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.

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.

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.

Changes in Synapsin Levels in the Millipede Gymnostreptus olivaceus Schubart, 1944 Exposed to Different Concentrations of Deltamethrin.

Millipedes are ecologically important soil organisms and may also be an economically threatening species in rural and urban areas when population outbreaks occur. In order to control infestations commercial formulations of deltamethrin have been commonly applied, even though there are few studies about the effects of such insecticide on millipedes. This paper describes the effects of this insecticide on millipedes showing neurotoxic effects assessed by synapsin labeling and confocal microscopy. Deltamethrin concentrations related to the DL50 of the active ingredient and a field concentration were applied topically in the diplopod Gymnostreptus olivaceus to evaluate the behavior, mortality rate, and synapsin levels in the brain 12, 24, and 48h after contact with deltamethin. The insecticide caused mortality at the higher concentrations employed, in which no change was observed in neurotransmission in the survivors. In contrast, at field concentrations, deltamethrin did not cause any deaths, but triggered significant changes in synapsin levels. The results obtained form the synapsin labeling provide several interpretations suggesting that the isolated application of this tool must be associated with additional tools in order to evaluate biologically induced effects of deltamethrin in an accurate way. In addition, the feasibility of chemical control of millipedes with deltamethrin is questioned.

Sublethal doses of fipronil intensify synapsin immunostaining in Atta sexdens rubropilosa (Hymenoptera: Formicidae) brains.

BACKGROUND: Although ants are common insects in agricultural ecosystems, few studies have considered how xenobiotics might induce physiological and morphological alterations in these insects. This study aimed to verify the neurotoxic action of sublethal doses of fipronil on the mushroom bodies of brains from the leaf-cutting ant Atta sexdens rubropilosa through immunocytochemistry analysis for the protein synapsin. RESULTS: The LD50 value was established as 1.42 ng ant(-1), and the sublethal doses used were LD50/10 and LD50/100. Synapsin labelling was more evident in the brains extracted from ants exposed to the insecticide, specifically in the regions of glia in the mushroom bodies, compared with the control group. It was possible to measure the intensity of emitted fluorescence in the areas of the mushroom bodies, and a statistical test showed differences between the control group and the treatment group. CONCLUSION: Thus, it is concluded that sublethal doses of the insecticide fipronil intensified synapsin immunostaining, suggesting an increased release of neurotransmitters, which may be linked to neurotoxicity and overexcitation. These sublethal doses may have two different effects: compromising the operation and maintenance of the colony and leading to the establishment of resistance in insects.

Impact of fipronil on the mushroom bodies of the stingless bee Scaptotrigona postica.

BACKGROUND: Studies on stingless bees are scarce, and little is known about these insects, especially regarding the effects of contamination by neurotoxic insecticides, which can cause damage to important structures of the insect brain. This study evaluated the morphological changes in the intrinsic neurons of the protocerebral mushroom bodies (Kenyon cells) of the stingless bee Scaptotrigona postica after exposure to different doses of fipronil, using light microscopy and transmission electron microscopy. This region of the brain was selected for analysis because of its importance as a sensory integration centre. RESULTS: In both oral and topical treatments, Kenyon cells presented pyknotic profiles, suggesting cell death. Statistical analysis showed significant differences among doses and exposure times. Transmission electron microscopy revealed changes in the nucleus and cellular organelles. Depending on the dose, the characteristics observed suggested apoptotosis or necrosis. CONCLUSION: This study demonstrates the toxic effects of fipronil. An increase in the number of pyknotic profiles of Kenyon cells of mushroom bodies was observed even at the sublethal doses of 0.27 ng AI bee(-1) and 0.24 ng AI µL(-1) in the topical and oral treatments respectively. Also, differences in the number of pyknotic profiles were dose and time dependent.

Effects of sublethal dose of fipronil on neuron metabolic activity of Africanized honeybees.

Fipronil is a neurotoxic insecticide that inhibits the gamma-aminobutyric acid receptor and can affect gustative perception, olfactory learning, and motor activity of the honeybee Apis mellifera. This study determined the lethal dose (LD50) and the lethal concentration (LC50) for Africanized honeybee and evaluated the toxicity of a sublethal dose of fipronil on neuron metabolic activity by way of histochemical analysis using cytochrome oxidase detection in brains from worker bees of different ages. In addition, the present study investigated the recovery mechanism by discontinuing the oral exposure to fipronil. The results showed that mushroom bodies of aged Africanized honeybees are affected by fipronil, which causes changes in metabolism by increasing the respiratory activity of mitochondria. In antennal lobes, the sublethal dose of fipronil did not cause an increase in metabolic activity. The recovery experiments showed that discontinued exposure to a diet contaminated with fipronil did not lead to recovery of neural activity. Our results show that even at very low concentrations, fipronil is harmful to honeybees and can induce several types of injuries to honeybee physiology.

Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera: Apidae) promoted by topical application of juvenile hormone.

The present study analyzed, the influence of the treatment with juvenile hormone on the ultrastructure of Apis mellifera L. workers' venom glands. Newly emerged workers received topical application of 1 microl of juvenile hormone diluted in hexane, in the concentration of 2 microg/pl. Two controls were used; one control received no treatment (group C1) and other received topical application of 1 microl of hexane (group C2). The aspect of the glandular cells, in not treated newly emerged workers, showed that they are not yet secreting actively. Cellular modifications happened according to the worker age and to the glandular area considered. The most active phase of the gland happened from the emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic, being the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera: Apidae) promoted by topical application of juvenile hormone

The present study analyzed, the influence of the treatment with juvenile hormone on the ultrastructure of Apis mellifera L. workers' venom glands. Newly emerged workers received topical application of 1 æl of juvenile hormone diluted in hexane, in the concentration of 2 æg/æl. Two controls were used; one control received no treatment (group C1) and other received topical application of 1 æl of hexane (group C2). The aspect of the glandular cells, in not treated newly emerged workers, showed that they are not yet secreting actively. Cellular modifications happened according to the worker age and to the glandular area considered. The most active phase of the gland happened from the emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic, being the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

O presente estudo analisou, através de estudos ultra-estruturais a influência do tratamento com hormônio juvenil sobre as glândulas de veneno de operárias de Apis mellifera L. Para tanto, operárias recém-emergidas receberam aplicação tópica de 1æl de hormônio juvenil, na concentração de 2 æg/æl, sendo usado o hexano como veículo. Foram feitos dois controles, um que não recebeu nenhum tipo de tratamento (grupo C1) e o outro que recebeu aplicação tópica de 1 æl de hexano (grupo C2). O aspecto das células glandulares, em operárias recém-emergidas, mostra que estas não estão ainda secretando ativamente. Observa-se que alterações celulares ocorrem de acordo com a idade da operária e da região glandular considerada no controle C1. Assim, a fase de secreção mais ativa da glândula ocorre entre a emergência e os 14 dias de idade; aos 25 dias as células já perderam sua característica secretora, sendo a região distal a primeira a sofrer degeneração. Os tratamentos com hormônio juvenil e com hexano alteram a cronologia do ciclo glandular, antecipando o início da secreção e da degeneração da glândula.