Bees and the Environmental Impact of the Rupture of the Fundão Dam.

The environmental consequences associated with the Fundão tailings dam failure in Mariana, Minas Gerais, Brazil on 5 November 2015 are still being investigated. Bees are the main pollinators believed to be most affected by the accident because they occur throughout the area affected by the dam rupture and build their nests in the soil. In this study, we evaluated the richness of different bee species in areas affected and not affected by the accident and measured the concentration of different metals on the pollen basket, or corbicula, located on the hind legs of Apis mellifera. Diversity indices were similar and there was no statistical difference in the diversity of bees sampled when comparing the sites affected and not affected by the tailings mud, either before or after the dam rupture. It is possible that the similarity is due to nearby forests that may be serving as areas of refuge. The levels of Al, Cu, Cd, Hg, and Pb in A. mellifera suggest no change in the level of metals in pollen collected by the bees that can be attributed to the rupture of the dam. Integr Environ Assess Manag 2020;16:631-635. © 2020 SETAC.

As consequências ambientais da ruptura da barragem de Fundão, ocorrida em Mariana, Minas Gerais, Brasil, em 5 de novembro de 2015, ainda estão sendo investigadas. As abelhas, principal grupo de polinizadores, podem ter sido afetadas direta e indiretamente, uma vez que a maioria das espécies constrói seus ninhos no solo. Neste estudo, avaliamos a riqueza de espécies de abelhas nas áreas afetadas e não afetadas e medimos a concentração de metais pesados nas corbículas de Apis mellifera, espécie que ocorre em toda a área afetada pela ruptura da barragem. Os índices de diversidade foram semelhantes e não houve diferença estatística na diversidade de abelhas amostradas quando comparamos locais afetados e não afetados pela lama de rejeitos após o rompimento da barragem. É possível que a semelhança se deva a florestas próximas que possam estar servindo como áreas de refúgio. Os resultados mostraram a presença de Al, Cu, Cd, Hg e Pb em A. mellifera. Nossos resultados indicam que não há efeito da contaminação de metais pesados nas abelhas devido à ruptura da barragem. Integr Environ Assess Manag 2020;16:631-635.

Buzz-pollination in Neotropical bees: genus-dependent frequencies and lack of optimal frequency for pollen release.

Over 50 genera of bees release pollen from flower anthers using thoracic vibrations, a phenomenon known as buzz-pollination. The efficiency of this process is directly affected by the mechanical properties of the buzzes, namely the duration, amplitude, and frequency. Nonetheless, although the effects of the former two properties are well described, the role of buzz frequency on pollen release remains unclear. Furthermore, nearly all of the existing studies describing vibrational properties of natural buzz-pollination are limited to bumblebees (Bombus) and carpenter bees (Xylocopa) constraining our current understanding of this behavior and its evolution. Therefore, we attempted to minimize this shortcoming by testing whether flower anthers exhibit optimal frequency for pollen release and whether bees tune their buzzes to match these (optimal) frequencies. If true, certain frequencies will trigger more pollen release and lighter bees will reach buzz frequencies closer to this optimum to compensate their smaller buzz amplitudes. Two strategies were used to test these hypotheses: (i) the use of (artificial) vibrational playbacks in a broad range of buzz frequencies and amplitudes to assess pollen release by tomato plants (Solanum lycopersicum L.) and (ii) the recording of natural buzzes of Neotropical bees visiting tomato plants during pollination. The playback experiment indicates that although buzz frequency does affect pollen release, no optimal frequency exists for that. In addition, the recorded results of natural buzz-pollination reveal that buzz frequencies vary with bee genera and are not correlated with body size. Therefore, neither bees nor plants are tuned to optimal pollen release frequencies. Bee frequency of buzz-pollination is a likely consequence of the insect flight machinery adapted to reach higher accelerations, while flower plant response to buzz-pollination is the likely result of its pollen granular properties.

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.

Karyotypic description of the stingless bee Meliponaquinquefasciata Lepeletier, 1836 (Hymenoptera, Meliponini) with emphasis on the presence of B chromosomes.

Stingless bees are distributed widely in the tropics, where they are major pollinators of several plant species. In this study, the karyotype of Meliponaquinquefasciata Lepeletier, 1836 was analysed, with emphasis on the presence of B chromosomes. Post-defecating larvae were analysed using Giemsa staining, the C-banding technique, sequential staining with fluorochromes, and FISH. The chromosome number ranged from 2n = 18 to 22 (females) and from n = 9 to 13 (males) due to the presence of 0-4 B chromosomes. This result demonstrates that M.quinquefasciata has the same chromosomal number as other Melipona Illiger, 1806 species. Considering the A complement, heterochromatin was located only in the pericentromeric region of pair 1. Staining with chromomycin A3 (CMA3) and labelling with rDNA probe, indicated that this region corresponded to the nucleolus organising region. The B chromosomes of M.quinquefasciata could be found in individuals from different localities, they were completely heterochromatic (C-banding) and uniformly stained by 4',6-diamidino-2-phenylindole (DAPI). Variations in the number of B chromosomes were detected between cells of the same individual, between individuals of the same colony, and between colonies from different localities.

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.

Melipona quadrifasciata (Hymenoptera: Apidae) fat body persists through metamorphosis with a few apoptotic cells and an increased autophagy.

Fat body, typically comprising trophocytes, provides energy during metamorphosis. The fat body can be renewed once the larval phase is complete or recycled and relocated to form the fat body of the adult insect. This study aims to identify the class of programmed cell death that occurs within the fat body cells during the metamorphosis of the stingless bee Melipona quadrifasciata. Using immunodetection techniques, the fat body of the post-defecating larvae and the white-, pink-, brown-, and black-eyed pupae were tested for cleaved caspase-3 and DNA integrity, followed by ultrastructural analysis and identification of autophagy using RT-PCR for the Atg1 gene. The fat body of M. quadrifasciata showed some apoptotic cells positive for cleaved caspase-3, although without DNA fragmentation. During development, the fat body cells revealed an increased number of mitochondria and free ribosomes, in addition to higher amounts of autophagy Atg1 mRNA, than that of the pupae. The fat body of M. quadrifasciata showed few cells which underwent apoptosis, but there was evidence of increased autophagy at the completion of the larval stage. All together, these data show that some fat body cells persist during metamorphosis in the stingless bee M. quadrifasciata.

Genetic diversity and structure of Atta robusta (Hymenoptera, Formicidae, Attini), an endangered species endemic to the restinga ecoregion.

The genetic diversity and structure of the ant Atta robusta were assessed by ISSR (inter-simple sequence repeats) in 72 colonies collected from 10 localities in the Brazilian states of Espírito Santo (48 colonies) and Rio de Janeiro (24 colonies). The ISSR pattern included 67 bands, 51 of them (76.1%) polymorphic. Analysis of molecular variance (AMOVA) revealed a high level (57.4%) of inter-population variation, which suggested a high degree of genetic structure that was confirmed by UPGMA (unweighted pair-group method using an arithmetic average) cluster analysis. The significant correlation between genetic and geographic distances (r = 0.64, p < 0.05) indicated isolation that reflected the distance between locations. Overall, the populations were found to be genetically divergent. This finding indicates the need for management plans to preserve and reduce the risk of extinction of A. robusta.

The antennal sensilla of Melipona quadrifasciata (Hymenoptera: Apidae: Meliponini): a study of different sexes and castes.

The sensilla of insects are integumental units that play a role as sensory structures and are crucial for the perception of stimuli and for communication. In this study, we compared the antennal sensilla of females (workers and queens), males (haploid (n) and diploid (2n)), and queen-like males (QLMs, resulting from 2n males after juvenile hormone (JH) treatment) in the stingless bee Melipona quadrifasciata. Images of the dorsal antenna surfaces were acquired using a scanning electron microscope. As reported for other hymenopterans, this species exhibits a heterogeneous sensillar distribution along the antennae. Thirteen different types of sensilla were found in the antennae of M. quadrifasciata: trichodea (subtypes I to VI), chaetica (subtypes I and II), placodea, basiconica, ampullacea, coeloconica, and coelocapitula. Sensilla trichodea I were the most abundant, followed by sensilla placodea, which might function in olfactory perception. Sensilla basiconica, sensilla chaetica I, sensilla coeloconica, and sensilla ampullacea were found exclusively in females. In terms of the composition and size of the sensilla, the antennae of QLMs most closely resemble those of the 2n male, although QLMs exhibit a queen phenotype. This study represents the first comparative analysis of the antennal sensilla of M. quadrifasciata. The differences found in the type and amount of sensilla between the castes and sexes are discussed based on the presumed sensillary functions.

Genetic divergence between Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) populations.

Melipona quadrifasciata is a stingless bee widely found throughout the Brazilian territory, with two recognized subspecies, M. quadrifasciata anthidioides, that exhibits interrupted metasomal stripes, and M. quadrifasciata quadrifasciata, with continuous metasomal stripes. This study aimed to estimate the genetic variability of these subspecies. For this purpose, 127 colonies from 15 Brazilian localities were analyzed, using nine species-specific microsatellite primers. At these loci, the number of alleles ranged from three to 15 (mean: 7.2), and the observed heterozygosity (Ho) ranged from 0.03-0.21, while the expected heterozygosity (He) ranged from 0.23-0.47. The genetic distances among populations ranged from 0.03-0.45. The FST multilocus value (0.23) indicated that the populations sampled were structured, and the clustering analysis showed the formation of two subgroups and two more distant populations. The first group contained the subspecies M. quadrifasciata quadrifasciata, and the other, the subspecies M. quadrifasciata anthidioides and the two M. quadrifasciata populations with continuous metasomal stripes from northern Minas Gerais. These results confirmed that the yellow metasomal stripes alone are not a good means for correctly identifying the different subspecies of M. quadrifasciata.

Imidacloprid-induced impairment of mushroom bodies and behavior of the native stingless bee Melipona quadrifasciata anthidioides.

Declines in pollinator colonies represent a worldwide concern. The widespread use of agricultural pesticides is recognized as a potential cause of these declines. Previous studies have examined the effects of neonicotinoid insecticides such as imidacloprid on pollinator colonies, but these investigations have mainly focused on adult honey bees. Native stingless bees (Hymenoptera: Apidae: Meliponinae) are key pollinators in neotropical areas and are threatened with extinction due to deforestation and pesticide use. Few studies have directly investigated the effects of pesticides on these pollinators. Furthermore, the existing impact studies did not address the issue of larval ingestion of contaminated pollen and nectar, which could potentially have dire consequences for the colony. Here, we assessed the effects of imidacloprid ingestion by stingless bee larvae on their survival, development, neuromorphology and adult walking behavior. Increasing doses of imidacloprid were added to the diet provided to individual worker larvae of the stingless bee Melipona quadrifasciata anthidioides throughout their development. Survival rates above 50% were only observed at insecticide doses lower than 0.0056 µg active ingredient (a.i.)/bee. No sublethal effect on body mass or developmental time was observed in the surviving insects, but the pesticide treatment negatively affected the development of mushroom bodies in the brain and impaired the walking behavior of newly emerged adult workers. Therefore, stingless bee larvae are particularly susceptible to imidacloprid, as it caused both high mortality and sublethal effects that impaired brain development and compromised mobility at the young adult stage. These findings demonstrate the lethal effects of imidacloprid on native stingless bees and provide evidence of novel serious sublethal effects that may compromise colony survival. The ecological and economic importance of neotropical stingless bees as pollinators, their susceptibility to insecticides and the vulnerability of their larvae to insecticide exposure emphasize the importance of studying these species.

The use of polliniferous resources by Melipona capixaba, an endangered stingless bee species.

Abstract Pollen types present in samples from corbiculae of Melipona capixaba (Moure and Camargo) (Hymenoptera: Apidae: Meliponina) worker bees were analyzed, as well as pollen samples from food pots inside the hives in three sites located at the bees' original habitat. The aim was to find out the sources used as a trophic resource by this species. The dominant pollen grains in the spectrum of the samples belonged to the families Myrtaceae and Melastomataceae. Eucalyptus was the most frequent pollen type in the corbiculae in Conceição do Castelo municipality; Eucalyptus, Myrcia, and Melastomatacea/Combretaceae in the Fazenda do Estado district; and Eucalyptus and Myrcia in the São Paulo de Aracê district, both in the Domingos Martins municipality. Eucalyptus and Melastomataceae/Combretaceae were the predominant pollen types in the food pots. Eucalyptus was the most prevalent type all year round or most of the year. The most common pollen types in the months that Eucalyptus was not present or dominant in the samples were of remaining native forest species, "ruderal" (field) plants, fruit-bearing plants, and introduced ornamental plants.

Prediction of social structure and genetic relatedness in colonies of the facultative polygynous stingless bee Melipona bicolor (Hymenoptera, Apidae).

Stingless bee colonies typically consist of one single-mated mother queen and her worker offspring. The stingless bee Melipona bicolor (Hymenoptera: Apidae) shows facultative polygyny, which makes this species particularly suitable for testing theoretical expectations concerning social behavior. In this study, we investigated the social structure and genetic relatedness among workers from eight natural and six manipulated colonies of M. bicolor over a period of one year. The populations of M. bicolor contained monogynous and polygynous colonies. The estimated genetic relatedness among workers from monogynous and polygynous colonies was 0.75 ± 0.12 and 0.53 ± 0.16 (mean ± SEM), respectively. Although the parental genotypes had significant effects on genetic relatedness in monogynous and polygynous colonies, polygyny markedly decreased the relatedness among nestmate workers. Our findings also demonstrate that polygyny in M. bicolor may arise from the adoption of related or unrelated queens.

Prediction of social structure and genetic relatedness in colonies of the facultative polygynous stingless bee Melipona bicolor (Hymenoptera, Apidae)

Stingless bee colonies typically consist of one single-mated mother queen and her worker offspring. The stingless bee Melipona bicolor (Hymenoptera: Apidae) shows facultative polygyny, which makes this species particularly suitable for testing theoretical expectations concerning social behavior. In this study, we investigated the social structure and genetic relatedness among workers from eight natural and six manipulated colonies of M. bicolor over a period of one year. The populations of M. bicolor contained monogynous and polygynous colonies. The estimated genetic relatedness among workers from monogynous and polygynous colonies was 0.75 ± 0.12 and 0.53 ± 0.16 (mean ± SEM), respectively. Although the parental genotypes had significant effects on genetic relatedness in monogynous and polygynous colonies, polygyny markedly decreased the relatedness among nestmate workers. Our findings also demonstrate that polygyny in M. bicolor may arise from the adoption of related or unrelated queens.

Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae) by fluorochromes.

Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae) were cytogenetically analyzed using conventional staining and the fluorochromes CMA(3) e DAPI. The females have 2n = 34 chromosomes (2K = 32 M¯+2 A¯). Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA (3) (+) bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.

Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae).

Due to their high degree of polymorphism, microsatellites are considered useful tools for studying population genetics. Nevertheless, studies of genetic diversity in stingless bees by means of these primers have revealed a low level of polymorphism, possibly the consequence of the heterologous primers used, since in most cases these were not specifically designed for the species under consideration. Herein we compared the number of polymorphic loci and alleles per locus, as well as observed heterozygosity in Melipona rufiventris and M. mondury populations, using specific and heterologous primers. The use of specific primers placed in evidence the greater frequency of polymorphic loci and alleles per locus, besides an expressive increase in observed heterozygosity in M. rufiventris and M. mondury, thereby reinforcing the idea that populational studies should be undertaken by preferably using species-specific microsatellite primers.

Genetic variability in five populations of Partamona helleri (Hymenoptera, Apidae) from Minas Gerais State, Brazil.

Partamona is a Neotropical genus of stingless bees that comprises 33 species distributed from Mexico to southern Brazil. These bees are well-adapted to anthropic environments and build their nests in several substrates. In this study, 66 colonies of Partamona helleri from five localities in the Brazilian state of Minas Gerais (São Miguel do Anta, Teixeiras, Porto Firme, Viçosa and Rio Vermelho) were analyzed using nine microsatellite loci in order to assess their genetic variability. Low levels of observed (H(o) = 0.099-0.137) and expected (H (e) = 0.128-0.145) heterozygosity were encountered and revealed discrete genetic differentiation among the populations (F (ST) = 0.025). AMOVA further showed that most of the total genetic variation (94.24%) in P. helleri was explained by the variability within local populations.

Genetic variability in five populations of Partamona helleri (Hymenoptera, Apidae) from Minas Gerais State, Brazil

Partamona is a Neotropical genus of stingless bees that comprises 33 species distributed from Mexico to southern Brazil. These bees are well-adapted to anthropic environments and build their nests in several substrates. In this study, 66 colonies of Partamona helleri from five localities in the Brazilian state of Minas Gerais (São Miguel do Anta, Teixeiras, Porto Firme, Viçosa and Rio Vermelho) were analyzed using nine microsatellite loci in order to assess their genetic variability. Low levels of observed (Ho = 0.099-0.137) and expected (He = 0.128-0.145) heterozygosity were encountered and revealed discrete genetic differentiation among the populations (F ST =0.025). AMOVA further showed that most of the total genetic variation (94.24 percent) in P. helleri was explained by the variability within local populations.

Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae)

Due to their high degree of polymorphism, microsatellites are considered useful tools for studying population genetics. Nevertheless, studies of genetic diversity in stingless bees by means of these primers have revealed a low level of polymorphism, possibly the consequence of the heterologous primers used, since in most cases these were not specifically designed for the species under consideration. Herein we compared the number of polymorphic loci and alleles per locus, as well as observed heterozygosity in Melipona rufiventris and M. mondury populations, using specific and heterologous primers. The use of specific primers placed in evidence the greater frequency of polymorphic loci and alleles per locus, besides an expressive increase in observed heterozygosity in M. rufiventris and M. mondury, thereby reinforcing the idea that populational studies should be undertaken by preferably using species-specific microsatellite primers.

Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae) by fluorochromes

Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae) were cytogenetically analyzed using conventional staining and the fluorochromes CMA3 e DAPI. The females have 2n = 34 chromosomes (2K=32+2). Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA3+ bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.

Microsatellite loci for the stingless bee Melipona rufiventris (Hymenoptera: Apidae).

Eight microsatellite primers were developed from ISSR (intersimple sequence repeats) markers for the stingless bee Melipona rufiventris. These primers were tested in 20 M. rufiventris workers, representing a single population from Minas Gerais state. The number of alleles per locus ranged from 2 to 5 (mean = 2.63) and the observed and expected heterozygosity values ranged from 0.00 to 0.44 (mean = 0.20) and from 0.05 to 0.68 (mean = 0.31), respectively. Several loci were also polymorphic in M. quadrifasciata, M. bicolor, M. mandacaia and Partamona helleri and should prove useful in population studies of other stingless bees.