Behavioral suites mediate group-level foraging dynamics in communities of tropical stingless bees

Competition for floral resources is a key force shaping pollinator communities, particularly among social bees. The ability of social bees to recruit nestmates for group foraging is hypothesized to be a major factor in their ability to dominate rich resources such as mass-flowering trees. We tested the role of group foraging in attaining dominance by stingless bees, eusocial tropical pollinators that exhibit high diversity in foraging strategies. We provide the first experimental evidence that meliponine group foraging strategies, large colony sizes and aggressive behavior form a suite of traits that enable colonies to improve dominance of rich resources. Using a diverse assemblage of Brazilian stingless bee species and an array of artificial “flowers” that provided a sucrose reward, we compared species’ dominance and visitation under unrestricted foraging conditions and with experimental removal of group-foraging species. Dominance does not vary with individual body size, but rather with foraging group size. Species that recruit larger numbers of nestmates (Scaptotrigona aff. depilis, Trigona hyalinata, Trigona spinipes) dominated both numerically (high local abundance) and behaviorally (controlling feeders). Removal of group-foraging species increased feeding opportunities for solitary foragers (Frieseomelitta varia, Melipona quadrifasciata and Nannotrigona testaceicornis). Trigona hyalinata always dominated under unrestricted conditions. When this species was removed, T. spinipes or S. aff. depilis controlled feeders and limited visitation by solitary-foraging species. Because bee foraging patterns determine plant pollination success, understanding the forces that shape these patterns is crucial to ensuring pollination of both crops and natural areas in the face of current pollinator declines.     

Intra- and interspecific nestmate recognition inMelipona workers (Hymenoptera: Apidae)

The possible significance of nestmate recognition in prevention of robbing and parasitism in three species of stingless bees was assessed. Nestmate discrimination abilities vary among them; Melipona quadrifasciataworkers attacked 74% of nonnestmate conspecifics that were encountered, while M. scutellarisand M. rufiventriswere less discriminating, attacking only 14 and 60% of non-nestmates, respectively. In tests of interspecific interactions, M. quadrifasciataand M. scutellariswere the least mutually tolerant of all species pairs tested. Tests with Apis melliferashowed a high degree of intolerance by two of the three Meliponaspecies.     

Variation in the ability to communicate three-dimensional resource location by stingless bees from different habitats

We evaluated the ability of two Brazilian stingless bee species, Melipona mandacaia andM. bicolor , to recruit nestmates to a specific three-dimensional location. We used experimental feeder arrays and provide the first detailed evidence demonstrating that recruitment communication in Melipona can lead to large, rapid and highly significant increases in the number of nestmates visiting a specific location. Melipona bicolor andM. mandacaia foragers both recruited nestmates to the correct distance and direction, but differed in their ability to recruit nestmates to the correct height. These differences may relate to their respective habitats. Melipona mandacaia inhabits semi-arid areas of Caatinga where most food sources occur close to the ground, and its foragers evidently cannot recruit nestmates to the correct height. Melipona bicolor, an Atlantic rainforest species, evidently does not communicate height when the food source is at ground level, but can communicate height when the food source is at the forest canopy level (12 m high), where major food sources occur. Species-specific variation in three-dimensional location communication is intriguing because it suggests that Melipona may be a good model for studying the evolution of recruitment communication systems in highly social bees.     

Spectral sensitivity of photoreceptors in the compound eye of stingless tropical bees

The spectral sensitivity of photoreceptors of the tropical bees Melipona quadrifasciata, M. marginata, and Trigona spinipes were measured on-line with a rapid constant response amplitude method. The results indicate that although these bees forage in different habitats they have the same set of photoreceptors with sensitivity maxima in ultra-violet, blue and green.     

Partitioning of pollen resources by two stingless bee species in the north Bahia,Brazil

Analysing the pollen stored by stingless bees allows identification of the diversity of flowering plants visited by these bees while collecting resources during a given period. However, few studies have focused on investigating the sharing of resources between species of native bees that coexist in transitional plant formations, which support conservation efforts and increase colony productivity. This study aims to describe the partitioning of pollen resources among colonies of two species of stingless bees in the state of the Bahia (Brazil) through the analysis of stored pollen. The study was conducted in neighbouring vegetation zones of the relevant species Melipona quadrifasciata anthidioides and Melipona scutellaris by collecting monthly pollen samples stored by three colonies of each species over one year. The collected samples were treated using the acetolysis technique, and the partitioning of pollen resources between the two species was investigated through quali-quantitative analysis of the treated pollen. The results show that among the 16 pollen types belonging to a particular species or group identified in the Melipona quadrifasciata anthidioides samples and the 18 identified in the Melipona scutellaris samples, 13 were shared by both species. The two most common pollen types stored by both species were from Mimosa arenosa (Fabaceae, Mimosoidae) and Eucalyptus sp. (Myrtaceae). These results demonstrate the niche similarity of pollen pasture between these two species and suggest that conservation efforts for these bee species should focus on the plant families most visited by bees.     

Ecdysteroid titers in pupae of highly social bees relate to distinct modes of caste development

Modifications in endocrine programs are common mechanisms that generate alternative phenotypes. In order to understand how such changes may have evolved, we analyzed the pupal ecdysteroid titers in two closely related, highly social bees: the honey bee, Apis mellifera, and a stingless bee, Melipona quadrifasciata. In both species, the ecdysteroid titers in queens reached their peak levels earlier than in workers. Titer levels at peak maxima did not differ for the honey bee castes, but in Melipona they were twofold higher in queens than in workers. During the second half of pupal development, when the ecdysteroid titers decrease and the cuticle progressively melanizes, the titer in honey bee queens remained higher than in workers, while the reverse situation was observed in Melipona. Application of the juvenile hormone analog Pyriproxyfen^® to spinning-stage larvae of Melipona induced queen development. Endocrinologically this was manifest in a queen-like profile of the pupal ecdysteroid titer. Comparing these data with previous results on preimaginal hormone titers in another stingless bee, we conclude that the timing and height of the pupal ecdysteroid peak may depend on the nature of the specific stimuli that initially trigger diverging queen/worker development. In contrast, the interspecific differences in the late pupal ecdysteroid titer profiles mainly seem to be related to caste-specific programs in tissue differentiation, including cuticle pigmentation.     

Deceptive orchids with Meliponini as pollinators

Visitation of orchids by Meliponini (stingless bees) is confirmed only in 13Melipona, Partamona andTrigona, forXylobium andMaxillaria, with the addition ofTrigona fulviventris visitingIonopsis. Some bees evinced multiple floral visitation by carrying several stipes and viscidia from pollinaria, thus may cause seed set. None foraged pseudopollen, nor is collection of this substance by bees verified. Meliponine-visited orchids had pollinia in quartets with emplacement on the bee's scutellum, possibly devices for pollinia survival on a social bee passing through its nest. Further, orchids produced no nectar, but bees repeatedly came to flowers. A testable basis for the orchid-meliponine relationship is mimicry of rewarding resources, or bee pheromone mimicry, recently documented for some honey bees. Meliponine pheromone analogs (nerol and 2-heptanol) are here noted forMaxillaria, but lack of foraging with pheromones byMelipona suggests multiple avenues of mimicry by orchids, including alarm pheromone and carrion mimicry.     

Effectiveness of recruitment behavior in stingless bees (Apidae,Meliponini)

Summary We examined the ability of stingless bees to recruit nest mates to a food source (i) in group foraging species laying pheromone trails from the food to the nest (Trigona recursa Smith, T. hypogea Silvestri, Scaptotrigona depilis Moure), (ii) in solitary foraging species with possible but still doubtful communication of food location inside the nest (Melipona seminigra Friese, M. favosa orbignyi Guérin), and (iii) in species with a less precise (Nannotrigona testaceicornis Lep., Tetragona clavipes Fab.) or no communication (Frieseomelitta varia Lep.). The bees were allowed to collect food (sugar solution or liver in the necrophageous species) ad libitum and the forager number to accumulate, as it would do under normal unrestrained conditions. The median number of bees collecting differed considerably among the species (1.0–1436.5). It was highest in the species employing scent trails. The time course of recruitment was characteristic for most of the species and largely independent of the number of foragers involved. The two Melipona species recruited other bees significantly faster than T. recursa, S. depilis, and N. testaceicornis during the first 10 to 30 minutes of an experiment. In species laying a scent trail to guide nestmates to a food source the first recruits appeared with a delay of several minutes followed by a quick increase in forager number. The median time required to recruit all foragers available differed among the species between 95.0 and 240.0 min. These differences can at least partly be explained by differences in the recruitment mechanisms and do not simply follow from differences in colony biomass.     

Effect of group size on the aggression strategy of an extirpating stingless bee, Trigona spinipes

Summary. Group aggression influences communication and defense strategies in many social insect communities. Such aggression plays a particularly significant role in the lives of stingless bees, important native Neotropical pollinators, in which the battle for food resources can be deadly and critical to colony survival. However, the effects of group size on individual aggression levels and the spatio-temporal aggression strategy of communal aggressors have not been fully explored. We therefore investigated how group size affects the aggression levels and the spatio-temporal attack strategy (which body parts, and the amount of time spent in attacking each part) in close combats between Trigona spinipes foragers and a natural competitor, Melipona rufiventris. In all trials, T. spinipes foragers competitively excluded M. rufiventris foragers from nearby feeders, exhibiting four levels of aggressive behavior ranging from threat displays to prolonged grappling and decapitation. Surprisingly, aggression levels and spatial strategy corresponded to the size of group attacks. Larger groups of attackers used individually lower aggression levels than small groups of attackers. Smaller groups also attacked appendages linked to escape (legs and wings) with greater frequency than larger groups, which focused on vital central body areas (abdomen, thorax, and head). Increased aggression corresponded to increased risks for attackers and the attacked. All combatants engaging at the highest level of aggression died (100% mortality). Thus the dominance style of T. spinipes may minimize attack risk and maximize victim harm with finely tuned hostility.Received 22 May 2004; revised 18 August 2004; accepted 26 October 2004.     

A new species of Syntretus Foerster (Hymenoptera: Braconidae: Euphorinae), a parasitoid of the stingless bee Trigona carbonaria Smith (Hymenoptera: Apidae: Meliponinae)

A new species of euphorine braconid, Syntretus trigonaphagus sp. nov., is described and illustrated. This is the first Australian Syntretus species to be described and the first record of braconids parasitising Trigona Jurine stingless bees. Egg-laying by adults and emergence of larvae from the host Trigona carbonaria is detailed. A 12S ribosomal rRNA gene fragment was sequenced to confirm the association of larvae and adults of the wasp parasitoid.     

Intracellular melanization of the larvae of Dirofilaria immitis in the malpighian tubules of the mosquito, Aedes sollicitans

Frequent melanization of larvae of the nematode Dirofilaria immitis parasitizing the Malpighian tubules of the mosquito, Aedes sollicitans, has been observed. Melanized and nonmelanized larvae in the Malpighian tubules were examined using light and electron microscopy. The results indicate that the pattern of melanin deposition and the ultrastructural characteristics of the pigment around the worms are identical to that observed on nematodes which have undergone humoral melanization in other dipteran insects. In the Malpighian tubules, no contact between the intracellular melanized nematodes and the hemolymph or hemocytes was observed. The results suggest that the Malpighian tubules of this species of mosquito are capable of inducing a melanotic response to invading nematode parasites. It is proposed that this is an example of “humoral” melanization at an intracellular site.     

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.     

The effect of infection with Dirofilaria immitis (dog heartworm) on fluid secretion rates in the Malpighian tubules of the mosquitoes Aedes taeniorhynchus and Anopheles quadrimaculatus

Dirofilaria immitis, the parasitic nematode causing the disease dog heartworm, is transmitted by female mosquitoes. During their development, larval nematodes reside in the cells of the Malpighian tubules of these mosquitoes for approx 13 days. We have examined the effect of the presence of these large intracellular parasites on the main physiological function of the Malpighian tubules, i.e. fluid secretion. Rates of fluid secretion were examined in vitro using both normal and infected tubules of the mosquito species Aedes taeniorhynchus and Anopheles quadrimaculatus. Tubules of A. quadrimaculatus show changes in trasport rate during the reproductive cycle. Those of A. taeniorhynchus do not. Infection with larvae of D. immitis had no effect on the rate of fluid secretion in tubules of A. quadrimaculatus. In A. taeniorhynchus by contrast, the tubules show decline in transport with time following infection. The reduction in transport capacity is proportional to the number of worms infecting the tubule. The present paper and separate ultrastructural studies demonstrate that parallel changes in microvillar ultrastructure and epithelial transport rates occur in response to infection by the parasite. In both species examined, the survival of the mosquitoes and their vector potential are determined by factors other than the transport capacities of the infected Malpighian tubules.     

Extinction of anciently associated gut bacterial symbionts in a clade of stingless bees

Animal-microbe symbioses are often stable for millions of years. An example is the clade consisting of social corbiculate bees—honeybees, bumblebees, and stingless bees—in which a shared ancestor acquired specialized gut bacteria that subsequently diversified with hosts. This model may be incomplete, however, as few microbiomes have been characterized for stingless bees, which are diverse and ecologically dominant pollinators in the tropics. We surveyed gut microbiomes of Brazilian stingless bees, focusing on the genus Melipona, for which we sampled multiple species and biomes. Strikingly, Melipona lacks Snodgrassella and Gilliamella, bacterial symbionts ubiquitous in other social corbiculate bees. Instead, Melipona species harbor more environmental bacteria and bee-specific Starmerella yeasts. Loss of Snodgrassella and Gilliamella may stem from ecological shifts in Melipona or the acquisition of new symbionts as functional replacements. Our findings demonstrate the value of broadly sampling microbiome biodiversity and show that even ancient symbioses can be lost.Subject terms: Symbiosis, Microbiome, Microbial ecology, Metagenomics, Microbial ecology     

Microsatellite loci for the stingless bee Melipona rufiventris (Hymenoptera: Apidae)

Eight microsatellite primers were developed from ISSR (intersimple sequence repeats) markers for the stingless bee Melipona rufiventris. These primers were tested in 20 M. rufiventris workers, representing a single population from Minas Gerais state. The number of alleles per locus ranged from 2 to 5 (mean = 2.63) and the observed and expected heterozygosity values ranged from 0.00 to 0.44 (mean = 0.20) and from 0.05 to 0.68 (mean = 0.31), respectively. Several loci were also polymorphic in M. quadrifasciata, M. bicolor, M. mandacaia and Partamona helleri and should prove useful in population studies of other stingless bees.     

Expression of methyl farnesoate epoxidase (mfe) and juvenile hormone esterase (jhe) genes and their relation to social organization in the stingless bee Melipona interrupta (Hymenoptera: Apidae)

Social organization in highly eusocial bees relies upon two important processes: caste differentiation in female larvae, and age polyethism in adult workers. Juvenile Hormone (JH) is a key regulator of both processes. Here we investigated the expression of two genes involved in JH metabolism - mfe (biosynthesis) and jhe (degradation) - in the context of social organization in the stingless bee Melipona interrupta. We found evidence that the expression of mfe and jhe genes is related to changes in JH levels during late larval development, where caste determination occurs. Also, both mfe and jhe were upregulated when workers engage in intranidal tasks, but only jhe expression was downregulated at the transition from nursing to foraging activities. This relation is different than expected, considering recent reports of lower JH levels in foragers than nurses in the closely related species Melipona scutellaris. Our findings suggest that highly eusocial bees have different mechanisms to regulate JH and, thus, to maintain their level of social organization.     

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca²⁺ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca²⁺ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca²⁺ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca²⁺ regulation. Basolateral Ca²⁺ transport across distal tubules of Drosophila varied throughout the life cycle; Ca²⁺ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca²⁺ uptake and Ca²⁺ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca²⁺ transport is bidirectional. Ca²⁺ uptake across the distal segment of tubules of young adults and Ca²⁺ release across the distal segment of tubules of older adults was also suggestive of reversible Ca²⁺ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca²⁺ sequestration during periods of high dietary calcium intake and passive Ca²⁺ release during periods of calcium deficiency.     

A molecular phylogeny of the Old World stingless bees (Hymenoptera: Apidae: Meliponini) and the non-monophyly of the large genus Trigona

Abstract.  We examined the inter- and infrageneric relationships of Old World Meliponini with a near-complete sampling of supra-specific taxa. DNA sequences for the taxa were collected from four genes (mitochondrial 16S rRNA, nuclear long-wavelength rhodopsin copy 1 (opsin), elongation factor-1α copy F2 and arginine kinase). Additional sampling of New World taxa indicated that Trigona sensu lato is not monophyletic: Trigona from the Indo-Malayan/Australasian Regions forms a large clade distantly related to the Neotropical Trigona . A separate clade comprises the Afrotropical meliponines, and includes the 'minute' species found in the Afrotropical, Indo-Malayan and Australasian Regions. The Neotropical genus Melipona , by contrast with previous investigations, is not the sister lineage to the remaining stingless bees, but falls within the strongly supported Neotropical clade. These results constitute the framework for a revised classification and ongoing biological investigations of Meliponini. A single taxonomic change, Heterotrigona bakeri stat.n. , is proposed on the basis of sequence divergence.     

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.     

Transepithelial transport of salicylate by the Malpighian tubules of insects from different orders

The organic anion salicylate is a plant secondary metabolite that protects plants against phytophagous insects. In this study, a combination of salicylate-selective microelectrodes and a radioisotope tracer technique was used to study the transepithelial transport of salicylate by the Malpighian tubules of 10 species of insects from five orders. Our results show that salicylate is transported into the lumen of the Malpighian tubules in all the species evaluated, except Rhodnius prolixus. The transepithelial transport of salicylate by the Malpighian tubules of Drosophila simulans, Drosophila erecta, Drosophila sechellia, and Acheta domesticus was saturable, Na⁺-dependent and inhibited by α-cyano-4-hydroxycinnamic acid. This transport system resembles that previously found in tubules of Drosophila melanogaster. In contrast, transepithelial transport of salicylate by Malpighian tubules of Tenebrio molitor, Plagiodera versicolora, Aedes aegypti, and Trichoplusia ni was unaffected by Na⁺-free bathing saline. The presence of both salicylate and salicylate metabolites in the secreted fluid samples from the Malpighian tubules of A. domesticus, R. prolixus, T. molitor, and T. ni indicates that insect Malpighian tubules may both transport and metabolize salicylate. The highest capacities to rid the hemolymph of salicylate were found in T. molitor, P. versicolora and Drosphila spp. Our results suggest that transport of salicylate by the Malpighian tubules might contribute to elimination of this organic anion from the hemolymph, particularly in some species that encounter high levels of organic anion in the diet.