Neotropical bee microbiomes point to a fragmented social core and strong species-level effects.

BACKGROUND: Individuals that band together create new ecological opportunities for microorganisms. In vertical transmission, theory predicts a conserved microbiota within lineages, especially social bees. Bees exhibit solitary to social behavior among and/or within species, while life cycles can be annual or perennial. Bee nests may be used over generations or only once, and foraging ecology varies widely. To assess which traits are associated with bee microbiomes, we analyzed microbial diversity within solitary and social bees of Apidae, Colletidae, and Halictidae, three bee families in Panama's tropical forests. Our analysis considered the microbiome of adult gut contents replicated through time, localities, and seasons (wet and dry) and included bee morphology and comparison to abdominal (dissected) microbiota. Diversity and distribution of tropical bee microbes (TBM) within the corbiculate bee clade were emphasized. RESULTS: We found the eusocial corbiculate bees tended to possess a more conserved gut microbiome, attributable to vertical transmission, but microbial composition varied among closely related species. Euglossine bees (or orchid bees), corbiculates with mainly solitary behavior, had more variable gut microbiomes. Their shorter-tongued and highly seasonal species displayed greater diversity, attributable to flower-visiting habits. Surprisingly, many stingless bees, the oldest corbiculate clade, lacked bacterial genera thought to predate eusociality, while several facultatively social, and solitary bee species possessed those bacterial taxa. Indeed, nearly all bee species displayed a range of affinities for single or multiple variants of the "socially associated" bacterial taxa, which unexpectedly demonstrated high sequence variation. CONCLUSIONS: Taken together, these results call into question whether specific bacterial associates facilitate eusocial behavior, or are subsequently adopted, or indicate frequent horizontal transmission between perennial eusocial colonies and other social, facultatively social, and solitary bees. Video Abstract.

Stingless Bee (Apidae: Apinae: Meliponini) Ecology.

Stingless bees form perennial colonies of honey-making insects. The >600 species of stingless bees, mainly Neotropical, live throughout tropical latitudes. Foragers influence floral biology, plant reproduction, microbe dispersal, and diverse ecosystem functions. As tropical forest residents since the upper Cretaceous, they have had a long evolutionary history without competition from honey bees. Most stingless bees are smaller than any Apis species and recruit nest mates to resources, while their defense strategies exclude stinging behavior but incorporate biting. Stingless bees have diversified ecologically; excel in nesting site selection and mutualisms with plants, arthropods, and microbes; and display opportunism, including co-opting plant defenses. As their biology becomes better known, applications to human endeavors are imposing selective pressures from exploitation and approaches to conservation that entail colony extraction from wildlands. Although some meliponines can adjust to new conditions, their populations shall require tropical diversity for survival and reproduction.

Size-dependent Scaling of Stingless Bee Flight Metabolism Reveals an Energetic Benefit to Small Body Size.

Understanding the effect of body size on flight costs is critical for development of models of aerodynamics and animal energetics. Prior scaling studies that have shown that flight costs scale hypometrically have focused primarily on larger (> 100 mg) insects and birds, but most flying species are smaller. We studied the flight physiology of thirteen stingless bee species over a large range of body sizes (1-115 mg). Metabolic rate during hovering scaled hypermetrically (scaling slope = 2.11). Larger bees had warm thoraxes while small bees were nearly ecothermic; however, even controlling for body temperature variation, flight metabolic rate scaled hypermetrically across this clade. Despite having a lower mass-specific metabolic rate during flight, smaller bees could carry the same proportional load. Wingbeat frequency did not vary with body size, in contrast to most studies that find wingbeat frequency increases as body size decreases. Smaller stingless bees have greater relative forewing surface area which may help them reduce the energy requirements needed to fly. Further, we hypothesize that the relatively larger heads of smaller species may change their body pitch in flight. Synthesizing across all flying insects, we demonstrate that the scaling of flight metabolic rate changes from hypermetric to hypometric at approximately 58 mg body mass with hypermetic scaling below (slope = 1.2) and hypometric scaling (slope = 0.67) above 58 mg in body mass. The reduced cost of flight likely provides selective advantages for the evolution of small body size in insects. The biphasic scaling of flight metabolic rates and wingbeat frequencies in insects supports the hypothesis that the scaling of metabolic rate is closely related to the power requirements of locomotion and cycle frequencies.

Description of a new ant- and stingless-bee-loving species of Cryptostigma Ferris (Hemiptera: Coccomorpha: Coccidae) from Ecuador living inside internodes of Cecropia (Urticaceae), with an updated key to the adult females and first-instar nymphs of the genus.

A new soft scale species, Cryptostigma cecropiaphilum Kondo & Roubik, sp. nov. (Hemiptera: Coccomorpha: Coccidae), is described from specimens collected from inside hollow branches (internodes) of Cecropia ficifolia Warb. ex Snethl and Cecropia sciadophylla Mart. (Urticaceae), in Orellana province, Ecuador. The new species was found in association with three mutualistic hymenopterans: two species of Plebeia Schwarz, subgenus Nanoplebeia Engel (Hymenoptera: Apidae: Meliponini), and Azteca sp. (Hymenoptera: Formicidae: Dolichoderinae). The new species of coccid is described based on the morphology of the adult female and first-instar nymph. Keys to the known adult females and first-instar nymphs of Cryptostigma Ferris are provided.

The importance of insects on land and in water: a tropical view.

Tropical insects are astonishingly diverse and abundant yet receive only marginal scientific attention. In natural tropical settings, insects are involved in regulating and supporting ecosystem services including seed dispersal, pollination, organic matter decomposition, nutrient cycling, herbivory, food webs and water quality, which in turn help fulfill UN Sustainable Development Goals (SDGs). Current and future global changes that affect insect diversity and distribution could disrupt key ecosystem services and impose important threats on ecosystems and human well-being. A significant increase in our knowledge of tropical insect roles in ecosystem processes is thus vital to ensure sustainable development on a rapidly changing planet.

Colony-level non-associative plasticity of alarm responses in the stingless honey bee, Tetragonisca angustula.

In ants, bees, and other social Hymenoptera alarm pheromones are widely employed to coordinate colony nest defense. In that context, alarm pheromones elicit innate species-specific defensive behaviors. Therefore, in terms of classical conditioning, an alarm pheromone could act as an unconditioned stimulus (US). Here we test this hypothesis by establishing whether repeated exposure to alarm pheromone in different testing contexts modifies the alarm response. We evaluate colony level alarm responses in the stingless bee, Tetragonisca angustula, which has a morphologically distinct guard caste. First, we describe the overall topology of defense behaviors in the presence of an alarm pheromone. Second, we show that repeated, regular exposure to synthetic alarm pheromone reduces different components of the alarm response, and memory of that exposure decays over time. This observed decrease followed by recovery occurs over different time frames and is consistent with behavioral habituation. We further tested whether the alarm pheromone can act as a US to classically condition guards to modify their defense behaviors in the presence of a novel (conditioned) stimulus (CS). We found no consistent changes in the response to the CS. Our study demonstrates the possibility that colony-level alarm responses can be adaptively modified by experience in response to changing environmental threats. Further studies are now needed to reveal the extent of these habituation-like responses in regard to other pheromones, the potential mechanisms that underlie this phenomenon, and the range of adaptive contexts in which they function at the colony level.

Coupling of pollination services and coffee suitability under climate change.

Climate change will cause geographic range shifts for pollinators and major crops, with global implications for food security and rural livelihoods. However, little is known about the potential for coupled impacts of climate change on pollinators and crops. Coffee production exemplifies this issue, because large losses in areas suitable for coffee production have been projected due to climate change and because coffee production is dependent on bee pollination. We modeled the potential distributions of coffee and coffee pollinators under current and future climates in Latin America to understand whether future coffee-suitable areas will also be suitable for pollinators. Our results suggest that coffee-suitable areas will be reduced 73-88% by 2050 across warming scenarios, a decline 46-76% greater than estimated by global assessments. Mean bee richness will decline 8-18% within future coffee-suitable areas, but all are predicted to contain at least 5 bee species, and 46-59% of future coffee-suitable areas will contain 10 or more species. In our models, coffee suitability and bee richness each increase (i.e., positive coupling) in 10-22% of future coffee-suitable areas. Diminished coffee suitability and bee richness (i.e., negative coupling), however, occur in 34-51% of other areas. Finally, in 31-33% of the future coffee distribution areas, bee richness decreases and coffee suitability increases. Assessing coupled effects of climate change on crop suitability and pollination can help target appropriate management practices, including forest conservation, shade adjustment, crop rotation, or status quo, in different regions.

Impact of Striped-Squirrel Nectar-Robbing Behaviour on Gender Fitness in Alpinia roxburghii Sweet (Zingiberaceae).

Nectar-robbing has the potential to strongly affect male and female reproductive fitness of plants. One example of nectar theft is that shown by striped-squirrels (Tamiops swinhoei) on a number of ginger species, including Alpinia roxburghii and A. kwangsiensis (Zingiberaceae). In this study, we used a fluorescent dye as a pollen analogue, and measured fruit and seed output, to test the effect of squirrel nectar-robbing on A. roxburghii reproductive fitness. Pollen transfer between robbed and unrobbed flowers was assessed by comparing 60 randomly established plots containing robbed and unrobbed flowers. The frequency of squirrel robbing visits and broken styles were recorded from a number of flowers for five consecutive days. Two bee species (Bombus eximius and Apis cerana), were the primary pollinators, and their visitation frequency was recorded for six consecutive days. The results showed that fluorescent powder from unrobbed flowers was dispersed further, and to a greater number of flowers than that placed on robbed flowers. Additionally, robbing flowers caused significant damage to reproductive organs, resulting in lower fruit and seed sets in robbed than in unrobbed flowers and influencing both male and female fitness. The frequency of the primary pollinator visits (B. eximius) was significantly higher for unrobbed plants than for robbed plants. The present study clearly shows the negative impact of squirrel robbing on A. roxburghii male reproductive fitness and neutral impact on female reproductive fitness.

Arthropod Distribution in a Tropical Rainforest: Tackling a Four Dimensional Puzzle.

Quantifying the spatio-temporal distribution of arthropods in tropical rainforests represents a first step towards scrutinizing the global distribution of biodiversity on Earth. To date most studies have focused on narrow taxonomic groups or lack a design that allows partitioning of the components of diversity. Here, we consider an exceptionally large dataset (113,952 individuals representing 5,858 species), obtained from the San Lorenzo forest in Panama, where the phylogenetic breadth of arthropod taxa was surveyed using 14 protocols targeting the soil, litter, understory, lower and upper canopy habitats, replicated across seasons in 2003 and 2004. This dataset is used to explore the relative influence of horizontal, vertical and seasonal drivers of arthropod distribution in this forest. We considered arthropod abundance, observed and estimated species richness, additive decomposition of species richness, multiplicative partitioning of species diversity, variation in species composition, species turnover and guild structure as components of diversity. At the scale of our study (2 km of distance, 40 m in height and 400 days), the effects related to the vertical and seasonal dimensions were most important. Most adult arthropods were collected from the soil/litter or the upper canopy and species richness was highest in the canopy. We compared the distribution of arthropods and trees within our study system. Effects related to the seasonal dimension were stronger for arthropods than for trees. We conclude that: (1) models of beta diversity developed for tropical trees are unlikely to be applicable to tropical arthropods; (2) it is imperative that estimates of global biodiversity derived from mass collecting of arthropods in tropical rainforests embrace the strong vertical and seasonal partitioning observed here; and (3) given the high species turnover observed between seasons, global climate change may have severe consequences for rainforest arthropods.

Setting the pace of life: membrane composition of flight muscle varies with metabolic rate of hovering orchid bees.

Patterns of metabolic rate variation have been documented extensively in animals, but their functional basis remains elusive. The membrane pacemaker hypothesis proposes that the relative abundance of polyunsaturated fatty acids in membrane phospholipids sets the metabolic rate of organisms. Using species of tropical orchid bees spanning a 16-fold range in body size, we show that the flight muscles of smaller bees have more linoleate (%18 : 3) and stearate (%18 : 0), but less oleate (%18 : 1). More importantly, flight metabolic rate (FlightMR) varies with the relative abundance of 18 : 3 according to the predictions of the membrane pacemaker hypothesis. Although this relationship was found across large differences in metabolic rate, a direct association could not be detected when taking phylogeny and body mass into account. Higher FlightMR, however, was related to lower %16 : 0, independent of phylogeny and body mass. Therefore, this study shows that flight muscle membrane composition plays a significant role in explaining diversity in FlightMR, but that body mass and phylogeny are other factors contributing to their variation. Multiple factors are at play to modulate metabolic capacity, and changing membrane composition can have gradual and stepwise effects to achieve a new range of metabolic rates. Orchid bees illustrate the correlated evolution between membrane composition and metabolic rate, supporting the functional link proposed in the membrane pacemaker hypothesis.

Detecting insect pollinator declines on regional and global scales.

Recently there has been considerable concern about declines in bee communities in agricultural and natural habitats. The value of pollination to agriculture, provided primarily by bees, is >$200 billion/year worldwide, and in natural ecosystems it is thought to be even greater. However, no monitoring program exists to accurately detect declines in abundance of insect pollinators; thus, it is difficult to quantify the status of bee communities or estimate the extent of declines. We used data from 11 multiyear studies of bee communities to devise a program to monitor pollinators at regional, national, or international scales. In these studies, 7 different methods for sampling bees were used and bees were sampled on 3 different continents. We estimated that a monitoring program with 200-250 sampling locations each sampled twice over 5 years would provide sufficient power to detect small (2-5%) annual declines in the number of species and in total abundance and would cost U.S.$2,000,000. To detect declines as small as 1% annually over the same period would require >300 sampling locations. Given the role of pollinators in food security and ecosystem function, we recommend establishment of integrated regional and international monitoring programs to detect changes in pollinator communities.

Arthropod diversity in a tropical forest.

Most eukaryotic organisms are arthropods. Yet, their diversity in rich terrestrial ecosystems is still unknown. Here we produce tangible estimates of the total species richness of arthropods in a tropical rainforest. Using a comprehensive range of structured protocols, we sampled the phylogenetic breadth of arthropod taxa from the soil to the forest canopy in the San Lorenzo forest, Panama. We collected 6144 arthropod species from 0.48 hectare and extrapolated total species richness to larger areas on the basis of competing models. The whole 6000-hectare forest reserve most likely sustains 25,000 arthropod species. Notably, just 1 hectare of rainforest yields >60% of the arthropod biodiversity held in the wider landscape. Models based on plant diversity fitted the accumulated species richness of both herbivore and nonherbivore taxa exceptionally well. This lends credence to global estimates of arthropod biodiversity developed from plant models.

Large-range movements of neotropical orchid bees observed via radio telemetry.

Neotropical orchid bees (Euglossini) are often cited as classic examples of trapline-foragers with potentially extensive foraging ranges. If long-distance movements are habitual, rare plants in widely scattered locations may benefit from euglossine pollination services. Here we report the first successful use of micro radio telemetry to track the movement of an insect pollinator in a complex and forested environment. Our results indicate that individual male orchid bees (Exaerete frontalis) habitually use large rainforest areas (at least 42-115 ha) on a daily basis. Aerial telemetry located individuals up to 5 km away from their core areas, and bees were often stationary, for variable periods, between flights to successive localities. These data suggest a higher degree of site fidelity than what may be expected in a free living male bee, and has implications for our understanding of biological activity patterns and the evolution of forest pollinators.

A molecular phylogeny of the stingless bee genus Melipona (Hymenoptera: Apidae).

Stingless bees (Meliponini) constitute a diverse group of highly eusocial insects that occur throughout tropical regions around the world. The meliponine genus Melipona is restricted to the New World tropics and has over 50 described species. Melipona, like Apis, possesses the remarkable ability to use representational communication to indicate the location of foraging patches. Although Melipona has been the subject of numerous behavioral, ecological, and genetic studies, the evolutionary history of this genus remains largely unexplored. Here, we implement a multigene phylogenetic approach based on nuclear, mitochondrial, and ribosomal loci, coupled with molecular clock methods, to elucidate the phylogenetic relationships and antiquity of subgenera and species of Melipona. Our phylogenetic analysis resolves the relationship among subgenera and tends to agree with morphology-based classification hypotheses. Our molecular clock analysis indicates that the genus Melipona shared a most recent common ancestor at least approximately 14-17 million years (My) ago. These results provide the groundwork for future comparative analyses aimed at understanding the evolution of complex communication mechanisms in eusocial Apidae.

Ecological impatt on native bees by the invasive africanized honey eee / Abejas cleptoparasitas, con énfasis en las abejas hospederas colectoras de aceites (Hymenoptera: Apoidea)

Very little effort has been made to investigate bee population dynamics among intact wilderness areas. The presence of newly-arrived feral Africanized honey bee (AHB), Apis mellifera (Apidae), populations was studied for 10-17 years in areas previously with few or no escaped European apiary honey bees. Here I describe and interpret the major results from studies in three neotropical forests: French Guiana, Panama and Yucatan, Mexico (5° to 19° N. latitude). The exotic Africanized honey bees did not produce a negative effect on native bees, including species that were solitary or highly eusocial. Major differences over time were found in honey bee abundance on flowers near habitat experiencing the greatest degree of disturbance, compared to deep forest areas. At the population level, sampled at nest blocks, or at flower patches, or at light traps, there was no sudden decline in bees after AHB arrival, and relatively steady or sinusoidal population dynamics. However, the native bees shifted their foraging time or floral species. A principal conclusion is that such competition is silent, in floristically rich habitats, because bees compensate behaviorally for competition. Other factors limit their populations.

Pocos estudios han considerado la dinámica de poblaciones de abejas en bosques o hábitats no alterados por el hombre. La presencia de abejas silvestres Africanizadas de Apis mellifera (Apidae) fue estudiado por 10-17 años en áreas previamente sin esta especie. Aquí presento e interpreto resultados de tres bosques neotropicales: Guyana Francesa, Panamá y Yucatán, México (5° a 19° N. latitud). La abeja Africanizada exótica no produjo efecto negativo en las abejas nativas, incluyendo especies altamente sociales y solitarias. Diferencias mayores a través del tiempo fueron encontradas en la abundancia de las abejas de miel en flores cerca de hábitat con mayor grado de disturbio, comparado con el bosque espeso. Al nivel poblacional, muestreado en bloques de nidos trampa, en flores o con trampas ultravioletas de insectos, no hubo disminución pronta de abejas, y sí hubo una población relativamente estable o sinusoidal. Sin embargo, las abejas nativas cambiaron su hora de buscar provisiones o su selección de especies florales. Una conclusión principal es que esta competencia por los recursos es ‘silenciosa';, en las áreas florísticamente ricas estudiadas, porque las mismas abejas compensan con su comportamiento. Otros factores rigen sus poblaciones.

Composition of stingles bee honey: setting quality standards

Se compilaron datos de composición de 152 mieles de abejas sin aguijón (Meliponini) en estudios realizados desde 1964, y se evaluaron para proponer requisitos de calidad para este producto. Dado que la miel de abejas sin aguijón tienen una composición distinta a la de Apis mellifera, algunos parámetros físico-químicos fueron presentados según la especie abejas sin aguijón. El origen entomológico de la miel se asignó a 17 especies de Meliponini de Brasil, una de Costa Rica, seis de México, 27 de Panamá, una de Surinam, dos de Trinidad & Tobago, y siete de Venezuela, mayormente del género Melipona. Los resultados variaron así: humedad (19,9-41,9g/100g), pH (3,15-4,66), acidez libre (5,9-109,0meq/Kg), cenizas (0,01-1,18g/100g), actividad de la diastasa (0,9-23,0DN), conductividad eléctrica (0,49-8,77mS/cm), HMF (0,9-78,4mg/Kg), actividad de la invertasa (19,8-90,1IU), nitrógeno (14,34-144,00mg/100g), azúcares reductores (58,0-75,7g/100g) y sacarosa (1,1-4,8g/100g), El contenido de humedad de las mieles de abejas sin aguijón es generalmente superior al máximo de 20 por ciento establecido para la miel de A. mellifera. Las directrices ofrecidas pueden ayudar a la expansión consistente de la base de datos físico-químicos de miel de abejas sin aguijón, para establecer sus requisitos de calidad en un futuro. El análisis de polen debería dirigirse hacia el reconocimiento de las mieles uniflorales producidas por las abejas sin aguijón, a fin de obtener productos estandarizados según las especies botánicas. Se necesita una campaña de control de calidad de miel tanto para los recolectores de miel de abejas sin aguijón como para los meliponicultores, junto con la armonización de los métodos analíticos

Energy metabolism in orchid bee flight muscles: carbohydrate fuels all.

The widely accepted idea that bees fuel flight through the oxidation of carbohydrate is based on studies of only a few species. We tested this hypothesis as part of our research program to investigate the size-dependence of flight energetics in Panamanian orchid bees. We succeeded in measuring rates of O(2) consumption and CO(2) production in vivo during hovering flight, as well as maximal activities (V(max) values) in vitro of key enzymes in flight muscle energy metabolism in nine species belonging to four genera. Respiratory quotients (ratios of rates of CO(2) production to O(2) consumption) in all nine species are close to 1.0. This indicates that carbohydrate is the main fuel used for flight. Trehalase, glycogen phosphorylase and hexokinase activities are sufficient to account for the glycolytic flux rates estimated from rates of CO(2) production. High activities of other glycolytic enzymes, as well as high activities of mitochondrial oxidative enzymes, are consistent with the estimated rates of carbohydrate-fueled oxidative metabolism. In contrast, hydroxyacylCoA dehydrogenase, an enzyme involved in fatty acid oxidation, was not detectable in any species. Thoracic homogenates displayed ADP-stimulated oxidition of pyruvate + proline, but did not oxidize palmitoyl l-carnitine + proline as substrates. A metabolic map, based on data reported herein and information from the literature, is presented. The evidence available supports the hypothesis that carbohydrate serves as the main fuel for flight in bees.

Allometric scaling of flight energetics in Panamanian orchid bees: a comparative phylogenetic approach.

The relationship between body size and flight energetics was studied in the clade of tropical orchid bees, in order to investigate energy metabolism and evolution. Body mass, which varied from 47 to 1065 mg, was found to strongly affect hovering flight mass-specific metabolic rates, which ranged from 114 ml CO(2) h(-1) g(-1) in small species to 37 ml CO(2) h(-1) g(-1) in large species. Similar variation of wingbeat frequency in hovering flight occurred among small to large species, and ranged from 250 to 86 Hz. The direct relationship between such traits was studied by the comparative method of phylogenetically independent contrasts (PIC), using a new molecular phylogeny generated from the cytochrome b gene partial sequences. We found wingbeat frequency variation is satisfactorily explained by variation in wing loading, after corrections for body mass and phylogeny. The correlated evolution of mass-specific metabolic rate, wingbeat frequency and wing loading was also revealed after correcting for phylogeny and body mass. Further, the effect of body size on flight energetics can be understood in terms of a relationship between wing form and kinematics, which directly influence and explain the scaling of metabolic rate in this group of bees.