Intrinsic colony conditions affect the provisioning and oviposition process in the stingless bee Melipona scutellaris

The cell provisioning and oviposition process (POP) is a unique characteristic of stingless bees (Meliponini), in which coordinated interactions between workers and queen regulate the filling of brood cells with larval resources and subsequent egg laying. Environmental conditions seem to regulate reproduction in stingless bees; however, little is known about how the amount of food affects quantitative sequences of the process. We examined intrinsic variables by comparing three colonies in distinct conditions (strong, intermediate and weak state). We predicted that some of these variables are correlated with temporal events of POP in Melipona scutellaris colonies. The results demonstrated that the strong colony had shorter periods of POP.

Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

Ovariole structure and oogenesis in queens and workers of the stingless bee Melipona quadrifasciata (Hymenoptera: Apidae, Meliponini) kept under different social conditions

The high variability in the reproductive biology of stingless bees makes them very amenable for comparative studies with other eusocial bee taxa. We investigated the structural organization of the ovaries of Melipona quadrifasciata queens and workers kept under different social conditions by analyzing their general histology, mitotic activity, and microfilament organization. The overall dynamics of ovarian activity were similar in the two castes, and at emergence their ovarioles contained a previtellogenic follicle. Stingless bees and honey bees differ in the structural organization in the lower germarium, but they have in common synchronized mitotic activity and putative germ line stem cells in the terminal filament. Unlike honey bees, stingless bee workers lay trophic eggs in addition to reproductive eggs. The overall similarities in oogenesis between the two taxa suggest that the decision to form trophic eggs should only occur in the late stages of oogenesis.

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.

Production of workers, queens and males in Plebeia remota colonies (Hymenoptera, Apidae, Meliponini), a stingless bee with reproductive diapause

Queen, male and worker production was studied during one year in three Plebeia remota colonies from Atlantic Rainforest in Cunha, Sao Paulo State, and two from a subtropical Araucaria forest in Prudentopolis, Parana State. All the colonies were kept in Sao Paulo city during our study. Plebeia remota has reproductive diapause during autumn and winter, which makes its biology of special interest. Brood production begins before spring, renewing the colony cycle. We sampled brood combs monthly in these five colonies. The number of cells in each comb varied significantly with time of the year; the smallest brood combs appear to be a consequence of reduced food availability. However, worker, queen and male frequencies did not differ significantly in time, and this presumably is due to the fact that they all are necessary for the growth, maintenance and reproduction of the colony. Although some molecular, morphological and behavioral differences have been detected in several studies comparing populations from Cunha and from Prudentopolis, we did not find significant differences between the colonies from these two localities in number of brood cells and worker, queen and male production.

Male sleeping aggregations of solitary oil-collecting bees in Brazil (Centridini, Tapinotaspidini, and Tetrapediini; Hymenoptera: Apidae)

Males of solitary bees usually spend the night in clusters on small branches of plants, cavities and flowers. The individuals usually return to the same location each evening during their life, exhibiting site fidelity to a particular plant. We report on the sleeping roosts of the males of some oil-collecting bees of the genera Centris, Paratetrapedia, Lanthanomelissa, Monoeca, and Tetrapedia, as well as the host plants. We discuss the role of the male clusters to the associated plants.

Inferences of evolutionary and ecological events that influenced the population structure of Plebeia remota, a stingless bee from Brazil

The present study characterised the population genetic structure of Plebeia remota through mitochondrial DNA (mtDNA) analysis and evaluated evolutionary and ecological processes that may have contributed to the species current genetic scenario. Seventy feral nests were sampled representing four geographic regions (Cunha, Curitiba, Prudentopolis, and Blumenau). Fifteen composite mtDNA haplotypes were determined and a high genetic structure was detected among all populations. The current population structure may be a result of queen philopatry and vegetation shifts caused by palaeoclimatic changes and uplift of Brazilian coastal ranges. Finally, this study strongly suggests a revision of the taxonomic status of P. remota from the Prudentopolis region.

Phylogeography and historical demography of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera, Apidae): incongruence between morphology and mitochondrial DNA

The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.

Palynological and physicochemical characterization of Apis mellifera L. bee pollen in the Southern region of Brazil

Bee pollen has been used for many years in both traditional medicine and supplementary nutrition, as well as in alternative diets, mainly due to its nutritional properties and health benefits. Bee pollen production is a recent activity in Brazil, having begun in the late 1980s. However, the country has the potential of being a large world producer of high quality pollen, particularly because of the great diversity of tropical flora and the resistance of the Brazilian Apis mellifera bee races. Thirty-six samples of bee pollen from the Southern region of Brazil were analyzed regarding pollen types and physicochemical and nutritional composition. Only one sample was considered monofloral, which was exclusively composed by pollen from the Asteraceae family). The State of Parana showed a greater variety of pollen types, 18 in total, representing 82% of the total number identified in this study. The bee pollen in the States of Rio Grande do Sul and Parana showed a higher number of samples with humidity content above the standard permitted by the Brazilian legislation, i.e. over 4%. The bee pollen was characterized by its high protein content with average values of 20.47%. The analysis regarding humidity, lipids and sugar showed no statistical differences among the samples (p<0.05). The pollen samples had a high concentration of reducible sugars (48%). The predominant minerals in the samples PR, SC and RS were phosphorus (7102.29, 6873.40, 6661.73 mg/kg of pollen), followed by potassium (5383.73, 4997.77, 4773.26 mg/kg of pollen), calcium (1179.05, 961.93, 848.36 mg/kg of pollen) and magnesium (818.02, 679.01, 725.89 mg/kg of pollen). Statistical analysis (Tukey test) demonstrated no significant difference between the contents of calcium, copper, iron, phosphorus and sodium in the pollen samples of the South of Brazil. However, the samples from the State of Parana contained the highest contents of potassium and differed statistically from the samples of the State of Rio Grande do Sul.

Bait and Habitat Preferences, and Temporal Variability of Social Wasps (Hymenoptera: Vespidae) Attracted to Vertebrate Carrion

In addition to feeding on carrion tissues and fluids, social wasps can also prey on immature and adult carrion flies, thereby reducing their populations and retarding the decomposition process of carcasses. In this study, we report on the occurrence and behavior of social wasps attracted to vertebrate carrion. The collections were made monthly from September 2006 to October 2007 in three environments (rural, urban, and forest) in six municipalities of southeast Brazil, using baited bottle traps. We collected Agelaia pallipes (Olivier, 1791) (n = 143), Agelaia vicina (Saussure, 1854) (n = 106), Agelaia multipicta (Haliday, 1836) (n = 18), and Polybia paulista Ihering, 1896 (n = 3). The wasps were observed feeding directly on the baits and preying on adult insects collected in the traps. Bait and habitat associations, temporal variability of social wasps, and possible forensic implications of their actions are discussed.

Nestmate recognition in a stingless bee: does the similarity of chemical cues determine guard acceptance?

The ability to discriminate nestmates from non-nestmates is critical to the maintenance of the integrity of social insect colonies. Guard workers compare the chemical cues of an incoming individual with their internal template to determine whether the entrant belongs to their colony. In contrast to honeybees, Apis mellifera, stingless bees have singly mated queens and, therefore, are expected to have a higher chemical homogeneity in their colonies. We tested whether aggressive behaviour of Frieseomelitta varia guards towards nestmate and non-nestmate foragers reflects chemical similarities and dissimilarities, respectively, of cuticular hydrocarbon profiles. We also introduced individuals of Lestrimelitta limao, an obligatory robber species, to test the ability of guards to react effectively to intruders from other taxa. We verified that foraging nestmates were almost invariably accepted, while heterospecific and conspecific non-nestmates were rejected at relatively high rates. However, non-nestmate individuals with higher chemical profile similarity were likely to be accepted by guards. We conclude that guards compare the chemical cuticular blend of incoming individuals and make acceptance decisions according to the similarity of the compounds between the colonies. (c) 2007 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae)

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORE of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pl of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.