Fungi microbiot of Melipona subnitida Ducke (Hymenoptera: Apidae)

This paper reports on the occurrence of filamentous fungi found on the surface of the bees body from the specie Melipona subnitida Ducke that inhabits rocky places on the semi-arid Northeastern Brazil. Bees with cause of natural death were collected of beehives belonging to the Centro de Multiplica??o de Animais Silvestres of the Universidade Federal Rural do Semi-Arido. We found the fungi: Aspergillus sp. 6 (37.5%); Aspergillus niger 2 (12.5%); Penicilium sp. 2 (12.5%); Aspergillus terreus 1 (6.3%); Curvularia sp. 1 (6.35%); Monilia sp. 1 (6.3%); Nigrospora sp. 1 (6.3%); Cladosporium sp. 1 (6.3%); Tricoderma sp. 1 (6.3%).     

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.     

Genetic divergence between Melipona quadrifasciata Lepeletier (Hymenoptera,Apidae) populations

Melipona quadrifasciata is a stingless bee widely found throughout the Brazilian territory, with two recognized subspecies, M. quadrifasciata anthidioides, that exhibits interrupted metasomal stripes, and M. quadrifasciata quadrifasciata, with continuous metasomal stripes. This study aimed to estimate the genetic variability of these subspecies. For this purpose, 127 colonies from 15 Brazilian localities were analyzed, using nine species-specific microsatellite primers. At these loci, the number of alleles ranged from three to 15 (mean: 7.2), and the observed heterozygosity (H_o) ranged from 0.03–0.21, while the expected heterozygosity (H_e) ranged from 0.23–0.47. The genetic distances among populations ranged from 0.03–0.45. The F_ST multilocus value (0.23) indicated that the populations sampled were structured, and the clustering analysis showed the formation of two subgroups and two more distant populations. The first group contained the subspecies M. quadrifasciata quadrifasciata, and the other, the subspecies M. quadrifasciata anthidioides and the two M. quadrifasciata populations with continuous metasomal stripes from northern Minas Gerais. These results confirmed that the yellow metasomal stripes alone are not a good means for correctly identifying the different subspecies of M. quadrifasciata.     

Age polyethism in Plebeia emerina (Friese) (Hymenoptera: Apidae) colonies related to propolis handling

Stingless bees collect plant resins and make it into propolis, although they have a wider range of use for this material than do honey bees (Apis spp.). Plebeia spp. workers employ propolis mixed with wax (cerumen) for constructing and sealing nest structures, while they use viscous (sticky) propolis for defense by applying it onto their enemies. Isolated viscous propolis deposits are permanently maintained at the interior of their colonies, as also seen in other Meliponini species. Newly-emerged Plebeia emerina (Friese) workers were observed stuck to and unable to escape these viscous propolis stores. We examined the division of labor involved in propolis manipulation, by observing marked bees of known age in four colonies of P. emerina from southern Brazil. Activities on brood combs, the nest involucrum and food pots were observed from the first day of life of the marked bees. However, work on viscous propolis deposits did not begin until the 13th day of age and continued until the 56th day (maximum lifespan in our sample). Although worker bees begin to manipulate cerumen early, they seem to be unable to handle viscous propolis till they become older.     

Harvesting dynamics of pollen sources by Melipona scutellaris Latreille (Hymenoptera: Apidae): a comparative analysis with Apis mellifera L. (Hymenoptera: Apidae) in the Atlantic Forest Domain

Generalist foraging behavior among stingless bees is accepted but untested, as well as the subsidiary hypothesis of floral preferences in the genus Melipona. Here we analyzed those hypotheses comparing the use of floral sources of pollen, through paired analyses of pollen samples from different colonies of Melipona scutellaris Latreille, in three areas of the Atlantic Forest Domain, in Northern Brazil. From August, 2004 to January, 2005, monthly samples of pollen were collected at the entrance of twelve colonies of M. scutellaris. In two places, four colonies of M. scutellaris were compared with four colonies of africanized Apis mellifera L. The main pollen sources chosen by the colonies of M. scutellaris were flowers of the following plant families, in decreasing order of importance: Myrtaceae, Mimosaceae, Anacardiaceae, Sapindaceae, Caesalpiniaceae and Fabaceae. Productive pollen sources of Asteraceae, Arecaceae e Rubiaceae were heavily exploited by the colonies of A. mellifera and discharged by the colonies of M.scutellaris. Often, both species shared the main productive pollen sources, as the flowers of Myrtaceae and Mimosaceae. On the other hand, no pollen sources were heavily exploited altogether by both of them, as a rule. In different places and periods, the colonies of M. scutellaris presented high intra-specific similarity and they formed distinct clusters apart from A. mellifera. Therefore, the selection of pollen sources by colonies was species dependent. The paired comparisons refute the hypothesis of random flower exploitation by colonies and give support to the subsidiary hypothesis of selectivity or floral preferences by M.scutellaris.     

Floral Lipid Chemistry of Byrsonima crassifolia (Malpigheaceae) and a Use of Floral Lipids by Centris Bees (Hymenoptera: Apidae)

Byrsonima rrassifolia (L.), a Neotropical malpighiaceous tree or treelet, has flowers that produce abundant lipids instead of nectar. Several species of Centris bees collect this oil. The floral oil shows variation between trees and can be separated into two types. One type is most common (11 of 14 samples) and consists of mono- and di-glycerides, some free fatty acids, a small amount of tri-glycerides and a trace of carbohydrate. The composition of the glycerides is predominantly (53–63%) esters of unsaturated 18C–22C fatty acids with the rest made up of 14C–18C saturated fatty acids. The second type of floral oil is similar to type one except that the samples contain large amounts of an unknown lipid more polar than the standards. Oil collected from the scopae of two representative Centris species, C. adanae and C. flavofasciata, was the same as the type one oil collected from B. rrassifolia floral elaiophores. The liquid provisions from the cells of several groundnesting Centris species was also found to be identical to B. crassifolia type one floral elaiophore oil with the exception of several minor ninhydrin positive compounds which may have been derived from the pollen which makes up part of the larval food provisions.     

Pollen analysis of honeys of Melipona (Michmelia) seminigra merrillae and Melipona (Melikerria) interrupta (Hymenoptera: Apidae) bred in Central Amazon,Brazil

The pollen present in honey in colonies of Melipona seminigra merrillae and Melipona interrupta bred in Manaus was analysed. Between August and October 2012, honey samples were collected from the Sucupira meliponary, located in Manaus, Amazonas, Brazil. We identified a total of 70 pollen types belonging to 35 botanical families. In the samples from Melipona seminigra merrillae, the most represented pollen types were: Miconia-type (Melastomataceae) was the dominant pollen (DP) in August (51.19%), September (76.83%) and October (58.33%); Triplaris-type (Polygonaceae) was an accessory pollen (AP) in August (20.5%); and the remaining pollen types were classified as isolated pollen (IP), with Talisia macrophylla (Sapindaceae) exhibiting the highest percentage in August (12.92%). For Melipona interrupta, the most frequent pollen types were as follows: Miconia-type was the DP in August (59.33%) and October (61.33%) and an AP in September (37.5%); and Triplaris-type was an AP in August (35.83%), September (34.16%) and October (23.33%). The diversity of pollen types was not significantly different between the bees in the months evaluated. However, there was a large significant niche overlap in the months studied, August (O_ik = 0.95), October (O_ik = 0.89) and September (O_ik = 0.69), revealing that of the 70 pollen types found in the samples, 22 were shared by the two bee species in large proportions, 28 were exploited exclusively by Melipona seminigra merrillae and 20 were collected only by Melipona interrupta.     

Pollen types from colonies of Melipona scutellaris Latreille, 1811 (Hymenoptera: Apidae) established in a coffee plantation

Melipona scutellaris is a eusocial bee with potential to be used as a crop pollinator. This study aimed to identify pollen types used by Melipona scutellaris through the analysis of pollen loads and honey samples in colonies established in a coffee plantation. We analysed 117 pollen loads and pollen content of 15 honey samples. In the pollen loads, we identified 40 pollen types. Poincianella pyramidalis, Coffea arabica, Commelina villosa, Mimosa tenuiflora, Myrcia, Senna macranthera, Solanum and Syagrus coronata stood out as predominant pollen. We found a negative correlation between the abundances of the pollen types Coffea arabica and Mimosa tenuiflora during coffee flowering. In the honey samples, we recorded 33 pollen types. Eucalyptus and Machaerium occurred in frequencies of between 16 and 45%. Although Melipona scutellaris gathers resources from several plant species, its diet is based on a relatively narrow subset of plants.     

Inheritance pattern of RAPD markers in Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae).

Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F(1) and BC(1) were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.     

Isoenzyme Variation in Melipona rufiventris (Hymenoptera: Apidae, Meliponina) in Minas Gerais State, Brazil

The stingless bee Melipona rufiventris is an important pollinator in several Brazilian ecosystems. Originally widely distributed in Minas Gerais (MG) state, this species is becoming very rare. Therefore this species was included in the endangered species list of MG. We used isoenzyme data for a better understanding of the genetic structure of several M. rufiventris colonies. Samples of 35 colonies were collected from 12 localities and evaluated by nine enzymatic systems, which yielded 17 loci. M. rufiventris genetic variation was found to be low, typical of an endangered species. The proportion of polymorphic loci was 5.88% in both ecosystems. Only Est-4 was polymorphic in colonies from the Forest and Mdh-1 in colonies from the Cerrado. The expected heterozygosity ranged from 0.0068 in the Cerrado to 0.0078 in the Forest. Despite this, enzyme electrophoretic analyses provided a good idea of the diversity between samples from Cerrado and Forest which reinforce the existence of two different forms of M. rufiventris in MG, one present in the Cerrado and the other in Forest. This information is of great importance for the conservation of, M. rufiventris in MG.     

The Panama microplate,island studies and relictual species of Melipona (Melikerria) (Hymenoptera: Apidae: Meliponini)

The endemic stingless honey‐making bee Melipona (Melikerria) insularis sp.n. on Coiba and Ranchería Islands in Pacific Panama is described, together with the proposed sister species, M. ambigua sp.n. from northeast Colombia. The Coiba Island group and Panama mainland were surveyed, yielding one meliponine endemic (M. insularis sp.n. ) and six meliponine genera and species. The poor Coiba fauna of amphibians and birds corresponds to the poor social bee fauna and suggests habitat barriers generally precluded recolonization from the mainland during glacial periods. Many animals became extinct, yet some remain as relicts. Melipona insularis sp.n. was isolated on accreted terranes of Coiba rainforest in the Panama microplate. Morphology suggests that M. insularis sp.n. is not a direct descendant of the San Blas‐E. Panama endemic Melikerria, M. triplaridis. A phylogenetic hypothesis corroborates disjunct distributions. Rainforest endemics such as Peltogyne purpurea (Fabaceae) and Ptilotrigona occidentalis (Apidae, Meliponini) also occur as relictual, disjunct populations in Central and South America. These may have been isolated before accelerated biotic exchange began 2.4 Ma. Our work supports the geological findings of both a volcanic arc and the San Blas massif providing a substantial bridge for Melikerria from Colombia and Panama in Eocene to Miocene times. We suggest there have been taxon cycles permitting recolonization during glaciations, whereby colonies of M. insularis sp.n. were able to recolonize Ranchería, a 250 ha island, 2 km from Coiba. However, rafting colonies nesting in trees, carried on vegetation mats, may have produced founding populations of Melipona in Central America and on oceanic islands such as Coiba.     

Evaluation of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera: Apidae) as pollinator of greenhouse tomatoes

The Neotropical stingless bee Melipona quadrifasciata Lepeletier was evaluated for pollinating tomatoes (variety Rodas; long-life hybrid) in greenhouses under plastic and with a hydroponic system and "organic concepts" in Minas Gerais State, Brazil. Flowers not pollinated did not set any fruit. Pollination by bees plus manual pollination did not differ from either bee or manual pollination. Maximum fruit diameter, fruit height, and roundness (quotient between maximum fruit diameter and fruit height) were not significantly different between treatments, but fruit visited by M. quadrifasciata had 10.8% less seeds (dry mass) than manual pollination. This apparently low efficiency of M. quadrifasciata pollination was attributed to the overlap of only 30 min between highest bee foraging activity and highest flower stigma receptivity. Thus, it was concluded that M. quadrifasciata is a feasible pollinator of greenhouse tomatoes because of 1) the observed increase in fruit quality with lower mechanical injury than traditional manual pollination, 2) no significant decrease in fruit size, and 3) high price of such product in the market. Some considerations for sustainable use of M. quadrifasciata as greenhouse pollinator are presented. Although techniques for keeping captive colonies of M. quadrifasciata are currently available, the sole current method for acquiring new colonies is removing them from the forest, and if demand was created for large numbers of colonies for commercial use, techniques for captive rearing must be developed to prevent serious declines in wild populations.     

Sperm utilisation by Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) queens subjected to multiple mating

Summary In most Hymenoptera species the queen mates once but in a small number of species, multiple matings can occur normally. So, in this study, physogastric M. quadrifasciata queens were mated with a second male to investigate how these queens, naturally inseminated and laying eggs, use spermatozoa stored in their spermatheca, when they are mated with a second male. Results demonstrate that spermatozoa of different males mix in the spermatheca of M. quadrifasciata queens and that there is a gradual increase in the utilisation of spermatozoa of the second male, which could be explained by a competition among spermatozoa of different drones over the way in which spermatozoa are stored in the spermatheca.     

Histochemistry of the cuticle from proventriculus in stingless bee, Melipona quadrifasciata anthidioides (Hymenoptera, Apidae)

Histochemical study of the proventriculus from stingless bee Melipona quadrifasciata anthidioides revealed for the first time the presence of cysteine-rich proteins in the cuticle that covers the bulb of the proventriculus and its associated hair-like process. The presence of tyrosine-rich proteins was observed in the same structure, but not in hair-like projections. The cuticle of the crop and a small portion of the proventriculus contained no acid carbohydrates or fats and small amounts of neutral carbohydrates. These results are discussed in relation to different composition of insect cuticle depending on its different functions.