Stingless Bee Melipona Quadrifasciata Research Articles

Neonicotinoid effects on tropical bees: Imidacloprid impairs innate appetitive responsiveness, learning and memory in the stingless bee Melipona quadrifasciata

Together with other anthropogenic factors, pesticides play a major role in pollinator decline worldwide. Most studies on their influence on pollinators have focused on honey bees given the suitability of this insect for controlled behavioral testing and raising. Yet, studies on pesticide impact should also contemplate tropical species, which contribute a major part of biodiversity and which have remained so far neglected. Here we focused on the stingless bee Melipona quadrifasciata and asked if the widely used neonicotinoid imidacloprid disrupts its learning and memory capabilities. We fed stingless bees with 0.1, 0.5 or 1 ng of imidacloprid, tested their innate appetitive responsiveness and trained them to associate odors and sucrose reward using the olfactory conditioning of the proboscis extension response. The same experiments were performed on Africanized honey bees. One hour after intoxication, both species decreased their innate responsiveness to sucrose but the effect was more accentuated in stingless bees. In both species, learning and memory were affected in a dose-dependent manner. These results indicate that pesticides have dramatic consequences on tropical bee species and claim for rational policies regulating their use in the tropics.

Geopropolis from stingless bee Melipona quadrifasciata quadrifasciata Lepeletier, 1836: chemical characterization and compounds of interest

Melipona quadrifasciata quadrifasciata Lepeletier, 1836 (Mandaçaia) is a species of stingless bee, native in Brazil, that produces geopropolis, a physical mixture of propolis and clay, instead of producing conventional propolis. The present work aimed to chemically and thermally characterize Mandaçaia’s geopropolis collected at Campo Alegre city, located in southern Brazil, and deepen the range of knowledge about its properties. The propolis extraction was carried out by hydroalcoholic maceration. Characterizations were performed using ultraviolet-visible spectrophotometry (UV-Vis), Fourier transformation infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), ultra-high performance liquid chromatography (UHPLC), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). UV-Vis results demonstrated a total polyphenol content of 10.15 ± 0.24 mg GAE.g−1 and total flavonoid content of 22.51 ± 3.47 mg QE.g−1. Chemical species from phenolic compounds such as terpenes, diterpenes, and fatty acids were identified by UHPLC. In conclusion, data showed that Mandaçaia’s geopropolis extract has a rich and diverse chemical composition which encourages new studies about its pharmaceutical, cosmetic, and food applications.

Synthesis of PMMA Nanoparticles Loaded with Geopropolis Extract from the Brazilian Stingless Bee Melipona quadrifasciata Lepeletier, 1836 by Miniemulsion Polymerization

AbstractThe present work aims to encapsulate the concentrated geopropolis extract from the Brazilian stingless bee Melipona quadrifasciata quadrifasciata Lepeletier, 1836 in poly(methyl methacrylate) (PMMA) nanoparticles produced by miniemulsion polymerization. The concentrated geopropolis extract is obtained by hydroalcoholic maceration and characterized by ultraviolet–visible spectrophotometry (UV–Vis) to quantify flavonoids and polyphenols in terms of quercetin (Q) and galic acid (GA), respectively. The PMMA‐Propolis nanoparticles is characterized by size exclusion chromatography (SEC), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), UV–Vis and nuclear magnetic resonance (NMR1H). UV–Vis results of the geopropolis extract revealed an average flavonoid content of 22.51 mg Q g–1 and an average polyphenol content of 10.15 mg GA g–1. TEM images showed that the produced PMMA nanoparticles are spherical, and their average diameter reduced from 72.62 to 44.60 nm by increasing the propolis concentration from 0% to 40%. Additionally, the kinetic data reveal a reduction of the polymerization reaction rate as well as the average molecular weight when raising the propolis extract concentration in the reaction media. These results suggest that propolis extract may be used as a biocompatible and biodegradable co‐stabilizer and chain transfer agent in miniemulsion polymerization reactions of vinyl monomers.

Glyphosate-Based Herbicide Causes Cellular Alterations to Gut Epithelium of the Neotropical Stingless Bee Melipona quadrifasciata quadrifasciata (Hymenoptera: Meliponini).

Glyphosate-based herbicides (GBH) are the best-selling pesticides in Brazil, with hundreds of thousands of tons sold per year. There is no study investigating morphological alterations caused by GBH on the epithelium of the gut in bees. Here, we aimed to demonstrate effects of chronic ingestion of GBH in the midgut digestive cells of the Brazilian stingless bee Melipona quadrifasciata quadrifasciata Lepeletier 1836. We kept forager workers of M. quadrifasciata in laboratory conditions and fed on food contaminated with three different concentrations of GBH for 10days, after which the midgut digestive cell structure and ultrastructure were analyzed. The presence of GBH in food did not affect food consumption, indicating that M. quadrifasciata bees do not reject food contaminated with GBH. As digestive cells of the midgut release apocrine secretion as a detoxication mechanism, we expected that the ingestion of food contaminated with GBH in the present study affect the height of midgut digestive cells. However, such reduction did not occur, probably because of the low-test concentrations. Although there were differences in digestive cell ultrastructure, ingestion of GBH impaired midgut digestive cell cohesion by disorganizing the smooth septate junctions between cells, which may probably be caused by the adjuvant "polyethoxylated tallow amine" present in the GBH. Previous studies demonstrated that GBH increase bees' sensibility to intestine infections, based on the present results we hypothesized that the loss of cell cohesion in the midgut epithelium favors pathogenic microbial infections and harms food absorption, increasing bees' mortality.

Pollen spectrum collected by Melipona quadrifasciata anthidioides Lepeletier, 1863 (Apidae: Meliponini) in an anthropized region of Caatinga

The stingless bee Melipona quadrifasciata anthidioides is a major pollinator of the Brazilian semi-arid region (Caatinga), it is widely used in meliponiculture due to the economic value of the honey. The goals of this study were to identify the floral resources exploited by these bees in a human-modified area from Caatinga in Bahia, and to provide a pollen calendar that might be helpful to beekeepers. Pollen loads were collected monthly from each colony over a 7-month period. Twenty pollen morphotypes were identified with high frequencies of pollen types belonging to Myrtaceae, Fabaceae-Caesalpinioideae and Solanaceae. The dominant pollens were represented by Mimosa 1 and Mimosa arenosa (Fabaceae-Caesalpinioideae). The accessory types were Mimosa tenuiflora, and Psidium guajava (Myrtaceae), while Solanum 1 and Solanum 2 represented major isolated pollen types. Some morphotypes, such as those from Mimosa are common in anthropized areas, thus playing a major role in the survival of bee colonies from areas where floral resources are limited. The present result revealed that Melipona quadrifasciata anthidioides interacts with several plant species, thus highlighting the importance of conserving the regional flora. Moreover, the pollen calendar obtained is particularly useful to the management of this stingless bee in meliponary since it provides information about the plants that can be used as pollen source throughout the year. This information is also helpful for both the conservation of regional biodiversity and beekeeping in local agriculture.

Toxicological assessment of agrochemicals on bees using machine learning tools

Machine learning (ML) is a branch of artificial intelligence (AI) that enables the analysis of complex multivariate data. ML has significant potential in risk assessments of non-target insects for modeling the multiple factors affecting insect health, including the adverse effects of agrochemicals. Here, the potential of ML for risk assessments of glyphosate (herbicide; formulation) and imidacloprid (insecticide, neonicotinoid; formulation) on the stingless bee Melipona quadrifasciata was explored. The collective behavior of forager bees was analyzed after in vitro exposure to agrochemicals. ML algorithms were applied to identify the agrochemicals that the bees have been exposed to based on multivariate behavioral features. Changes in the in situ detection of different proteins in the midgut were also studied. Imidacloprid exposure leads to the greatest changes in behavior. The ML algorithms achieved a higher accuracy (up to 91%) in identifying agrochemical contamination. The two agrochemicals altered the detection of cells positive for different proteins, which can be detrimental to midgut physiology. This study provides a holistic assessment of the sublethal effects of glyphosate and imidacloprid on a key pollinator. The procedures used here can be applied in future studies to monitor and predict multiple environmental factors affecting insect health in the field.

Longitudinal survey reveals delayed effects of low gene expression on stingless bee colony health

Bee populations are declining globally due to different environmental stressors, such as pathogens, malnutrition, and agrochemicals. Brazil is the home of hundreds of stingless bee species, some of them now considered endangered, though very little is known about the impact of disease on native bees. In Southern Brazil the endangered stingless bee Melipona quadrifasciata is affected by an annual syndrome that causes sudden death of workers, eventually leading colonies to collapse. Although novel viruses were found in foragers from diseased colonies, none has been consistently implicated in the outbreak. Here we conducted an integrative longitudinal survey on M. quadrifasciata managed colonies, measuring individual- and colony-level traits, to identify the causes behind the syndrome. We found that key genes related to xenobiotic metabolization, nutrition and immune responses are downregulated in foragers from colonies that became diseased two months later. The period that preceded the outbreak was also marked by pronounced forager weight loss as well as behavioural changes. Our findings reveal the syndrome may result from the stingless bee failure to respond to sublethal stressors and disruption of colony dynamics. These results support the proposition that worldwide bee mortality is influenced by a combination of diverse sublethal factors, and increase awareness of the long-term effects of genetic diversity erosion in stingless bee species, which can be enhancing their vulnerability to environmental stressors.

Impact of glues used for RFIDs on the longevity and flight muscles of the stingless bee Melipona quadrifasciata (Apidae: Meliponini)

We conducted several tests on the stingless bee Melipona quadrifasciata aiming to determine the impact of the glue used for applying radio frequency identification (RFID) tags on this bee species. The study was organized in three experimental sets, in which we evaluated the effects of a synthetic glue, a natural glue (shellac), and the effects of the bee manipulation alone (control group). We performed (i) an in vitro experiment (bioassay), in which we tested five different experimental treatments in triplicate: chip plus synthetic glue, chip plus shellac, synthetic glue, shellac, and control, totaling 150 bees (n = 30 per treatment); (ii) field experiments, in which we tested the RFID tracking system composed of RFID tags, reading units, antennas, and circuit boards; and (iii) the morphological and histochemical analyses of the flight muscles of bees collected from each experimental treatment (n = 5 per treatment) at 48 h after the beginning of bioassay. Use of the natural glue, as opposed to the synthetic glue, promoted an increase of the bees’ longevity while inhibiting detrimental impacts on their foraging activities, as observed by both the bioassay and field experiments. We found negative responses to the synthetic glue treatment combined with the electronic tags, showing that the natural glue induces less morphological damage to the flight muscles of tagged stingless bees.

The Neonicotinoid Insecticide Imidacloprid Disrupts Bumblebee Foraging Rhythms and Sleep.

SummaryNeonicotinoids have been implicated in the large declines observed in insects such as bumblebees, an important group of pollinators. Neonicotinoids are agonists of nicotinic acetylcholine receptors that are found throughout the insect central nervous system and are the main mediators of synaptic neurotransmission. These receptors are important for the function of the insect central clock and circadian rhythms. The clock allows pollinators to coincide their activity with the availability of floral resources and favorable flight temperatures, as well as impact learning, navigation, and communication. Here we show that exposure to the field-relevant concentration of 10 μg/L imidacloprid caused a reduction in bumblebee foraging activity, locomotion, and foraging rhythmicity. Foragers showed an increase in daytime sleep and an increase in the proportion of activity occurring at night. This could reduce foraging and pollination opportunities, reducing the ability of the colony to grow and reproduce, endangering bee populations and crop yields.

Changes in innate immune response and detoxification in Melipona quadrifasciata (Apinae: Meliponini) on oral exposure to azadirachtin and spinosad

Exposure to agrochemicals and pathogens has been indicated as critical factors of declining pollinator populations, including species of native tropical bees. In the present study, we investigated the sublethal effects of acute oral exposure to azadirachtin and spinosad bioinsecticides on the expression of immunity-related genes and the number of hemocytes in uninfected and Escherichia coli–infected workers of the stingless bee Melipona quadrifasciata. Expression of the immune-related gene vitellogenin decreased and the number of circulating hemocytes increased in bioinsecticide-exposed uninfected bees and in unexposed E. coli–infected bees as compared with unexposed uninfected bees. Our results show that though bioinsecticides are considered safe, contamination by sublethal doses of azadirachtin and spinosad can interfere with the immune system of bees.

Essential and Potentially Toxic Elements from Brazilian Geopropolis Produced by the Stingless Bee Melipona quadrifasciata anthidioides Using ICP OES.

Melipona quadrifasciata anthidioides is a species of stingless bee popularly known in Brazil as "mandaçaia". Products derived from bees for food and therapeutic uses, have stimulated the evaluation of the chemical composition of geopropolis. Concentrations of 24 essential and potentially toxic elements were determined in geopropolis samples, using a sequential optical emission spectrometer with inductively coupled plasma (ICP OES) after microwave-assisted acid digestion. Principal component analysis (PCA) and Hierarchical cluster analysis (HCA) were used to carry out an exploratory analysis of the samples. The following elements were quantified (in mg Kg-1): Al (320,414.40-36,911.1), As (<LoQ-4.37), Ba (38.36-211.11), Ca (672.38-94,527), Co (<LoQ-14.12), Cr (17.41-38.07), Cu (10.63-28.73), Fe (21,973.96-11,536.47), K (1974.38-9198.91), Mg (1961.17-7481.79), Mn (50.51-310.51), Na (154.55-340.46), Ni (2.28-21.74), P (16.59-51.07), Pb (3.45-8.55), Sb (<LoQ-1.64), Se (<LoQ-1.01), Sn (4.92-16.14), Sr (9.21-36.29), V (28.77-78.73) and Zn (24.34-50.31). Cd and Mo were found to be below the limit of detection (LoD) and quantification (LoQ) values of ICP OES in all investigated samples. Geopropolis can be a potential source of macro- and microelements for colonies and products derived from these bees for human consumption, contributing to their geographical origin and quality control, besides being an indicator of environmental monitoring. Graphical Abstract.

Aximopsis masneri (Chalcidoidea: Eurytomidae), a parasitoid of Euglossa bees (Apidae: Euglossini): life-history traits and experience of rearing in captivity on Melipona larvae (Apidae: Meliponini)

In the present study, we provide data on the biology of Aximopsis masneri, a parasitoid of Euglossa townsendi. We found a nest of E. townsendi built into a knife sheath, in Viçosa/MG/Brazil. The Euglossa nests are difficult to find and maintain in the laboratory, to solve this problem, we used closed brood cells containing prepupa of the stingless bee Melipona quadrifasciata as hosts. The brood cells of M. quadrifasciata are easy to obtain and handle, which allowed us to rear the parasitoids for more than one generation. Here, we provided information on the oviposition behavior, average survival time of each generation, and sex ratio. Therefore, we suggest that M. quadrifasciata can be used as host species for studies on the biology of parasitoids of solitary bees.

How landscape composition affects pollen collection by stingless bees?

Human disturbances are one of the main reasons for the recent deficit of pollinators, which can cause severe habitat loss, decreasing food availability and nesting resources for pollinators. However, few studies show how landscapes composition and configuration can impact the pollen foraging of bees. We aimed to evaluate how landscape structure and local characteristics can influence the pollen foraging patterns of the stingless bee Melipona quadrifasciata anthidioides in agricultural landscapes of Chapada Diamantina, a region of the Bahia state in Brazil. Pollen samples were collected from fourteen M. quadrifasciata anthidioides colonies to evaluate how pollen collection varies according to different proportions of human land-use, landscape heterogeneity, plant richness and distance from crops. To explain the richness and diversity of pollen collected by bees, we used generalized linear models, and linear models, compared through the Akaike information criteria corrected for small samples (AICc). Pollen richness can be explained by the distance from crops, plant richness and human land-use proportion. Bees will collect pollen from more plant species when they are in a natural and diverse landscape. Pollinator friendly crop areas must be planned to be surrounded by diverse natural nearby environments, guaranteeing yearlong pollen resources available for bees’ maintenance.

Cover Caption

Tomato flowers are pollinated by bees that vibrate their thorax for pollen collecting, behavior known as buzz‐pollination. The phenomenon is common and a specimen of the Neotropical stingless bee Melipona quadrifasciata is here depicted visiting a tomato flower for pollen collecting. While testing whether flower anthers exhibit optimal frequency for pollen release and whether flower bees tune their buzzes to match these frequencies, we recognized that neither bees nor plants are tuned to optimal pollen release frequencies (see pages 133–142). Photo provided José Lino‐Neto.

Post-embryonic Development of the Seminal Vesicle in the Stingless Bee Melipona quadrifasciata Lepeletier, 1836 (Apidae: Meliponini)

The male accessory glands of stingless bees (Apidae: Meliponini) are absent and the morphology of their seminal vesicle indicate probable secretory function by this organ. This study investigated the post-embryonic development of the seminal vesicles in males of the stingless bee Melipona quadrifasciata by histology and histochemistry. White-eyed pupae, pink-eyed pupae, brown-eyed pupae, black-eyed pupae, newly emerged and sexually mature males were studied. Seminal vesicle has a wall with a single layered epithelium onto a thin basement membrane, followed by a well-developed muscle layer. The epithelium is polarized in the pupal stage with basal cell region strongly positive for glucoconjugates and carbohydrates. The seminal vesicle has an enlarged lumen from the young pupal stages with luminal content increasing gradually with glucoconjugates along the pupal development. In the newly emerged and mature males, the histochemical tests to carbohydrates were negative. In the sexually mature males, spermatozoa clusters are embedded by the glucoconjugates content of the seminal vesicle lumen. In conclusion, the seminal vesicle of M. quadrifasciata has a secretory function during the pupal stage and in newly emerged males, whereas in adult males this organ stores the spermatozoa..

The virome of an endangered stingless bee suffering from annual mortality in southern Brazil.

Meliponiculture - the management of stingless bee colonies - is an expanding activity in Brazil with economic, social and environmental potential. However, unlike in apiculture, the pathogens that impact on meliponiculture remain largely unknown. In southern Brazil, every year at the end of the summer, managed colonies of the stingless bee Melipona quadrifasciata manifest a syndrome that eventually leads to collapse. Here we characterize the M. quadrifasciata virome using high-throughput sequencing, with the aim of identifying potentially pathogenic viruses, and test whether they are related to the syndrome outbreaks. Two paired viromes are explored, one from healthy bees and another from unhealthy ones. Each virome is built from metagenomes assembled from sequencing reads derived either from RNA or DNA. A total of 40 621 reads map to viral contigs of the unhealthy bees' metagenomes, whereas only 11 reads map to contigs identified as viruses of healthy bees. The viruses showing the largest copy numbers in the virome of unhealthy bees belong to the family Dicistroviridae - common pathogenic honeybee viruses - as well as Parvoviridae and Circoviridae, which have never been reported as being pathogenic in insects. Our analyses indicate that they represent seven novel viruses associated with stingless bees. PCR-based detection of these viruses in individual bees (healthy or unhealthy) from three different localities revealed a statistically significant association between viral infection and symptom manifestation in one meliponary. We conclude that although viral infections may contribute to colony collapses in the annual syndrome in some meliponaries, viruses spread opportunistically during the outbreak, perhaps due to colony weakness.

Ultramorphology of the peritrophic matrix in bees (Hymenoptera: Apidae)

The midgut is a region of the alimentary canal of bees with the lumen lined by a peritrophic matrix (PM) that is composed of chitin and proteins, secreted in response to feeding stimulus. The PM in adult bees has been described only under histological aspects. The objective of this work was to describe the PM in different bee species with a scanning electron microscope. The PMs have different features among the species analyzed. In the stingless bee Melipona quadrifasciata (Meliponini), the PM has two layers and the surface associated with the ectoperitrophic space has a hexagonal sculpture, whereas the endoperitrophic surface shows some wrinkles. In the honey bee Apis mellifera (Apini), and in the solitary bees Centris fuscata (Centridini) and Epicharis flava (Centridini) the PM has a laminate aspect with thin and irregular layers with dense material covering their surface, likely found for non Apidae bees Megachile rotundata (Megachilidae) and Oxaea flavescens (Andrenidae). These data suggest that the PM varies according to the bee species, which may be due to phylogeny other than feeding habits.

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.

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.

Vibrating donor-partners during trophallaxis modulate associative learning ability of food receivers in the stingless bee Melipona quadrifasciata

Multiple sensory modalities within a communication context lead to an increased responsiveness of the receptor. Due to this increase in attention when exposed to multiple stimuli, it is possible that the learning ability of a receiver is modulated through communication signals. Stingless bees (Apidae; Meliponini) are a good model to use in order to investigate communication systems because they display a much wider behavioural repertoire regarding foraging and communication than the thoroughly studied honeybee Apis mellifera. Similar to honeybees, meliponine bees show appetitive unconditioned responses (extension of the proboscis after contacting sugar with the antennae) that may be conditioned to odour. Such associative learning events take place within the nest, for instance, when a successful forager unloads the nectar collected in the field to receiver partners (trophallaxis). During these contacts, receivers obtain multimodal information about the collected resource, such as its sugar content, its specific scent, and, in case of several meliponine species such as Melipona quadrifasciata, thoracic pulsed vibrations produced by the food donors. These vibrations correlate with the profitability of the resource exploited by the forager. With this in mind, we asked whether and to what extent food receivers in stingless bee colonies (M. quadrifasciata) integrate this multimodal information – vibratory–olfactory–gustatory – while receiving food from foragers. We found that the scented food elicited stronger vibrations (longer and more frequent vibrations with an overall higher duty cycle) than unscented food of equal sugar content. We also observed a positive correlation between the amount of vibrations a bee received during trophallaxis and its subsequent learning performance in a proboscis extension response conditioning procedure. Our findings suggest that the vibrations during trophallaxis affect the receiver's behaviour by modulating its ability to establish the food-odour association.