Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.     

The influence of swimming demand on phenotypic plasticity and morphological integration: a comparison of two polymorphic charr species

In northern freshwater lakes, several fish species have populations composed of discrete morphs, usually involving a divergence between benthic and limnetic morphs. Although it has been suggested that swimming demand plays an important role in morphological differentiation, thus influencing habitat selection, it is unclear how it affects reaction norms, patterns in character correlation, and levels of morphological integration. We examined whether swimming demand could induce morphological plasticity in the directions expected under divergent habitat selection, and evaluated its influence on the morphological integration in Arctic charr (Salvelinus alpinus) and brook charr (S. fontinalis), two congeneric species exhibiting conspicuous and subtle resource polymorphism, respectively. We found that changes in morphology were induced by differential swimming demands in both species. The length of the pectoral fin was the character that responded most strongly according to the predicted morphological expectations under divergent habitat selection. High levels of morphological plasticity, relatively low levels of integration, and differences found in the morphological correlation structure among water velocity treatments suggest that constraints on morphological change are unlikely in either species, thus allowing great potential for phenotypic flexibility in both species. The magnitude of character integration, however, was larger for Arctic charr than for brook charr. This latter result is discussed in the light of the differences in the level of polymorphism between the two species in the wild. The results of the present study indicate that swimming demand alone may not be sufficient to generate the polymorphism encountered in nature. Given that both diet and swimming demands can induce morphological changes, it would be important to conduct experiments targeting the interaction between the morphological modules related to trophic and swimming demands.     

Anatomy and ultrastructure of the salivary gland in the thorax of the honeybee worker,Apis mellifera (Insecta,Hymenoptera)

Summary The thoracic salivary gland of the worker honeybee was investigated by dissection, light microscopy, scanning electron microscopy, and transmission electron microscopy. The glands are paired and each lateral half consists of two parts, a smaller external and a larger internal lobe. The lobes are composed of densely packed secretory tubes and ducts, the tubes of which often show ramifications. A reservoir is packed within the anterior medial part of the gland. The secretory tubes are composed of two types of cells, secretory cells, which are most frequent, and parietal cells. Secretory cells are characterized by a basal labyrinth, abundant rough endoplasmic reticulum, dark secretory vesicles, light vesicles of different sizes, and apical microvilli. Parietal cells are smaller and have a characteristically lobed nucleus and no secretory vesicles. Between the cells there are intercellular canaliculi. In the center of each tube there is an extracellular space with a central cuticular channel. The abundance of rough endoplasmic reticulum and the rare occurrence of smooth endoplasmic reticulum implies a saliva with proteins but rarely with pheromones. Between the secretory tubes there are frequently neuronal profiles which are partly in contact with the secretory cells. Thus a nervous control of this gland is, in contrast to previous investigations, clearly demonstrated. The axonal endings contain dark neurosecretory vesicles as well as light synaptic vesicles. Large parts of the glands are surrounded by a thin tissue sheath which has a smooth surface towards the secretory tubes and shows irregular protrusions towards the outer side. This sheath is considered to be a tracheal air sac, and due to its large extension is probably of importance for the hemolymph flow in the thorax.     

Assessment of genetic variation in Apis mellifera jemenitica (Hymenoptera: Apidae) using Cytochrome Oxidase I gene sequences

The Arabian Honeybee Apis mellifera jemenitica is endemic to the Arabian Peninsula. It is highly adapted to temperature extremes and drought dominating the region. In this study, the mitochondrial Cytochrome Oxidase I (COI) was analyzed in 133 specimens of A. m. jemenitica from eight localities along the Red Sea cost of Saudi Arabia. Results revealed 33 synonymous, and 6 non-synonymous mutations within the COI sequences, resulting in change of 4 amino acids. Phylogenetic analysis based on either type of mutations revealed two main haplogroups accounting for 94% of the samples. In total Eighteen new haplotypes were identified and uploaded in the genebank, Fourteen of them are restricted to one/both haplogroups. All haplotypes identified in this study clustered with reference COI sequences of the sub-lineag Z (African Lineage). However one Haplotype (MW428270) represents high COI variability compared to other haplotypes and may resemble different evolutionary sub-lineage. Tajima's Neutrality Test (Ps = 0.025; D = -1.5) indicated population size expansion that took place after selective sweep and/or purifying selection.     

Antennal malformations in light ocelli drones of Apis mellifera (Hymenoptera, Apidae)

Malformed antennae of Apis mellifera light ocelli drones were drawn, dissected and mounted permanently on slides containing Canada balsam, in order to count the olfactory discs present in each segment, in comparison with the number of those structures in normal antennae of their brothers. Some drones presented morphological abnormalities in a single segment of the right or left antenna, but others had two or more malformed segments in a same antenna. Drones with malformations in both antennae were also observed. The 4th and 5th flagellum segments were the most frequently affected. In a low number of cases the frequency of olfactory discs in malformed segments did not differ from that one recorded for normal segments. However, in most cases studied, the antennal malformations brought about a significant reduction in the number of olfactory discs from malformed segments.     

Differences in volatile composition and sexual morphs in rambutan (Nephelium lappaceum L.) flowers and their effect in the Apis mellifera L. (Hymenoptera,Apidae) attraction

We studied the volatile composition and sexual morphs of Nephelium lappaceum flowers from two orchards, and investigated the choice behavior of the honey bee, Apis mellifera toward the floral extracts from both locations. Our results showed significant differences in chemical composition and sexual morphs; only the hermaphrodite flowers from the Herradero orchard produced limonene and α-pinene and had longer peduncle and sepal than flowers from the Metapa orchard; on the other hand, the hermaphrodite flowers from the Metapa orchard had longer gynoecium. In the behavioral experiment the extracts from the Herradero orchard seemed to give A. mellifera foragers better cues for orientation to food sources, perhaps due to the presence of limonene and α-pinene, which are absent in the samples from Metapa. Such differences in both orchards could affect pollinator attraction and ultimately seed set and productivity.     

Kinetic properties of arginine phosphokinase from honeybees, Apis mellifera L. (Hymenoptera, Apidae)

The kinetic properties of honeybee arginine phosphokinase (APK), which catalyzes the reaction: Arginine phosphate + ADP + H⁺ ? arginine + ATP, have been studied.In the direction of ATP synthesis, the pH optimum was around pH 7.2 and the activation energy over the range 18–44 °C was about 10,500 cal/mole. The optimum ratio of Mg²⁺:ADP was about 4:1.In the direction of arginine phosphate (AP) synthesis, the enzyme had a pH optimum around pH 8.3. The energy of activation for the reaction over the range 22–39 °C was about 7500 cal/mole. The optimum ratio of Mg²⁺:ATP was about 1:1.The initial velocities of the reactions in the direction of ATP and AP synthesis were measured at varying concentrations of one substrate while the concentration of the other substrate was held constant at several levels. The double reciprocal plots of the data obtained yielded a series of intersecting lines, indicating that the enzyme has a sequential mechanism. Radioisotope exchange experiment showed that arginine phosphokinase did not catalyze ATP ? ADP exchange in the absence of arginine. Product inhibition studies showed that arginine was competitive with AP and noncompetitive with ADP; whereas ATP was competitive with ADP and noncompetitive with arginine. The results from initial velocity, radioisotope exchange, and product inhibition studies suggested that the enzyme has a rapid equilibrium, random mechanism.     

Cranial evolution in sakis (Pithecia, Platyrrhini). II: Evolutionary processes and morphological integration

Patterns of interspecific differentiation in saki monkeys (Pithecia) are quantitatively described and possible evolutionary processes producing them are examined. The comparison of species correlation matrices to expected patterns of morphological integration reveal significant and similar patterns of development-based cranial integration among species. Aspects of the facial region are more heavily influenced by general size variation than features of the neural region. The comparison of pooled within- and between-groups V/CV matrices suggests that genetic drift might be a sufficient explanation for saki cranial evolution. Differential natural selection gradients are also reconstructed because selection may also have caused population differentiation through evolutionary time. These gradients illustrate the inherent multivariate nature of selection, being a consequence of the interaction between existing morphological integration (correlation) among traits and the action of natural selection. Yet, our attempt to interpret selection gradients in terms of their functional significance did not result in any clear association between selection and function. Perhaps this is also an indication that morphological evolution in sakis was mostly neutral.     

Inter-individual variation in nutrient balancing in the honeybee (Apis mellifera)

The Geometric Framework approach in nutritional ecology postulates that animals attempt to balance the consumption of different nutrients rather than simply maximizing energetic gain. The intake target with respect to each nutrient maximizes fitness in a specific dimension and any difference between individuals in intake target therefore represents alternative behavioral and fitness maximization strategies. Nutritional interactions are a central component of all social groups and any inter-individual variation in intake target should therefore have a significant influence on social dynamics. Using the honeybee colony as an experimental model, we quantified differences in the carbohydrate intake target of individual foragers using a capillary feeder (CAFE) assay. Our results show that the bees did not simply maximize their net energetic gain, but combined sugar and water in their diet in a way that brought them to an intake target equivalent to a 33% sucrose solution. Although the mean intake target with respect to the nutrients sucrose and water was the same under different food choice regimens, there was significant inter-individual variation in intake target and the manner in which individuals reached this target, a variation which suggests different levels of tolerance to nutrient imbalance. We discuss our results in the context of how colony performance may be influenced by the different nutrient balancing strategies of individual members and how such nutritional constraints could have contributed to the evolution of sociality.     

On the comparison of the strength of morphological integration across morphometric datasets

Evolutionary morphologists frequently wish to understand the extent to which organisms are integrated, and whether the strength of morphological integration among subsets of phenotypic variables differ among taxa or other groups. However, comparisons of the strength of integration across datasets are difficult, in part because the summary measures that characterize these patterns (RV coefficient and r_PLS) are dependent both on sample size and on the number of variables. As a solution to this issue, we propose a standardized test statistic (a z‐score) for measuring the degree of morphological integration between sets of variables. The approach is based on a partial least squares analysis of trait covariation, and its permutation‐based sampling distribution. Under the null hypothesis of a random association of variables, the method displays a constant expected value and confidence intervals for datasets of differing sample sizes and variable number, thereby providing a consistent measure of integration suitable for comparisons across datasets. A two‐sample test is also proposed to statistically determine whether levels of integration differ between datasets, and an empirical example examining cranial shape integration in Mediterranean wall lizards illustrates its use. Some extensions of the procedure are also discussed.     

Effect of entomopathogens on Africanized Apis mellifera L. (Hymenoptera: Apidae)

This study aimed to evaluate the effect of commercially used entomopathogens on Africanized Apis mellifera L. (Hymenoptera: Apidae). Four bioassays were performed: 1) pulverized entomopathogens on A. mellifera; 2) entomopathogens sprayed on a smooth surface; 3) entomopathogens sprayed on soy leaves; and 4) entomopathogens mixed with candy paste (sugar syrup). Five treatments were prepared: sterile distilled water (control), distilled water sterilized with Tween^® 80 (0.01%), and the commercial entomopathogens Metarhizium anisopliae E9 (1.0 × 10⁹ conidia mL^?1), Beauveria bassiana PL63 (1.0 × 10⁸ conidia mL^?1) and Bacillus thuringiensis var. kurstaki HD-1 (3.0 × 10⁸ spores mL^?1). Each treatment consisted of five repetitions, with 20 workers per repetition, which were stored in a plastic box and, later, in a biological oxygen demand (B.O.D.) incubator (27 ± 2 °C, RH of 60% ± 10%, 12-h photophase). The mortality of the workers was evaluated from 1 h to 240 h, and the data were analyzed using Bayesian inference. The workers killed by the ingestion of candy paste contaminated with the pathogens (products) were randomly separated and selected for the removal of the midgut. Each midgut was fixed in Bouin's solution and prepared for histology. B. bassiana was verified to reduce the survival of A. mellifera workers in all bioassays. Moreover, M. anisopliae reduced the survival of A. mellifera workers directly sprayed, on a smooth surface and mixed with candy. B. thuringiensis reduced A. mellifera survival on a smooth surface and mixed with candy paste. However, its effects were lower than that observed by B. bassiana. The treatments with the biological products did not induce morphometric alterations in the midgut of A. mellifera.     

Geographical patterns of mitochondrial DNA variation in Apis mellifera iberiensis (Hymenoptera: Apidae)

An extensive survey of mitochondrial haplotypes in honeybee colonies from the Iberian Peninsula has corroborated previous hypotheses about the existence of a joint clinal variation of African (A) and west European (M) evolutionary lineages. It has been found that the Iberian Peninsula is the European region with the highest haplotype diversity (12 haplotypes detected of the M lineage and 10 of the A lineage). The frequency of A haplotypes decreases in a SW-NE trend, while that of M haplotypes increases. These results are discussed in relation to hypotheses about the African origin of Apis mellifera and an early colonization of west Europe during intermediate Pleistocene glaciation events, followed by a regional differentiation. The extant pattern of haplotype frequency and distribution seems to be influenced at a regional scale by adaptation to local climatic conditions and the mobile beekeeping that has become a large-scale practice during the last decades. Other previous anthropogenic influences (Greek, Roman and Arab colonizations) are thought to be of minor importance in present day populations.     

Crystal structure of peptidoglycan recognition protein SA in Apis mellifera (Hymenoptera: Apidae)

Peptidoglycan recognition protein SA (PGRP‐SA) is a key pattern recognition receptor in the insect innate immune system. PGRP‐SA can bind to bacterial PGN and activate the Toll pathway, which triggers the expression and release of antimicrobial peptides to prevent bacterial infection. Here, we report the first structure of Apis mellifera PGRP‐SA from Hymenoptera at 1.86 Å resolution. The overall architecture of Am‐PGRP‐SA was similar to the Drosophila PGRP‐SA; however, the residues involved in PGN binding groove were not conserved, and the binding pocket was narrower. This structure gives insight into PGN binding characteristics in honeybees.     

Genetic pollution and number of matings in a black honey bee (Apis mellifera mellifera) population

Summary In the south-east of France, local honey bees possess only the B allele at the MDH locus, whereas the races which are usually imported into this area do not have this allele. The proportion of non-B genes in a sample of drones was used to measure the genetic pollution in the local population. Within the course of a breeding scheme of local bees, 99 queens, whose genotypes are BB, were naturally mated between April 25 and June 10, 1985 at la Tave (Gard, France). Twenty daughters-workers of each queen were analysed at the MDH locus. The frequency of the B allele in drones that mated with these queens is estimated by the proportion of workers with genotype BB and the genetic pollution by the cumulated frequency of the other alleles. The sampling variances of these frequencies involve a coefficient which is a function of the average number of drones mated with a queen. This latter parameter is estimated through the maximum likelihood method. In addition to the three well-known alleles, a rare allele (frequency=0.0055), possibly equivalent to the S1 allele described by Badino et al. (1983), has been found in three different colonies. Cumulating the frequencies of the non-B alleles results in an estimation of the genetic pollution equal to 0.0394 (±0.0071). This low value allows us to proceed to the next step of the selection project. The mean number of drones mated to a queen is 12.4 with a (10.4–19.3) confidence interval at the 90% level.     

Hydrolases in the hypopharyngeal glands of workers of Scaptotrigona postica and Apis mellifera (Hymenoptera, Apinae)

Hydrolytic enzymes from hypopharyngeal gland extracts of newly emerged, nurse and foraging workers of two eusocial bees, Scaptotrigona postica, a native Brazilian stingless bee, and the Africanized honey bee (Apis mellifera) in Brazil, were compared. The hypopharyngeal gland is rich in enzymes in both species. Fifteen different enzymes were found in the extracts, with only a few quantitative differences between the species. Some of the enzymes present in the extracts may have intracellular functions, while others seem to be digestive enzymes. Scaptotrigona postica, had lower beta-glucosidase and higher lipase esterase activities than A. mellifera. The differences may be due to different feeding habits and behavioral peculiarities of the two species.     

Flower visitation patterns of the coexisting honey bees Apis cerana japonica and Apis mellifera (Hymenoptera: Apidae)

We compared flower visitation patterns of two coexisting honey bees, Apis mellifera Linnaeus and Apis cerana japonica Radoszkowski, on 20 plant species, including three exotics, under natural conditions in Nara, Japan, from April to August 2012. We also measured flower color based on bee color vision (15 flower species), nectar volume (nine species) and nectar concentration (eight species). Flowers colored white, pink, red, purple and cream were classified as bee‐blue‐green, and yellow was classified as bee‐green. Apis cerana visited 14 plant species and A. mellifera visited 11. Although the two Apis species are similar in morphology, they visited different plants: in particular, A. cerana visited native plant species more often than did A. mellifera. Both A. mellifera and A. cerana visited not only nectariferous flowers but also those with no nectar. We also found different visitation patterns between A. cerana and A. mellifera: Apis cerana more often visited flowers with smaller color angle (bee‐blue‐green), lower chroma and higher brightness, and flowers secreting nectars of higher concentration and smaller volume than did A. mellifera.     

Vibration Signal Behavior of Waggle-dancers in Swarms of the Honey Bee,Apis mellifera (Hymenoptera: Apidae)

We hypothesize two functions of the vibration signal (dorsal ventral abdominal vibration = DVAV) during swarming in honey bees: 1. it enhances recruitment to the specific sites advertised by the waggle dancers which also perform the vibration signal; and 2. it acts as a nonspecific modulatory signal to stimulate activity in other bees. The stimulation of activity invoked by the second hypothesis might include increasing nest-site scouting and dance following early in the house-hunting process or rousing quiescent bees to prepare them for lift-off late in the process, or both. In studies of neotropical African bee swarms in Costa Rica and European bees in California we tested these hypotheses by looking for associations between production of vibration signals by nest-site recruiters and site attractiveness (indicated by which site was ultimately chosen and by distance from the swarm since swarms may have a distance preference). Overall, bees dancing for the chosen sites performed vibration signals to the same extent as those dancing for the other sites. There were no distance differences between sites whose scouts did and did not vibrate other bees. These results are inconsistent with the hypothesis that the vibration signal enhances recruitment to especially high quality sites and they support the hypothesis that it plays a general excitatory role in the context of house hunting by swarming bees.