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Stingless bees are a diverse group with a relevant role in pollinating native species. Its diet is rich in carbohydrates and proteins, by collecting pollen and nectar supplies the development of its offspring. Fermentation of these products is associated with microorganisms in the colony. However, the composition of microorganisms that comprise this microbiome and its fundamental role in colony development is still unclear. To characterize the colonizing microorganisms of larval food in the brood cells of stingless bees Frieseomelitta varia , Melipona quadrifasciata , Melipona scutellaris , and Tetragonisca angustula , we have utilized molecular and culture-based techniques. Bacteria of the phyla Firmicutes, Proteobacteria, Actinobacteria, and fungi of the phyla Ascomycota, Basidiomycota, Mucoromycota, and Mortierellomycota were found. Diversity analysis showed that F. varia had a greater diversity of bacteria in its microbiota, and T. angustula had a greater diversity of fungi. The isolation technique allowed the identification of 189 bacteria and 75 fungi. In summary, this research showed bacteria and fungi associated with the species F. varia , M. quadrifasciata , M. scutellaris , and T. angustula , which may play an essential role in the survival of these organisms. Besides that, a biobank with bacteria and fungus isolates from LF of Brazilian stingless bees was created, which can be used for different studies and the prospection of biotechnology compounds.

The stingless bees of tribe Meliponini are efficient pollinators playing a key role in ecosystem services. Frieseomelitta Friese (Hymenoptera: Apidae: Meliponini) includes 16 Neotropical species, of which 6 are found in the state of Bahia, northeastern Brazil. In order to provide a refined cytotaxonomic analysis, we characterized the heterochromatin composition and variation among 6 Frieseomelitta species. All species shared a diploid number (2n) of 30 chromosomes. Frieseomelitta dispar Moure, Frieseomelitta francoi Moure, and Frieseomelitta meadewaldoi Cockerell (Hymenoptera: Apidae), presented GC-rich heterochromatic regions while Frieseomelitta sp.n., Frieseomelitta varia Lepelitier, and Frieseomelitta doederleini Friese (Hymenoptera: Apidae) were characterized by homogenous heterochromatin, without evidence of AT or GC-rich sites. The number and location of microsatellite repeats mapped by fluorescence in situ hybridization revealed interspecific variation. These data were useful to identify each species based on chromosomal markers, and represent important tools for clarifying the interspecific differentiation among Frieseomelitta species and for understanding the genome evolution in bees as a whole.

The stingless bee Frieseomelitta varia Lepeletier 1836 (Hymenoptera: Apidae) is an essential pollinator in natural and agricultural ecosystems in the Neotropical region. However, these bees may be exposed to pesticides during foraging, which can affect both individuals and their colonies. One example comes from the use of pyraclostrobin (a fungicide) and thiamethoxam (an insecticide) for pest control in pepper crops, which F. varia visits. This study aimed to evaluate the isolated and combined sublethal effects of thiamethoxam (TMX) (0.000543 ng a.i./µL) and pyraclostrobin (PYR) (1.5 ng i.a./µL) on the morphology of the midgut and Malpighian tubules of F. varia workers. Results showed that both pesticides, regardless of the exposure time (through feeding during 48 h or 96 h), disturbed the morphology of the analyzed organs. Specifically, F. varia exposed orally to sublethal concentrations of thiamethoxam and pyraclostrobin, either alone or in combination, exhibited a higher rate of damage to the midgut (e.g., vacuolization, apocrine secretion, and cellular elimination) compared to the bees in the control groups, both after 48 h and 96 h of exposure. In Malpighian tubules, vacuolation is the only damage present. As the observed morphological alterations likely compromise the excretion and absorption functions, exposure to pyraclostrobin and thiamethoxam may lead to disturbances at both the individual and colony levels. These results highlight the urgent need for a future reassessment of the safety of fungicides and insecticides regarding their potential effects on bee populations.

Microbes have been identified as fundamental for the good health of bees, acting as pathogens, protective agent against infection/inorganic toxic compounds, degradation of recalcitrant secondary plant metabolites, definition of social group membership, carbohydrate metabolism, honey and bee pollen production. However, study of microbiota associated with bees have been largely confined to the honeybees and solitary bees. Here, I characterized the microbiota of indoor surface nest of four brazilian stingless bee species ( Apidae : Meliponini ) with different construction behaviors and populations. Bees that use predominantly plant material to build the nest ( Frieseomelitta varia and Tetragonisca angustula ) have a microbiome dominated by bacteria found in the phylloplane and flowers such as Pseudomonas sp. and Sphingomonas sp. Species that use mud and feces (Trigona spinipes) possess a microbiome dominated by coliforms such as Escherichia coli and Alcaligenes faecalis. Melipona quadrifasciata , which uses both mud / feces and plant resin, showed a hybrid microbiome with microbes found in soil, feces and plant material. These findings indicate that indoor surface microbiome varies widely among bees and reflects the materials used in the construction of the nests.

As pollinators, bees are key to maintaining the biodiversity of angiosperm plants, and for agriculture they provide a billion-dollar ecosystem service. But they also compete for resources (primarily nectar and pollen), especially the highly social bees that live in perennial colonies. So, how do they organize their daily temporal activities? Here, we present a versatile, low-cost device for the continuous, automatic recording and data analysis of the locomotor activity in the colony-entrance tube of highly eusocial bees. Consisting of an in-house built block containing an infrared detector, the passage of bees in the colony entrance tunnel is registered and automatically recorded in an Arduino environment, together with concomitant recordings of temperature and relative humidity. With a focus on the highly diverse Neotropical stingless bees (Meliponini), we obtained 10-day consecutive recordings for two colonies each of the species Melipona quadrifasciata and Frieseomelitta varia, and also for the honey bee. The Lomb-Scargle periodogram analysis identified a predominant circadian rhythmicity for all three species, but also indications of ultradian rhythms. For M. quadrifasciata, which is comparable in size to the honey bee, we found evidence for a possibly anticipatory activity already before sunrise. As all three species also presented activity at night in the colony entrance tube, this also raises questions about sleep organization in social insects. The cost and versatility of the device and the open-source options for data analysis make this an attractive system for conducting studies on circadian rhythms in social bees under natural conditions, complementing studies on flower visits by these important pollinators.

Background Meiotic recombination is a fundamental genetic process that shuffles allele combinations and promotes accurate segregation of chromosomes. Analyses of the ubiquitous variation of recombination rates within and across species suggest that recombination is evolving adaptively. All studied insects with advanced eusociality have shown exceptionally high recombination rates, which may represent a prominent case of adaptive evolution of recombination. However, our understanding of the relationship between social evolution and recombination rates is incomplete, partly due to lacking empirical data. Here, we present a linkage map of the monandrous, advanced eusocial Brazilian stingless bee, Frieseomelitta varia , providing the first recombination analysis in the diverse Meliponini (Hymenoptera, Apidae). Results Our linkage map includes 1417 markers in 19 linkage groups. This map spans approximately 2580 centimorgans, and comparisons to the physical genome assembly indicate that it covers more than 75 % of the 275 Megabasepairs (Mbp) F. varia genome. Thus, our study results in a genome-wide recombination rate estimate of 9.3–12.5 centimorgan per Mbp. This value is higher than estimates from nonsocial insects and comparable to other highly social species, although it does not support our prediction that monandry and strong queen-worker caste divergence of F. varia lead to even higher recombination rates than other advanced eusocial species. Conclusions Our study expands the association between elevated recombination and sociality in the order Hymenoptera and strengthens the support for the hypothesis that advanced social evolution in hymenopteran insects invariably selects for high genomic recombination rates.

Background Most of our understanding on the social behavior and genomics of bees and other social insects is centered on the Western honey bee, Apis mellifera. The genus Apis, however, is a highly derived branch comprising less than a dozen species, four of which genomically characterized. In contrast, for the equally highly eusocial, yet taxonomically and biologically more diverse Meliponini, a full genome sequence was so far available for a single Melipona species only. We present here the genome sequence of Frieseomelitta varia , a stingless bee that has, as a peculiarity, a completely sterile worker caste. Results The assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275 Mb composed of 2173 scaffolds. A BUSCO analysis for the 10,526 predicted genes showed that these represent 96.6% of the expected hymenopteran orthologs. We also predicted 169,371 repetitive genomic components, 2083 putative transposable elements, and 1946 genes for non-coding RNAs, largely long non-coding RNAs. The mitochondrial genome comprises 15,144 bp, encoding 13 proteins, 22 tRNAs and 2 rRNAs. We observed considerable rearrangement in the mitochondrial gene order compared to other bees. For an in-depth analysis of genes related to social biology, we manually checked the annotations for 533 automatically predicted gene models, including 127 genes related to reproductive processes, 104 to development, and 174 immunity-related genes. We also performed specific searches for genes containing transcription factor domains and genes related to neurogenesis and chemosensory communication. Conclusions The total genome size for F. varia is similar to the sequenced genomes of other bees. Using specific prediction methods, we identified a large number of repetitive genome components and long non-coding RNAs, which could provide the molecular basis for gene regulatory plasticity, including worker reproduction. The remarkable reshuffling in gene order in the mitochondrial genome suggests that stingless bees may be a hotspot for mtDNA evolution. Hence, while being just the second stingless bee genome sequenced, we expect that subsequent targeting of a selected set of species from this diverse clade of highly eusocial bees will reveal relevant evolutionary signals and trends related to eusociality in these important pollinators.

Background/Objectives: A striking feature of the karyotypes of stingless bees is the large amount of heterochromatin present in most species. Cytogenomic studies performed in some Meliponini species have suggested that evolutionary events related to the diversification and amplification of satellite DNA families in the heterochromatin may reflect the structuring of phylogenetic clades in this tribe. In this study, we performed a genomic analysis in Frieseomelitta varia to characterize different satDNA families in its genome. We also investigated the presence of the most abundant satDNA family of F. varia in its own chromosomes, in two other Frieseomelitta species, and in other Meliponini genera encompassing the three main clades of Neotropical Meliponini, according to the available molecular phylogeny. Methods: Genomic analyses were performed using RepeatExplorer2 on the Galaxy platform, and chromosomal investigations were conducted using fluorescent in situ hybridization. Results: Seven satDNA families were recovered, which together totaled an abundance of 11.223% of the analyzed F. varia genomic fraction. The most abundant satDNA family, FvarSat01-306, predominates in the analyzed repetitive fraction (representing around 89%) and was recently amplified and homogenized in almost all the heterochromatin of F. varia. In addition, the data revealed an unprecedented sharing of this satDNA family in the centromeric/pericentromeric heterochromatin among different Meliponini genera, with independent amplifications and loss of this sequence in some taxa. Conclusions: One family of satellite DNA makes up most of the heterochromatin in this species and is shared with other Meliponini.

In vitro rearing protocols already established for honeybees are currently being adapted to assess the risk of pesticides, and to conduct comparative developmental biology studies on stingless bees. However, differences in critical life-history traits (development time and the type of larval nutrition leading to caste differentiation process) among social bees require the development of an in vitro rearing protocol for each species and caste. We generated a protocol to produce workers of Frieseomelitta varia (Lepeletier, 1836), a non-endangered and highly eusocial pollinator species with wide geographical distribution. We tested the viability of using either the eggs or the first instar larvae as the starting point for in-vitro transfer. In vitro rearing was performed in acrylic plates at 30 °C and 99% relative humidity during the larval feeding phase. The humidity was subsequently reduced to 75% during the following days of development. The experimental larvae were offered either 25 µL or 27 µL of larval food. The development time, emergence and mortality rates, and morphological parameters of the emerged workers were assessed. In the process of validating the protocol, the adults that emerged after in vitro rearing were compared with colony-reared adults. In our results, 27 µL of larval food allowed 90% of workers to emerge. No significant differences were found between the emerging workers reared in vitro and those reared in the colony. The described protocol is a useful method for rearing F. varia workers in vitro, which can be used for diverse types of experimental approaches.

Chemical compounds on the cuticle are a rich source of information used during interactions among social insects. Despite the multitude of studies on these substances and their function in ants, wasps, and honeybees, little is known about this subject in stingless bees (Hymenoptera: Apidae, Meliponini). We studied the chemical composition of the cuticle of the stingless bee, Frieseomelitta varia, by gas chromatography-mass spectrometry (GC-MS), to investigate potential chemical variation among castes, gender, age, and reproductive status. We found differences in the cuticular hydrocarbon composition among workers, males, and queens, recording both qualitative and quantitative differences among individuals of different ages and gender. The cuticle of physogastric queens presented a chemical profile that was distinct from all other groups in the analysis, with high relative abundances of alkenes and alkadienes with 27, 29, and 31 carbon atoms. We discuss the possibility that these compounds signal a queen's presence to the colony, thereby initiating all vital worker-queen interactions.