Morphology and structure of the tarsal glands of the stingless bee Melipona seminigra

Footprint secretions deposited at the nest entrance or on food sources are used for chemical communication by honey bees, bumble bees, and stingless bees. The question of the glandular origin of the substances involved, however, has not been unequivocally answered yet. We investigated the morphology and structure of tarsal glands within the fifth tarsomeres of the legs of workers of Melipona seminigra in order to clarify their possible role in the secretion of footprints. The tarsal gland is a sac-like fold forming a reservoir. Its glandular tissue is composed of a unicellular layer of specialized epidermal cells, which cover the thin cuticular intima forming the reservoir. We found that the tarsal glands lack any openings to the outside and therefore conclude that they are not involved in the secretion of footprint substances. The secretion produced accumulates within the glands reservoir and reaches as far as into the arolium. Thus it is likely that it serves to fill and unfold the arolium during walking to increase adhesion on smooth surfaces, as is known for honey bees and weaver ants.     

Skimming behaviour and spreading potential of Stenus species and Dianous coerulescens (Coleoptera: Staphylinidae)

Rove beetles of the genus Stenus Latreille and the genus Dianous Leach possess pygidial glands containing a multifunctional secretion of piperidine and pyridine-derived alkaloids as well as several terpenes. One important character of this secretion is the spreading potential of its different compounds, stenusine, norstenusine, 3-(2-methyl-1-butenyl)pyridine, cicindeloine, α-pinene, 1,8-cineole and 6-methyl-5-heptene-2-one. The individual secretion composition enables the beetles to skim rapidly and far over the water surface, even when just a small amount of secretion is emitted. Ethological investigations of several Stenus species revealed that the skimming ability, skimming velocity and the skimming behaviour differ between the Stenus species. These differences can be linked to varied habitat claims and secretion saving mechanisms. By means of tensiometer measurements using the pendant drop method, the spreading pressure of all secretion constituents as well as some naturally identical beetle secretions on the water surface could be established. The compound 3-(2-methyl-1-butenyl)pyridine excelled stenusine believed to date to be mainly responsible for skimming relating to its surface activity. The naturally identical secretions are not subject to synergistic effects of the single compounds concerning the spreading potential. Furthermore, evolutionary aspects of the Steninae’s pygidial gland secretion are discussed.     

A novel marine silk

The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex β-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements.     

A comparative analysis of colour preferences in temperate and tropical social bees

The spontaneous occurrence of colour preferences without learning has been demonstrated in several insect species; however, the underlying mechanisms are still not understood. Here, we use a comparative approach to investigate spontaneous and learned colour preferences in foraging bees of two tropical and one temperate species. We hypothesised that tropical bees utilise different sets of plants and therefore might differ in their spontaneous colour preferences. We tested colour-naive bees and foragers from colonies that had been enclosed in large flight cages for a long time. Bees were shortly trained with triplets of neutral, UV-grey stimuli placed randomly at eight locations on a black training disk to induce foraging motivation. During unrewarded tests, the bees’ responses to eight colours were video-recorded. Bees explored all colours and displayed an overall preference for colours dominated by long or short wavelengths, rather than a single colour stimulus. Naive Apis cerana and Bombus terrestris showed similar choices. Both inspected long-wavelength stimuli more than short-wavelength stimuli, whilst responses of the tropical stingless bee Tetragonula iridipennis differed, suggesting that resource partitioning could be a determinant of spontaneous colour preferences. Reward on an unsaturated yellow colour shifted the bees’ preference curves as predicted, which is in line with previous findings that brief colour experience overrides the expression of spontaneous preferences. We conclude that rather than determining foraging behaviour in inflexible ways, spontaneous colour preferences vary depending on experimental settings and reflect potential biases in mechanisms of learning and decision-making in pollinating insects.     

Honeybee glucose oxidase—its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H₂O₂), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H₂O₂ content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H₂O₂ in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H₂O₂ content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.     

Reproductive disturbance of Japanese bumblebees by the introduced European bumblebee Bombus terrestris

The European bumblebee, Bombus terrestris, is an invasive eusocial species whose distribution is expanding greatly beyond its native range because numerous colonies are imported to or locally produced in non-native countries for pollination of agricultural crops. Closely related species exist in Japan where the unrestricted import and use of B. terrestris has resulted in the establishment of wild colonies. Laboratory studies previously showed that B. terrestris and Japanese native species can copulate and produce fertilized eggs. Although these eggs do not hatch, the interspecific mating can cause a serious reproductive disturbance to native bumblebees. In this study, we determined the frequencies of interspecies mating between B. terrestris males and native bumblebee queens in the wild on the islands of Hokkaido and Honshu by analyzing the DNA sequences of spermatozoa stored in spermathecae of native queens. We found that 20.2% of B. hypocrita hypocrita queens and 30.2% of B. hypocrita sapporoensis queens had spermatozoa of B. terrestris males in their spermathecae. Given that a Bombus queen generally mates only once in her life, such high frequencies of interspecific mating with B. terrestris pose serious threats to the populations of native bumblebees in Japan.     

The influence of pigmentation patterning on bumblebee foraging from flowers of Antirrhinum majus

Patterns of pigmentation overlying the petal vasculature are common in flowering plants and have been postulated to play a role in pollinator attraction. Previous studies report that such venation patterning is significantly more attractive to bee foragers in the field than ivory or white flowers without veins. To dissect the ways in which venation patterning of pigment can influence bumblebee behaviour, we investigated the response of flower-naïve individuals of Bombus terrestris to veined, ivory and red near-isogenic lines of Antirrhinum majus. We find that red venation shifts flower colour slightly, although the ivory background is the dominant colour. Bees were readily able to discriminate between ivory and veined flowers under differential conditioning but showed no innate preference when presented with a free choice of rewarding ivory and veined flowers. In contrast, both ivory and veined flowers were selected significantly more often than were red flowers. We conclude that advantages conferred by venation patterning might stem from bees learning of their use as nectar guides, rather than from any innate preference for striped flowers.     

Characteristics of volatile compounds removal in biogas slurry of pig manure by ozone oxidation and organic solvents extraction

Biogas slurry is not suitable for liquid fertilizer due to its high amounts of volatile materials being of complicated composition and peculiar smell. In order to remove volatiles from biogas slurry efficiently, the dynamic headspace and gas chromatography-mass spectrometry were used to clear the composition of volatiles. Nitrogen stripping and superfluous ozone were also used to remove volatiles from biogas slurry. The results showed that there were 21 kinds of volatile compounds in the biogas slurry, including sulfur compounds, organic amines, benzene, halogen generation of hydrocarbons and alkanes, some of which had strong peculiar smell. The volatile compounds in biogas slurry can be removed with the rate of 53.0% by nitrogen stripping and with rate of 81.7% by the oxidization and stripping of the superfluous ozone. On this basis, the removal rate of the volatile compounds reached 99.2% by chloroform and n-hexane extraction, and almost all of odor was eliminated. The contents of some dissolved organic compounds decreased obviously and however main plant nutrients had no significant change in the biogas slurry after being treated.     

More than euglossines: the diverse pollinators and floral scents of Zygopetalinae orchids

Floral volatile organic compounds (VOCs) play important roles in plant-pollinator interactions. We investigated the reproductive ecology and floral VOCs of Zygopetalinae orchids to understand the relationship between floral scents and pollinators. We performed focal observations, phenological censuses and breeding system experiments in eight species in southeast Brazil. Floral scents were collected and analysed using SPME/GC-MS. We performed multivariate analyses to group species according to affinities of their VOCs and define compounds associated to each plant. Dichaea cogniauxiana was pollinated by weevils which use their developing ovules, while D. pendula was pollinated by the same weevils and perfume-collecting male euglossine bees. The other species were deceit-pollinated by bees. Zygopetalum crinitum was pollinated by carpenter bees, while W. warreana, Z. mackayi and Z. maxillare were bumblebee-pollinated. The latter was also pollinated by Centris confusa. Breeding system varied widely with no association to any pollinator group. Most VOCs are common to other floral scents. Zygopetalum crinitum presented an exclusive blend of VOCs, mainly composed of benzenoids. The scents of Pabstia jugosa, Promenaea xanthina and the Zygopetalum spp. were similar. The bumblebee-pollinated species have flowering periods partially overlapped, thus neither phenology nor pollinators constitute hybridization barriers among these species. Euglossines are not the only pollinators of Zygopetalinae. Different VOCs, size and lifespan of flowers are associated with distinct pollinators. A distinctive VOC bouquet may determine specialisation in carpenter bees or male euglossines within bee-pollinated flowers. Finally, visitation of deceit-pollinated flowers by perfume-collecting euglossines allows us to hypothesise how pollination by this group of bees had evolved.     

Potential increase in mating frequency of queens in feral colonies of Bombus terrestris introduced into Japan

With the exception of several species, bumblebees are monandrous. We examined mating frequency in feral colonies of the introduced bumblebee Bombus terrestris in Japan. Using microsatellite markers, genotyping of sperm DNA stored in the spermatheca of nine queens detected multiple insemination paternities in one queen; the others were singly mated. The average effective paternity frequency estimated from the genotypes of queens and workers was 1.23; that estimated from the workers’ genotype alone was 2.12. These values were greater than those of laboratory-reared colonies in the native ranges of B. terrestris. The genotypes of one or two workers did not match those of their queens or showed paternities different from those of their nestmates; this may have arisen from either queen takeover or drifting of workers. These alien workers were responsible for the heterogeneous genotype distribution within each B. terrestris colony, resulting in higher estimates of paternity frequency than of insemination frequency. The high mating frequency of introduced B. terrestris may have occurred by artificial selection through mass breeding for commercialization. Moreover, polyandrous queens may be selectively advantageous, because reproduction by such queens is less likely to be disturbed by interspecific mating than that by monandrous queens.     

有机粘土疏水性吸附过程的分子模拟:烷基碳原子数和有机物logK_(ow)的影响

基于从分子尺度上解析改性剂烷基碳原子数和有机物疏水性对有机粘土吸附能力的影响机制,以从头算的量子化学计算方法(MP2/6-31g(d))模拟了有机粘土在水溶液中的吸附过程.通过计算吸附前后几何构型、能量、化学键和电荷分布等参数的变化,分析了影响有机粘土疏水性吸附的关键因素,并对疏水性吸附作用力的化学本质进行了初步探讨.计算结果表明,烷基碳原子数增加对提高有机物吸附能的作用并不显著,长链烷基提高有机粘土吸附容量的真正原因在于其可提供更多的有机物吸附位.有机物的疏水性(以正辛醇-水分配系数logKow表征)是影响疏水性吸附过程的关键因子,水分子的引力作用对疏水性吸附具有决定性影响,理论上解释了普遍存在的有机物吸附量与logKow呈正相关的实验现象.疏水性吸附力的作用前提是有机物为疏水性,即当水分子的引力作用较弱时疏水性作用力才可以表现出来;而对于亲水性有机物,疏水性吸附力会被水分子的引力所抵消,吸附强度减弱.模拟红外光谱和电荷分布结果表明,吸附前后无化学键断裂和电子得失的发生,只有原子电荷密度的微小变化,据此推测疏水性吸附力以分子间弱相互作用力——London色散力为主.     

Flower-visiting behavior of male bees is triggered by nectar-feeding insects

Bees are important pollinators for many flowering plants. Female bees are thought to be more effective pollinators than male bees because they carry much more pollen than males. Males of some solitary bee species are known to patrol near flowers that females visit. Because patrolling males visit flowers to mate or defend their territories, they may function as pollinators. However, the significance of patrolling males to pollination has not been studied. We studied males of a solitary bee, Heriades fulvohispidus (Megachilidae), patrolling near flowers and visiting flowers that attracted nectar-feeding insects, including conspecifics, on the Ogasawara (Bonin) Islands. To test the hypothesis that patrolling male bees may function as pollen vectors, we compared the frequency of visits by H. fulvohispidus to flowers of an endemic plant, Schima mertensiana (Theaceae); comparisons were made among flowers with a dead H. fulvohispidus, a dead beetle, a piece of plastic, and nothing (control flowers). Patrolling H. fulvohispidus more frequently visited flowers with a dead conspecific, a dead beetle, or a piece of plastic than the control flowers. Our experiment demonstrates that nectar-feeding insects (including conspecifics and other insects) enhance the flower-visiting frequency of patrolling H. fulvohispidus males on S. mertensiana flowers. Furthermore, we observed S. mertensiana pollen on patrolling males as well as females, suggesting that male bees may also function as pollen vectors.     

Mimicry of queen Dufour's gland secretions by workers of Apis mellifera scutellata and A. m. capensis

The development of the Dufour's gland of workers of the two honey bee races Apis mellifera scutellata and A. m. capensis was measured. The Dufour's glands of A. m. capensis workers were longer and increased in length more rapidly than the glands of workers of A. m. scutellata at comparable ages. Analysis of the Dufour's gland secretions of workers and queens of both races revealed that there were caste and racial differences. Secretions of queenright A. m. scutellata workers were dominated by a series of long-chain hydrocarbons. In contrast the secretions of the A. m. capensis workers both under queenright and queenless conditions were a mixture of hydrocarbons and wax-type esters, as were those of queens. Multivariate analysis of the secretion profiles indicated that laying workers of both races mimic queens. The secretions of the A. m. capensis laying workers mimicked queen secretions most closely, enabling them to act as successful social parasites.     

Background levels of atmospheric hydrocarbons,sulphate and nitrate over the western Mediterranean

Aerosols collected on top of Alfabia ridge (∼1100 m; Mallorca Island) have been analysed for chloride, nitrate, sulphate, aliphatic, and polycyclic aromatic hydrocarbons. The results exhibit concentrations in the level of the lowest previously reported for the western Mediterranean atmosphere. However, they are significantly higher than those described for very remote sites, thus defining a background level of particulate atmospheric compounds in this region. Two natural and anthropogenic sources, namely higher plant waxes and coal combustion particles, have been recognized as the main organic inputs to these background aerosols. The former is represented by the major n-alkanes and the latter by the polycyclic aromatic hydrocarbons.     

Identification of cuticular lipids eliciting interspecific courtship in the German cockroach, Blattella germanica

The cuticular surface of sexually mature females of the German cockroach contains a sex pheromone that, upon contact with the male's antennae, elicits a characteristic species-specific courtship behavior. This female-specific pheromone is a blend of several long-chain methyl ketones, alcohols and aldehydes, all derived from prominent cuticular hydrocarbons found in all life stages of this cockroach. We found that contact with the antennae of 5 out of 20 assayed cockroach species elicited courtship behavior in German cockroach males. The heterospecific courtship-eliciting compounds were isolated by behaviorally guided fractionation of the active crude extracts and compared to the native sex pheromone components. We identified two active compounds from the cuticular extract of the Oriental cockroach, Blatta orientalis -- 11-methylheptacosan-2-one and 27-oxo-11-methylheptacosan-2-one; the former compound was confirmed by synthesis and proved to independently stimulate courtship in German cockroach males. These compounds share common features with, but are distinct from, any of the known contact sex pheromone components. This suggests that sex pheromone reception in the male German cockroach is unusually promiscuous, accepting a wide range of compounds that share certain features with its native pheromone, thus resulting in a broad spectrum of behavioral response to other species. We propose that several characteristics of their mating system -- chiefly, absence of closely related species in the anthropogenic environment, resulting in relaxation of selection on sexual communication, and a highly male-biased operational sex ratio -- have driven males to respond with extremely low thresholds to a wide spectrum of related compounds.     

Monogamy in large bee societies: a stingless paradox

High genetic diversity is important for the functioning of large insect societies. Across the social Hymenoptera (ants, bees, and wasps), species with the largest colonies tend to have a high colony-level genetic diversity resulting from multiple queens (polygyny) or queens that mate with multiple males (polyandry). Here we studied the genetic structure of Trigona spinipes, a stingless bee species with colonies an order of magnitude larger than those of polyandrous honeybees. Genotypes of adult workers and pupae from 43 nests distributed across three Brazilian biomes showed that T. spinipes colonies are usually headed by one singly mated queen. Apart from revealing a notable exception from the general incidence of high genetic diversity in large insect societies, our results reinforce previous findings suggesting the absence of polyandry in stingless bees and provide evidence against the sperm limitation hypothesis for the evolution of polyandry. Stingless bee species with large colonies, such as T. spinipes, thus seem promising study models to unravel alternative mechanisms to increase genetic diversity within colonies or understand the adaptive value of low genetic diversity in large insect societies.     

Larval salivary glands are a source of primer and releaser pheromone in honey bee (Apis mellifera L.)

A brood pheromone identified in honeybee larvae has primer and releaser pheromone effects on adult bees. Using gas chromatography–mass spectrometry (GC–MS) to evaluate fatty acid esters—the pheromonal compounds—in different parts of the larvae, we have localized the source of the esters as the larval salivary glands. A histochemical study describes the glands and confirms the presence of lipids in the glands. Epithelial cells of the gland likely secrete the fatty acids into the lumen of the gland. These results demonstrate the salivary glands to be a reservoir of esters, components of brood pheromone, in honeybee larvae.     

1-Tridecene—male-produced sex pheromone of the tenebrionid beetle <Emphasis Type="Italic">Parastizopus transgariepinus</Emphasis>

Males of the genus Parastizopus (Coleoptera: Tenebrionidae) exhibit a special pheromone-emitting behaviour. They do a headstand, expose the aedeagus and remain in this posture for a few seconds. The pheromone emitted by P. transgariepinus was collected by solid-phase micro-extraction (100 microm polydimethylsiloxane fibre) and identified as 1-tridecene by gas chromatography/mass spectrometry. Presumably, this compound originates from the aedeagal gland, a special feature in Parastizopus, as 1-tridecene is the main compound in the gland reservoirs (23.6+/-3.8%), accompanied by various less volatile fatty acid esters (25.2+/-2.0%) and hydrocarbons (51.2+/-5.7%). 1-Tridecene is also part of the pygidial defensive secretion of both sexes, together with other 1-alkenes, monoterpene hydrocarbons and 1,4-benzoquinones, but as none of these other compounds was detected during calling, the pygidial gland could be ruled out as pheromone source. Extracts of the aedeagal gland reservoirs and the pygidial defensive secretion contained comparable amounts of 1-tridecene, 1.24+/-0.41 and 1.88+/-0.54 microg/male, respectively. Chemo-orientation experiments using a servosphere showed that 1 microg of 1-tridecene was attractive to females but not to males.     

Genetic diversity affects colony survivorship in commercial honey bee colonies

Honey bee (Apis mellifera) queens mate with unusually high numbers of males (average of approximately 12 drones), although there is much variation among queens. One main consequence of such extreme polyandry is an increased diversity of worker genotypes within a colony, which has been shown empirically to confer significant adaptive advantages that result in higher colony productivity and survival. Moreover, honey bees are the primary insect pollinators used in modern commercial production agriculture, and their populations have been in decline worldwide. Here, we compare the mating frequencies of queens, and therefore, intracolony genetic diversity, in three commercial beekeeping operations to determine how they correlate with various measures of colony health and productivity, particularly the likelihood of queen supersedure and colony survival in functional, intensively managed beehives. We found the average effective paternity frequency (m _e ) of this population of honey bee queens to be 13.6?±?6.76, which was not significantly different between colonies that superseded their queen and those that did not. However, colonies that were less genetically diverse (headed by queens with m _e ?≤?7.0) were 2.86 times more likely to die by the end of the study when compared to colonies that were more genetically diverse (headed by queens with m _e ?>?7.0). The stark contrast in colony survival based on increased genetic diversity suggests that there are important tangible benefits of increased queen mating number in managed honey bees, although the exact mechanism(s) that govern these benefits have not been fully elucidated.     

Biosynthesis of the defensive alkaloid cicindeloine in Stenus solutus beetles

To protect themselves from predation and microorganismic infestation, rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine, 3-(2-methyl-1-butenyl)pyridine, and cicindeloine in their pygidial glands. The biosynthesis of stenusine and 3-(2-methyl-1-butenyl)pyridine was previously investigated in Stenus bimaculatus and Stenus similis, respectively. Both molecules follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from l-lysine and the side chain from l-isoleucine. The different alkaloids are finally obtained by slight modifications of shared precursor molecules. The piperideine alkaloid cicindeloine occurs as a main compound additionally to (E)-3-(2-methyl-1-butenyl)pyridine and traces of stenusine in the pygidial gland secretion of Stenus cicindeloides and Stenus solutus. Feeding of S. solutus beetles with [D,¹⁵N]-labeled amino acids followed by GC/MS analysis techniques showed that cicindeloine is synthesized via the identical pathway and precursor molecules as the other two defensive alkaloids.