Silk formation mechanisms in the larval salivary glands of<Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae)

The mechanism of silk formation inApis mellifera salivary glands, during the 5th instar, was studied. Larval salivary glands were dissected and prepared for light and polarized light microscopy, as well as for scanning and transmission electron microscopy. The results showed that silk formation starts at the middle of the 5th instar and finishes at the end of the same instar. This process begins in the distal secretory portion of the gland, going towards the proximal secretory portion; and from the periphery to the center of the gland lumen. The silk proteins are released from the secretory cells as a homogeneous substance that polymerizes in the lumen to form compact birefringent tactoids. Secondly, the water absorption from the lumen secretion, carried out by secretory and duct cells, promotes aggregation of the tactoids that form a spiral-shape filament with a zigzag pattern. This pattern is also the results of the silk compression in the gland lumen and represents a high concentration of macromolecularly well-oriented silk proteins.     

Morphological changes in the cephalic salivary glands of females and males of <Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Scaptotrigona postica</Emphasis> (Hymenoptera,Apidae)

The cephalic salivary glands of some species of bees are exclusive and well developed only in Apinae. These glands were studied with light and scanning electron microscopy in workers, queens and males from the honey bee Apis mellifera, and the stingless bee Scaptotrigona postica in different life phases. The results show that the cephalic salivary glands are present in females of both the species, and in males of S. postica. Nevertheless, they are poorly developed in young males of A. mellifera. In both species, gland growth is progressive from the time of emergence to the oldest age but, in A. mellifera males, the gland degenerates with age. Scanning electron microscopy shows that the secretory units of newly emerged workers are collapsed while in older workers they are turgid. Some pits on the surface of the secretory units correspond to open intercellular spaces. The possible functions of these glands in females and males of both species are discussed.     

Medium for development of bee cell cultures (<Emphasis Type="Italic">Apis mellifera</Emphasis>: Hymenoptera: Apidae)

A media for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and modified Hert–Hunter 70 media. Cell culture systems for bees solves an impasse that has hindered efforts to isolate and screen pathogens which may be influencing or causing colony collapse disorder of bees. Multiple life stages of maturing larvae to early pupae were used to successfully establish cell cultures from the tissues of the head, thorax, and abdomen. Multiple cell types were observed which included free-floating suspensions, fibroblast-like, and epithelia-like monolayers. The final culture medium, WH2, was originally developed for hemipterans, Asian citrus psyllid, Diaphorina citri, and leafhopper, Homalodisca vitripennis cell cultures but has been shown to work for a diverse range of insect species such as bees. Bee cell cultures had various doubling times at 21–23°C ranging from 9–15 d. Deformed wing virus was detected in the primary explanted tissues, which tested negative by rt-PCR for Israeli acute paralysis virus (IAPV), Kashmir bee virus, acute bee paralysis virus, and black queen cell virus. Culture inoculation with IAPV from an isolate from Florida field samples, was detectable in cell cultures after two subcultures. Cell culture from hymenoptera species, such as bees, greatly advances the approaches available to the field of study on colony collapse disorders.     

Presence of <Emphasis Type="Italic">Ascosphaera apis</Emphasis>, the causative agent of chalkbrood disease,in honeybees <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae) in Japan

The presence of Ascosphaera apis, a fungus that is the causative agent of chalkbrood disease, was surveyed in Japan using a diagnostic polymerase chain reaction (PCR). A total of 336 individual European honeybees Apis mellifera were taken from 25 different apiaries in various regions of Japan. Of the 112 colonies surveyed, A. apis was detected in 27 colonies (24.1%). Positive results by PCR were obtained from 49 out of 336 surveyed individuals (14.6%). Based on these results, the distribution of A. apis in A. mellifera is widespread across Japan and does not exhibit significant differences between geographic areas. DNA sequences of the ITS and 5.8S rRNA region from all 17 isolates of A. apis were identical, even though they were from geographically distinct areas in Japan. It is suggested that no intra-species variation may be due to a recent bottleneck effect probably caused by humans before geographical expansion of the fungus.     

Sociality in <Emphasis Type="Italic">Euglossa</Emphasis> (<Emphasis Type="Italic">Euglossa</Emphasis>) <Emphasis Type="Italic">viridissima</Emphasis> Friese (Hymenoptera,Apidae, Euglossini)

Euglossa viridissima is an orchid bee that forms both solitary and multiple female nests, making it a suitable species for the study of factors leading to diverse degrees of sociality in Euglossines. We conducted observations in eight reused nests (where a first generation of bees had been produced) kept in artificial boxes from the Yucatan Peninsula, Mexico. Five nests were reused (reactivated) by a single female (SFN), two nests reused by a mother and one daughter (MFN1) and one nest reused by the mother and two daughters (MFN2). No single nest was reactivated by unrelated females. The number of foraging trips, their duration and the duration of cell provisioning was not different between SFN and MFN. The overall production of cells per female was not different either between both types of nest. However, in MFN although all females did lay eggs, there was a reproductive skew in favor of the mother (95 and 45% of the brood produced in MFN1 and MFN2 respectively). She showed reproductive control of her daughters through oophagy and displaying threatening behavior when the daughters tried to open a cell where she had laid an egg. Brood losses to parasites (Anthrax sp. (Bom-byliidae) and Hoplostelis bivittata (Megachilidae)) were only found in SFN which possibly reflects and advantage of MFN in this respect. Our results coupled with other studies in Euglossa, reveal that a wide range of social behaviors occur in this genus, from solitary and communal to primitive reproductive division of labor. Multiple factors involving different levels of pressure imposed by food availability and parasites may favor such a diverse range of nesting behaviors. Interestingly, female associations in E. viridissima seem a result of kin selection that is enforced by coercion from mother females on their daughters. More studies are needed to shed light upon the social organization of Euglossa and other Euglossines and on their phylogenetic relationships in order to trace the origins of eusociality in Apidae. Received 12 February 2008; revised 25 June 2008; accepted 17 July 2008.     

Intraspecies differences in mechanisms of formation of protective processes in the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Presented in the work are data on interrace differences in mechanisms of formation of antioxidant and phenoloxidase complexes in honeybees of the Middle Russian (Apis mellifera L.) and Caucasian (Apis mellifera caucasica Gorb.) geographic races. Different degree has been revealed of activation of these complexes depending on their localization in the insect organism and providing different strategy of adaptation to temperature stress. A diverse role of ascorbic acid in regulation of the honeybee protective system has been shown.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

Reproductive biology of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Africanized honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Since its first contact with Apis mellifera, the population dynamics of the parasitic mite Varroa destructor varies from one region to another. In many regions of the world, apiculture has come to depend on the use of acaricides, because of the extensive damage caused by varroa to bee colonies. At present, the mite is considered to contribute to the recent decline of honey bee colonies in North America and Europe. Because in tropical climates worker brood rearing and varroa reproduction occurs all year round, it could be expected that here the impact of the parasite will be even more devastating. Yet, this has not been the case in tropical areas of South America. In Brazil, varroa was introduced more than 30 years ago and got established at low levels of infestation, without causing apparent damage to apiculture with Africanized honey bees (AHB). The tolerance of AHB to varroa is apparently attributable, at least in part, to resistance in the bees. The low fertility of this parasite in Africanized worker brood and the grooming and hygienic behavior of the bees are referred as important factors in keeping mite infestation low in the colonies. It has also been suggested that the type of mite influences the level of tolerance in a honey bee population. The Korea haplotype is predominant in unbalanced host-parasite systems, as exist in Europe, whereas in stable systems, as in Brazil, the Japan haplotype used to predominate. However, the patterns of varroa genetic variation have changed in Brazil. All recently sampled mites were of the Korea haplotype, regardless whether the mites had reproduced or not. The fertile mites on AHB in Brazil significantly increased from 56% in the 1980s to 86% in recent years. Nevertheless, despite the increased fertility, no increase in mite infestation rates in the colonies has been detected so far. A comprehensive literature review of varroa reproduction data, focusing on fertility and production of viable female mites, was conducted to provide insight into the Africanized bee host-parasite relationship.     

The Mating System of <Emphasis Type="Italic">Amegilla</Emphasis> (<Emphasis Type="Italic">Asarapoda</Emphasis>) <Emphasis Type="Italic">paracalva</Emphasis> Brooks (Hymenoptera: Apidae)

Males of the solitary bee Amegilla (Asarapoda) paracalva employ two mate-locating tactics: aggressive defense of sites from which virgin females are emerging and patrolling flower patches that are visited by conspecific females. At one study site, a single male was able to control an entire emergence area for one or more days. Multiple males patrolled one flower patch, interacting aggressively on occasion but no one individual was able to monopolize this resource. Territorial males at the emergence site secured mates by waiting by tunnels for receptive virgin females to emerge after metamorphosis. Males patrolling the flower patch pounced upon flower visiting conspecifics and mated with receptive females there. Territorial males at the emergence site were larger than average individuals, probably because of the advantage larger males have when grappling with opponents. Flower patrolling males were smaller than territorial males at the emergence sites, perhaps because of the advantages gained by these males from rapid, agile flight.     

Polymorphism analysis of <Emphasis Type="Italic">csd</Emphasis> gene in six <Emphasis Type="Italic">Apis mellifera</Emphasis> subspecies

The complementary sex determination (csd) gene is the primary gene determining the gender of honey bees (Apis spp). In this study we analyzed the polymorphism of csd gene in six Apis mellifera subspecies. The genomic region 3 of csd gene in these six A. mellifera was cloned, and identified. A total of 79 haplotypes were obtained from these six subspecies. Analysis showed that region 3 of csd gene has a high level of polymorphism in all the six A. mellifera subspecies. The A. m. anatolica subspecies has a slightly higher nucleotide diversity (π) than other subspecies, while the π values showed no significant difference among the other five subspecies. The phylogenetic tree showed that all the csd haplotypes from different A. mellifera subspecies are scattered throughout the tree, without forming six different clades. Population differentiation analysis showed that there are significant genetic differentiations among some of the subspecies. The NJ phylogenetic tree showed that the A. m. caucasica and A. m. carnica have the closest relationship, followed by A. m. ssp, A. m. ligustica, A. m. carpatica and A. m. anatolica that were gathered in the tree in turn.     

Biological activity of some plant essential oils against <Emphasis Type="Italic">Varroa destructor</Emphasis> (Acari: Varroidae), an ectoparasitic mite of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae)

This experiment was conducted to evaluate acaricidal activity of the essential oils of Thymus kotschyanus, Ferula assa-foetida and Eucalyptus camaldulensis against Varroa destructor under laboratory conditions. Moreover, fumigant toxicity of these oils was tested on Apis mellifera. After preliminary dose-setting experiments, mites and honey bees were exposed to different concentrations of the oil, with 10 h exposure time. Essential oil of T. kotschyanus appeared the most potent fumigant for V. destructor (LC₅₀ = 1.07, 95% confidence limit (CL) = 0.87–1.26 μl/l air), followed by E. camaldulensis (LC₅₀ = 1.74, 95% CL = 0.96–2.50 μl/l air). The lowest acaricidal activity (LC₅₀ = 2.46, 95% CL = 2.10–2.86 μl/l air) was attributed to essential oil of F. assa-foetida. Surprisingly, among the three oils tested, essential oil of T. kotschyanus had the lowest insecticidal activity against A. mellifera (LC₅₀ = 5.08, 95% CL = 4.54–5.06 μl/l air). These findings proved that essential oil of T. kotschyanus has potential of practical value for use as alternative acaricide in the management of varroa in apiaries.     

Larval oophagy in the ant <Emphasis Type="Italic">Amblyopone silvestrii</Emphasis> (Hymenoptera,Formicidae)

Summary Larvae pass through five instars in the temperate, subterranean ponerine ant, Amblyopone silvestrii. Field colonies displayed a large decrease in the number of eggs during mid-summer, despite the fact that queens maintained fully developed ovaries with mature eggs. Observations of laboratory colonies indicate that cannibalism by 1st and 2nd instar larvae caused this decrease in egg number. These instars consumed a total of 66–75% of eggs in the nest, with each larva consuming 2–3 eggs before molting to the 3rd instar. At that time the larvae began to feed on arthropods. The preferred prey of A. silvestrii consists of entire centipedes; the large size of these prey items relative to the size of early instar larvae makes it difficult for the larvae to feed on these prey. Additionally, workers of A. silvestrii do not engage in oral trophallaxis. Consequently, oophagy is a plausible method to feed these very small larvae.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

Genetic structure of <Emphasis Type="Italic">Varroa destructor</Emphasis> populations infesting <Emphasis Type="Italic">Apis mellifera</Emphasis> colonies in Argentina

Although mitochondrial DNA mapping of Varroa destructor revealed the presence of several haplotypes, only two of them (Korean and Japanese haplotypes) were capable to infest Apis mellifera populations. Even though the Korean haplotype is the only one that has been reported in Argentina, these conclusions were based on mites sampled in apiaries from a specific geographical place (Buenos Aires province). To study mites from several sites of Argentina could reveal the presence of the Japanese genotype, especially considering sites near to Brazil, where Japanese haplotype was already detected. The aim of this work was to study the genetic structure of V. destructor populations from apiaries located in various provinces of Argentina, in order to determine the presence of different haplotypes. The study was carried out between January 2006 and December 2009. Phoretic adult Varroa mites were collected from honey bee workers sampled from colonies of A. mellifera located in Entre Ríos, Buenos Aires, Corrientes, Río Negro, Santa Cruz and Neuquén provinces. Twenty female mites from each sampling site were used to carry out the genetic analysis. For DNA extraction a nondestructive method was used. DNA sequences were compared to Korean haplotype (AF106899) and Japanese haplotype (AF106897). All DNA sequences obtained from mite populations sampled in Argentina, share 98% of similitude with Korean Haplotype (AF106899). Taking into account these results, we are able to conclude that Korean haplotype is cosmopolite in Argentina.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Real-time observation of autophagic programmed cell death of<Emphasis Type="Italic"> Drosophila</Emphasis> salivary glands in vitro

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.Edited by N. Satoh     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.     

Recognition of a familiar place by the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Recent work shows that at any one place bees detect a limited variety of simple cues in parallel. At each choice point, they recognize a few cues in the range of positions where the cues occurred during the learning process. There is no need to postulate that they re-assemble the surrounding panorama in memory; only that they retain memories of the coincidences of cues in the expected retinotopic directions. The cues could be stimuli that excite groups of peripheral visual neurons. All the experimentally known cues are described, including modulation of the receptors, the locations of areas of black or colour, the nearness, size, averaged edge orientation, and radial and tangential edges. Cues of each type are separately summed within large fields, the size of which varies with the cue. Local orientation cues from edges at right angles cancel each other within each field, which also suggests that the discrimination of shape and texture is limited. Resolution depends on lateral interactions and the number of ommatidia required for each cue. To identify a new place, a few sparse cues, together with their directions, are learned in orientation flights. When the bee returns, the cues in the panorama are progressively matched as they coincide with the cues in memory. The limited number of cues, though economical for memory, may restrict the foraging behaviour and lead to flower constancy. This kind of a visual system is a candidate model for other animals or machines with economical processing systems.