Sublethal doses of imidacloprid and pyraclostrobin impair fat body of solitary bee Tetrapedia diversipes (Klug, 1810).

Solitary bees present greater species diversity than social bees. However, they are less studied than managed bees, mainly regarding the harmful effects of pesticides present in agroecosystems commonly visited by them. This study aimed to evaluate the effect of residual doses of imidacloprid and pyraclostrobin, alone and in combination, on the fat body (a multifunctional organ) of the neotropical solitary bee Tetrapedia diversipes by means of morphological and histochemical evaluation of oenocytes and trophocytes. Males and females of newly-emerged adults were submitted to bioassays of acute topical exposure. Experimental groups were essayed: control (CTR), solvent control (ACT), imidacloprid (IMI, 0.0028 ng/µL), pyraclostrobin (PYR, 2.7 ng/µL) and imidacloprid + pyraclostrobin (I + P). The data demonstrated that the residual doses applied in T. diversipes adults are sublethal at 96 h. Both oenocytes and trophocytes cells responded to topical exposure to the pesticides, showing morphological changes. In the IMI group, the bee oenocytes showed the greatest proportion of vacuolization and altered nuclei. The pyraclostrobin exposure increased the intensity of PAS-positive labeling (glycogen) in trophocytes. This increase was also observed in the I + P group. Changes in energy reserve (glycogen) of trophocytes indicate a possible mobilization impairment of this neutral polysaccharide to the hemolymph, which can compromise the fitness of exposed individuals. Also, changes in oenocytes can compromise the detoxification function performed by the fat body. This is the first study to show sublethal effects in neotropical solitary bees and highlight the importance of studies with native bees.

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.

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach.

Bees are important pollinators that help to maintain the biodiversity of wild and cultivated plants. However, the increased and inappropriate use of agrochemicals has caused an imbalance in the populations of these insects visiting flowers for pollen and nectar collection. Therefore, new research methods for understanding the mechanisms of action of pesticides and their impacts on the brains of bees, such as neurotoxicity and cellular changes, in response to different active characteristics and dosages of insecticides are necessary. Thus, with the aim of developing tests with greater specificity at the level of cells or tissues, this study sought to standardize a method for the in vitro culture of the nervous tissue of Apis mellifera. For this purpose, the brains of six foragers bees were transferred to three different insect cell culture media and it supplementation with 10% foetal bovine serum (FBS): Grace, Schneider, Leibovitz, Grace + FBS, Schneider + FBS and Leibovitz + FBS media for each collection time. Nervous tissue was collected after 1, 6, 12 and 24 h of incubation in a humidified CO2 incubator at 32 °C, and histological sections of the organs were analysed. The results showed that Leibovitz medium and Leibovitz medium + serum are potential culture media for the cultivation of nervous tissue, since they resulted in less tissue spacing and tissue disarrangement. Therefore, additional supplements are necessary to obtain an ideal medium for the cultivation of A.mellifera nervous tissue.

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.].

Semi-quantitative analysis of morphological changes in bee tissues: A toxicological approach.

During foraging, bees are exposed to sublethal doses of insecticides, which can cause morphological changes to various organs, such as the midgut, Malpighian tubules, and mushroon body. Thus, the purpose of this study was to establish a scoring system to evaluate these alterations based on the damage caused and its reversibility. Therefore, a grade 1 score indicates a minimal and easily reversible lesion, increased apocrine secretion, increased cell elimination into the lumen, and a larger quantity of spherocrystals; grade 2 was assigned to moderate and typically reversible injuries, such as changes in the brush border, vacuolation/loss of cytoplasmic material, presence/height of the brush border, and cell swelling; and grade 3 was assigned to serious and irreversible, loss of cell nests of regenerative cells, pyknosis, and loss of contact between Kenyon cells. In addition, frequency values were assigned since the alterations can occur at different frequencies according to the insecticide and the bees exposed; the frequency ranges from 0 to 6, with 0 representing the absence of an alteration and 6 representing a high-frequency occurrence. Based on the analyses, we conclude that each change causes morphological damage, which may or may not be irreversible and could affect the health of the colony.

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.