An Overview of Tsetse Fly Repellents: Identification and Applications.

Tsetse flies are vectors of the parasite trypanosoma that cause the neglected tropical diseases human and animal African trypanosomosis. Semiochemicals play important roles in the biology and ecology of tsetse flies. Previous reviews have focused on olfactory-based attractants of tsetse flies. Here, we present an overview of the identification of repellents and their development into control tools for tsetse flies. Both natural and synthetic repellents have been successfully tested in laboratory and field assays against specific tsetse fly species. Thus, these repellents presented as innovative mobile tools offer opportunities for their use in integrated disease management strategies.

Differences in the suitability of published honey bee (Apis mellifera) reference genes between the African subspecies Apis mellifera scutellata and European derived Apis mellifera.

Quantitative real-time polymerase chain reaction (qPCR) is a method widely used to determine changes and differences in gene expression. As target gene expression is most often quantified relative to the expression of reference genes, the validation of suitable reference genes is of critical importance. In practice, however, such validation might not be thoroughly conducted if the same species and the same tissue or body parts are used for qPCR experiments. Here we show, that qPCR reference genes published for workers of European honey bee (Apis mellifera) subspecies fail to be stably expressed in workers of the African subspecies Apis mellifera scutellata. This is the case even when the sampled workers are in the same life stage, the same organ was dissected and the same reagents were used. Thus, reference genes need to be thoroughly re-tested before they can be used as suitable references even when the only thing that changes is the subspecies used.

Chemical Cues From Honeydew and Cuticular Extracts of Trialeurodes Vaporariorum Serve as Kairomones for The Parasitoid Encarsia Formosa.

Kairomones are semiochemicals that are emitted by an organism and which mediate interspecific interaction that is of benefit to an organism of another species that receives these chemical substances. Parasitoids find and recognize their hosts through eavesdropping on the kairomones emitted from the by-products or the body of the host. Hemipteran insect pests feed on plant sap and excrete the digested plant materials as honeydew. Honeydew serves as a nutritional food source for parasitoids and a medium for micro-organisms whose activity induces the release of volatiles exploited by parasitoids for host location. The parasitoid Encarsia formosa preferentially parasitizes its host, the greenhouse whitefly, Trialeurodes vaporariorum, on tomato Solanum lycopersicum, but little is known about the chemicals that mediate these interactions. We investigated the olfactory responses of the parasitoid E. formosa to odours from honeydew and nymphs of T. vaporariorum in a Y-tube olfactometer. Arrestment behaviour of the parasitoid to honeydew and nymph extracts, as well as to synthetic hydrocarbons, was also observed in Petri-dish bioassays. We found that T. vaporariorum honeydew volatiles attracted the parasitoid E. formosa but odours from the whitefly nymphs did not. We also found that the parasitoid spent more time searching on areas treated with extracts of honeydew and nymphs than on untreated areas. Gas-chromatography-mass spectrometric analysis revealed that the honeydew volatiles contained compounds such as (Z)-3-hexenol, δ-3-carene, 3-octanone, α-phellandrene, methyl salicylate, ß-ocimene, ß-myrcene, and (E)-ß-caryophyllene which are known to be attractive to E. formosa. The cuticular extracts of the nymphs predominantly contained alkanes, alkenes, and esters. Among the alkanes, synthetic nonacosane arrested the parasitoid. Our findings are discussed in relation to how the parasitoid E. formosa uses these chemicals to locate its host, T. vaporariorum.

Functional response of the hypopharyngeal glands to a social parasitism challenge in Southern African honey bee subspecies.

Hypopharyngeal gland (HPG) development in honey bee workers is primarily age-dependent and changes according to the tasks performed in the colony. HPG activity also depends on colony requirements and is flexible in relation to the need for feeding brood. Very little is known about HPG development in the honey bee subspecies found in Southern Africa. We examined HPG development in Apis mellifera scutellata and A. m. capensis, including A. m. scutellata colonies infested with an invasive parasitic clonal lineage of A. m. capensis known to manipulate food provisioning to the parasitic larvae by their A.m. scutellata hosts, under natural in-hive conditions in bees aged 0 to 14 days using light microscopy. We found marked differences in acini size (berry-like clusters of secretory cells) and the age at which maximum HPG development occurred between the subspecies and in the presence of the parasite. In A. m. scutellata workers, acini reached maximum size at 6 days. The acini of A. m. capensis workers were larger (up to double) than those of A. m. scutellata and reached maximum size at 8 days, while the HPG acini in A. m. scutellata workers infested with A. m. capensis clones reached development sizes similar to those of A. m. capensis at day 10 and were 1.5 times larger than those of uninfested A. m. scutellata. This provides foundational insights into a functional response affecting the development of the HPG most likely associated with brood pheromone composition and how this is altered in the presence of a social parasite.

Prisoners receive food fit for a queen: honeybees feed small hive beetles protein-rich glandular secretions through trophallaxis.

The honeybee nest parasite Aethina tumida (small hive beetle) uses behavioural mimicry to induce trophallactic feeding from its honeybee hosts. Small hive beetles are able to induce honeybee workers to share the carbohydrate-rich contents of their crops, but it is not clear whether the beetles are able to induce to workers to feed them the protein-rich hypopharyngeal glandular secretions fed to the queen, larvae and other nest mates. Protein is a limiting macronutrient in an insect's diet, essential for survival, growth and fecundity. Honeybees obtain protein from pollen, which is consumed and digested by nurse bees. They then distribute the protein to the rest of the colony in the form of hypopharyngeal gland secretions. Using 14C-phenylalanine as a qualitative marker for protein transfer, we show that small hive beetles successfully induce worker bees to feed them the protein-rich secretions of their hypopharyngeal glands during trophallaxis, and that females are more successful than males in inducing the transfer of these protein-rich secretions. Furthermore, behavioural observations demonstrated that female beetles do not preferentially interact with a specific age cohort of bees when soliciting food, but males tend to be more discriminant and avoid the more aggressive and active older bees.

The Role of Trialeurodes vaporariorum-Infested Tomato Plant Volatiles in the Attraction of Encarsia formosa (Hymenoptera: Aphelinidae).

Natural enemies locate their herbivorous host and prey through kairomones emitted by host plants and herbivores. These kairomones could be exploited to attract and retain natural enemies in crop fields for insect pest control. The parasitoid Encarsia formosa preferentially parasitises its whitefly host, Trialeurodes vaporariorum, a major pest of tomato Solanum lycopersicum, thus offering an effective way to improve whitefly control. However, little is known about the chemical interactions that occur in E. formosa-T. vaporariorum-S. lycopersicum tritrophic system. Using behavioural assays and chemical analyses, we investigated the kairomones mediating attraction of the parasitoid to host-infested tomato plants. In Y-tube olfactometer bioassays, unlike volatiles of healthy tomato plants, those of T. vaporariorum-infested tomato plants attracted E. formosa, and this response varied with host infestation density. Coupled gas chromatography/mass spectrometric analyses revealed that host infestation densities induced varying qualitative and quantitative differences in volatile compositions between healthy and T. vaporariorum adult-infested tomato plants. Bioassays using synthetic chemicals revealed the attractiveness of 3-carene, ß-ocimene, ß-myrcene and α-phellandrene to the parasitoid, and the blend of the four compounds elicited the greatest attraction. Our results suggest that these terpenes could be used as an attractant lure to recruit the parasitoid E. formosa for the control of whiteflies in tomato crop fields.

A Single SNP Turns a Social Honey Bee (Apis mellifera) Worker into a Selfish Parasite.

The evolution of altruism in complex insect societies is arguably one of the major transitions in evolution and inclusive fitness theory plausibly explains why this is an evolutionary stable strategy. Yet, workers of the South African Cape honey bee (Apis mellifera capensis) can reverse to selfish behavior by becoming social parasites and parthenogenetically producing female offspring (thelytoky). Using a joint mapping and population genomics approach, in combination with a time-course transcript abundance dynamics analysis, we show that a single nucleotide polymorphism at the mapped thelytoky locus (Th) is associated with the iconic thelytokous phenotype. Th forms a linkage group with the ecdysis-triggering hormone receptor (Ethr) within a nonrecombining region under strong selection in the genome. A balanced detrimental allele system plausibly explains why the trait is specific to A. m. capensis and cannot easily establish itself into genomes of other honey bee subspecies.

Odor-Mediated Group Organization and Coordination in the Termite-Raiding Ant Megaponera analis (Mayr).

Visual and olfactory communications are vital for coordinated group hunting in most animals. To hunt for prey, the group-raiding termite specialist ant Megaponera analis, which lacks good vision, must first confirm the presence or absence of conspecific raiders. Here, we show that M. analis uses olfactory cues for intraspecific communication and showed greater preference for conspecific odors over clean air (blank) or odors from its termite prey. Chemical analysis of ant volatiles identified predominantly short-chained hydrocarbons. Electrophysiological analysis revealed differential sensory detection of the odor compounds, which were confirmed in behavioral olfactometric choice assays with odor bouquets collected from major and minor castes and the 2 most dominant volatiles and n-undecane n-tridecane. A comparative analysis of the cuticular hydrocarbon profile with those of the short-chained odor bouquet of different populations shows a high divergence in the long-chained profile and a much-conserved short-chained odor bouquet. This suggests that there is less selection pressure for divergence and individual recognition in the short- than the long-chained odor profiles. We conclude that olfactory communication serves as an alternative to visual or sound communication, especially during group raids in M. analis when ants are not in direct contact with one another.

Modelling the effect of temperature on the biology and demographic parameters of the African coffee white stem borer, Monochamus leuconotus (Pascoe) (Coleoptera: Cerambycidae).

The African coffee white stem borer Monochamus leuconotus (Pascoe) (Coleoptera: Cerambycidae) is a destructive insect pest of Arabica coffee trees in African highlands. Our study aims to provide information on the pest biology as influenced by temperature, determine thermal thresholds, and provide life table parameters for M. leuconotus reared in the laboratory. The life cycle of M. leuconotus was studied at seven constant temperatures in the range 15-35 °C, with 80 ± 5% RH and a photoperiod of L:D 12:12. Linear and nonlinear models were fitted to laboratory data to describe the impact of temperature on M. leuconotus development, mortality, fecundity and senescence. The complete life cycle was obtained between 18 and 30 °C, with the egg incubation period ranging 10.8-29.2 days. The development time was longest for the larva, with 194.2 days at 30 °C and 543.1 days at 18 °C. The minimum temperature threshold (Tmin) was estimated at 10.7, 10.0 and 11.5 °C, for egg, larva and pupa, respectively. The maximum temperature threshold (Tmax) was estimated at 37.4, 40.6 and 40.0 °C for egg, larva and pupa, respectively. The optimum temperature for immature stage survival was estimated between 23.0 and 23.9 °C. The highest fecundity was 97.8 eggs per female at 23 °C. Simulated life table parameters showed the highest net reproductive rate (Ro) of 11.8 daughters per female at 26 °C and maximal intrinsic rate of increase (rm) between 26 and 28 °C, with a value of 0.008. Our results will help understanding M. leuconotus biology as influenced by temperature and may be used to predict the distribution and infestation risk under climate warming for this critical coffee pest.

Turning workers into false queens: the role of exogenous pheromones in regulating reproduction in worker honey bees.

One of the responses that honey bee workers can make in the event of queen loss is to develop into false queens. False queens are workers that exhibit both behavioural and physiological traits similar to those of a true queen. However, the presence of more than one false queen in a colony distorts the established hierarchies. As transformation into a false queen occurs after emergence as an adult, we tested the effect of worker mobile pheromone carriers (PCs) treated with exogenously supplied pheromones on their nestmates. The PCs carried either synthetic mandibular gland pheromones or pheromones extracted from Apis melliferacapensis parasitic workers. Only the PCs attracted retinues of workers, increased pheromone production and activated their ovaries, becoming false queens. Pheromones from A. m.capensis workers were more effective than extracts of commercially available synthetic queen pheromones in eliciting these effects. Using this simple mobile pheromone delivery system, we have shown that carrying amounts of exogenous pheromone can induce pheromone production in the carrier, resulting in the production of false queens within experimental groups. Possible implications of using this technique to modify and regulate worker reproduction in colonies are discussed.

The transcriptomic changes associated with the development of social parasitism in the honeybee Apis mellifera capensis.

Social insects are characterized by the division of labor. Queens usually dominate reproduction, whereas workers fulfill non-reproductive age-dependent tasks to maintain the colony. Although workers are typically sterile, they can activate their ovaries to produce their own offspring. In the extreme, worker reproduction can turn into social parasitism as in Apis mellifera capensis. These intraspecific parasites occupy a host colony, kill the resident queen, and take over the reproductive monopoly. Because they exhibit a queenlike behavior and are also treated like queens by the fellow workers, they are so-called pseudoqueens. Here, we compare the development of parasitic pseudoqueens and social workers at different time points using fat body transcriptome data. Two complementary analysis methods-a principal component analysis and a time course analysis-led to the identification of a core set of genes involved in the transition from a social worker into a highly fecund parasitic pseudoqueen. Comparing our results on pseudoqueens with gene expression data of honeybee queens revealed many similarities. In addition, there was a set of specific transcriptomic changes in the parasitic pseudoqueens that differed from both, queens and social workers, which may be typical for the development of the social parasitism in A. m. capensis.

Low fertility, fecundity and numbers of mated female offspring explain the lower reproductive success of the parasitic mite Varroa destructor in African honeybees.

Although Varroa destructor is the most serious ecto-parasite to the honeybee, Apis mellifera L., some honeybee populations such as Apis mellifera scutellata in Kenya can survive mite infestations without treatment. Previously, we reported that grooming behaviour could be a potential tolerant mechanism expressed by this honeybee subspecies towards mite infestation. However, both hygienic and grooming behaviours could not explain the lower mite-infestation levels recorded in these colonies. Here, we investigated the involvement of other potential resistant mechanisms including suppression of mite reproduction in worker brood cells of A. m. scutellata to explain the low mite numbers in their colonies. High infertility rates (26-27%) and percentages of unmated female offspring (39-58%) as well as low fecundity (1.7-2.2, average offspring produced) were identified as key parameters that seem to interact with one another during different seasons to suppress mite reproduction in A. m. scutellata colonies. We also identified offspring mortality in both sexes and absence of male offspring as key factors accounting for the low numbers of mated daughter mites produced in A. m. scutellata colonies. These results suggest that reduced mite reproductive success could explain the slow mite population growth in A. m. scutellata colonies.

Effect of Brood Pheromone on Survival and Nutrient Intake of African Honey Bees (Apis mellifera scutellata) under Controlled Conditions.

The influence of pheromones on insect physiology and behavior has been thoroughly reported for numerous aspects, such as attraction, gland development, aggregation, mate and kin recognition. Brood pheromone (BP) is released by honey bee larvae to indicate their protein requirements to the colony. Although BP is known to modulate pollen and protein consumption, which in turn can affect physiological and morphological parameters, such as hypopharyngeal gland (HPG) development and ovarian activation, few studies have focused on the effect of BP on nutritional balance. In this study, we exposed newly emerged worker bees for 14 d and found that BP exposure increased protein intake during the first few days, with a peak in consumption at day four following exposure. BP exposure decreased survival of caged honey bees, but did not affect either the size of the HPG acini or ovarian activation stage. The uncoupling of the BP releaser effect, facilitated by working under controlled conditions, and the presence of larvae as stimulating cues are discussed.

Resistance rather than tolerance explains survival of savannah honeybees (Apis mellifera scutellata) to infestation by the parasitic mite Varroa destructor.

Varroa destructor is considered the most damaging parasite affecting honeybees (Apis mellifera L.). However, some honeybee populations such as the savannah honeybee (Apis mellifera scutellata) can survive mite infestation without treatment. It is unclear if survival is due to resistance mechanisms decreasing parasite reproduction or to tolerance mechanisms decreasing the detrimental effects of mites on the host. This study investigates both aspects by quantifying the reproductive output of V. destructor and its physiological costs at the individual host level. Costs measured were not consistently lower when compared with susceptible honeybee populations, indicating a lack of tolerance. In contrast, reproduction of V. destructor mites was distinctly lower than in susceptible populations. There was higher proportion of infertile individuals and the reproductive success of fertile mites was lower than measured to date, even in surviving populations. Our results suggest that survival of savannah honeybees is based on resistance rather than tolerance to this parasite. We identified traits that may be useful for breeding programmes aimed at increasing the survival of susceptible populations. African honeybees may have benefited from a lack of human interference, allowing natural selection to shape a population of honeybees that is more resistant to Varroa mite infestation.

High Royal Jelly-Producing Honeybees (Apis mellifera ligustica) (Hymenoptera: Apidae) in China.

China is the largest producer and exporter of royal jelly (RJ) in the world, supplying >90% of the global market. The high production of RJ in China is principally owing to the high RJ-producing lineage of honeybees (Apis mellifera ligustica Spinola, 1806) established by beekeepers in the 1980s. We describe the development of high royal jelly-producing honeybees and the management of this lineage today. Previous research and recent advances in the genetic characterization of this lineage, and the molecular markers and mechanisms associated with high RJ production are summarized. The gaps in our knowledge and prospects for future research are also highlighted.

Effects of age and Reproductive Status on Tergal Gland Secretions in Queenless Honey bee Workers, Apis mellifera scutellata and A. m. capensis.

Secretions from tergal glands are part of a queen's pheromonal control of worker reproduction in honey bees. However, in queenless honey bee colonies, workers compete to gain pheromonal, and hence reproductive dominance, over nestmates with ontogenetic changes in their glandular secretions that affect the behavioral or physiological responses of other individuals. Using gas chromatography/mass spectrometry, we investigated for the first time the age-dependent changes in tergal gland secretions of queenless workers of the clonal lineage of Apis mellifera capensis and workers of A. m. scutellata. The reproductive status of honey bee workers was determined by recording the presence of spermathecae and the level of ovarian activation. The tergal gland chemicals identified in both A. m. scutellata workers and A. m. capensis clone workers were oleic acid, n-tricosene, n-pentacosene, and n-heptacosene, with three additional compounds, palmitic acid, n-heneicosene, and n-nonacosene, in A. m. capensis clones. We report ethyl esters as new compounds from honey bee worker tergal gland profiles; these compounds increased in amount with age. All A. m. capensis clone workers dissected had spermathecae and showed ovarian activation from day 4, while ovarian activation only started on day 7 for A. m. scutellata workers that had no spermathecae. Tergal gland secretions were present in higher quantities in bees with activated, rather than inactive ovaries. This suggests that tergal gland secretions from reproductive workers could act as releaser and primer pheromones in synergy with other glandular compounds to achieve pheromonal and reproductive dominance.

Impact of Varroa destructor on honeybee (Apis mellifera scutellata) colony development in South Africa.

The devastating effects of Varroa destructor Anderson & Trueman on European honeybee colonies (Apis mellifera L.) have been well documented. Not only do these mites cause physical damage to parasitised individuals when they feed on them, they also transmit viruses and other pathogens, weaken colonies and can ultimately cause their death. Nevertheless, not all honeybee colonies are doomed once Varroa mites become established. Some populations, such as the savannah honeybee, A. m. scutellata, have become tolerant after the introduction of the parasite and are able to withstand the presence of these mites without the need for acaricides. In this study, we measured daily Varroa mite fall, Varroa infestation rates of adult honeybees and worker brood, and total Varroa population size in acaricide treated and untreated honeybee colonies. In addition, honeybee colony development was compared between these groups in order to measure the cost incurred by Varroa mites to their hosts. Daily Varroa mite fall decreased over the experimental period with different dynamics in treated and untreated colonies. Varroa infestation rates in treated adult honeybees and brood were lower than in untreated colonies, but not significantly so. Thus, indicating a minimal benefit of treatment thereby suggesting that A. m. scutellata have the ability to maintain mite populations at low levels. We obtained baseline data on Varroa population dynamics in a tolerant honeybee over the winter period. Varroa mites appeared to have a low impact on this honeybee population, given that colony development was similar in the treated and untreated colonies.

Olfactory detection of prey by the termite-raiding antPachycondyla analis.

The African termiteraiding ant Pachycondyla analis Latreille (Hymenoptera: Formicidae) organizes group raids on termites of the subfamily Macrotermitinae. Termites and ants occupy and share similar habitats, resulting in a co-evolutionary arms race between termites as prey and ants as predators. The present study explored whether P. analis uses semio- chemical signaling cues to detect potential termite prey prior to and during raids. Ants' responses to odors emitted from termites alone, termite gallery soil, and termites inside their galleries were tested using Y-tube olfactometer assays. The results showed that P. analis detected odors of termites and those of their galleries, and odors from termites inside their galleries were more attractive to both minor and major ant workers than odors from termites alone. The composition of these odor sources was identified using gas chromatography-mass spectrometry analysis. While the odors from termite gallery soils were compositionally richer (containing 13 compounds rather than nine from termites alone), those from the termites alone were quantitatively richer, releasing about six times more odors than gallery soil. Most of the compounds in the odor profiles were identified as hydrocarbons. Naphthalene, previously identified as an insect repellent, was also identified as a component of the odors from the gallery soil. These results demonstrate that odors play an important role in prey detection by P. analis.

Expression of honey bee (Apis mellifera) sterol homeostasis genes in food jelly producing glands of workers.

Adult workers of Western honey bees (Apis mellifera L.) acquire sterols from their pollen diet. These food sterols are transported by the hemolymph to peripheral tissues such as the mandibular and the hypopharyngeal glands in the worker bees' heads that secrete food jelly which is fed to developing larvae. As sterols are obligatory components of biological membranes and essential precursors for molting hormone synthesis in insects, they are indispensable to normal larval development. Thus, the study of sterol delivery to larvae is important for a full understanding of honey bee larval nutrition and development. Whereas hypopharyngeal glands only require sterols for their membrane integrity, mandibular glands add sterols, primarily 24-methylenecholesterol, to its secretion. For this, sterols must be transported through the glandular epithelial cells. We have analyzed for the first time in A. mellifera the expression of genes which are involved in intracellular movement of sterols. Mandibular and hypopharyngeal glands were dissected from newly emerged bees, 6-day-old nurse bees that feed larvae and 26-day-old forager bees. The expression of seven genes involved in intracellular sterol metabolism was measured with quantitative real-time PCR. Relative transcript abundance of sterol metabolism genes was significantly influenced by the age of workers and specific genes but not by gland type. Newly emerged bees had significantly more transcripts for six out of seven genes than older bees indicating that the bulk of the proteins needed for sterol metabolism are produced directly after emergence.

Honeybees prefer warmer nectar and less viscous nectar, regardless of sugar concentration.

The internal temperature of flowers may be higher than air temperature, and warmer nectar could offer energetic advantages for honeybee thermoregulation, as well as being easier to drink owing to its lower viscosity. We investigated the responses of Apis mellifera scutellata (10 colonies) to warmed 10% w/w sucrose solutions, maintained at 20-35°C, independent of low air temperatures, and to 20% w/w sucrose solutions with the viscosity increased by the addition of the inert polysaccharide Tylose (up to the equivalent of 34.5% sucrose). Honeybee crop loads increased with nectar temperature, as did the total consumption of sucrose solutions over 2 h by all bees visiting the feeders. In addition, the preference of marked honeybees shifted towards higher nectar temperatures with successive feeder visits. Crop loads were inversely proportional to the viscosity of the artificial nectar, as was the total consumption of sucrose solutions over 2 h. Marked honeybees avoided higher nectar viscosities with successive feeder visits. Bees thus showed strong preferences for both warmer and less viscous nectar, independent of changes in its sugar concentration. Bees may benefit from foraging on nectars that are warmer than air temperature for two reasons that are not mutually exclusive: reduced thermoregulatory costs and faster ingestion times due to the lower viscosity.