweetpotato in most areas of cultivation, the feeding of which induces production in the sweetpotato root of extremely bitter tasting and toxic sesquiterpenes which can render the sweetpotato unfit for consumption. A significant step towards improved management of this weevil species was the identification of a female-produced sex pheromone [(Z)-3-dodecenyl (E)-2-butenoate] to which males are highly attracted. Reported here are results of research that documents a nearly 5-fold increase in male sweetpotato weevil catch in traps baited with this pheromone and a green light provided by a solar-powered, light-emitting diode (LED). The combination of olfactory and night-visible visual cues significantly enhanced trap effectiveness for this nighttime-active insect species. These results provide promise for improved sweetpotato weevil detection and suppression in mass trapping programs.

The potential of irradiation as a postharvest disinfestation treatment against Phlyctinus callosus (Coleoptera: Curculionidae).

Phlyctinus callosus (Boheman) (Coleoptera: Curculionidae) is a pest of major phytosanitary concern for some of South Africa's biggest export markets such as the United States and Europe because this pest does not occur there. At present, fumigation with methyl bromide is the only postharvest disinfestation treatment against this pest; therefore, sustainable alternatives are needed. One such alternative is irradiation treatment of whole pallets of packed fruit to sterilize insects that may be present within the cartons. Wild adult P. callosus weevils were treated with 5, 10, 20, 40, and 80 Gy of gamma-irradiation and then cross mated to breed with either treated or nontreated adults of the opposite sex. Fecundity and fertility were monitored and recorded. Trials were conducted during the 2009-2010 and 2010-2011 fruit harvesting seasons. The results from both seasons indicated that irradiation did not affect fecundity but fertility was significantly affected, decreasing as irradiation doses increased. Egg hatch was zero for mating crosses that involved females weevils treated with a dose of 80 Gy gamma-irradiation. Probit analysis indicated that in the first season, the estimated LD95 for crosses involving treated males and treated females was 30 Gy, while in the second season it was 49.5 Gy. Respective estimated LD99S were 47.9 and 169.4 Gy. Ultimately, a dose lower than the current generic dose of 400 Gy, approved for irradiation disinfestation treatments, would control P. callosus should they occur in packed export fruit.

Large-scale confirmatory tests of a phytosanitary irradiation treatment against Sternochetus frigidus (Coleoptera: Curculionidae) in Philippine mango.

The mango pulp weevil, Sternochetus frigidus (F.), is an important quarantine pest preventing the export of mangoes from the Philippines to the United States and other countries. Previously, a radiation dose of 100 Gy was proposed for phytosanitary treatment of S. frigidus based on dose-response studies with larvae, pupae, and adult weevils. To validate an irradiation treatment, large-scale confirmatory tests were conducted with adults (the most radiation-tolerant stage) in mangoes at 100 and 150 Gy. After treatment, adults were removed from fruit, sexed, and mated in pairs to observe any reproduction. At 100 Gy, adults laid a small number of eggs but none of the eggs hatched. At 150 Gy (measured doses 96.7-164.1 Gy),4,559 treated weevils laid no eggs, indicating that this dose caused complete sterility. Irradiation treatment with a minimum absorbed dose of 165 Gy will therefore provide quarantine security for S. frigidus in exported Philippine mangoes.

Could sterile males be used to vector a microbiological control agent? The case of Rhynchophorus ferrugineus and Beauveria bassiana.

Rhynchophorus ferrugineus (Coleoptera, Curculionidae) is the most threatening pest of palms worldwide. The potential of gamma-irradiated males to spread a pathogenic strain of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Clavicipitaceae) to control this pest was studied. First, the effects of gamma irradiation (15 and 25 Gy) on the mating success and performance of adult males irradiated at age one day were studied in the laboratory. Although male longevity decreased after irradiation (118.6 vs. 244.7 days for irradiated and control males, respectively) and their testes suffered from the treatment, fecundity of mated females did not depend on the irradiation status of the male (86.8 ± 5.5 eggs in 15 days). However, egg hatching was significantly lower in couples with irradiated males (31.4% vs. 86.5% for irradiated and control couples, respectively), and this value decreased after a second mating (6.1% vs. 85.9%). Therefore, irradiation did not affect male sexual competiveness but sperm quality. Second, a semi-field assay was carried out to evaluate infestation in young Phoenix canariensis caused by different combinations of couples with irradiated and/or B. bassiana-challenged males. The number of immature stages found in infested palms was significantly higher when females mated with untreated males and lower when mated with irradiated males (either B. bassiana-infected or not). Some females from the fungus-challenged treatments showed post-mortem hyphal growth, and this horizontal transmission proves that irradiated males could act as a vector for B. bassiana and should be considered as a new method to improve the biological control of R. ferrugineus.

Age- and time interval-specific gamma radiation-induced DNA damage in adult maize weevils, Sitophilus zeamais Motschulsky, assessed using comet assays.

The gamma radiation-induced DNA damage in adult maize weevils, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), was assessed using single-cell electrophoresis (comet assay). Analysis of DNA damage following 0.5 and 1.0 kGy of gamma radiation was performed using cells from 1- and 15-day-old adults. Gamma-irradiated adults from both age groups showed typical DNA fragmentation, whereas cells from non-irradiated adults showed more intact DNA than young S. zeamais. Investigations using the comet assay showed that tail length, % tail DNA and % DNA damage all increased in adults of both age groups when compared to the control insects. A maximum comet length of 227.33 µm was recorded for 15-day-old adults at 24h after irradiation with 1.0 kGy and a minimum of 50.12 µm for 1-day-old adults at 0 h after irradiation with 0.5 kGy. The percentage of DNA damage increased up to 57.31% and 68.15% for 1- and 15-day-old adults, respectively, at 24h after irradiation with 1.0 kGy, whereas only 8.58% and 12.22% DNA damage were observed in the control batches. The results also showed that percentage of DNA damage increased at 24h after irradiation compared to that at 0 h. However, further studies are needed to confirm these results.

Assessment of effect of partial sterility on mating performance in sweetpotato weevil (Coleoptera: Curculionidae).

The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations. Although the effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females, the use of gamma radiation to induce sterility negatively impacts reproductive cells as well as somatic cells. Consequently, sterilization by irradiation drastically diminishes mating performance over time. In the current study, we evaluated the effect of irradiation dose intensity on fertility, mating propensity, and mating competitiveness in sweetpotato weevil, Cylas formicarius elegantulus (Summers) (Coleoptera: Curculionidae), for 16 d after irradiation. Although the mating propensity of males irradiated with 200 Gy, the dose currently used to induce complete sterility of C. f. elegantulus in the SIT program in Okinawa Prefecture, was equal to that of nonirradiated weevils for the first 6 d, the mating propensity of males irradiated with doses between of 75 and 150 Gy was maintained for the first 12 d. The potential fertilization ability of weevils was highly depressed compared with the control weevils, even in those treated with 75 Gy. Mating performance was severely compromised in weevils that were irradiated with a dose of 100 Gy or more. These results demonstrate that partial sterilization can be highly advantageous in eradication programs for the sweetpotato weevil. We discuss the advantages of the application of partial irradiation in insect eradication programs.

Sperm storage and viability within females of Euscepes postfasciatus: effect of irradiation on sperm abundance and viability within female.

Irradiation has been widely used in suppression or eradication programs that use the sterile insect technique (SIT) or in studying sperm competition. Although it is well known that irradiation has negative impacts on reproductive (sperm) cells, previous studies have assumed that sperm from irradiated males behave identically to normal sperm in the female reproductive tract after mating. In this study, we used the West Indian sweetpotato weevil Euscepes postfasciatus to investigate the effect of irradiation on the abundance and viability of sperm in female spermatheca for 14 days after copulation. The abundance of sperm in females did not change throughout the experimental period, and sperm viability gradually decreased regardless of irradiation. In this weevil, irradiated sperm appear to behave identically to normal sperm in females for 14 days following irradiation/copulation. Therefore, the effect of irradiation on sperm viability within the female spermatheca is considered to be insignificant.

Detection of gamma radiation-induced DNA damage in maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) assessed using the comet assay.

PURPOSE: The Deoxyribonucleic Acid (DNA) Comet assay, being a quick, simple, sensitive, reliable and fairly inexpensive method for measuring DNA strand breaks, has been used to assess DNA damage caused by gamma radiation in developmental stages of maize weevil Sitophilus zeamais Motschulsky. MATERIALS AND METHODS: An analysis of DNA damage following gamma radiation ((60)Cobalt) treatments at a dose of 0.5 and 1.0 kGy (Kilo Gray) has been carried out with cells obtained from the larvae, pupae and adults of S. zeamais. RESULTS: Gamma radiation induced significant damage at DNA level in the experimental larvae, pupae and adults as manifested by increased strand breaks compared to intact cells from non-irradiated ones. Comet assay revealed that tail length and % tail DNA varied significantly for all developmental stages of S. zeamais. CONCLUSIONS: This study suggests that the comet assay would be a potentially useful tool for detecting DNA damage in insect pest control strategies due to its advantages in the requirement of small numbers of cells per sample and sensitivity in detecting low levels of DNA damage.

Effect of irradiation on mating ability in the male sweetpotato weevil (Coleoptera: Curculionidae).

The sterile insect technique (SIT) is widely used for suppressing or eradicating target pest insect populations. The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. Irradiation is the effective manner to sterilize mass-reared insects. The negative impacts of this procedure are not limited to damage on reproductive cells. Gamma-radiation damages the epithelial tissue of midgut, which affects the alimentation in insects. Irradiated males alter their mating behavior over time because of the depression of metabolic activity by sterilization. In this study, we evaluated the male mating performance and sexually compatibility of irradiated male Cylas formicarius elegantulus (Summers) (Coleoptera: Curculionidae) with a 200-Gy dose, as currently used in the SIT program in Okinawa Prefecture, throughout 16 d after irradiation in the laboratory. The mating ability of irradiated males did not differ from that of control males for about a week. However, the mating ability of irradiated male drastically decreased thereafter. We consider that irradiated male C. formicarius elegantulus with a 200-Gy dose had no major effect on male mating behavior approximately for a week after irradiation.

Effect of irradiation on dispersal ability of male sweetpotato weevils (Coleoptera: Brentidae) in the field.

We used the mark-and-recapture method in the field to test the effect of gamma radiation on the dispersal ability of the male sweetpotato weevil, Cylas formicarius elegantulus (Summers) (Coleoptera: Brentidae), a serious sweetpotato pest in Japan. To evaluate the dispersal ability of male sweetpotato weevil, we released 27,218 males (13,302 males irradiated with a dose of 200 Gy and 13,916 nonirradiated males) in two replications (September and October 2005). Each replication lasted 5 d from the release of weevils to the removal of traps, and male weevils were released twice (1 and 3 d before trap setting). Forty pheromone traps were placed in lines corresponding to eight compass directions and five distance classes (20, 50,100, 200, and 500 m) in each replication. We captured 2,263 irradiated males (17.0%) and 2,007 nonirradiated males (14.4%) in the two replications. Six irradiated and eight nonirradiated males were captured in the traps 500 m far from the release point. All parameters to evaluate the dispersal ability of irradiated male sweetpotato weevil (recapture rate, dispersal distance, and dispersal direction) were similar to nonirradiated males in three of the four trials. However, parameters were different between irradiated males and nonirradiated males in one trial. Because the majority of parameters consistently show that the similarity of the dispersal ability, we considered that male sweetpotato weevil irradiated with a dose of 200 Gy possessed equal dispersal ability to that of nonirradiated males in the field.

Irradiation as a methyl bromide alternative for postharvest control of Omphisa anastomosalis (Lepidoptera: Pyralidae) and Euscepes postfasciatus and Cylas formicarius elegantulus (Coleoptera: Curculionidae) in sweet potatoes.

Irradiation studies were conducted with three sweet potato, Ipomoea batatas (L.) Lam., pests to determine an effective dose for quarantine control. Dose-response tests indicated that the most radiotolerant stage occurring in roots was the pupa of sweetpotato vine borer, Omphisa anastomosalis (Guenee), and the adult of West Indian sweetpotato weevil, Euscepes postfasciatus (Fairmaire), and sweetpotato weevil, Cylas formicarius elegantulus (Summers). In large-scale confirmatory tests, irradiation of 60,000 C. formicarius elegantulus adults, 62,323 E. postfasciatus adults, and 30,282 O. anastomosalis pupae at a dose of 150 Gy resulted in no production of F1 adults, demonstrating that this dose is sufficient to provide quarantine security.