Phenolic Content and Bioactivity as Geographical Classifiers of Propolis from Stingless Bees in Southeastern Mexico.

Propolis collected by stingless bees is a valuable biocultural resource and a source of bioactive compounds. Methodologies to establish both the geographic origin and the potential pharmacological activity of propolis of stingless bees are required to regulate their sustainable use. The aim of this study was to classify Melipona beecheii propolis according to its phenolic compound content and potential pharmacological activity, using in vitro assays and statistical methodologies of multivariate analysis, hierarchical cluster analysis, and principal component analysis. Propolis samples were collected from seven states in southeastern Mexico. Total phenolic content and flavonoids were determined spectrophotometrically, and antioxidant, anti-inflammatory, and antimicrobial activities were evaluated. Both total phenolic content and flavonoids, and in vitro bioactivity potential of propolis extracts showed significant variations. Multivariate analysis, hierarchical cluster analysis, and principal component analysis enabled us to distinguish and classify propolis produced by M. beecheii according to similarity in terms of total phenolic content, in vitro bioactivity potential, and geographical origin. This strategy could be used to establish regulations for sustainable use, marketing, and industrial applications.

Pollination service provided by honey bees to buzz-pollinated crops in the Neotropics.

Generalist honey bees grant significant pollination services worldwide. Although honey bees can provide compensatory pollination services, their service to buzz-pollinated crops, compared to specialized pollinators, is not clear. In this study, we assessed the contribution of Africanized honey bees (AHB) and native sonicating bees (NBZ) to the pollination of eggplant (Solanum melongena) and annatto (Bixa orellana) in Yucatan, Mexico, one of the largest producers of these crops in the Americas and a region with one of the largest densities of honey bees in the world. We first compared the relative frequency and abundance of both bee types on flowers of both crops. Secondly, we controlled access to flowers to compare the number and weight of fruit and number of seed produced after single visits of AHB and native bees. For a better assessment of pollination services, we evaluated the productivity of individual flowers multiply visited by AHB. The results were compared against treatments using pollinator-excluded flowers and flowers that were supplied with additional pollen, which allowed an overall measure of pollination service provision (PSP). Our results showed that AHB were the predominant flower visitors in both crops and that were poorly efficient on individual visits. Notably, fruit quantity and seed number increased concomitantly with the number of AHB visits per flower on eggplant, but not on annatto. Estimation of PSP revealed no pollination deficit on eggplant but that a deficit existed on the pollination services to annatto. We found that AHB numerical predominance compensates their poor individual performance and can complement the services of native bees on eggplant, but not on annatto. We discuss possible explanations and implications of these results for buzz-pollinated crops in the neotropics an area with little assessment of pollination services and a high density of honey bees.

Anthropogenic effects on the body size of two neotropical orchid bees.

To accommodate an ever-increasing human population, agriculture is rapidly intensifying at the expense of natural habitat, with negative and widely reported effects on biodiversity in general and on wild bee abundance and diversity in particular. Cities are similarly increasing in area, though the impact of urbanisation on wild bees is more equivocal and potentially positive in northern temperate regions. Yet agriculture and urbanisation both lead to the loss and alteration of natural habitat, its fragmentation, a potential reduction in floral availability, and warmer temperatures, factors thought to be drivers of wild bee decline. They have also been shown to be factors to which wild bee populations respond through morphological change. Body size is one such trait that, because of its relation to individual fitness, has received growing attention as a morphological feature that responds to human induced modification in land use. Here, we investigated the change in body size of two sympatric orchid bee species on the Yucatan Peninsula of Mexico in response to urbanization and agricultural intensification. By measuring 540 male individuals sampled from overall 24 sites, we found that Euglossa dilemma and Euglossa viridissima were on average smaller in urban and agricultural habitats than in natural ones. We discuss the potential role of reduced availability of resources in driving the observed body size shifts. Agricultural and urban land management in tropical regions might benefit wild bees if it encompassed the planting of flowering herbs and trees to enhance their conservation.

Nesting substrates and nest density of stingless bees in the Petenes Biosphere Reserve, Mexico / Sustratos de nidificación y densidad de nidos de abejas sin aguijón en la reserva de la biósfera de los petenes, méxico

RESUMEN Las abejas sin aguijón (ASA) son organismos relevantes en los ecosistemas por su desempeño como polinizadores. En México, la mayor diversidad de ASA se encuentra en el sureste. En este estudio se determinaron las especies de ASA y densidad de nidos en dos sitios con diferente perturbación dentro de la Reserva de la Biósfera de los Petenes (RBLP) Campeche (México). Asimismo, se identificaron los sustratos y las especies arbóreas de nidificación. En total se realizaron diez muestreos en transectos entre los meses de enero a mayo de 2018. Se registraron 56 nidos pertenecientes a ocho especies diferentes de ASA (Cephalotrigona zexmeniae, Frieseomelitta nigra, Lestrimellita niitkib, Nannotrigona perilampoides, Partamona bilineata, Plebeia frontalis, Scaptotrigona pectoralis y Trígona fulviventris). Las especies con mayor número de nidos correspondieron a F. nigra (41,07 %) y S. pectoralis (28,57 %). No se encontraron diferencias significativas en la densidad de nidos entre los sitios Hecelchakán (2,15 nidos/ha) y Calkiní (0,19 nidos/ha). El sustrato preferido para anidación son las cavidades en árboles vivos (78,57 %). En total 15 especies arbóreas fueron identificadas como sustrato de nidificación y la especie más abundante correspondió a Crescentia cujete (n = 18, 40,9 %). El grado de perturbación tuvo poco efecto en el número y densidad de nidos de ASA, sin embargo, en general, la RBLP presentó una composición de especies y densidad de nidos reducida. Por lo tanto, este estudio permitió establecer las bases para futuros estudios sobre estrategias de manejo y conservación de estas especies para evitar su desaparición en la RBLP.

ABSTRACT Stingless bees (ASA) are relevant organisms in ecosystems because of their performance as pollinators. In Mexico, the higher diversity of ASA is found in the southeast. In this study, the ASA species and nest density of nests were identified at two sites with different disturbances (Hecelchakán and Calkiní) within the Petenes Biosphere Reserve (RBLP), Campeche (Mexico). Also, the substrates and the nesting tree species were identified. In total, ten sampling transects were performed between January and May 2018. In total 56 nests belonging to eight different species of ASA (Cephalotrigonazexmeniae, Frieseomelitta nigra, Lestrimellita niitkib, Nanotrigonaperilampoides, Partamona bilineata, Plebeia frontalis, Scaptotrigona pectoralis and Trigona fulviventris) were found. The species with the highest number of nests corresponded to F. nigra (41.07 %) and S. pectoralis (28.57 %). No significant differences were found in nest density between the Hecelchakán (2.15 nests/ha) and Calkiní (0.19 nests/ha) site. The preferred substrate for nesting is cavities in living trees (78.57 %). In total 15 tree species were identified as nesting substrate and the most abundant species corresponded to Crescentia cujete (n = 8, 40.90 %). The degree of disturbance had little effect on the number and density of ASA nest, however, in general, the RBLP presented low species composition and nest density. Therefore, this study allowed to establish the bases for future studies on management and conservation strategies of these species to avoid their loss in the RBLP.

Sympatric cleptobiotic stingless bees have species-specific cuticular profiles that resemble their hosts.

Stingless bees are the largest group of eusocial pollinators with diverse natural histories, including obligate cleptobionts (genus Lestrimelitta) that completely abandoned flower visitation to rely on other stingless bees for food and nest materials. Species of Lestrimeliita are thought to specialize upon different host species, and deception through chemical similarity has been proposed as a mechanism to explain this phenomenon. In the Yucatan Peninsula of Mexico, Scaptotrigona pectoralis is a species chemically distinct from, and not preferred as a host by, locally widespread Lestrimeliita niitkib; witnessing attacks on S. pectoralis colonies offered the opportunity to test the sensory deception hypothesis to cletoparasitism. Analysis of cuticular profiles revealed that the Lestrimelitta attacking S. pectoralis differed significantly in odour bouquet to L. niitkib and, in contrast, it resembled that of S. pectoralis. Further analyses, including morphometrics, mtDNA barcoding, and the examination of taxonomic features, confirmed the existence of two sympatric Lestrimelitta species. The results give support to the hypothesis of chemical deception as a cleptobiotic strategy in Lestrimelitta sp. This is the first evidence that sympatric cleptobionts of the same genus select hosts in accordance with species-specific cuticular profiles, with possible consequences for ecological adaptation and the evolution of these remarkable organisms and the community of stingless bee hosts.

Tragedy of the commons in Melipona bees revisited.

Melipona stingless bees display a paradoxical overproduction of queens, which are later eliminated by nest-mate workers. Mechanistically, it was suggested that the monoterpenoid geraniol deposited into newly provisioned cells by adult bees would cause larvae to develop into queens in Melipona beecheii. This system could be evolutionarily stable if many of these new queens were to leave the nest and parasitize other genetically unrelated colonies nearby, as was shown to occur in a congeneric species. Here, we use microsatellite markers to test whether queen overproduction could be a strategy by which adult workers control the caste fate of the developing larvae to export copies of their own genes to the rest of the population via queen parasitism in M. beecheii. In addition, we re-examined whether artificially increasing the levels of geraniol indeed caused larvae to develop as queens rather than workers. Contrary to our prediction, we found no evidence for queen parasitism in M. beecheii and observed no effect of geraniol on the rearing of new queens. Together, these results support the original 'tragedy of the commons' hypothesis for queen overproduction in Melipona bees, where individual larvae selfishly bias their development towards the queen pathway according to their best evolutionary interests.

Transcriptomic Signatures of Ageing Vary in Solitary and Social Forms of an Orchid Bee.

Eusocial insect queens are remarkable in their ability to maximize both fecundity and longevity, thus escaping the typical trade-off between these two traits. Several mechanisms have been proposed to underlie the remolding of the trade-off, such as reshaping of the juvenile hormone (JH) pathway, or caste-specific susceptibility to oxidative stress. However, it remains a challenge to disentangle the molecular mechanisms underlying the remolding of the trade-off in eusocial insects from caste-specific physiological attributes that have subsequently arisen. The socially polymorphic orchid bee Euglossa viridissima represents an excellent model to address the role of sociality per se in longevity as it allows direct comparisons of solitary and social individuals within a common genetic background. We investigated gene expression and JH levels in young and old bees from both solitary and social nests. We found 902 genes to be differentially expressed with age in solitary females, including genes involved in oxidative stress, versus only 100 genes in social dominant females, and 13 genes in subordinate females. A weighted gene coexpression network analysis further highlights pathways related to ageing in this species, including the target of rapamycin pathway. Eleven genes involved in translation, apoptosis, and DNA repair show concurrent age-related expression changes in solitary but not in social females, representing potential differences based on social status. JH titers did not vary with age or social status. Our results represent an important step in understanding the proximate mechanisms underlying the remodeling of the fecundity/longevity trade-off that accompanies the evolutionary transition from solitary life to eusociality.

Heat stress during development affects immunocompetence in workers, queens and drones of Africanized honey bees (Apis mellifera L.) (Hymenoptera: Apidae).

Though social insects generally seem to have a reduced individual immunoresponse compared to solitary species, the impact of heat stress on that response has not been studied. In the honey bee, the effect of heat stress on reproductives (queens and males/drones) may also vary compared to workers, but this is currently unknown. Here, we quantified the activity of an enzyme linked to the immune response in insects and known to be affected by heat stress in solitary species: phenoloxidase (PO), in workers, queens and drones of Africanized honey bees (AHBs) experimentally subjected to elevated temperatures during the pupal stage. Additionally, we evaluated this marker in individuals experimentally infected with the entomopathogenic fungus Metarhizium anisopliae. Differences in PO activity were found between sexes and castes, with PO activity generally higher in workers and lower in reproductives. Such differences are associated with the likelihood of exposure to infection and the role of different individuals in the colony. Contrary to our expectation, heat stress did not cause an increase in PO activity equally in all classes of individual. Heat stress during the pupal stage significantly decreased the PO activity of AHB queens, but not that of workers or drones, which more frequently engage in extranidal activity. Experimental infection with Metarhizium anisopliae reduced PO activity in queens and workers, but increased it in drones. Notably, heat stressed workers lived significantly shorter after infection despite exhibiting greater PO activity than queens or drones. We suggest that this discrepancy may be related to trade-offs among immune response cascades in honey bees such as between heat shock proteins and defensin peptides used in microbial defence. Our results provide evidence for complex relationships among humoral immune responses in AHBs and suggest that heat stress could result in a reduced life expectancy of individuals.

Patterns of sexual size dimorphism in stingless bees: Testing Rensch's rule and potential causes in highly eusocial bees (Hymenoptera: Apidae, Meliponini).

Eusocial insects offer a unique opportunity to analyze the evolution of body size differences between sexes in relation to social environment. The workers, being sterile females, are not subject to selection for reproductive function providing a natural control for parsing the effects of selection on reproductive function (i.e., sexual and fecundity selection) from other kinds of natural selection. Patterns of sexual size dimorphism (SSD) and testing of Rensch's rule controlling for phylogenetic effects were analyzed in the Meliponini or stingless bees. Theory predicts that queens may exhibit higher selection for fecundity in eusocial taxa, but contrary to this, we found mixed patterns of SSD in Meliponini. Non-Melipona species generally have a female-biased SSD, while all analyzed species of Melipona showed a male-biased SSD, indicating that the direction and magnitude of the selective pressures do not operate in the same way for all members of this taxon. The phylogenetic regressions revealed that the rate of divergence has not differed between the two castes of females and the males, that is, stingless bees do not seem to follow Rensch's rule (a slope >1), adding this highly eusocial taxon to the various solitary insect taxa not conforming with it. Noteworthy, when Melipona was removed from the analysis, the phylogenetic regressions for the thorax width of males on queens had a slope significantly smaller than 1, suggesting that the evolutionary divergence has been larger in queens than males, and could be explained by stronger selection on female fecundity only in non-Melipona species. Our results in the stingless bees question the classical explanation of female-biased SSD via fecundity and provide a first evidence of a more complex determination of SSD in highly eusocial species. We suggest that in highly eusocial taxa, additional selection mechanisms, possibly related to individual and colonial interests, could influence the evolution of environmentally determined traits such as body size.

Developmental stability, age at onset of foraging and longevity of Africanized honey bees (Apis mellifera L.) under heat stress (Hymenoptera: Apidae).

Beekeeping with the western honey bee (Apis mellifera) is important in tropical regions but scant information is available on the possible consequences of global warming for tropical beekeeping. We evaluated the effect of heat stress on developmental stability, the age at onset of foraging (AOF) and longevity in Africanized honey bees (AHBs) in the Yucatan Peninsula of Mexico, one of the main honey producing areas in the Neotropics, where high temperatures occur in spring and summer. To do so, we reared worker AHB pupae under a fluctuating temperature regime, simulating current tropical heatwaves, with a high temperature peak of 40.0 °C for 1 h daily across six days, and compared them to control pupae reared at stable temperatures of 34.0-35.5 °C. Heat stress did not markedly affect overall body size, though the forewing of heat-stressed bees was slightly shorter than controls. However, bees reared under heat stress showed significantly greater fluctuating asymmetry (FA) in forewing shape. Heat stress also decreased AOF and reduced longevity. Our results show that changes occur in the phenotype and behavior of honey bees under heat stress, with potential consequences for colony fitness.

Sweat bees on hot chillies: provision of pollination services by native bees in traditional slash-and-burn agriculture in the Yucatán Peninsula of tropical Mexico.

Traditional tropical agriculture often entails a form of slash-and-burn land management that may adversely affect ecosystem services such as pollination, which are required for successful crop yields. The Yucatán Peninsula of Mexico has a >4000 year history of traditional slash-and-burn agriculture, termed 'milpa'. Hot 'Habanero' chilli is a major pollinator-dependent crop that nowadays is often grown in monoculture within the milpa system.We studied 37 local farmers' chilli fields (sites) to evaluate the effects of landscape composition on bee communities. At 11 of these sites, we undertook experimental pollination treatments to quantify the pollination of chilli. We further explored the relationships between landscape composition, bee communities and pollination service provision to chilli.Bee species richness, particularly species of the family Apidae, was positively related to the amount of forest cover. Species diversity decreased with increasing proportion of crop land surrounding each sampling site. Sweat bees of the genus Lasioglossum were the most abundant bee taxon in chilli fields and, in contrast to other bee species, increased in abundance with the proportion of fallow land, gardens and pastures which are an integral part of the milpa system.There was an average pollination shortfall of 21% for chilli across all sites; yet the shortfall was unrelated to the proportion of land covered by crops. Rather, chilli pollination was positively related to the abundance of Lasioglossum bees, probably an important pollinator of chilli, as well indirectly to the proportion of fallow land, gardens and pastures that promote Lasioglossum abundance. Synthesis and applications. Current, low-intensity traditional slash-and-burn (milpa) agriculture provides Lasioglossum spp. pollinators for successful chilli production; fallow land, gardens and pasture therefore need to be valued as important habitats for these and related ground-nesting bee species. However, the negative impact of agriculture on total bee species diversity highlights how agricultural intensification is likely to reduce pollination services to crops, including chilli. Indeed, natural forest cover is vital in tropical Yucatán to maintain a rich assemblage of bee species and the provision of pollination services for diverse crops and wild flowers.

Comparative toxicity of pesticides to stingless bees (Hymenoptera: Apidae: Meliponini).

Stingless bees are potential pollinators of commercial tropical crops and their use may increase in the short term. However, studies comparing the toxicity of pesticides to different individuals and species are lacking, making it difficult to evaluate their short- and long-term effects on colonies and populations of these insects. In this work, we tested the lethality of compounds from the main pesticide groups on stingless bees of the species Melipona beecheii Bennett, Trigona nigra Provancher, and Nannotrigona perilampoides Cresson. The LDo (in micrograms per bee) for each pesticide was calculated for callow workers and foragers of the three species as well as for gynes and drones of M. beecheii. The results showed that all species were highly susceptible to the evaluated compounds. Nicotinoid pesticides were the most toxic, followed in descending order by permethrin, diazinon, and methomyl. We found evidence of a relationship between the body weight of the species and their LD50 for permethrin and methomyl (r = 0.91 and 0.90, respectively) but not for diazinon (r = -0.089). An analysis of contingency tables showed that within each species, callow workers had higher mortalities than foragers (P < 0.01). In M. beecheii at similar pesticide dose more males died compared with females [chi2((0.0),1) = 10.16]. However, gynes were less resistant than workers [chi2((0.01),1)) = 8.11]. The potential negative consequences of pesticides to native stingless bees are discussed considering the reproductive biology of these insects. It is important to take actions to prevent damage to these key species for the ecology and agriculture of Mexico and Latin America

Potencial de la abejas nativas en la polinización de cultivos / Native Bees potential on crop polinization

En esta nota se discute sobre la polinización de cultivos como una actividad potencial del cultivo de abejas nativas.

This note refers to the potential of native bees cultivation in crop polinization.

Comparative efficiency of Nannotrigona perilampoides, Bombus impatiens (Hymenoptera: Apoidea), and mechanical vibration on fruit production of enclosed habanero pepper.

The native bee Nannotrigona perilampoides Cresson (Apidae: Meliponini) has been evaluated with promising results in greenhouse pollination of Solanaceae in Mexico. However, no comparison has been done with imported bumble bees (Apidae: Bombini), which are the most common bees used for greenhouse pollination. We compared the foraging activity and fruit production of habanero pepper. Capsicum chinense Jacquin, by using N. perilampoides and Bombus impatiens Cresson in pollination cages. Both bee species collected pollen on a similar number of flowers per unit time, but N. perilampoides visited significantly more flowers per trip, lasted longer on each flower, and spent more time per foraging trip. Ambient temperature and light intensity significantly affected the foraging activity of N. perilampoides. Light intensity was the only environmental variable that affected B. impatiens. Except for the fruit set, there were not significant differences in the quality of fruit produced by both bee species; however, N. perilampoides and B. impatiens performed better than mechanical vibration for all the variables measured. The abortion of fruit caused the low fruit set produced by B. impatiens, and we speculate it might be due to an excessive visitation rate. Pollination efficiency per visit (Spear's pollination efficiency index) was similar for both bee species in spite of the significantly lower amount of pollen removed by N. perilampoides. We suggested that the highest number of flowers visited per foraging trip coupled with adequate amounts of pollen transported, and transferred between flowers, could explain the performance of N. perilampoides as a good pollinator of habanero pepper. Our experiments confirm that N. perilampoides could be used as an alternative pollinator to Bombus in hot pepper under tropical climates.

Behavior and pollination efficiency of Nannotrigona perilampoides (Hymenoptera: Meliponini) on greenhouse tomatoes (Lycopersicon esculentum) in subtropical México.

The acclimation, foraging behavior, and pollination efficiency of stingless bees of the species Nannotrigona perilampoides Cresson were evaluated in tomato (Lycopersicon esculentum Mill.) plants cultivated in two greenhouses. The greenhouses were divided into three areas of 16 m2, and one of the following treatments was used for pollination: stingless bees (SB), mechanical vibration (MV), and no pollination (NP). Observations were conducted once a week from 0800 to 1600 hours during 2 mo. The acclimation of the bees to the greenhouses was estimated by the number of bees that did not return to the hive (lost bees) and by comparing the population of the colonies (brood and adults). The foraging activity of the bees across the day was evaluated by comparing the number of foragers per hour. The influence of environmental variables on the foraging activity was also analyzed. The pollination efficiency was compared among treatments through the percentage of fruit set, weight of individual fruit, kilograms of fruit produced per square meter, and the number of seed per fruit. The bees started foraging on the flowers approximately 7 d after the colonies were introduced to the greenhouse. There was a decline in the population of the colonies across the experiment, but colonies did not die out. Correlations of environmental variables with the foraging activity of the bees showed that none of them had a significant influence on pollen foraging. However, water collection was positively correlated with the temperature and negatively correlated with the humidity inside the greenhouse. The estimation of the pollination efficiency per treatment showed that there were significant differences in fruit set in SB (83 +/- 4.2) and MV (78.5 +/- 6.4) compared with NP (52.6 +/- 7.6). However, the average weight of the fruit was similar for the three treatments (65 g). There were significant differences for seed number in SB (200 +/- 15.3) and MV (232 +/- 21.4) compared with NP (120 +/- 16.6). The productivity in kilograms of fruit per square meter was higher in SB (5.72 +/- 0.61) and MV (5.66 +/- 0.58 kg) compared with NP (3.34 +/- 0.72). The number of seed was positively correlated with the weight of the fruit. We conclude that the use of Nannotrigona testaceicornis Rondani, for pollinating greenhouse tomatoes in tropical climates, could be an alternative to the use of highly defensive African-derived Apis mellifera or non-native bumble bees (Bombus spp.). However, more research is needed to evaluate the cost/benefit on large-scale greenhouse pollination using N. perilampoides Cresson against other bee species and pollination methods.

Paternity frequency and maternity of males in some stingless bee species.

In monogynous hymenopteran societies, the number of mates of a queen strongly influences the potential for conflict between workers and queens over the maternity of males. Queens always 'prefer' their own sons to sons of workers, regardless of queen mating frequency. When a queen mates once, workers are more closely related to, and therefore are expected to prefer, their own sons and then sons of sisters to sons of the queen. However, if effective paternity frequency exceeds 2, workers on average should prefer queen-produced males to males produced by their sisters. We studied the queen mating frequency of seven stingless bee species: the Mexican species Scaptotrigona mexicana, S. pectoralis and the Australian species Austroplebeia symei, Trigona clypearis, T. hockingsi, T. mellipes and T. sapiens. We then determined whether males arise from eggs laid by workers or queens in A. symei, T. clypearis, T. hockingsi and T. mellipes. We show that all seven species investigated are most likely singly mated and that the queen dominates reproduction. This indicates that the queen's mating frequency alone does not determine whether workers or the queen produces the males.