Fungivorous mites enhance the survivorship and development of stingless bees even when exposed to pesticides.

Stingless bees are the largest group of eusocial bees in the world. They play an essential role as crop pollinators and have been considered for inclusion in pesticide risk assessments (RAs). Beyond the mutualism involving stingless bee larvae and fungi, the fungivorous mite Proctotydaeus (Neotydeolus) alvearii proved to be interesting for studies of associations with stingless bees. Their presence is related to colony strength and health, showing a permanent-host-association level. Here, we tested whether the coexistence with P. (N.) alvearii affects stingless bee larvae survivorship and development, including when fed pesticide-dosed food. We chose dimethoate, the reference standard for toxicity tests, and thiamethoxam, widely used in neotropical crops and listed to be reassessed in RAs. Bees associated with the mites showed higher larval survivorship rates, even in the dosed ones, and revealed changes in the developmental time and body size. Our study represents the first approach to stingless bee responses to the coexistence of fungivorous mites inside brood cells, leading us to believe that these mites play a beneficial role in stingless bees, including when they are exposed to pesticides.

A food-ingested sublethal concentration of thiamethoxam has harmful effects on the stingless bee Melipona scutellaris.

In recent years, the importance of bee's biodiversity in the Neotropical region has been evidencing the relevance of including native bees in risk assessments. Therefore, the sublethal effects of the insecticide thiamethoxam on the survival and morphological parameters of the stingless bee Melipona scutellaris were investigated in the present study. Cells from both non-target organs (Malpighian tubules and midgut) and target organs (brain) were analyzed for morphological alterations using light microscopy and transmission electron microscopy. The findings showed that when M. scutellaris foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100 = 0.000543 ng a. i./µL), longevity was not reduced but brain function was affected, even with the non-target organs attempting to detoxify. The cellular damage in all the organs was mostly reflected in irregular nuclei shape and condensed chromatin, indicating cell death. The most frequent impairments in the Malpighian tubules were loss of microvilli, disorganization of the basal labyrinth, and cytoplasmic loss. These characteristics are related to an attempt by the cells to increase the excretion process, probably because of the high number of toxic molecules that reach the Malpighian tubules and need to be secreted. In general, damages that compromise the absorption of nutrients, excretion, memory, and learning processes, which are essential for the survival of M. scutellaris, were found. The present results also fill in gaps on how these bees respond to thiamethoxam exposure and will be useful in future risk assessments for the conservation of bee biodiversity.

Larvae of stingless bee Scaptotrigona bipunctata exposed to organophosphorus pesticide develop into lighter, smaller and deformed adult workers.

Organophosphorus pesticides such as chlorpyrifos are often used in agriculture due to their broad spectrum of action. However, this insecticide and acaricide is considered highly toxic to the environment and can cause toxicity in nontarget insects such as bees. In addition to adult individuals, immature can also be exposed to residues of this insecticide by larval food. Thus, we investigated the effects of chlorpyrifos concentrations on the larval development of stingless bee Scaptotrigona bipunctata workers reared in vitro. We evaluated four different biomarkers: a) survival, b) development time, c) body mass and d) morphological characteristics (head width, intertegular distance, wing area and proportion of deformed bees). The exposure of the larvae to different doses of chlorpyrifos significantly reduced survival probability but did not cause changes in the development time. Regarding morphometric analysis, bees exposed to chlorpyrifos showed a reduction in body mass and size, and 28% of the emerged adults showed a reduction in wing area and deformations. Therefore, this work shows that S. bipunctata larvae exposed to the sublethal effects of chlorpyrifos are likely to have reduced chances of survival. However, if they emerge, they will be lighter, smaller and less able than equivalent but not exposed workers. These impaired attributes have the potential to compromise the future workforce in colonies exposed to this pesticide.

Is the Water Supply a Key Factor in Stingless Bees' Intoxication?

Water is an important resource for stingless bees, serving for both honey dilution and the composition of larval food inside nests, yet can be an important route of exposure to pesticides. Assuming bees can forage naturally on pesticide-contaminated or noncontaminated areas, we investigated whether water supply influences the choice between neonicotinoid-dosed or nondosed feeders and on mortality of the stingless bee, Melipona scutellaris (Latreille, Hymenoptera, Apidae). At the field concentration, there was no significant mortality; however, the bees were not able to distinguish the feeders. In the cages containing high-concentration feeders, with water supply, the bees preferred nondosed food, and with no water, the mortality increased. Considering that in the field it is common to find extrapolated concentrations, our work suggested that water may allow avoidance of high dosed food and minimize mortality.

What is the most suitable native bee species from the Neotropical region to be proposed as model-organism for toxicity tests during the larval phase?

Currently, Brazil has a full framework for pesticide risk assessment established for Apis mellifera, based on the North American approach. However, the use of Apis mellifera as model-organism as a surrogate for Brazilian native species of stingless bees has been questioned. Assessments on other stages of development than adult individual are essential. Our study aimed to standardize in vitro larval rearing method for the stingless bee species Scaptotrigona postica and Tetragonisca angustula, comparing the results to those obtained for M. scutellaris (previously described), for proposing the most suitable one for using in toxicological larval tests. We used the most efficient method for determining the toxicity of dimethoate on S. postica larvae. We presented the first comparative approach of responses to in vitro larval rearing methods among native bee species from Neotropical region, for use in risk assessment. Our results showed that S. postica was the most suitable native species to be proposed as model-organism. In addition, our results are also very useful for a ring test to validate the method, in accordance to OECD.

Correction: In vitro larval rearing protocol for the stingless bee species Melipona scutellaris for toxicological studies.

[This corrects the article DOI: 10.1371/journal.pone.0213109.].

In vitro larval rearing protocol for the stingless bee species Melipona scutellaris for toxicological studies.

Brazil has the highest biodiversity of native stingless bees in the world. However, Brazilian regulations are based on protocols standardized by the Organization for Economic Cooperation and Development (OECD), which uses Apis mellifera as a model organism. The safety of the use of an exotic species as a substitute for a native species is a problem that concerns members of the academy and the government agencies responsible for studies of this nature in the neotropical regions where there are occurrences of stingless bee species. Regarding the exposure of larvae to pesticides, several indicators suggest that the same rearing method for A. mellifera cannot be applied to stingless bees, mainly because of their different feeding systems. Thus, it is necessary to establish an in vitro rearing method for native social bees. We developed a larval rearing method for the stingless bee species Melipona scutellaris and evaluated parameters such as the defecation rate, pupation, emergence, mortality and morphometry of the newly emerged workers. The control was represented by the morphometry of individuals that emerged from natural combs (in vivo). In addition, we determined the average lethal concentration (LC50) of the insecticide dimethoate, the standard active ingredient used for the validation of toxicity tests. Procedures conducted prior to the in vitro bioassays allowed us to obtain the actual dimensions of the rearing cells for making acrylic plates for use in establishing how much each larva consumes during its development, that is, determining how much larval food should be placed in every artificial cell. Tests performed with M. scutellaris indicated an average of 80.2% emergence of individuals relative to the larvae, 92.61% relative to the pupae and a mean of 7.42% larval mortality. The mean of the intertegular distance, head width and wing asymmetry parameters were not significantly different between individuals from the in vitro and in vivo rearing methods. The LC50 value determined was 27.48 ng dimethoate / µL diet. The method described for M. scutellaris showed development rates above OECD standards, which requires at least 75% emergence, and produced newly emerged workers with similar dimensions to those produced under natural conditions; thus these results enable their use as a rearing protocol for this species (or genus) and, consequently, their use in toxicity tests. The results produced with M. scutellaris are the first steps for a proposed toxicity test protocol for stingless bee larvae that can be standardized and included as a protocol in the OECD.

Pesticide Exposure Assessment Paradigm for Stingless Bees.

Although the importance of bees as the pollinators responsible for maintaining gene flow for many native and cultivated plants in ecosystems around the world is recognized, much of their biodiversity and behavior remains to be discovered. Stingless bees are considered key pollinators for several plant species in tropical and subtropical ecosystems and they also provide pollination services for economically important agricultural crops. Many countries are using the honey bee (Apis mellifera Linnaeus, 1758, Hymenoptera: Apidae) as a surrogate to evaluate the risk of pesticides to all species of bees. However, there is uncertainty regarding the extent to which honey bees can serve as surrogates for non-Apis bee species in the risk assessment for pesticides. This paper provides a short overview of the life history traits relevant in risk assessment of stingless bees. It summarizes what is known about stingless bee exposure to pesticides compared to that of honey bees and presents criteria for potential candidate species from Brazil for use in pesticide risk assessment in tropical environments. This paper also identifies gaps in knowledge of bee biology and pesticide exposure routes not covered by the current honey bee exposure assessment paradigm. Based on these gaps, research is needed on life history traits, estimates of nectar and pollen consumption, mud, resin, and water collection and available protocols to adequately assess toxic effects of pesticides to stingless bees. This review is part of a series of papers on the risk of exposure of non-Apis bees to pesticides.