Stingless Bee Melipona Scutellaris Research Articles

Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and fat body morphology and physiology

The stingless bee Melipona scutellaris performs buzz pollination, effectively pollinating several wild plants and crops with economic relevance. However, most research has focused on honeybees, leaving a significant gap in studies concerning native species, particularly regarding the impacts of pesticide combinations on these pollinators. Thus, this study aimed to evaluate the sublethal effects of imidacloprid (IMD), pyraclostrobin (PYR), and glyphosate (GLY) on the behavior and fat body cell morphology and physiology of M. scutellaris. Foragers were orally exposed to the different pesticides alone and in combination for 48 h. Bees fed with contaminated solution walked less, moved slower, presented morphological changes in the fat body, including vacuolization, altered cell shape and nuclei morphology, and exhibited a higher count of altered oenocytes and trophocytes. In all exposed groups, alone and in combination, the number of cells expressing caspase-3 increased, but the TLR4 number of cells expressing decreased compared to the control groups. The intensity of HSP70 immunolabeling increased compared to the control groups. However, the intensity of the immunolabeling of HSP90 decreased in the IMD, GLY, and I + G (IMD + GLY) groups but increased in I + P-exposed bees (IMD + PYR). Alternatively, exposure to PYR and P + G (PYR + GLY) did not affect the immunolabeling intensity. Our findings demonstrate the hazardous effects and environmental consequences of isolated and combined pesticides on a vital neotropical pollinator. Understanding how pesticides impact the fat body can provide crucial insights into the overall health and survival of native bee populations, which can help develop more environmentally friendly approaches to agricultural practices.

Ultrastructural Changes in the Midgut of Brazilian Native Stingless Bee Melipona scutellaris Exposed to Fungicide Pyraclostrobin.

Melipona scutellaris is a Brazilian stingless bee that is important for pollinating wild flora and agriculture crops. Fungicides have been widely used in agriculture, and floral residues can affect forager bees. The goal of our study was to evaluate the effects of sublethal concentrations of pyraclostrobin on the midgut ultrastructure of M. scutellaris forager workers. The bees were collected from three non-parental colonies and kept under laboratory conditions. The bees were orally exposed continuously for five days to pyraclostrobin in syrup at concentrations of 0.125 ng a.i./µL (FG1) and 0.005 ng a.i./µL (FG2). The control bees (CTL) were fed a no-fungicide sucrose solution, and the acetone solvent control bees (CAC) received a sucrose solution containing acetone. At the end of the exposure, the midguts were sampled, fixed in Karnovsky solution, and routinely processed for transmission electron microscopy. Ultrastructural analysis demonstrated that both the fungicide concentrations altered the midgut, such as cytoplasmic vacuolization (more intense in FG1), the presence of an atypical nuclear morphology, and slightly dilated mitochondrial cristae in the bees from the FG1 and FG2 groups (both more intense in FG1). Additionally, there was an alteration in the ultrastructure of the spherocrystals (FG1), which could be the result of cellular metabolism impairment and the excretion of toxic metabolites in the digestive cells as a response to fungicide exposure. The results indicate that ingested pyraclostrobin induced cytotoxic effects in the midgut of native stingless bees. These cellular ultrastructural responses of the midgut are a prelude to a reduced survival rate, as observed in previous studies.

Chemical Profile of Elements in the Stingless Bee Melipona scutellaris (Hymenoptera: Apidae: Meliponini) from Sites with Distinct Anthropogenic Activities.

Stingless bees are pollinators in forests and crops that, during foraging, may be exposed to several environmental xenobiotics, including metallic elements. This study evaluated the presence of magnesium (Mg), aluminum (Al), calcium (Ca), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), molybdenum (Mo), cadmium (Cd), barium (Ba), and lead (Pb) over the body surface of the stingless bee Melipona scutellaris visiting areas with different anthropogenic activity levels. The analyses were carried out using scanning electron microscopy with X-ray energy-dispersive spectroscopy (SEM/XEDS) and all tested elements were identified. Lead was the most abundant element in all samples, whereas Mn had the lowest abundance. High amounts of Cu and Zn were detected in the areas with metallurgic industries. The presence of metals on the body surface of this stingless bee varied according to the level of human activities in the studied areas.

Modified QuEChERS Method for Extracting Thiamethoxam and Imidacloprid from Stingless Bees: Development, Application, and Green Metrics.

In the present study, a method for the determination of residues of the neonicotinoid insecticides thiamethoxam and imidacloprid in the stingless bee Melipona scutellaris Latreille (1811) was optimized through a factorial design, tested using green metrics, and then applied to exposed bees. It combines the extraction with a modified quick, easy, cheap, effective, rugged, and safe method and the determination by liquid chromatography-tandem mass spectrometry analysis. Different parameters such as the mass of the sample, dispersive sorbents, and elution solvents were assessed. Method validation parameters were checked and include sensitivity, specificity, and linearity. The limit of quantification of 0.0025 μg g-1 was obtained for both insecticides, where accuracy was 94%-100% with satisfactory intraday and interday precisions (relative standard deviation <10%). The qualified method was applied to orally and topically exposed bee samples, and the results indicated that it is suitable for the determination and quantification of neonicotinoid pesticide residues in this species. Moreover, green analytical metrics like the National Environmental Methods Index, Eco Scale score, high-performance liquid chromatography with an environmental assessment tool (HPLC-EAT), waste generation, and amount of sample were compared with methods described in the literature involving neonicotinoid analysis in honeybees. As a result, the present study displayed the highest Eco Scale score and HPLC-EAT score and the second smallest amount of sample and waste generated. Thus, the method meets green analytical metrics more than other methods. In this sense, besides the application, the multicriteria decision analysis tool employed suggests that this is a good option as a green analytical method. Environ Toxicol Chem 2022;41:2365-2374. © 2022 SETAC.

Transcriptomic analysis of Malpighian tubules from the stingless bee Melipona scutellaris reveals thiamethoxam-induced damages

The concern about pesticide exposure to neotropical bees has been increasing in the last few years, and knowledge gaps have been identified. Although stingless bees, (e.g.: Melipona scutellaris), are more diverse than honeybees and they stand out in the pollination of several valuable economical crops, toxicity assessments with stingless bees are still scarce. Nowadays new approaches in ecotoxicological studies, such as omic analysis, were pointed out as a strategy to reveal mechanisms of how bees deal with these stressors. To date, no molecular techniques have been applied for the evaluation of target and/or non-target organs in stingless bees, such as the Malpighian tubules (Mt). Therefore, in the present study, we evaluated the differentially expressed genes (DEGs) in the Mt of M. scutellaris after one and eight days of exposure to LC50/100 (0.000543 ng a.i./μL) of thiamethoxam (TMX). Through functional annotation analysis of four transcriptome libraries, the time course line approach revealed 237 DEGs (nine clusters) associated with carbon/energy metabolism and cellular processes (lysosomes, autophagy, and glycan degradation). The expression profiles of Mt were altered by TMX in processes, such as detoxification, excretion, tissue regeneration, oxidative stress, apoptosis, and DNA repair. Transcriptome analysis showed that cell metabolism in Mt was mainly affected after 8 days of exposure. Nine genes were selected from different clusters and validated by RT-qPCR. According to our findings, TMX promotes several types of damage in Mt cells at the molecular level. Therefore, interference of different cellular processes directly affects the health of M. scutellaris by compromising the function of Mt.

A food-ingested sublethal concentration of thiamethoxam has harmful effects on the stingless bee Melipona scutellaris

In recent years, the importance of bee's biodiversity in the Neotropical region has been evidencing the relevance of including native bees in risk assessments. Therefore, the sublethal effects of the insecticide thiamethoxam on the survival and morphological parameters of the stingless bee Melipona scutellaris were investigated in the present study. Cells from both non-target organs (Malpighian tubules and midgut) and target organs (brain) were analyzed for morphological alterations using light microscopy and transmission electron microscopy. The findings showed that when M. scutellaris foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100 = 0.000543 ng a. i./μL), longevity was not reduced but brain function was affected, even with the non-target organs attempting to detoxify. The cellular damage in all the organs was mostly reflected in irregular nuclei shape and condensed chromatin, indicating cell death. The most frequent impairments in the Malpighian tubules were loss of microvilli, disorganization of the basal labyrinth, and cytoplasmic loss. These characteristics are related to an attempt by the cells to increase the excretion process, probably because of the high number of toxic molecules that reach the Malpighian tubules and need to be secreted. In general, damages that compromise the absorption of nutrients, excretion, memory, and learning processes, which are essential for the survival of M. scutellaris, were found. The present results also fill in gaps on how these bees respond to thiamethoxam exposure and will be useful in future risk assessments for the conservation of bee biodiversity.

Efficiency of two digestion methods in determining the presence of metals (Cd, Cu, Cr, Pb and Zn) in geopropolis produced by Melipona scutellaris

This study evaluates the efficiency of acid extraction and total digestion to determine the presence of metals in geopropolis produced by the stingless bee Melipona scutellaris. Geopropolis samples were collected at five meliponaries in the city and in the metropolitan region of Salvador, Bahia State, Brazil. The sample treatment methods comprised acid extraction and total digestion. The Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) technique was used to quantify Cd, Cu, Cr, Pb, and Zn. Geopropolis samples submitted to both digestion methods showed statistical differences. For both methods, Cr and Zn showed the highest concentrations, while those of Cd were the lowest. The Cr concentration for determination by total digestion was 37.53 mg/kg, while for acid extraction it was 32.90 mg/kg. For Zn, the concentration was 17.65 mg/kg and 8.85 mg/kg for total digestion and acid extraction, respectively. The total digestion method showed the highest concentrations of the metals evaluated; however, acid extraction (USEPA 3050b) is a more straightforward procedure for metal evaluation in geopropolis samples and presented values that support the use of geopropolis as a bioindicator. The acid extraction method USEPA 3050b, in combination with detection using ICP OES, showed efficiency in analyses carried out to determine metals in geopropolis.

Acute toxicity of the insecticide abamectin and the fungicide difenoconazole (individually and in mixture) to the tropical stingless bee Melipona scutellaris.

Stingless bees have been recognized as essential plant pollinators and producers of various natural products in neotropical areas. Research into the potential risks of pesticides they may be exposed to in agricultural fields, however, remains meagre. Especially the toxicity of pesticide mixtures likely to occur under real-world conditions and that are likely to exert synergetic effects has been poorly studied. The aim of the present study was therefore to evaluate the single and mixture acute contact and oral toxicity of commercial products containing the insecticide abamectin and the fungicide difenoconazole in laboratory bioassays with the Brazilian native stingless bee Melipona scutellaris. In addition, a comparison of the insecticide sensitivity of stingless bees relative to the honeybee Apis mellifera was made based on previously published toxicity data. Except for oral exposure to abamectin, M. scutellaris appeared to be more sensitive that A. mellifera in the single compound toxicity tests. A difenoconazole concentration at the NOEC (no observed effect concentration) level indicated a synergetic toxic interaction with abamectin. A sensitivity comparison based on published toxicity data for A. mellifera and stingless bees indicated several insecticidal modes of action having a high relative sensitivity to stingless bees that need especial consideration in future studies. The research findings highlight the need for testing native bee species and environmentally relevant pesticide mixtures in risk assessments to avoid underestimation of potential risks to bee populations and the subsequent loss of pollination ecosystem services.

Physical characterization of geopropolis produced by Melipona scutellaris (Hymenoptera: Apidae)

Geopropolis has medicinal potential and has been studied due to its complex chemical composition, with more than 300 compounds already identified. However, its physical components are still little known, mainly the soil fraction. In this study, we characterized components of geopropolis produced by stingless bee Melipona scutellaris to know its matrix considering, both the granulometric characteristics and the organic matter (OM) content in the soil. We collected geopropolis and soil samples around bee colonies in an urban-industrial area in the city and in the metropolitan region of Salvador, Bahia State, Brazil. The geopropolis composition in the different study sites is only 9–9.3% of OM and the main matrix was composed of soil mineral material or sediments from the surrounding areas, contributing with approximately 91% on average. The mineral fraction of geopropolis shows a predominance of sandy material, common in soils of the region. Soil contribution to this stingless bee product highlights the importance of this study, because it is the first to characterize geopropolis matrix through the analysis of OM content and granulometry of the soil.

Fungicide pyraclostrobin affects midgut morphophysiology and reduces survival of Brazilian native stingless bee Melipona scutellaris

Native stingless bees are key pollinators of native flora and important for many crops. However, the loss of natural fragments and exposure to pesticides can hinder the development of colonies and represent a high risk for them. Nevertheless, most studies are conducted with honeybees and there are not many studies on native species, especially in relation to the effects of fungicides on them. Therefore, the objective of this paper is to evaluate the effects of sublethal concentrations of pyraclostrobin, on Melipona scutellaris forager workers. These Brazilian native stingless bees were submitted to continuous oral exposure to three concentrations of pyraclostrobin in sirup: 0.125 ng a.i./µL (P1), 0.025 ng a.i./µL (P2), and 0.005 ng a.i./µL (P3). Histopathological and histochemical parameters of midgut, as well as survival rate were evaluated. All concentrations of fungicide showed an increase in the midgut lesion index and morphological signs of cell death, such as cytoplasmic vacuolizations, presence of atypical nuclei or pyknotic nuclei. Histochemical analyzes revealed a decreased marking of polysaccharides and neutral glycoconjugates both in the villi and in peritrophic membrane in all exposed-groups in relation to control-groups. P1 and P2 groups presented a reduction in total protein marking in digestive cells in relation to control groups. As a consequence of alteration in the midgut, all groups exposed to fungicide showed a reduced survival rate. These findings demonstrate that sublethal concentrations of pyraclostrobin can lead to significant adverse effects in stingless bees. These effects on social native bees indicate the need for reassessment of the safety of fungicides to bees.

Determination and uptake of abamectin and difenoconazole in the stingless bee Melipona scutellaris Latreille, 1811 via oral and topic acute exposure

Bees are considered as important providers of ecosystem services, acting via pollination process in crops and native plants, and contributing significantly to the maintenance of biodiversity. However, the decrease of bee's population has been observed worldwide and besides other factors, this collapse is also related to the extensive use of pesticides. In this sense, studies involving the assessment of adverse effects and the uptake of pesticides by bees are of great concern. This work presents an analytical method for the determination of the insecticide abamectin and the fungicide difenoconazole in the stingless bee Melipona scutellaris exposed via oral and topic to endpoints concentrations of active ingredients (a.i.) alone and in commercial formulations and the discussion about its mortality and uptake. For this purpose, QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) acetate modified method was used for extraction and pesticides were determined by LC-MS/MS. The validation parameters have included: a linear range between 0.01 and 1.00 μg mL−1; and LOD and LOQ of 0.038 and 0.076 μg g−1 for abamectin and difenoconazole, respectively. The uptake of tested pesticides via oral and topic was verified by the accumulation in adult forager bees, mainly when the commercial product was tested. Mortality was observed to be higher in oral exposure than in topic tests for both pesticides. For abamectin in a commercial formulation (a.i.) no differences were observed for oral or topic exposure. On the other hand, for difenoconazole, topic exposure had demonstrated higher accumulation in bees, according to the increase of received dose. Through the results, uptake and the possible consequences of bioaccumulated pesticides are also discussed and can contribute to the knowledge about the risks involving the exposure of bees to these compounds.

Meliponamycins: Antimicrobials from Stingless Bee-Associated Streptomyces sp.

Social insects establish complex interactions with microorganisms, some of which play defensive roles in colony protection. The important role of pollinators such as the stingless bee Melipona scutellaris in nature encouraged us to pursue efforts to study its associated microbiota. Here we describe the discovery of two novel cyclic hexadepsipeptides, meliponamycin A (1) and meliponamycin B (2), from Streptomyces sp. ICBG1318 isolated from M. scutellaris nurse bees. Their structures were established by interpretation of NMR and MS data, and the absolute configuration of the constituent amino acids was determined by the advanced Marfey's method. Compounds 1 and 2 showed strong activity against the entomopathogen Paenibacillus larvae and human pathogens Staphylococcus aureus and Leishmania infantum.

Scientific Note: Warming Nurses, a New Worker Role Recorded for the First Time in Stingless Bees.

Nest temperature is a crucial variable that determines colony survival in social insects. The successful production and development of a new brood, therefore, depends on stable thermal conditions and limited temperature oscillations. Thermoregulatory processes are useful in controlling both individual activity and for the maintenance of colony temperature. We measured heat production generated by nurse bees working on brood combs of the stingless bee Melipona scutellaris (Hymenoptera, Apidae, Meliponini) in this study; our results enabled us to identify the existence of a new task performed by nurse bees, referred to here as 'hot bees' because of their higher thorax surface temperature (4°C above that of other bees within the brood area). This additional heat has been little studied in stingless bees but is likely the result of thorax muscle contractions or, indeed, the development of this musculature as these are recently emerged individuals. We hypothesize that these 'hot bees' contribute to the maintenance of warmth within the nest brood area.

Paenibacillus polymyxa Associated with the Stingless Bee Melipona scutellaris Produces Antimicrobial Compounds against Entomopathogens.

Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(-)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

Epigenetic modifications and their relation to caste and sex determination and adult division of labor in the stingless bee Melipona scutellaris.

Stingless bees of the genus Melipona, have long been considered an enigmatic case among social insects for their mode of caste determination, where in addition to larval food type and quantity, the genotype also has a saying, as proposed over 50 years ago by Warwick E. Kerr. Several attempts have since tried to test his Mendelian two-loci/two-alleles segregation hypothesis, but only recently a single gene crucial for sex determination in bees was evidenced to be sex-specifically spliced and also caste-specifically expressed in a Melipona species. Since alternative splicing is frequently associated with epigenetic marks, and the epigenetic status plays a major role in setting the caste phenotype in the honey bee, we investigated here epigenetic chromatin modification in the stingless bee Melipona scutellaris. We used an ELISA-based methodology to quantify global methylation status and western blot assays to reveal histone modifications. The results evidenced DNA methylation/demethylation events in larvae and pupae, and significant differences in histone methylation and phosphorylation between newly emerged adult queens and workers. The epigenetic dynamics seen in this stingless bee species represent a new facet in the caste determination process in Melipona bees and suggest a possible mechanism that is likely to link a genotype component to the larval diet and adult social behavior of these bees.

The relationship between queen execution and cuticular hydrocarbons in stingless bee Melipona scutellaris (Hymenoptera: Meliponini)

In queenright colonies of stingless bees of the genus Melipona, workers recognize, attack, and kill young virgin queens. For Melipona scutellaris, we observed that virgin queens were executed when they were between 5 and 9 days old, while newly emerged queens were not attacked. The faster movements of old virgin in relation to newly emerged might be responsible for attacks. It has been also hypothesized that cuticular hydrocarbons are the source of the signal used by workers to recognize virgin queens. We investigated whether newly emerged, 8 days old virgin and physogastric queens of M. scutellaris have different cuticular hydrocarbon profiles. Cuticular hydrocarbons of three ages were compared using gas chromatography-mass spectrometry. The cuticular hydrocarbon profiles varied by reproductive status and age. Changes in the cuticular hydrocarbons in virgin queens during aging suggest that these compounds, together with change in movement, may play a role in the recognition of virgin queens by workers prior to regicide.

Foraging behavior: the food choice and quality of honey from Melipona scutellaris latreille (hymenoptera: Apidae)

Data on the preferred foraging times and identification of pollen collected by stingless bees has led to further insights into the bees’ behavioral and ecological habits, thus enabling the analysis of the honey produced which in turn will facilitate quality control of this product. Present study reveals the foraging times and food preferences of the stingless bee Melipona scutellaris (hymenoptera: Apidae) together with a physicochemical evaluation of the honey produced by this bee. M. scutellaris tended to be more active in the morning during the winter, visited a wide variety of plants in their search for food, and produced a good quality honey. In winter the bees showed generalist behavior foraging many different plants.

Sexual dimorphism and phenotypic plasticity in the antennal lobe of a stingless bee, Melipona scutellaris.

Among social insects, the stingless bees (Apidae, Meliponini), a mainly tropical group of highly eusocial bees, present an intriguing variety of well-described olfactory-dependent behaviors showing both caste- and sex-specific adaptations. By contrast, little is known about the neural structures underlying such behavioral richness or the olfactory detection and processing abilities of this insect group. This study therefore aimed to provide the first detailed description and comparison of the brains and primary olfactory centers, the antennal lobes, of the different members of a colony of the stingless bee Melipona scutellaris. Global neutral red staining, confocal laser scanning microscopy, and 3D reconstructions were used to compare the brain structures of males, workers, and virgin queens with a special emphasis on the antennal lobe. We found significant differences between both sexes and castes with regard to the relative volumes of olfactory and visual neuropils in the brain and also in the number and volume of the olfactory glomeruli. In addition, we identified one (workers, queens) and three or four (males) macroglomeruli in the antennal lobe. In both sexes and all castes, the largest glomerulus (G1) was located at a similar position relative to four identified landmark glomeruli, close to the entrance of the antennal nerve. This similarity in position suggests that G1s of workers, virgin queens, and males of M. scutellaris may correspond to the same glomerular entity, possibly tuned to queen-emitted volatiles since all colony members need this information.

Major chemical contituents of geopropolis from Melipona scutellaris

Geopropolis is a different kind of propolis collected by the stingless bee Melipona scutellaris that is made up of plant resins, wax and soil. Its crude extract was cytotoxic to colon cancer cells, however its chemical profile has not been thoroughly studied so far. We used bioguided fractionation against two colon cancer cell lines to purify the major bioactive compounds. We isolated and identified prenylated coumarins, benzophenones and cinnamic acid derivatives from geopropolis. Most of those compounds are found in plants from the genus Kielmeyera (Guttiferae), which is widely distributed in the Northeast Brazil, the area of collection of geopropolis.

Sneaky queens in Melipona bees selectively detect and infiltrate queenless colonies

Insect societies are characterized by advanced cooperation, but at the same time the complexity of their colonies renders them susceptible to reproductive parasitism. Recently, a genetic study on the Brazilian stingless bee Melipona scutellaris showed that unrelated queens frequently invade and take over colonies in which the mother queen had died. In the present study, we investigated this phenomenon using radio frequency identification (RFID) tags. We confirmed that alien queen take-overs are common within this species, and demonstrated that mated queens actively seek out colonies without a queen to reproduce in. Furthermore, we found that queens only penetrate their target colonies in the evening, when guarding efficiency is significantly reduced. We hypothesize that this strategy reduces the chance of the queens being attacked by entrance guards, thus maximizing their chance of successful infiltration.