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Over the last few decades, agrochemicals have been partially associated with a global reduction in bees. Toxicological assessment is crucial for understanding the overall agrochemical risks to stingless bees. Therefore, the lethal and sublethal effects of agrochemicals commonly used in crops (copper sulfate, glyphosate, and spinosad) on the behavior and gut microbiota of the stingless bee, Partamona helleri, were assessed using chronic exposure during the larval stage. The recommended field doses of CuSO4 (200 Active Ingredient, a.i., µg) and spinosad (8,16 a.i. µg) reduced survival, but not glyphosate (148 a.i. µg). No significant adverse effects on bee development were observed in any treatment with CuSO4 or glyphosate, but spinosad increased the number of deformed bees and reduced their body mass. Agrochemicals changed the behavior of bees and composition of the gut microbiota of adult bees, and metals such as copper accumulated in the bees’ bodies. The response of bees to agrochemicals depends on the class or dose of the ingested compound. In vitro rearing of stingless bees larvae is a useful tool to elucidate the sublethal effects of agrochemicals.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Paratuberculosis mainly affecting domestic ruminants. The interaction between MAP and sperm and/or germ cells has not yet been established, however the adherence between MAP and the host cell surface is associated to the 85 complex proteins that bind to the host cell’s fibronectin. Therefore, this study aimed to evaluate the binding of MAP to bovine sperm and to verify changes in these cells by the presence of MAP before and after sperm cryopreservation. Polyclonal antibodies to MAP 85 complex proteins were produced and utilized in the analyzes. Two Nelore bulls were used for semen collection and MAP dilutions (103–108 CFU/mL) were inoculated in the samples; sperm motility and vigor were evaluated using light microscopy at different times before and after cryopreservation and in the presence and absence of the antibodies 85A and 85B. Interaction of MAP and sperm, interaction of MAP and sperm in the presence of Ab 85A and in the presence of Ab 85B were analyzed by scanning electron microscopy. The viability of MAP after sperm cryopreservation were evaluated by plating the samples after thawing. It was observed that sperm in the presence of MAP shows a decrease in motility and vigor, and that the higher the MAP concentration, the lower the sperm performance. It was possible to determine the viability of MAP after cryopreservation in samples of higher concentrations, which demonstrates the potential of transmission of this pathogen through artificial insemination. The interaction of MAP with bovine sperm occurs mainly in the midpiece and may be linked to the proteins 85A and 85B present in the MAP membrane.

Africanized and wild bees are sensitive to synthetic insecticides, but may not be sensitive to botanical extracts. In this work, we evaluated the toxicity of botanical extracts with homemade preparations used in agroecological crops and their constituents on the bees Apis mellifera and Partamona helleri. Toxicity bioassays of adult bees were done by means of oral exposure and ingestion, using the insecticide imidacloprid as a positive control. Dietary consumption, respiration rate and bee flight were evaluated as sublethal parameters. Although some extracts were toxic to bees, survival was always higher compared to the results obtained with the imidacloprid, which was lethal to 100% of bees. In dietary consumption, P. helleri consumed less (5 mg/bee) in 3 h than A. mellifera (11 mg/bee), and P. helleri consumed less (7 mg/bee) in 24 h than A. mellifera (22 mg/bee). There was no difference in consumption of food containing plant extracts or food containing water only. We did not detect any adverse effects of the botanical extracts on bee respiration rates or flight. The major constituent of N. tabacum is nicotine (8.4–15.1%), in A. americana it is β-caryophyllene (11.3%), and in A. colubrina, lupeol (12.2%). Imidacloprid and nicotine were more toxic to bees (LC50 ≤ 1.3 and LC50 ≤ 44.3). Botanical extracts were selective to A. mellifera and the native bee P. helleri, and therefore, have the potential for ecofriendly pest control.

Paratransgenesis consists of genetically engineering an insect symbiont to control vector-borne diseases. Biosafety assessments are a prerequisite for the use of genetically modified organisms (GMOs). Assessments rely on the measurement of the possible impacts of GMOs on different organisms, including beneficial organisms, such as pollinators. The bacterium Serratia AS1 has been genetically modified to express anti-Plasmodium effector proteins and does not impose a fitness cost on mosquitoes that carry it. In the present study, we assessed the impact of this bacterium on the native bee Partamona helleri (Meliponini), an ecologically important species in Brazil. Serratia eGFP AS1 (recombinant strain) or a wild strain of Serratia marcescens were suspended in a sucrose solution and fed to foragers, followed by measurements of survival, feeding rate, and behavior (walking and flying). These bacteria did not change any of the variables measured at 24, 72, and 144 h after the onset of the experiment. Recombinant and wild bacteria were detected in the homogenates of digestive tract during the 144 h period analyzed, but their numbers decreased with time. The recombinant strain was detected in the midgut at 24 h and in the hindgut at 72 h and 144 h after the onset of the experiment under the fluorescent microscope. As reported for mosquitoes, Serratia eGFP AS1 did not compromise the foragers of P. helleri, an ecologically relevant bee.

Populations of stingless bees have declined around the world and pesticides have been indicated as one of the possible causes of this decrease. Spinosad, which is synthesized from the fermentation process produced by the soil actinomycete Saccharopolyspora spinosa, is one of the most used bioinsecticides today. This study aimed to evaluate the possible effects of spinosad (formulation) on survival, general group activity and the processes of autophagy, apoptosis and oxidative stress in two organs (midgut and brain) of workers of Partamona helleri, after 24 h of oral exposure. Workers were orally exposed to different concentrations of spinosad. The concentration (8.16 × 10−3 mg a.i./mL) that led to the mortality of approximately half the number of treated bees was considered LC50 and was used in behavior, histology and immunofluorescence bioassays. The results revealed that bee survival was substantially reduced with increasing spinosad concentrations. The LC50 of the bioinsecticide compromised general group activity, caused morphological alterations in the midgut and intensified the processes of autophagy, apoptosis and oxidative stress in this organ. The brain, on the other hand, did not present significant alterations under the tested conditions. The data obtained demonstrate, therefore, that spinosad negatively affects individual survival, general group activity and the midgut epithelium of P. helleri.

The maize weevil (Sitophilus zeamais) is a serious worldwide pest of stored products frequently requiring control measures to reduce its infestation and minimize grain losses. The fumigant phosphine and insecticides including pyrethroids and organophosphates have been the most used management tools against this stored product pest, but the over-reliance on these chemical products has led to insecticide resistance in maize weevil populations. A recent alternative, the actinomycete-based insecticide spinosad, has been considered highly effective against several stored grain pests, including maize weevils, and has sparked increasing attention. Nonetheless, the sublethal effects of spinosad have yet to be studied, particularly regarding its potential effect on insect dispersal within contaminated grain masses since exposure may be either enhanced or compromised. Thus, we carried out laboratory experiments using a digital tracking system and commercial insecticide formulations with the objective to assess if deltamethrin- or spinosad-contaminated grains may affect the walking activity and dispersal movements of adult maize weevils. The overall activity of groups of weevils was enhanced with insecticide exposure. However, insecticide exposure did not affect resting time and distance walked, but velocity and number of stops were affected compromising walking in individual walking bioassays. Insecticide avoidance by either irritability and repellence were not detected. Dispersal of adult maize weevils on treated grains differed among insecticidal treatments with individuals spending more time at the edges of arenas in non-treated and deltamethrin-treated grains. In contrast, insects on spinosad-treated grains were more evenly distributed within the arena. These findings reinforce the potential of spinosad as an alternative for managing maize weevils because this insecticide minimizes the risk of dispersal away from treated grains.

Rhodnius prolixus is the principal vector of Trypanosoma cruzi , the aetiological agent of Chagas disease in American countries. This insect is haematophagous during all life cycles and, to antagonize its haemostatic, inflammatory and immune systems, it secretes saliva while feeding on the vertebrate host's blood. Here, we investigated characteristic changes of the salivary glands (SG) that occur during insect development. Two pairs of lobules and ducts comprise the SG of R. prolixus . The organ's size increases over time, but the microanatomical structures are preserved during insect development. Both lobules have a single layer epithelium formed by binucleated cells, which surrounds the saliva reservoir. The principal lobule presents higher polysaccharide and total protein contents than the accessory lobe. A network of external muscle layers is responsible for organ contraction and saliva release. Apocrine, merocrine and holocrine secretion types occur in the secretory epithelium. Dopamine, serotonin and tyrosine-hydroxylase are neural-related molecules that regulate SG function both during and after feeding.

Paratransgenesis consists of genetically engineering an insect symbiont to control vector-borne diseases. Biosafety assessments are a prerequisite for the use of genetically modified organisms (GMOs). Assessments rely on the measurement of the possible impacts of GMOs on different organisms, including beneficial organisms, such as pollinators. The bacterium Serratia AS1 has been genetically modified to express anti-Plasmodium effector proteins and does not impose a fitness cost on mosquitoes that carry it. In the present study, we assessed the impact of this bacterium on the native bee Partamona helleri (Meliponini), an ecologically important species in Brazil. Serratia eGFP AS1 (recombinant strain) or a wild strain of Serratia marcescens were suspended in a sucrose solution and fed to bees, followed by measurements of survival, feeding rate, and behavior (walking and flying). These bacteria did not change any of the variables measured at 24, 72, and 144 h after ingestion. Recombinant and wild bacteria were detected in the digestive tract up to 144 h after ingestion, but their numbers decreased with time. The recombinant strain was detected in the midgut at 24 h and in the hindgut at 72 h and 144 h after ingestion. As reported for mosquitoes, Serratia eGFP AS1 is a safe candidate for combating vector-borne diseases, as it did not compromise the native and ecologically relevant bee, P. helleri.

The resistance of Aedes aegypti to chemical insecticides has been reported and our work proposes the use of biosurfactants as an alternative larvicide. We evaluated the effect of rhamnolipids against larvae of pyrethroid-resistant and susceptible A. aegypti strains. Time-mortality and sublethal effects were evaluated via survival analysis and swimming behavior, respectively. Rhamnolipids showed larvicidal effect at all tested concentrations. Rhamnolipids at 300 mg L(−1) killed 100% of both susceptible and resistant larvae within 24 h of exposure and 99% after 30-days stored (pyrethroid-susceptible larvae). Regarding the sublethal effects, the swimming rate was reduced in 50 and 100 mg L(−1) of rhamnolipids in grouped (pyrethroid-susceptible) larvae. Rhamnolipids at 50 mg L(−1) reduced the distance and speed and increased the number of stops and resting time of individualized pyrethroid-susceptible larvae. The larvicidal effect of the rhamnolipids evaluated demonstrates that these compounds represent an alternative to control A. aegypti.

International audience; Exposure to agrochemicals and pathogens has been indicated as critical factors of declining pollinator populations, including species of native tropical bees. In the present study, we investigated the sublethal effects of acute oral exposure to azadirachtin and spinosad bioinsecticides on the expression of immunity-related genes and the number of hemocytes in uninfected and Escherichia coli–infected workers of the stingless bee Melipona quadrifasciata. Expression of the immune-related gene vitellogenin decreased and the number of circulating hemocytes increased in bioinsecticide-exposed uninfected bees and in unexposed E. coli–infected bees as compared with unexposed uninfected bees. Our results show that though bioinsecticides are considered safe, contamination by sublethal doses of azadirachtin and spinosad can interfere with the immune system of bees.

Large-scale pesticide application poses a major threat to bee biodiversity by causing a decline in bee populations that, in turn, compromises ecosystem maintenance and agricultural productivity. Biopesticides are considered an alternative to synthetic pesticides with a focus on reducing potential detrimental effects to beneficial organisms such as bees. The production of healthy queen stingless bees is essential for the survival and reproduction of hives, although it remains unknown whether biopesticides influence stingless bee reproduction. In the present study, we investigated the effects of the biopesticide azadirachtin on the survival, behavior, morphology, development, and reproduction of queens of the stingless bee Partamona helleri (Friese, 1900). The neonicotinoid imidacloprid was used as a toxic reference standard. Queens were orally exposed in vitro to a contaminated diet (containing azadirachtin and imidacloprid) during development. Azadirachtin resulted in reduced survival, similarly to imidacloprid, altered development time, caused deformations, and reduced the size of the queens' reproductive organs. All of these factors could potentially compromise colony survival. Results from the present study showed azadirachtin posed a toxicological hazard to P. helleri queens.

Insecticide resistance is a ubiquitous consequence in arthropod pest species subjected to insecticide use in agricultural fields. The widespread use of insecticides also allows selection of insecticide resistance among non-target arthropod species, such as natural enemies. Nonetheless, the potential consequences of sublethal insecticide exposure in resistant natural enemies are frequently neglected. The detected pyrethroid resistance in the lady beetle Eriopis connexa (Germar) in Brazilian agricultural fields afford the opportunity of assessing the consequences of the sublethal exposure of the broadly used pyrethroid insecticide lambda-cyhalothrin in the mating behavior and reproductive output of susceptible and resistant populations of this species. Survival bioassays with lambda-cyhalothrin allowed estimation of sublethal exposure times at the maximum labeled rate to assess the reproductive consequences of such exposure in pyrethroid-susceptible and resistant strains of E. connexa. Such sublethal exposures led to significant difficulties in female mounting by male of both populations. Pyrethroid exposure also extended the duration of female body tremulation and of coupling, while latency to mate, tremulation, coupling and female shaking to dislodge the males after coupling differed between strains with interacting effect of insecticide exposure only for the latter behavior. As a consequence mainly of latency to mate, progeny production was significantly smaller among pyrethroid-resistant females where lambda-cyhalothrin exposure exhibited a negligible effect. Thus, population rather than exposure itself prevailed in determining reproductive output of E. connexa and pyrethroid resistance incurred in reproductive costs in this species that may counterweight the benefits of its survival.

Insecticides can affect biological control by parasitoids. Here, we examined the lethal and sublethal effects of two conventional insecticides, imidacloprid and lambda-cyhalothrin, and a reduced-risk bioinsecticide, spinosad, on the aphid parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae). Concentration-mortality curves generated from insecticide residue bioassays found that wasps were nearly 20-fold more susceptible to spinosad than imidacloprid and lambda-cyhalothrin. Imidacloprid and lambda-cyhalothrin compromised adult parasitoid longevity, but not as dramatically as spinosad: concentrations >200 ng spinosad/cm2 reduced wasp longevity by half. Imidacloprid and lambda-cyhalothrin also compromised aphid parasitism by wasps. Although increasing imidacloprid concentrations led to increased host viability and reduced progeny production, lambda-cyhalothrin did not affect viability of parasitized hosts or parasitoid progeny production in a dose-dependent manner. Our results demonstrate that reduced risk bioinsecticide products like spinosad can be more toxic to biological control agents than certain conventional insecticides.

Assessments of acute insecticide toxicity frequently focus on the lethal effects on individual arthropod pest species and populations neglecting the impacts and consequences of sublethal exposure. However, the sublethal effects of insecticides may lead to harmful, neutral, or even beneficial responses that may affect (or not) the behavior and sexual fitness of the exposed insects. Intriguingly, little is known about such effects on stored product insect pests in general and the maize weevil in particular. Thus, we assessed the sublethal effects of spinosad and deltamethrin on female mate-searching, mating behavior, progeny emergence, and grain consumption by maize weevils. Insecticide exposure did not affect the resting time, number of stops, and duration of mate-searching by female weevils, but their walking velocity was compromised. Maize weevil couples sublethally exposed to deltamethrin and spinosad exhibited altered reproductive behavior (walking, interacting, mounting, and copulating), but deltamethrin caused greater impairment. Curiously, higher grain consumption and increased progeny emergence were observed in deltamethrin-exposed insects, suggesting that this pyrethroid insecticide elicits hormesis in maize weevils that may compromise control efficacy by this compound. Although spinosad has less of an impact on weevil reproductive behavior than deltamethrin, this bioinsecticide also benefited weevil progeny emergence, but did not affect grain consumption. Therefore, our findings suggest caution using either compound, and particularly deltamethrin, for controlling the maize weevil, as they may actually favor this species population growth when in sublethal exposure requiring further assessments. The same concern may be valid for other insecticides as well, what deserves future attention.

The granary and maize weevils are serious worldwide threats to stored products and their control has heavily relied on synthetic insecticides, which are largely recommended based on studies with acute lethal doses neglecting the importance of potential sublethal effects of insecticides. Deltamethrin has been widely used for managing grain weevils and other stored grain pest species, but reported control failures with this insecticide sparked the search for alternative insecticidal compounds. The bioinsecticide spinosad is one of such alternatives whose use against stored grain insect pests is relatively recent, but encompasses the control of grain weevils. Nonetheless, little is known about the sublethal effects of spinosad on these insect pest species. Here we assessed the insecticidal effects of commercial formulations of spinosad and deltamethrin against the weevil species Sitophilus granarius and S. zeamais. Both spinosad and deltamethrin were able to effectively control the insects, although the latter caused a faster mortality than the former. Behavioral pattern changes were caused by both insecticides, especially deltamethrin, triggering irritability (i.e., avoidance after contact). Different feeding and drinking responses were also detected for both weevil species, which exhibited significant avoidance to deltamethrin and to spinosad, but with a milder response to the latter. Apparently spinosad is not as easily recognizable as deltamethrin by S. zeamais and S. granarius, remaining effective against both species without minimizing as much the potential exposure as deltamethrin.

The ongoing concern about bee decline has largely focused on honey bees and neonicotinoid insecticides, while native pollinators such as Neotropical stingless bees and agrochemicals such as other insecticide groups, pesticides in general, and fertilizers-especially leaf fertilizers-remain neglected as potential contributors to pollination decline. In an effort to explore this knowledge gap, we assessed the lethal and sublethal behavioral impact of heavy metal-containing leaf fertilizers in a native pollinator of ecological importance in the Neotropics: the stingless bee Friesella schrottkyi (Friese). Two leaf fertilizers-copper sulfate (24% Cu) and a micronutrient mix (Arrank L: 5% S, 5% Zn, 3% Mn, 0.6% Cu, 0.5% B, and 0.06% Mo)-were used in oral and contact exposure bioassays. The biopesticide spinosad and water were used as positive and negative controls, respectively. Copper sulfate compromised the survival of stingless bee workers, particularly with oral exposure, although less than spinosad under contact exposure. Sublethal exposure to both leaf fertilizers at their field rates also caused significant effects in exposed workers. Copper sulfate enhanced flight take-off on stingless bee workers, unlike workers exposed to the micronutrient mix. There was no significant effect of leaf fertilizers on the overall activity and walking behavior of worker bees. No significant effect was observed for the respiration rate of worker bees under contact exposure, but workers orally exposed to the micronutrient mix exhibited a reduced respiration rate. Therefore, leaf fertilizers do affect F. schrottkyi , what may also occur with other stingless bees, potentially compromising their pollination activity deserving attention.

Bee pollination is crucial for ecosystem maintenance and crop production. The ubiquity of bee pollinators in agricultural landscapes frequently results in their exposure to agrochemicals, which has been associated with their decline. Stingless bees are wild pollinators restricted to the Pantropical region, and like honey bees, are suffering colony losses. However, stingless bees and honey bees do not show the same behaviors, therefore, methods used for risk assessment of honey bees cannot be utilized on stingless bees. Herein, we describe protocols to standardize methods that allow for the exploration of lethal and sublethal effects of agrochemicals via acute and chronic exposure of stingless bees. The in vitro rearing used for chronic exposure from the egg to the adult stage proved to be effective in obtaining relevant screenings. In addition, we performed a meta‐analysis and summarized the results of toxicological studies conducted with the protocols described. The meta-analyses indicated a reduction in survival under acute and chronic exposures to agrochemicals, and revealed that our protocols for toxicological assessments did not have publication bias for either acute or chronic exposure. These findings proved that these standardized protocols are reliable for toxicological research on stingless bee., Graphical abstract Image, graphical abstract

The insect immune system faces various challenges; particularly in social bees, caste system and age polyethism expose individuals to numerous environmental and working conditions. However, little is known about how cellular defenses in social bees may be organized to respond to a variety of immune challenges. Here, we describe the morphological features and the total and differential counts of hemocytes in different female classes (newly emerged workers, nurses, foragers, and virgin queens) of the eusocial stingless bee Melipona quadrifasciata. Granulocytes and prohemocytes were, respectively, the most and the least abundant cells among all classes of females. Furthermore, there were more prohemocytes in virgin queens than in foragers. The total number of hemocytes was smaller in foragers, whereas the largest number was observed in nurse workers. This reduced amount of hemocytes in foragers might allow energy savings to perform colony activities such as foraging and defense. Foragers also had the biggest hemocytes (either prohemocytes, granulocytes, or plasmatocytes) in comparison to the other classes of females, which might have arisen as a compensation for the reduction in number of these cells during aging. These results suggest that profiles of hemocytes of M. quadrifasciata vary according to the caste and age of this eusocial bee.

In the present work, the effects of the neonicotinoid imidacloprid formulation on the total hemocyte counts (THC) and differential hemocyte counts (DHC) were investigated in foraging workers of the stingless bee Melipona quadrifasciata under (or not) the challenge with the bacteria Escherichia coli. The THC was not altered with the insecticide exposure and/or bacterial infection. However, the DHC of the bees changed with the imidacloprid exposure and/or bacterial infection. The number of prohemocytes (stem cells) increased in bees exposed to imidacloprid, but it did not change after the bacterial infection. The number of plasmatocytes (phagocytic cells) increased in imidacloprid-exposed and uninfected bees and decreased in infected bees regardless of the exposure to imidacloprid. On the other hand, there was a reduction of granulocytes, the most active immune cells, after imidacloprid exposure and an increase of granulocytes after the infection. Previous studies have shown that the neonicotinoids exposure can impair the immune system of bees. Our findings showed that the relative number of granulocytes declined with imidacloprid exposure, but the overall capacity of hemocyte responses in terms of total numbers after bacterial infection persisted even after the insecticide exposure.

Because of their natural origin, biopesticides are assumed to be less harmful to beneficial insects, including bees, and therefore their use has been widely encouraged for crop protection. There is little evidence, however, to support this ingrained notion of biopesticide safety to pollinators. Because larval exposure is still largely unexplored in ecotoxicology and risk assessment on bees, an investigation was performed on the lethal and sublethal effects of a diet treated with 2 bioinsecticides, azadirachtin and spinosad, on the stingless bee, Melipona quadrifasciata, which is one of the most important pollinators in the Neotropics. Survival of stingless bee larvae was significantly compromised at doses above 210 ng a.i./bee for azadirachtin and 114 ng a.i./bee for spinosad. No sublethal effect was observed on larvae developmental time, but doses of both compounds negatively affected pupal body mass. Azadirachtin produced deformed pupae and adults as a result of its insect growth regulator properties, but spinosad was more harmful and produced greater numbers of deformed individuals. Only spinosad compromised walking activity of the adult workers at doses as low as 2.29 ng a.i./bee, which is 1/5000 of the maximum field recommended rate. In conclusion, the results demonstrated that bioinsecticides can pose significant risks to native pollinators with lethal and sublethal effects; future investigations are needed on the likelihood of such effects under field conditions. Environ Toxicol Chem 2015;34:2149–2158. © 2015 SETAC

Although invertebrates generally have a low public profile, the honey bee, Apis mellifera L., is a flagship species whose popularity likely derives from the products it provides and its perceived ecological services. Therefore, the raging debate regarding honey bee decline has surpassed the realm of beekeepers, academia, industry and regulatory agencies and now also encompasses non-governmental agencies, media, fiction writers and the general public. The early interest and concern about honey bee colony collapse disorder (CCD) soon shifted to the bigger issue of pollinator decline, with a focus on the potential involvement of pesticides in such a phenomenon. Pesticides were previously recognised as the potential culprits of the reported declines, particularly the neonicotinoid insecticides owing to their widespread and peculiar use in agriculture. However, the evidence for the potential pivotal role of these neonicotinoids in honey bee decline remains a matter of debate, with an increased recognition of the multifactorial nature of the problem and the lack of a direct association between the noted decline and neonicotinoid use. The focus on the decline of honey bee populations subsequently spread to other species, and bumblebees became another matter of concern, particularly in Europe and the United States. Other bee species, ones that are particularly important in other regions of the world, remain the object of little concern (unjustifiably so). Furthermore, the continuous focus on neonicotinoids is also in need of revision, as the current evidence suggests that a broad spectrum of compounds deserve attention. Here we address both shortcomings. © 2015 Society of Chemical Industry

Enhanced reproductive output after sublethal insecticide exposure, including neonicotinoid exposure, has been reported in a diversity of arthropods. Suspicions of such a phenomenon in the Neotropical brown stink bug, Euschistus heros (Hemiptera: Pentatomidae), were sparked by the increasing densities of naturally occurring popula- tions of this insect pest species in Brazilian soybean fields. Here, we tested whether the sublethal exposure to imida- cloprid would induce changes in the survival and repro- ductive performances of E. heros adult females. The imidacloprid estimated LC50 was 0.83 (0.60-1.25) lg a.i./ cm 2 , and the dose recommended for field applications (4.2 lg a.i./cm 2 ) was within the concentration range of the imidacloprid estimated LC80 (2.66 (1.65-5.49) lg a.i./ cm 2 ). Newly emerged (B24 h) adult females were exposed for 48 h to dry imidacloprid residues (0.042 lg/cm 2 , equivalent to 1 % of the field rate dose) and exhibited higher levels of cell damage, greater ovariole length, and a larger area of the most developed follicle in their ovaries up to the 6th day of adulthood. Furthermore, these females exhibited reduced rates of survival but higher fecundity and fertility rates compared with untreated females. Our results thus suggest that females of E. heros increased their re- productive output in response to the imidacloprid sublethal exposure. These findings suggest a potential involvement of sublethal exposure to neonicotinoids in the recent out- breaks of the Neotropical brown stink bug E. heros ob- served in Brazilian soybean-producing regions.

As honeybees are the main pollinator subject to an intense research regarding effects of pesticides, other ecologically important native bee pollinators have received little attention in ecotoxicology and risk assessment of pesticides in general, and insecticides in particular, some of which are perceived as reduced-risk compounds. Here, the impact of three reduced-risk insecticides – azadirachtin, spinosad and chlorantraniliprole – was assessed in two species of stingless bees, Partamona helleri and Scaptotrigona xanthotrica ,w hich are important native pollinators in Neotropical America. The neonicotinoid imidacloprid was used as a positive control. Spinosad exhibited high oral and contact toxicities in adult workers of both species at the recommended label rates, with median survival times (LT50s) ranging from 1 to 4 h, whereas these estimates were below 15 min for imidacloprid. Azadirachtin and chlorantraniliprole exhibited low toxicity at the recommended label rates, with negligible mortality that did not allow LT50 estimation. Sublethal behavioural assessments of these two insecticides indicated that neither one of them affected the overall group activity of workers of the two species. However, both azadirachtin and chlorantraniliprole impaired individual flight take-off of P. helleri and S. xanthotrica worker bees, which may compromise foraging activity, potentially leading to reduced colony survival. These findings challenge the common perception of non-target safety of reduced-risk insecticides and bioinsecticides, particularly regarding native pollinator species.

Sublethal insecticide exposure may affect foraging of insects, including natural enemies, although the subject is usually neglected. The lady beetle Eriopis connexa (Germar, 1824) (Coleoptera: Coccinelidae) is an important predator of aphids with existing pyrethroid-resistant populations that are undergoing scrutiny for potential use in pest management systems characterized by frequent insecticide use. However, the potential effect of sublethal pyrethroid exposure on this predator’s foraging activity has not yet been assessed and may compromise its use in biological control. Therefore, our objective was to assess the effect of sublethal lambda-cyhalothrin exposure on three components of the prey foraging activity (i.e., walking, and prey searching and handling), in both pyrethroid-susceptible and -resistant adults of E. connexa. Both lady beetle populations exhibited similar walking patterns without insecticide exposure in noncontaminated arenas, but in partially contaminated arenas walking differed between strains, such that the resistant insects exhibited greater walking activity. Behavioral avoidance expressed as repellence to lambda-cyhalothrin was not observed for either the susceptible or resistant populations of E. connexa, but the insecticide caused avoidance by means of inducing irritability in 40% of the individuals, irrespective of the strain. Insects remained in the insecticide-contaminated portion of the arena for extended periods resulting in greater exposure. Although lambda-cyhalothrin exposure did not affect prey searching by susceptible lady beetles, prey searching was extended for exposed resistant predators. In contrast, prey handling was not affected by population or by lambda-cyhalothrin exposure. Thus, sublethal exposure to the insecticide in conjunction with the insect resistance profile can affect prey foraging with pyrethroid-exposed resistant predators exhibiting longer prey searching time associated with higher walking activity reducing its predatory performance.

International audience; AbstractThe ongoing debate regarding the role of pesticides in the global decline of bee populations is increasing the demand for use of biopesticides, compounds generally believed to be less harmful to pollinators. However, there is lack of evidence justifying such perceptions, particularly regarding native pollinator species like Neotropical stingless bees. Here, we investigated whether azadirachtin, a neem-based biopesticide, causes significant lethal and sublethal effects on adult workers of the Neotropical stingless bee species Melipona quadrifasciata and Partamona helleri. Susceptibility to azadirachtin varied with several factors, including the route of exposure, the concentration of the biopesticide, and the bee species. We found that although azadirachtin did not affect worker bee mortality, flight, or respiration rate, it did, however, induce a significant antifeeding effect on the stingless bee species.

Azadirachtin is a biorational insecticide commonly reported as selective to a range of beneficial insects. Nonetheless, only few studies have been carried out with pollinators, usually emphasizing the honeybee Apis mellifera and neglecting other important pollinator species such as the bumblebee Bombus terrestris. Here, lethal and sublethal effects of azadirachtin were studied on B. terrestris via oral exposure in the laboratory to bring out the potential risks of the compound to this important pollinator. The compound was tested at different concentrations above and below the maximum concentration that is used in the field (32 mg L(-1)). As most important results, azadirachtin repelled bumblebee workers in a concentration-dependent manner. The median repellence concentration (RC50) was estimated as 504 mg L(-1). Microcolonies chronically exposed to azadirachtin via treated sugar water during 11 weeks in the laboratory exhibited a high mortality ranging from 32 to 100 % with a range of concentrations between 3.2 and 320 mg L(-1). Moreover, no reproduction was scored when concentrations were higher than 3.2 mg L(-1). At 3.2 mg L(-1), azadirachtin significantly inhibited the egg-laying and, consequently, the production of drones during 6 weeks. Ovarian length decreased with the increase of the azadirachtin concentration. When azadirachtin was tested under an experimental setup in the laboratory where bumblebees need to forage for food, the sublethal effects were stronger as the numbers of drones were reduced already with a concentration of 0.64 mg L(-1). Besides, a negative correlation was found between the body mass of male offspring and azadirachtin concentration. In conclusion, our results as performed in the laboratory demonstrated that azadirachtin can affect B. terrestris with a range of sublethal effects. Taking into account that sublethal effects are as important as lethal effects for the development and survival of the colonies of B. terrestris, this study confirms the need to test compounds on their safety, especially when they have to perform complex tasks such as foraging. The latter agrees with the recent European Food Safety Authority guidelines to assess 'potentially deleterious' compounds for sublethal effects on behavior.

The preservation of natural enemies is one of the basic foundations for integrated pest management. Botanical insecticides have shown low impact on beneficial arthropods in relation to survival. Insecticides studies usually focus on the direct physiological effects of insecticides, whereas relatively little attention is placed on the behavioral response to exposure. A study was conducted to evaluate the effect of the botanical insecticide neem (Azadirachta indica A. Juss.; Meliaceae) on the mating behavior of the predatory stinkbug Podisus nigrispinus (Heteroptera: Pentatomidae). Unmated 5 to 7 d-old adults, separate by sex, were exposed to azadirachtin per contact on the treated surface. The treatments were composed for: untreated male and female; untreated male and treated female; treated male and untreated female; and treated male and female. Azadirachtin affected the duration of first mating (Wilcoxon test, χ2 = 13.38, df = 3, p = 0.004), which resulted in a higher effective average time of mating (EATM50) for treatment whose only female was treated with azadirachtin. This finding points to a sublethal effect of azadirachtin on mating behavior of P. nigrispinus that may compromise its reproduction.La preservación de los enemigos naturales es la base fundamental para el manejo integrado de las plagas. Los insecticidas botánicos han demostrado un bajo impacto sobre los artrópodos benéficos en relación a la supervivencia. Se desarrolló un estudio para evaluar el efecto del insecticida botánico neem (Azadirachta indica A. Juss.; Meliaceae) sobre el comportamiento de apareamiento del chinche depredador Podisus nigrispinus (Heteroptera: Pentatomidae). Se expusieron adultos vírgenes de 5-7 días de edad, separados por sexo, a residuos secos de este extracto. Machos y hembras vírgenes entre 5 y 7 d de edad fueron expuestos a la azadiractina, por contacto directo con superficies tratadas. Los tratamientos fueron: machos y hembras no tratados; macho tratado y hembra no tratada; macho no tratado y hembra tratada y macho y hembras tratadas. Los resultados demostraron que la azadiractina afectó la duración de la primera cópula (test de Wilcoxon, χ² = 13.38, df = 3, p = 0.004) lo que se traduce en un alto tiempo medio efectivo de cópula (EATM50) en el tratamiento en que sólo la hembra fue tratada con azadiractina. Esta constatación apunta a un efecto subletal de la azadiractina sobre el comportamiento de apareamiento de P. nigrispinus que probablemente compromete su reproducción.

As brocas-das-cucurbitáceas Diaphania spp. são as principais pragas das cucurbitáceas, podendo ocasionar perdas de até 100% na produção. A fim de reduzir o uso de inseticidas, o controle biológico, aplicado com parasitoides do gênero Trichogramma, tem-se destacado. Objetivou-se avaliar a seletividade dos ingredientes ativos abamectina, tiacloprido e clorfenapir, para Trichogramma atopovirilia Oatman & Platner (Hymenoptera: Trichogrammatidae). Para isso, 20 fêmeas recém-emergidas de T. atopovirilla foram individualizadas, em tubos de vidro (2,5 cm de diâmetro x 8,5 cm de comprimento), e oferecidas cartelas com 30 ovos de Diaphania hyalinata (Linnaeus) (Lepidoptera: Pyralidae), previamente imersas por cinco segundos em calda química. Os ingredientes ativos abamectina, tiacloprido e clorfenapir não afetaram o parasitismo de T. atopovirilia. Clorfenapir reduziu a emergência. Abamectina e tiacloprido são os mais recomendados no manejo integrado de pragas, pois foram os que se mostraram mais seletivos a T. atopovirilia em ovos de D. hyalinata. The cucurbit borers Diaphania spp. are major cucurbit pests, causing losses of up to 100% in production. Biological control with the parasitoid Trichogramma is an alternative for reducing the use of insecticides. The objective of this study was to evaluate the selectivity of the active ingredients abamectin, chlorfenapyr and thiacloprid to Trichogramma atopovirilia Oatman & Platner (Hymenoptera: Trichogrammatidae). For this purpose, 20 newly emerged females of T. atopovirilla were confined individually in glass tubes (2.5 cm diameter x 8.5 cm in length), with 30 eggs of Diaphania hyalinata (Linnaeus) (Lepidoptera: Pyralidae) glued on a small cardboard strip that was previously immersed for five seconds in chemical solution. The active ingredients abamectin, chlorfenapyr and thiacloprid did not affect the T. atopovirilia parasitism. However, chlorfenapyr reduced larval emergence. Abamectin and thiacloprid can be therefore recommended for the Integrated Pest Management, as they proved to be the most selective to T. atopovirilia in D. hyalinata eggs.

Trichogramma spp. (Hymenoptera: Trichogrammatidae) can control Diaphania hyalinata Linnaeus (Lepidoptera: Pyralidae). On the other hand, pesticides may reduce the efficiency of natural enemies. The objective was to evaluate the side-effects of fungicides used in the production of cucurbitaceous crops on Trichogramma atopovirilia Oatman & Platner parasitizing D. hyalinata eggs. The fungicides used in bioassays were: azoxystrobin (0.08 g active ingredient [ai] L-1), chlorothalonil (2.00 g ai L-1), mancozeb (1.60 g ai L-1), tebuconazole (0.25 g ai L-1) and thiophanate-methyl (0.49 g ai L-1). Cardboards with 30 D. hyalinata eggs previously immersedin fungicide solutions and distilled water (control) were offered separately to 20 newly emerged T. atopovirilia females in glass tubes. Parasitism, parasitism reduction, emergence, sex ratio, and number of individuals per egg were evaluated. The fungicides chlorothalonil, thiophanate-methyl and tebuconazole reduced parasitism of T. atopovirilia by 43.37, 27.64 and 18.51%, respectively. However, parasitism with azoxystrobin (79.21%) was higher than the control (67.37%) (P ≤ 0.05). Chlorothalonil, thiophanate-methyl and tebuconazole reduced emergence by 73.77, 75.62 and 79.35% (P ≤ 0.05), respectively. Azoxystrobin and thiophanate-methyl reduced the sex ratio by 0.77 and 0.76 (P ≤ 0.05), respectively. Fungicides did not reduce the number of individuals per egg. The fungicides azoxystrobin and mancozeb were selective for T. atopovirilia for most studied parameters suggesting that these products must have the priority in crop disease management to allow efficient biological control of T. atopovirilia against D. hyalinata.

The predator Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) has been used as a natural enemy to control herbivorous insects in eucalyptus plantations. However, because of the short co-evolutionary history of the predator with this imported plant species, the defensive compounds of eucalyptus may have a negative effect on the predator. Therefore, the survival and development of P. nigrispinus were studied on eucalyptus (Eucalyptus urophylla; exotic host plant) and guava (Psidium guajava; native host plant). P. nigrispinus performance was indeed superior on guava plants than on eucalyptus. The number of fertile eggs and nymphal survival were 52.6% and 12.0% on guava compared with 20.0% and 1.3% on eucalyptus, respectively. Longevity of P. nigrispinus was 115 days on guava and 98 days on eucalyptus. This shows that the predator is not yet fully adapted to the exotic host plant. One possible reason is that the plant toxic compounds in eucalyptus act on this predator by antibiosis.

The risks imposed by novel insecticides, mainly bioinsecticides, are largely unknown despite their increased use and their perceived environmental safety, which is based on their natural origin. Furthermore, unlike honeybees, native pollinator species have received little attention. In the present study, the lethal and sublethal effects of the neonicotinoid imidacloprid and the bioinsecticide spinosad were assessed in the stingless bee species Melipona quadrifasciata, an important native pollinator in the Neotropical region. The adult stingless bee workers exhibited high oral insecticide susceptibility, with LD50s of 23.54 and 12.07 ng a.i./bee for imidacloprid and spinosad, respectively. Imidacloprid also impaired worker respiration and overall group activity and flight, while spinosad significantly impaired only worker flight despite exhibiting higher oral toxicity to adult workers than imidacloprid. These findings indicate the hazardous nature not only of imidacloprid but also the bioinsecticide spinosad to adult workers of the native pollinator M. quadrifasciata. Therefore, bioinsecticides should not be exempted from risk assessment analysis due to their lethal and sublethal components.