Determination of acute lethal doses (LD50 and LC50) of imidacloprid for the native bee Melipona scutellaris Latreille, 1811 (Hymenoptera: Apidae)

The bee species Melipona scutellaris (L.) is native to Brazil, stingless and belongs to the Apidae family. In Brazil, stingless bees are responsible for 40 % to 90% of tree species pollination, depending on the considered ecosystem. However, their survival has been threatened since the country has been standing out as the biggest pesticides consumer in the world. Many of the pesticides used are considered toxic to bees, including imidacloprid (all existing studies were conducted with the Apis mellifera (L.) species). Although the bees are not the target of these substances, they are highly vulnerable to contamination. Thereby, the objective of this study was to establish the mean lethal dose (LD50) and the mean lethal concentration (LC50) of imidacloprid in the M. scutellaris fodder. In order to carry out this experiment, bees were collected and the test was performed according to OECD's protocol (1998a, 1998b), developed for A. mellifera. For the determination of LD50 and LC50, data was analyzed through the Probit method, using the BioStat software. The topical LD50 established in this study was 2.41 ng/bee for 24 hours and 1.29 ng/bee for 48 hours. The oral LC50 was 2.01 ng i.a./μL for 24 hours and 0.81 ng i.a./μL for 48 hours. This data showed that native bees of M. scutellaris species are more sensitive to this pesticide than A. mellifera. Thus, it is important to establish management methods which take this higher susceptibility into account to protect native species

Weakness and collapse of bee colonies in Brazil: are there cases of CCD?

Nas últimas décadas, o declínio de populações de abelhas silvestres e o colapso de colônias de Apis mellifera têm preocupado pesquisadores e apicultores. O objetivo deste artigo de revisão foi compilar alguns dos estudos mais relevantes relacionados às possíveis causas desses problemas – como nutrição, manejo, patógenos, parasitas e efeitos de agrotóxicos –, além de apresentar um breve histórico da síndrome “colony collapse disorder” (CCD) nos Estados Unidos e na Europa. Abordaram-se com mais detalhes os estudos desenvolvidos no Brasil, principalmente sobre agrotóxicos utilizados na agricultura e patógenos e parasitas que acometem as colônias de A. mellifera africanizada. Casos de enfraquecimento, declínio e colapso têm sido registrados, principalmente nos estados de São Paulo e Santa Catarina, que somam grandes perdas. Resultados das ocorrências analisadas indicaram que as mortalidades em massa registradas no Brasil não estavam associadas a patógenos ou a parasitas. Somente dois casos com características semelhantes às descritas para CCD foram registrados, porém sem causas definidas. Para que se conheçam as causas de enfraquecimentos e perdas de colônias de abelhas, é urgente a implementação de programas oficiais de levantamento sistemático da sanidade apícola, associados a pesquisas com foco nas avaliações dos possíveis impactos da fragmentação de habitats e das práticas agrícolas sobre as comunidades de abelhas.In the last decades, the decline of wild bee populations and the collapse of Apis mellifera colonies have concerned researchers and beekeepers. The objective of this review was to compile some of the most relevant studies related to the possible causes of these problems – such as nutrition, management, pathogens, parasites, and the effects of pesticides –, besides presenting a brief history of the colony collapse disorder syndrome (CCD) in the United States and Europe. The studies developed in Brazil were presented in more detail, mainly those on pesticides used in agriculture and on pathogens and parasites that attack the colonies of Africanized A. mellifera. Cases of weakening, decline, and collapse have been recorded in Brazil, mainly in the states of São Paulo and Santa Catarina, which add up to great losses. Results of the analyzed occurrences indicated that the recorded mass mortalities were not associated with pathogens or parasites. Only two cases with characteristics similar to those described for CCD were recorded, but without defined causes. In order to know the causes of the weakening and collapses of colonies, the implementation of official programs is urgent for the systematic survey of bee health, associated with research focusing on assessments of possible impacts of habitat fragmentation and agricultural practices on bee communities.

Contribuição à análise do processo de africanização de Apis mellifera (Hymenoptera, apidae): características do desenvolvimento das glândulas de Dufour e de veneno

Neste estudo foram analisados, histologicamente, o desenvolvimento pós-embrionário das glândulas de veneno e de Dufour, bem como os efeitos da aplicação tópica de hormônio juvenil, sobre a glândula de veneno do híbrido de Apis mellifera L., conhecido como abelha africanizada. Análises morfométricas comparativas das glândulas de veneno e de Dufour e bioquímicas da glândula de veneno da abelha africanizada e de três subespécies, A. m. ligustica, A. m. carnica e A. m. caucasica, foram, também, realizadas. As análises histológicas do desenvolvimento pós-embrionário mostraram que existem três pares de discos imaginais presentes no 7 , 8 e 9 segmentos larvais. Durante a metamorfose o par de discos do 7 segmento forma a placa subgenital, aquele do 8 origina as lancetas do ferrão e o par presente no 9 segmento origina as glândulas de veneno e de Dufour, além dos palpos do ferrão e bainha. Conforme mostrado histologicamente, as transformações glandulares que ocorrem durante a metamorfose, não foram influenciadas pelo hormônio juvenil exógeno, mas houve aceleração do ciclo secretor glandular em relação ao controle, em todas as idades analisadas nas glândulas de operárias que receberam aplicação de hormônio juvenil diluído em hexano e de hexano puro, durante o 5º estágio e na emergência. Estes dados indicam que o hormônio juvenil atua sobre a glândula de veneno e que esta atuação pode estar sendo potencializada pelo solvente utilizado. As análises morfométricas e bioquímicas comparativas da glândula de veneno mostraram que, as glândulas de abelhas africanizadas, tanto de operárias quanto de rainhas, apesar de maiores do que as das subespécies, possuem menor teor de proteína total. O perfil eletroforético protéico de tais glândulas revelaram que a única diferença entre as operárias ocorreu nas recém-emergidas de... .The present histological investigation analyzed the post-embryonary development of the Dufour and venom glands, as well as the juvenile hormone effect on the venom gland in Apis mellifera hybrids, which are also known as Africanized bees. Comparative morphometric and biochemical analyses of the venom gland in Africanized bee and in three subspecies, A. m. ligustica, A. m. carnica and A. m. caucasica, were also done. The histological post-embryonary development results showed three pair of imaginal discs present in segments 7th, 8th and 9th. During the metamorphosis, the pair of imaginal discs of segment 7th originates the subgenital plate, those of segment 8th originates the sting lancets and the pair of segment 9th originates the Dufour and venom glands, besides originates the oblong plates and sheath lobes of the sting. As shown by the histology, the glandular changes that occur in the venom gland during metamorphosis of workers treated with diluted juvenile hormone and pure hexane during 5th instars and emergence are not influenced by the exogenous juvenile hormone, but there was an increasing of the glandular cycle in relationship to the control. These data suggest that the juvenile hormone actuate on the venom gland and the solvent used may enhance such juvenile hormone action. The comparative morphometric and biochemical analyses of the venom gland showed that although the Africanized bee glands are larger than the subspecies, both workers and queens presented less amount of total protein. The eletrophoretic profile of the venom glands showed that, among workers, only A. m. caucasia does not present the 105.4 kDa band. Among queens, only A. m. ligustica does not present the 161.3 kDa band. The morphometric analyses showed a gradual increasing of the Dufourþs gland length from newly emerged to forager workers, and from virgin to fecundated queens, indicating that the venom gland is more developed in forager workers and in fecundated queens

Toxicity of imidacloprid to the stingless bee Scaptotrigona postica Latreille, 1807 (Hymenoptera: Apidae)

The stingless bee Scaptotrigona postica is an important pollinator of native and cultivated plants in Brazil. Among the factors affecting the survival of these insects is the indiscriminate use of insecticides, including the neonicotinoid imidacloprid. This work determined the toxicity of imidacloprid as the topical median lethal dose (LD50) and the oral median lethal concentration (LC50) as tools for assessing the effects of this insecticide. The 24 and 48 h LD50 values were 25.2 and 24.5 ng of active ingredient (a.i.)/bee, respectively. The 24 and 48 h LC50 values were 42.5 and 14.3 ng a.i./mu L of diet, respectively. Ours results show the hazard of imidacloprid and the vulnerability of stingless bees to it, providing relevant toxicological data that can used in mitigation programs to ensure the conservation of this species.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A 4

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs

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 tissue189COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP001; 88887.318482/2019–002014/12488–0; 2013/09555–5; 2017/21097–3We thank the Centro de Estudos de Insetos Sociais - CEIS (UNESP – Rio Claro -SP/Brazil) and Professor Mário Sergio Palma for providing the infrastructure and Carolina Grando for useful comments on a previous version of the manuscript. We also thank Cordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Grant Numbers 001 and 88887.318482/2019–00, and the São Paulo Research Foundation (FAPESP) for financing projects 2014/12488–0, 2013/09555–5 and 2017/21097–

Viruses, microsporidia and glyphosate residues in Africanized honey bees <i>Apis mellifera</i>, under field conditions

Bees are affected by several factors, including pathogens and pesticides, which may cause mortality and consequent population decline. Considering the ecological and economic importance of these pollinators, this study evaluated the occurrence of the microsporidia Nosema apis and Nosema ceranae, the viruses ABVP, DWV, BQCV, KBV, IAPV and CBPV and residues of the pesticides atrazine, chlorantraniliprole, deltamethrin, fipronil and glyphosate in workers from field colonies of Africanized honey bee Apis mellifera across one year. The presence of pesticide residues was also evaluated in honey and pollen samples. All tested viruses were found in the workers, with BQCV showing the highest copy number. BQCV levels were correlated with relative humidity. Nosema ceranae was the only microsporidian with higher intensities of infection during summer and autumn; seasons with high availability of food resources, which can act as dispersers of this pathogen. Small amounts of glyphosate and its metabolite were detected in the bees tested. These data show that honey bees are contaminated with pathogens and pesticides in the field and present correlative data for these threats.</p