Assessment of lethal and sublethal effects of imidacloprid, ethion, and glyphosate on aversive conditioning, motility, and lifespan in honey bees (Apis mellifera L.).

Honeybees (Apis mellifera) play an important role in agriculture worldwide. Several factors including agrochemicals can affect honey bee health including habitat fragmentation, pesticide application, and pests. The growing human population and subsequent increasing crop production have led to widespread use of agrochemicals and there is growing concern that pollinators are being negatively impacted by these pesticides. The present study compares acute exposure to imidacloprid (0.2 and 0.4 mgL-1), ethion (80 and 106.7 mgL-1) or glyphosate (0.12 and 0.24 mgL-1) on aversive learning and movement, to chronic exposure at these and higher concentrations on movement, circadian rhythms, and survival in honey bee foragers. For acute learning studies, a blue/yellow shuttle box experiment was conducted; we observed honey bee choice following aversive and neutral stimuli. In learning studies, control bees spent >50% of the time on yellow which is not consistent with previous color bias literature in the subspecies or region of the study. The learning apparatus was also used to estimate mobility effects within 20 min of exposure. Chronic exposure (up to 2 weeks) with the above metrics was recorded by an automated monitoring system. In chronic exposure experiments, RoundUp®, was also tested to compare to its active ingredient, glyphosate. We found that imidacloprid and ethion have negative impacts on aversive learning and movement following a single-dose and that chronic exposure effects were dose-dependent for these two insecticides. In contrast, glyphosate had no effect on learning and less of an effect on movement; RoundUp® showed dose-dependent results on circadian rhythmicity. Overall, the results suggest that short-term exposure to imidacloprid and ethion adversely affect honey bee foragers and chronic exposure to glyphosate may affect pollination success.

Are Entomopathogenic Nematodes Effective Biological Control Agents Against the Carob Moth, Ectomyelois ceratoniae?

The carob moth (Ectomyelois ceratoniae) is the key pest of pomegranate, which causes a significant percentage of losses in pomegranate orchards and warehouses of Iran annually. The pest larvae are characterized by displaying a cryptic behavior within the fruit, which avoids most routine control techniques, especially chemical method. The low efficiency of traditional measurements and also the rich species diversity of natural enemies within the infested fruits highlight the necessity of exploring effective control methods, especially environmental friendly approaches. Entomopathogenic nematodes (EPNs) are a group of biological control agents that actively search for the host, including those in a cryptic habitat like the carob moth larvae within infested fruits. Here, we assumed that treatment of the infested and dropped fruits with EPNs may provide new insight into the management of the carob moth. Three species of EPNs, Steinernema feltiae, S. carpocapsae, and Heterorhabditis bacteriophora were selected and used in a series of in vitro and in vivo experiments. In preliminary assays, the EPNs species were used with different concentrations of infective juveniles (IJs) (0, 1, 5, 10, 25, and 50 IJ/larvae) in 2-cm diam. plates. The mortality rates of the laboratory tests were 79.75% and 76.5% for S. feltiae and S. carpocapsae, corresponded to LC50 value of 2.02 IJ/larva for S. feltiae and 2.05 IJ/larva for S. carpocapsae. On the contrary, H. bacteriophora demonstrated low virulence on the pest larvae in petri tests with a LC50 = 426.92 IJ/larva. Hence, both Steinernema species were selected for subsequent experiments. The penetration rate for S. feltiae and S. carpocapsae into the hemocoel of the pest was 43% and 31%, respectively, and the corresponding reproduction rate was 15,452 IJ/larva for S. feltiae and 18,456 IJ/larva for S. carpocapsae. The gathered data from those in vitro tests were used for a field assay. Different concentrations (5, 10, 50, 100, and 160 IJ/cm2 of the arena) of S. feltiae and S. carpocapsae were applied in the field test. The mean mortality results from the last test were 10.89% and 26.65% for S. feltiae and S. carpocapsae, respectively. Finally, we found that these low virulence rates of the nematodes were attributed to inhibitory/repellency effects of saprophytic fungi within the infested pomegranates, a usual status of the infested fruits in autumn or winter seasons. Future work on additional EPN populations more adapted to the extreme conditions of the pomegranate production area in Iran may provide sufficient evidence to continue the further investigation on the best EPN species populations and advanced formulations with high durability.

Role of symbiotic bacteria in the growth and development of the Sunn pest, Eurygaster integriceps.

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is the most important pest of wheat and barley in wide areas of the world. Different aspects of the insect's life history have been studied, but to date nothing is known about their microbial symbionts. Here, the contribution of symbiotic bacteria to the fitness of the bug was investigated by combining two different approaches to manipulate the host's microbial community: the supplementation of antibiotics into the insects' diet and egg surface sterilization. First, bacteria cultured from gut homogenates were subjected to antibiotic screening tests using 20 different antibiotics. Norfloxacin was the most effective antibiotic, with the greatest inhibition zone among all antibiotics tested. Feeding norfloxacin to adult E. integriceps individuals significantly impaired growth and development of the offspring in a dose-dependent manner, i.e., higher antibiotic doses increased the negative effects on nymphal growth and development. Total developmental time from first nymphal instars to adult emergence in control animals was 30.1 days, but when adults had been offered diets with 10, 20, and 30 µg antibiotic per mg diet, the offspring's developmental time was prolonged to 32.8, 34.0, and 34.8 days, respectively. In the highest two doses of norfloxacin, all of the nymphs died before reaching the fifth nymphal instar. Similar results as for the antibiotic treatment were obtained when egg surface sterilization was used to manipulate the microbial community of E. integriceps. These results indicate that bacterial symbionts play a crucial role in the successful development of the host.

Differential expression of heat shock proteins and antioxidant enzymes in response to temperature, starvation, and parasitism in the Carob moth larvae, Ectomyelois ceratoniae (Lepidoptera: Pyralidae).

Insects face diverse biotic and abiotic stresses that can affect their survival. Many of these stressors impact cellular metabolism, often resulting in increased accumulation of reactive oxygen species (ROS). Consequently, insects will respond to these stressors by increasing antioxidant activity and increased production of heat shock proteins (HSPs). In this study, the effect of heat, cold, starvation, and parasitism by Habroacon hebetor wasps was examined in the carob moth, Ectomyelois ceratoniae, to determine which responses were common to different stresses. For all stressors, malondialdehyde levels increased, indicative of oxidative stress in the insects. The activity of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), increased with each stress, suggesting that these enzymes were serving a protective role for the insects. Heat (46°C for 100 min) and cold (-15°C for 30 min) treatments caused significant mortalities to all developmental stages, but pretreatments of moderate heat (37°C for 10 min) or cold (10°C for 10 min) induced thermotolerance and reduced the mortality rates when insects were subsequently exposed to lethal temperatures. Quantitative RT-PCR confirmed that heat and cold tolerance were associated with up-regulation of two HSPs, HSP70 and HSP90. Interestingly, HSP70 transcripts increased to a greater extent with cold treatment, while HSP90 transcripts increased more in response to high temperatures. RNA interference (RNAi)-mediated knockdown of either HSP70 or HSP90 transcripts was achieved by injecting larvae with dsRNA targeting each gene's transcripts, and resulted in a loss of acquired thermotolerance in insects subjected to the heat or cold pretreatments. These observations provide convincing evidence that both HSP70 and HSP90 are important mediators of the acquired thermotolerance. Starvation and parasitism by wasps caused differential expression of the HSP genes. In response to starvation, HSP90 transcripts increased to a greater extent than HSP70, while in contrast, HSP70 transcripts increased to a greater extent than those of HSP90 during the first 48 h of wasp parasitism. These results showed the differential induction of the two HSPs' transcripts with variable stresses. As well as, heat, cold, starvation, and parasitism induce oxidative stress, and antioxidant enzymes likely play an important role in reducing oxidative damage in E. ceratoniae.

Field Attraction of Carob Moth to Host Plants and Conspecific Females.

The carob moth, Ectomyelois ceratoniae (Zeller; Lepidoptera: Pyralidae), is a devastating pest in high-value crops around the world. An efficient sex pheromone attractant is still missing for the management of this pest, because the major pheromone component is unstable. Host plant volatiles attract herbivore insects and have shown to have good potential to be exploited as alternatives or supplements to sex pheromones. To explore this possibility in carob moth, we assessed the attraction of moths to the volatiles of mature pistachio and different fruit stages of pomegranate, alone and in combination with virgin females, using sticky delta traps in pomegranate orchards of Iran. Traps baited with mature pomegranates, whether uncracked or cracked, infested or uninfested, caught significantly larger numbers of male and both mated and virgin female carob moths than unbaited traps. Traps baited with headspace extract of cracked pomegranate only caught mated females, while mature pistachio only attracted males. Pomegranate flowers, unripe pomegranate, and headspace extract of pistachio did not attract moths. Traps baited with cracked fruit caught more mated females than traps baited with uncracked fruit. Males were attracted similarly to traps baited with cracked-infested pomegranate as to traps baited with virgin females alone. Interestingly, the combination of cracked pomegranate and virgin female enhanced the attraction of virgin females. Together, our results show that volatiles from cracked pomegranates alone or in combination with female sex pheromone have great potential for application in pest management programs of carob moth.

Decrease of memory retention in a parasitic wasp: an effect of host manipulation by Wolbachia?

Several factors, such as cold exposure, aging, the number of experiences and viral infection, have been shown to affect learning ability in different organisms. Wolbachia has been found worldwide as an arthropod parasite/mutualist symbiont in a wide range of species, including insects. Differing effects have been identified on physiology and behavior by Wolbachia. However, the effect of Wolbachia infection on the learning ability of their host had never previously been studied. The current study carried out to compare learning ability and memory duration in 2 strains of the parasitoid Trichogramma brassicae: 1 uninfected and 1 infected by Wolbachia. Both strains were able to associate the novel odors with the reward of an oviposition into a host egg. However, the percentage of females that responded to the experimental design and displayed an ability to learn in these conditions was higher in the uninfected strain. Memory duration was longer in uninfected wasps (23.8 and 21.4 h after conditioning with peppermint and lemon, respectively) than in infected wasps (18.9 and 16.2 h after conditioning with peppermint and lemon, respectively). Memory retention increased in response to the number of conditioning sessions in both strains, but memory retention was always shorter in the infected wasps than in the uninfected ones. Wolbachia infection may select for reduced memory retention because shorter memory induces infected wasps to disperse in new environments and avoid competition with uninfected wasps by forgetting cues related to previously visited environments, thus increasing transmission of Wolbachia in new environments.

Experimental evidence for chemical mate guarding in a moth.

In polyandrous species, males seek to maximize their reproductive output by monopolizing their mate. Often the male transfers substances to the female that suppress her sexual receptivity or antagonize the behavior of competing males; both are usually transferred in seminal fluids and represent forms of chemical mate guarding. In moths, more long-range female sex pheromones have been identified than in any other animal group, and males often display with close-range sex pheromones, yet odor-based post-copulatory mate guarding has not been described in moths so far. We tested the hypothesis that the male sex pheromone in the noctuid moth Heliothis virescens perfumes the female and functions as an anti-aphrodisiac. Indeed, virgin females perfumed with male pheromone extract, or with its main component, mated significantly less than control virgin females, and this effect persisted for two successive nights. This chemical mate guarding strategy was disadvantageous for H. virescens females, because the reproductive output of twice-mated females was significantly higher than that of once-mated females. Since the female and male sex pheromones are biosynthetically related in this and other moth species, chemical mate guarding may also impose selection pressure on the long-range female sex pheromone channel and consequently affect the evolution of sexual communication.