A mixture of mesotrione and atrazine harms adults and larvae of the predatory wasp Polistes satan.

Herbicides have been intensively used for weed control, raising concerns about their potentially adverse effects on non-target organisms. Research on the effects of these common agrochemicals on beneficial insects and the ecosystem services they provide (e.g., predation and pollination) is scarce. Therefore, we tested whether a commercial formulation comprising a mixture of mesotrione and atrazine was detrimental to adult females and larvae of the Neotropical predatory social wasp Polistes satan, which is an effective natural enemy of crop pests. Wasps were individually fed syrups contaminated with different concentrations of the herbicide above and below the maximum label rate (MLR = 12 mL/L). Survival was assessed. The locomotor activity, immune response, and midgut morphology of adults as well as the immune response of the larvae were also studied. Herbicide concentrations far above the MLR (12, 40, and 100 times) caused adult mortality, whereas lower concentrations (0.5, 1, and 6 times) did not. Herbicide exposure at 0.5 to 12 times the MLR increased adult activity. Adult exposure at 0.1 or 0.5 times the MLR did not affect melanotic encapsulation of foreign bodies but led to changes in the morphology of the midgut epithelium and peritrophic matrix. In larvae, the ingestion of herbicide at 0.1 or 0.2 times the MLR (corresponding to 9.6 and 19.2 ng of herbicide per individual) did not cause mortality but decreased their melanization-encapsulation response. Increased locomotor activity in herbicide-exposed adults can affect their foraging activity. The altered midgut morphology of adults coupled with the decreased immune response in larvae caused by herbicide exposure at realistic concentrations can increase the susceptibility of wasps to infections. Therefore, herbicides are toxic to predatory wasps.

Toxicological Assessments of Agrochemicals in Stingless Bees in Brazil: a Systematic Review.

The growing concern with the decline of pollinators worldwide is centered on honey bees, due to their wide distribution, economic, and ecological importance. This type of concern remained less evident for stingless bees, which are widely distributed in the Neotropics, until recently. Since exposure to agrochemicals has been identified as one of the potential threats to bees, the present systematic review compiled information from toxicological evaluations in stingless bees in Brazil, home to a considerable portion of the existing species. This systematic review was performed considering species, research institutions, scientific journals, metrics, experimental set ups, and agrochemicals. The first article in this topic was published in 2010. Since then, 93 scientific papers were published, which showed that there are few species of stingless bees used for toxicological evaluations and Brazilian institutions lead these evaluations. Only 1.5% of the stingless bees' species that occur in Brazil were assessed through chronic exposure in the larval stage. The Universidade Federal de Viçosa (UFV) is responsible for 37% of the total publications. The main route of exposure was acute, using adults in laboratory conditions. The main group of agrochemicals studied were insecticides, in particular the neonicotinoids. The current results reveal the advances achieved and point out the gaps that still need to be filled considering toxicological evaluations in stingless bees.

Ingesting microplastics or nanometals during development harms the tropical pollinator Partamona helleri (Apinae: Meliponini).

The disposal of plastics and metal-derived compounds results in the contamination of the environment with nano/microparticles, leading to the exposure of various organisms to these harmful particles. However, the impacts of these particles on pollinating insects, which provide relevant ecosystem services, are not well understood. The aim of this study was to assess the effects of microscopic particles on the tropical pollinator Partamona helleri (Apinae: Meliponini), specifically evaluating the toxicity of plastic microparticles (polystyrene - PS, and polyethylene terephthalate - PET) and nanoparticles of a metal oxide (titanium dioxide - TiO2) via larval ingestion by bees reared in vitro. The survival rate of P. helleri larvae was not affected by the ingestion of particles of PS (500 ng/bee), PET (500 ng/bee), or TiO2 (10 µg/bee) compared to the non-treated diet (control or diet without the particles). Adults derived from treated larvae had increased body weight compared to the control, and the walking behavior of adults was altered by the ingestion of particles. Adults that ingested PET or TiO2 as larvae tended to rest for a longer time and interact more with other bees than the control. Hemocyte counts also changed, with a shift in the proportion of plasmatocytes and prohemocytes in treated individuals. Our findings suggest that even levels considered low for honey bees of exposure to plastic microparticles or metal nanoparticles can harm the health and behavior of stingless bees.

The impact of early-life exposure to three agrochemicals on survival, behavior, and gut microbiota of stingless bees (Partamona helleri).

Over the last few decades, agrochemicals have been partially associated with a global reduction in bees' population. Toxicological assessment is therefore 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. When used at the field-recommended rates, both copper sulfate (200 µg of active ingredient/bee; a.i µg bee-1) and spinosad (8.16 a.i µg bee-1) caused a decrease in bee survival, while glyphosate (148 a.i µg bee-1) did not show any significant effects. No significant adverse effects on bee development were observed in any treatment with CuSO4 or glyphosate, but spinosad (0.08 or 0.03 a.i µg bee -1) 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.

Ingestion of polystyrene microparticles impairs survival and defecation in larvae of Polistes satan (Hymenoptera: Vespidae).

Microplastics (MPs) are widespread pollutants of emerging concern, and the risks associated with their ingestion have been reported in many organisms. Terrestrial environments can be contaminated with MPs, and terrestrial organisms, including arthropods, are predisposed to the risk of ingesting MPs. In the current study, the larvae of the paper wasp Polistes satan were fed two different doses (6 mg or 16 mg at once) of polystyrene MPs (1.43 mm maximum length), and the effects of these treatments on immature development and survival till adult emergence were studied. Ingestion of the two doses resulted in mortality due to impaired defecation prior to pupation. The survival of larvae that ingested 16 mg of MPs was significantly lower than that of the control. The ingestion of 16 mg of MPs also reduced the adult emergence (11.4%) in comparison to the control (44.4%). MPs were not transferred from the larvae to the adults that survived. These findings demonstrate that MP ingestion can be detrimental to P. satan, e.g. larval mortality can decrease colony productivity and thus the worker force, and that MPs can potentially affect natural enemies that occur in crops, such as predatory social wasps.

A Genetically Modified Anti-Plasmodium Bacterium Is Harmless to the Foragers of the Stingless Bee Partamona helleri.

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.

Microanatomical and secretory characterization of the salivary gland of the Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), a main vector of Chagas disease.

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.

Rhamnolipids on Aedes aegypti larvae: a potential weapon against resistance selection.

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.

Interaction of Mycobacterium avium subsp. paratuberculosis with bovine sperm.

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.

Spinosad-mediated effects on survival, overall group activity and the midgut of workers of Partamona helleri (Hymenoptera: Apidae).

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.

Acute and oral exposure to imidacloprid does not affect the number of circulating hemocytes in the stingless bee Melipona quadrifasciata post immune challenge.

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.

Variations in circulating hemocytes are affected by age and caste in the stingless bee Melipona quadrifasciata.

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.

The reduced-risk insecticide azadirachtin poses a toxicological hazard to stingless bee Partamona helleri (Friese, 1900) queens.

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.

Lethal and sublethal effects of azadirachtin on the bumblebee Bombus terrestris (Hymenoptera: Apidae).

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.