Chronic larval and adult honey bee laboratory testing: Which dietary additive should be considered when a test substance is not solubilized in acetone?

Chronic toxicity tests on adult and larval honey bees (Apis mellifera) can require the use of dietary additives (solvents, emulsifiers, adjuvants and viscosifier agents) when the active ingredient of plant protection products cannot be dissolved or does not remain stable and homogeneous within the test diets. Acetone is the widely used and accepted solvent allowed within the international regulatory guidelines, but it can be ineffective in keeping certain compounds in solution and can cause toxicity to adults and larvae. In this publication, we present an evaluation of alternative additives in adult and larval diets. Six dietary additives including five solvents (ethanol, isopropanol, n-propanol, propylene glycol and triethylene glycol) and a viscosifier agent (xanthan gum) at five concentrations along with a negative control and a solvent control (acetone) were investigated at seven laboratories. The safe levels for bees were determined for each of the additives used in the 10-day chronic adult and 22-day chronic larval tests. In the 10-day chronic adult study, ethanol and isopropanol were found to be safe at concentrations ≤ 5.0 %, while xanthan gum can be reliably used at concentrations ≤ 0.1 %. Greater variability across laboratories was observed for N-propanol, propylene glycol, and triethylene glycol and these agents may cause mortality when added to diets at concentrations above 0.25-0.5 %. The safe levels of additives to larval diet in the 22-day chronic larval test had a greater variability and were generally lower than what were observed for adult diet. Our results do not recommend the inclusion of ethanol or n-propanol into the larval diet, and isopropanol, propylene glycol, and triethylene glycol may cause mortality at concentrations above 0.25-0.5 %. Safe levels for xanthan gum were more variable than what was observed for adults, but it can be used reliably at concentrations ≤ 0.05 %. Our analyses conclude that several additives can be integrated successfully in honey bee laboratory bioassays at levels that cause low mortality to adults and larvae.

Frequently encountered pesticides can cause multiple disorders in developing worker honey bees.

Pesticide exposure is regarded as a contributing factor to the high gross loss rates of managed colonies of Apis mellifera. Pesticides enter the hive through contaminated nectar and pollen carried by returning forager honey bees or placed in the hive by beekeepers when managing hive pests. We used an in vitro rearing method to characterize the effects of seven pesticides on developing brood subjected dietary exposure at worse-case environmental concentrations detected in wax and pollen. The pesticides tested included acaricides (amitraz, coumaphos, fluvalinate), insecticides (chlorpyrifos, imidacloprid), one fungicide (chlorothalonil), and one herbicide (glyphosate). The larvae were exposed chronically for six days of mimicking exposure during the entire larval feeding period, which is the worst possible scenario of larval exposure. Survival, duration of immature development, the weight of newly emerged adult, morphologies of the antenna and the hypopharyngeal gland, and gene expression were recorded. Survival of bees exposed to amitraz, coumaphos, fluvalinate, chlorpyrifos, and chlorothalonil was the most sensitive endpoint despite observed changes in many developmental and physiological parameters across the seven pesticides. Our findings suggest that pesticide exposure during larvae development may affect the survival and health of immature honey bees, thus contributing to overall colony stress or loss. Additionally, pesticide exposure altered gene expression of detoxification enzymes. However, the tested exposure scenario is unlikely to be representative of real-world conditions but emphasizes the importance of proper hive management to minimize pesticide contamination of the hive environment or simulates a future scenario of increased contamination.

Essential oil of Siparuna guianensis as an alternative tool for improved lepidopteran control and resistance management practices.

Although the cultivation of transgenic plants expressing toxins of Bacillus thuringiensis (Bt) represents a successful pest management strategy, the rapid evolution of resistance to Bt plants in several lepidopteran pests has threatened the sustainability of this practice. By exhibiting a favorable safety profile and allowing integration with pest management initiatives, plant essential oils have become relevant pest control alternatives. Here, we assessed the potential of essential oils extracted from a Neotropical plant, Siparuna guianensis Aublet, for improving the control and resistance management of key lepidopteran pests (i.e., Spodoptera frugiperda and Anticarsia gemmatalis). The essential oil exhibited high toxicity against both lepidopteran pest species (including an S. frugiperda strain resistant to Cry1A.105 and Cry2Ab Bt toxins). This high insecticidal activity was associated with necrotic and apoptotic effects revealed by in vitro assays with lepidopteran (but not human) cell lines. Furthermore, deficits in reproduction (e.g., egg-laying deterrence and decreased egg viability), larval development (e.g., feeding inhibition) and locomotion (e.g., individual and grouped larvae walking activities) were recorded for lepidopterans sublethally exposed to the essential oil. Thus, by similarly and efficiently controlling lepidopteran strains susceptible and resistant to Bt toxins, the S. guianensis essential oil represents a promising management tool against key lepidopteran pests.

Sublethal exposure to deltamethrin reduces the abilities of giant water bugs to prey upon Aedes aegypti larvae.

Freshwater ecosystems provide environmental conditions for many arthropod species, including pests like mosquitoes and beneficial insects. Giant water bugs, Belostoma anurum (Hemiptera: Belostomatidae), are aquatic insects that provide biological control of mosquitoes and small vertebrates in freshwater environments. However, the application of insecticides aiming to control mosquitoes can lead to insecticide exposures of aquatic predators that can result in their death or significant reductions in their behavioral abilities. Here, we assessed the susceptibilities of B. anurum to the pyrethroid insecticide deltamethrin and evaluated whether sublethal exposure to deltamethrin would change the abilities of B. anurum to prey upon larvae of Aedes aegypti (Diptera: Culicidae). Bioassays of predator performance were conducted at three prey densities (i.e., 3, 6 and 9 larvae/100 mL of water) just after insecticide exposure and on the three following days. Our results revealed that B. anurum (LC50 = 90.9 µg a. i./L) was approximately 32-fold less susceptible to deltamethrin than A. aegypti larvae (LC50 = 2.8 µg a. i./L). However, the number of larvae eaten by B. anurum sublethally exposed to deltamethrin (at 13 µg a. i./L for 24 h) was significantly (P < 0.05) smaller than that recorded for unexposed predators. Furthermore, the deltamethrin-mediated behavioral changes were higher at the highest availability of prey and, as expected, just after insecticide exposure. Thus, sublethal exposure to deltamethrin reduces the ability of B. anurum to capture and prey upon A. aegypti larvae, compromising the efficacy of these insects as naturally occurring mosquito control agents.

Safety of methionine, a novel biopesticide, to adult and larval honey bees (Apis mellifera L.).

Methionine is an essential/indispensible amino acid nutrient required by adult and larval honey bees (Apis mellifera L. [Hymenoptera: Apidae]). Bees are unable to rear broods on pollen deficient in methionine, and reportedly behaviorally avoid collecting pollen or nectar from florets deficient in methioinine. In contrast, it has been demonstrated that methionine is toxic to certain pest insects; thus it has been proposed as an effective biopesticide. As an ecofriendly integrated pest management agent, methionine boasts a novel mode of action differentiating it from conventional pesticides, while providing non-target safety. Pesticides that minimize collateral effects on bees are desirable, given the economic and ecological concerns about honey bee health. The aim of the present study was to assess the potential impact of the biopesticide methionine on non-target adult and larval honey bees. Acute contact adult toxicology bioassays, oral adult assessments and chronic larval toxicity assessments were performed as per U.S. Environmental Protection Agency (EPA) requirements. Our results demonstrated that methionine fits the U.S. EPA category of practically nontoxic (i.e. lethal dose to 50% mortality or LD50 > 11µg/bee) to adult honey bees. The contact LD50 was > 25µg/bee and the oral LD50 was > 100µg/bee. Mortality was observed in larval bees that ingested DL-methionine (effective concentration to 50% mortality or EC50 560µg/bee). Therefore, we conclude that methionine poses little threat to the health of the honey bee, due to unlikely exposure at concentrations shown to elicit toxic effects.

Bti-based insecticide enhances the predatory abilities of the backswimmer Buenoa tarsalis (Hemiptera: Notonectidae).

The backswimmer Buenoa tarsalis (Hemiptera: Notonectidae) is a naturally occurring predator of immature stages of mosquitoes. These aquatic predators can suffer from non-targeted exposure to insecticides that are commonly used in aquatic environments to control mosquitoes. Here, we evaluated whether insecticide formulations containing the bacterium Bacillus thuringiensis var. israelensis (Bti) or the organophosphate pirimiphos-methyl would affect the survival and the predatory abilities of B. tarsalis. First, we conducted survival bioassays to estimate the median survival time (LT50) of B. tarsalis when exposed to Bti-based insecticide (at 0.25 and 25 mg a.i./L) and pirimiphos-methyl (at 1, 10 and 1000 mg a.i./L). The highest concentrations of the insecticides were equivalent to the label-recommended field rates. Second, the predatory abilities of B. tarsalis exposed to insecticides were evaluated at three prey densities (3, 6 and 9 mosquito larvae/100 mL water) just after insecticide exposure or after a 24 h recovery time. While the survival of B. tarsalis was significantly reduced with pirimiphos-methyl concentrations ≥10 mg a.i./L, the Bti-exposed predators exhibited similar survival as unexposed predators. Interestingly, after a recovery time of 24 h, B. tarsalis sublethally exposed to pirimiphos-methyl or Bti-based insecticide consistently killed more A. aegypti larvae (at the intermediate density) than unexposed predators. However, for the without-recovery bioassays, the pirimiphos-methyl-exposed predators exhibited reduced predatory abilities at the lowest prey density. Because they do not reduce the survival or the predatory abilities of B. tarsalis, Bti-based insecticides can be considered a safe insecticide to use in the presence of backswimmers.

Detection of a new pyrethroid resistance mutation (V410L) in the sodium channel of Aedes aegypti: a potential challenge for mosquito control.

The yellow fever mosquito, Aedes aegypti, particularly in Neotropical regions, is the principal vector of dengue, yellow fever, Zika and Chikungunya viruses. Pyrethroids remain one of the most used insecticides to control Aedes mosquitoes, despite the development of pyrethroid resistance in many mosquito populations worldwide. Here, we report a Brazilian strain of A. aegypti with high levels (approximately 100-60,000 fold) of resistance to both type I and type II pyrethroids. We detected two mutations (V410L and F1534C) in the sodium channel from this resistant strain. This study is the first report of the V410L mutation in mosquitoes. Alone or in combination with the F1534C mutation, the V410L mutation drastically reduced the sensitivity of mosquito sodium channels expressed in Xenopus oocytes to both type I and type II pyrethroids. The V410L mutation presents a serious challenge for the control of A. aegypti and will compromise the use of pyrethroids for the control of A. aegypti in Brazil; therefore, early monitoring of the frequency of the V410L mutation will be a key resistance management strategy to preserve the effectiveness of pyrethroid insecticides.

Agrochemical synergism imposes higher risk to Neotropical bees than to honeybees.

Bees are key pollinators whose population numbers are declining, in part, owing to the effects of different stressors such as insecticides and fungicides. We have analysed the susceptibility of the Africanized honeybee, Apis mellifera, and the stingless bee, Partamona helleri, to commercial formulations of the insecticides deltamethrin and imidacloprid. The toxicity of fungicides based on thiophanate-methyl and chlorothalonil were investigated individually and in combination, and with the insecticides. Results showed that stingless bees were more susceptible to insecticides than honeybees. The commercial fungicides thiophanate-methyl or chlorothalonil caused low mortality, regardless of concentration; however, their combination was as toxic as imidacloprid to both species, and over 400-fold more toxic than deltamethrin for A. mellifera. There were highly synergistic effects on mortality caused by interactions in the mixture of imidacloprid and the fungicides thiophanate-methyl, chlorothalonil and the combined fungicide formulation in A. mellifera, and also to a lesser extent in P. helleri. By contrast, mixtures of the deltamethrin and the combined fungicide formulation induced high synergy in P. helleri, but had little effect on the mortality of A. mellifera. Differences in physiology and modes of action of agrochemicals are discussed as key factors underlying the differences in susceptibility to agrochemicals.

Deltamethrin toxicity and impaired swimming behavior of two backswimmer species.

Backswimmers (Hemiptera: Heteroptera: Notonectidae) are insect predators in a wide variety of freshwater habitats. These insects are well known through their role as mosquito biocontrol agents, their ability to prey on immature fishes and frogs, and because they are often the first to colonize aquatic habitats. As a consequence, these predators may face intended or unintended insecticide exposures that may lead to death or to impairment of essential behaviors (e.g., swimming and position in the water column). The toxicity of deltamethrin (a type II pyrethroid insecticide stressor) and the swimming activity of the backswimmers Buenoa tarsalis and Martarega bentoi were evaluated. Concentration-mortality and survival bioassays were conducted with the insecticide, which were compared with controls without deltamethrin. Deltamethrin was 26-fold more toxic to B. tarsalis (median lethal concentration [LC50] = 4.0 ng a.i./L) than to M. bentoi (LC50 = 102.5 ng a.i./L). The pattern of occupation of B. tarsalis, but not of M. bentoi, in the water column was also disrupted, and B. tarsalis was forced to stay near the water surface longer with exposure to deltamethrin. Thus, based on the findings, B. tarsalis was less resilient to deltamethrin exposure compared with M. bentoi, and the efficacy of swimming-dependent processes might be negatively affected (e.g., prey catching, partner encounter, and antipredator behaviors) for B. tarsalis under deltamethrin exposure. Environ Toxicol Chem 2017;36:1235-1242. © 2016 SETAC.