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.

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.

Mandibular glands secrete 24-methylenecholesterol into honey bee (Apis mellifera) food jelly.

Honey bee (Apis mellifera) workers feed their larvae with food jelly that is secreted by specialized glands in their heads - the hypopharyngeal and the mandibular glands. Food jelly contains all the nutrients the larvae need to develop into adult honey bees, including essential dietary sterols. The main sterol in food jelly, 24-methylenecholesterol (24MC), is pollen-derived and delivered in food jelly to the larvae in a complex with two proteins, major royal jelly protein 1 (MRJP1) and apisim. Whereas the proteins are synthesized in the hypopharyngeal glands, the sterol-secreting gland has not been identified. We here identified the mandibular glands as sterol-secreting gland for food jelly production by direct detection of the four main honey bee sterols (24MC, campesterol, ß-sitosterol and isofucosterol). Furthermore, 24MC seems to be specifically enriched in the mandibular glands, thereby ensuring that food jelly contains the amounts of 24MC necessary for complex formation with MRJP1 and apisimin.

Spatial distribution of Glossina morsitans (Diptera: Glossinidae) in Zambia: A vehicle-mounted sticky trap survey and Maxent species distribution model.

BACKGROUND: Tsetse-transmitted African trypanosomiasis is a debilitating and fatal disease of humans and livestock if left untreated. While knowledge of the spatial distribution patterns of tsetse is essential for the development of risk-based vector control strategies, existing distribution maps in Zambia are more than 40 years old and were based on coarse spatial resolution data. The recently developed vehicle-mounted sticky trap (VST) provides an alternative sampling device to aid in updating existing distribution maps but has not been applied outside an experimental setting and is limited to motorable tracks. Therefore, the objective of the present study was to demonstrate the effectiveness of utilizing the VST for area-wide surveys of Glossina morsitans and to use the occurrence records to predict its spatial distribution in Zambia under current environmental conditions using Maxent. METHODOLOGY/PRINCIPAL FINDINGS: Two-sided all-blue VST baited with butanone and 1-octen-3-ol was used to survey 692 and 1020 km of transect routes in G. m. centralis Machado and G. m. morsitans Westwood previously published distribution in Zambia. Maxent species distribution technique was used to predict the potential distribution of the two subspecies using current climatic and environmental data which was then compared to the historical distribution. A total of 15,602 tsetse were captured with G. m. morsitans (58%) being the most abundant. G. m. centralis and G. pallidipes Austin represented 39 and 2% of the catch respectively, and G. brevipalpis Newstead was also detected. The predicted potential distribution for G. m. centralis was 80,863 km2 while that of G. m. morsitans was 70,490 km2 representing a 47 and 29% reduction compared to their historical distributions, respectively. CONCLUSION/SIGNIFICANCE: The VST is effective for sampling G. morsitans outside experimental settings and is recommended for use as an additional tsetse survey tool. The spatial distribution of G. morsitans in Zambia has reduced by 101,051 km2 due to temperature and land cover changes.

Seasonal and elevational changes of plant-pollinator interaction networks in East African mountains.

Across an elevation gradient, several biotic and abiotic factors influence community assemblages of interacting species leading to a shift in species distribution, functioning, and ultimately topologies of species interaction networks. However, empirical studies of climate-driven seasonal and elevational changes in plant-pollinator networks are rare, particularly in tropical ecosystems. Eastern Afromontane Biodiversity Hotspots in Kenya, East Africa. We recorded plant-bee interactions at 50 study sites between 515 and 2600 m asl for a full year, following all four major seasons in this region. We analysed elevational and seasonal network patterns using generalised additive models (GAMs) and quantified the influence of climate, floral resource availability, and bee diversity on network structures using a multimodel inference framework. We recorded 16,741 interactions among 186 bee and 314 plant species of which a majority involved interactions with honeybees. We found that nestedness and bee species specialisation of plant-bee interaction networks increased with elevation and that the relationships were consistent in the cold-dry and warm-wet seasons respectively. Link rewiring increased in the warm-wet season with elevation but remained indifferent in the cold-dry seasons. Conversely, network modularity and plant species were more specialised at lower elevations during both the cold-dry and warm-wet seasons, with higher values observed during the warm-wet seasons. We found flower and bee species diversity and abundance rather than direct effects of climate variables to best predict modularity, specialisation, and link rewiring in plant-bee-interaction networks. This study highlights changes in network architectures with elevation suggesting a potential sensitivity of plant-bee interactions with climate warming and changes in rainfall patterns along the elevation gradients of the Eastern Afromontane Biodiversity Hotspot.

Honey bees save energy in honey processing by dehydrating nectar before returning to the nest.

Honey bees process nectar into honey by active evaporation on the tongue and passive evaporation involving nest ventilation and fanning behaviour, as well as enzymatic action. The elimination of excess water from nectar carries considerable energetic costs. The concentration of the nectar load is assumed to remain constant during transport. However, some of this water elimination may occur before foragers return to the nest and pass their nectar loads to receiver bees. In honey bees captured while foraging in Macadamia orchards, we show that the nectar in their crops has approximately twice the sugar concentration of the fresh nectar in flowers. This was true for four Macadamia cultivars, with up to 75% of the initial water content being removed. There is a further concentration increase in the crops of returning bees captured at the hive entrance. The only possible route of water elimination from the crop is via evaporation from the mouthparts. We calculate the savings in honey processing costs to be on average 35 times more than the reduction in flight costs due to reduced body mass. Pre-concentration of nectar in foraging honey bees may be widespread, and of crucial importance for honey storage.

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.

Efficiencies of stationary sampling tools for the tsetse fly Glossina fuscipes fuscipes in western Kenya.

Monitoring the effectiveness of tsetse fly control interventions that aim to reduce transmission of African trypanosomiasis requires highly efficient sampling tools that can catch flies at low densities. The sticky small target (StS-target) has previously been shown to be more effective in sampling Glossina fuscipes fuscipes compared to the biconical trap. However, its efficiency in terms of the proportion of flies it catches out of those that visit it has not been reported. Furthermore, there are no reports on whether tsetse samples caught using the StS-target can be used for subsequent processes such as molecular tests. In this study, we evaluated the efficiency of the biconical trap and targets for sampling G. f. fuscipes. All targets were tiny (0.25 × 0.50 m) but varied in their capture system. We used targets with sticky surface (StS-targets) and those with an electrified surface (ES-targets). We also assessed the suitability of flies caught on the StS-target for molecular tests by amplifying DNA of bacterial communities. Randomized block design experiments were undertaken in Mbita area and Manga Island on Lake Victoria of western Kenya. Fly catches of each sampling tool were compared to those of the sampling tool flanked by electric (E) nets and analyzed using a negative binomial regression. The total catch for each sampling tool alone was divided by the total catch of the sampling tool flanked by two E-nets to obtain its efficiency expressed as a percentage. A proportion of flies caught on the StS-target was preserved for molecular tests. Overall, the efficiencies of the biconical trap, ES-target and StS-target were 7.7%, 13.3% and 27.0%, respectively. A higher proportion of females (69 to 79%) than males approached all the sampling tools, but the trap efficiency was greater for male G. f. fuscipes than females. Furthermore, sequencing the 16S rRNA gene from fly samples caught on the StS-target revealed the presence of Spiroplasma. Our results indicate that the SS-target is the most efficient trap to monitor G. f. fuscipes population during interventions, with the biconical trap being the least efficient, and samples collected from StS-targets are suitable for molecular studies.

A novel vehicle-mounted sticky trap; an effective sampling tool for savannah tsetse flies Glossina morsitans morsitans Westwood and Glossina morsitans centralis Machado.

BACKGROUND: Black screen fly round (BFR) is a mobile sampling method for Glossina morsitans. This technique relies on the ability of operator(s) to capture flies landing on the screen with hand nets. In this study, we aimed to evaluate a vehicle-mounted sticky panel trap (VST) that is independent of the operator's ability to capture flies against BFR, for effective and rapid sampling of G. m. morsitans Westwood and G. m. centralis Machado. We also determined the influence of the VST colour (all-blue, all-black or 1:1 blue-black), orientation and presence of odour attractants on tsetse catch. METHODOLOGY/PRINCIPAL FINDINGS: Using randomised block design experiments conducted in Zambia, we compared and modelled the number of tsetse flies caught in the treatment arms using negative binomial regression. There were no significant differences in the catch indices of the three colour designs and for in-line or transversely oriented panels for both subspecies (P > 0.05). When baited with butanone and 1-octen-3-ol, VST caught 1.38 (1.11-1.72; P < 0.01) times more G. m. centralis flies than the un-baited trap. Attractants did not significantly increase the VST catch index for G. m. morsitans (P > 0.05). Overall, the VST caught 2.42 (1.91-3.10; P < 0.001) and 2.60 (1.50-3.21; P < 0.001) times more G. m. centralis and G. m. morsitans respectively, than the BFR. The VST and BFR took 10 and 35 min respectively to cover a 1 km transect. CONCLUSION/SIGNIFICANCE: The VST is several times more effective for sampling G. m. morsitans and G. m. centralis than the BFR and we recommend its use as an alternative sampling tool.

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.

Plant nutrient quality impacts survival and reproductive fitness of the dengue vector Aedes aegypti.

BACKGROUND: In a recent study using DNA barcoding, we identified the plants fed upon by four Afro-tropical mosquito species that vector dengue, malaria, and Rift Valley fever. Herein, we have expanded on this study by investigating the role of three of the plants, Pithecellobium dulce (Fabaceae), Leonotis nepetifolia (Lamiaceae), and Opuntia ficus-indica (Cactaceae), on the survival, fecundity, and egg viability of the dengue vector Aedes aegypti. METHODS: We tested these effects using females that received (i) an initial three rations of blood meals and (ii) no blood meal at all. Two controls were included: age-matched females fed on glucose solution with or without an initial blood meal and those fed exclusively on blood meals. Data were collected daily over a 30-day period. The amino acid contents of Ae. aegypti guts and their respective diets were detected by coupled liquid chromatography-mass spectrometry. RESULTS: Females fed on P. dulce and an exclusively blood meal diet had a shorter survival than those fed on glucose. On the other hand, females fed on L. nepetifolia survived longer than those fed exclusively on blood meals, whereas those fed on O. ficus-indica had the shortest survival time. With an initial blood meal, females fed on L. nepetifolia laid 1.6-fold more eggs while those fed on the other diets laid fewer eggs compared to those fed exclusively on blood meals. Hatching rates of the eggs laid varied with the diet. Mass spectroscopic analysis of gut contents of mosquitoes exposed to the different diets showed qualitative and quantitative differences in their amino acid levels. CONCLUSION: Our findings highlight the central role of plant nutrients in the reproductive fitness of dengue vectors, which may impact their disease transmission potential.

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.

Biological traits of wild-caught populations of Aedes aegypti in dengue endemic and non-endemic regions of Kenya.

Variation in vector traits can modulate local scale differences in pathogen transmission. Here, we compared seasonal variation in the wing length (proxy for body size) and energy reserves of adult wild-caught Aedes aegypti populations from a dengue endemic (Kilifi) and non-endemic (Isiolo) area of Kenya. Vector sampling in the dengue endemic site was conducted during the dry and wet seasons. In the non-endemic area, it was limited to the dry season which characterizes this ecology where sporadic or no rainfall is commonplace during the year. We found variation by site in the body size of both sexes, with an overall smaller size of Ae. aegypti populations collected from Isiolo than those from Kilifi. Our results show that although total carbohydrates and lipids levels were highest in both sexes during the dry season, they were two-fold higher in males than females. However, we found weak correlations between body size and energy reserves for both sexes, with body size being more sensitive in identifying differences at a population level. These results provide insights into the determinants of the vectoring potential of Ae. aegypti populations in dengue endemic and non-endemic ecologies in Kenya.

Effect of zebra skin-derived compounds on field catches of the human African trypanosomiasis vector Glossina fuscipes fuscipes.

The riverine tsetse fly Glossina fuscipes fuscipes is a major vector of trypanosome pathogens causing African trypanosomiasis. This fly species uses a combination of olfactory and visual cues to locate its hosts. Previously, traps and targets baited with visual cues have been used in vector control, but the development of olfactory-based tools has been challenging. Recently, repellents have shown promise as olfactory-based tools in tsetse vector control. Here, we evaluated a three-component blend comprising 6-methyl-5-hepten-2-one, acetophenone and geranyl acetone (blend K), previously identified as a repellent for savannah tsetse flies in zebra skin odor, on G. f. fuscipes populations. Using a series of 6 × 6 randomized Latin square-designed experiments, G. f. fuscipes catches in biconical traps were monitored on four islands of Lake Victoria in western Kenya between July and September 2019, after the long rainy season. Traps were baited with blend K and individual components of this blend. The known tsetse repellent blend WRC (waterbuck repellent compounds) and trap alone were included as controls. Daily catch data in thirty-six replicate trials were analyzed using generalized linear model with negative binomial error structure using the package "MASS" in R. Treatment, day and site were set as predictor variables. Our results showed that, blend K significantly reduced G. f. fuscipes catches by 25.6% (P < 0.01) compared to the control trap alone but was not significantly different from WRC which reduced catches by 20.7% (P < 0.05). Of the individual compounds, geranyl acetone solely significantly reduced catches by 29.1% (P < 0.01) which did not differ from blend K or WRC. We conclude that geranyl acetone accounts for the repellent effect of blend K on the riverine tsetse fly, G. f. fuscipes, demonstrating the ecological importance of animal skin odors in the host-seeking behavior of medically-important tsetse fly vectors.

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.

Hydroxylation patterns associated with pheromone synthesis and composition in two honey bee subspecies Apis mellifera scutellata and A. m. capensis laying workers.

Colony losses due to social parasitism in the form of reproductive workers of the Apis mellifera capensis clones results from the production of queen-like pheromonal signals coupled with ovarian activation in these socially parasitic honey bees. While the behavioral attributes of these social parasites have been described, their genetic attributes require more detailed exploration. Here, we investigate the production of mandibular gland pheromones in queenless workers of two sub-species of African honey bees; A. m. scutellata (low reproductive potential) and A. m. capensis clones (high reproductive potential). We used standard techniques in gas chromatography to assess the amounts of various pheromone components present, and qPCR to assess the expression of cytochrome P450 genes cyp6bd1 and cyp6as8, thought to be involved in the caste-dependent hydroxylation of acylated stearic acid in queens and workers, respectively. We found that, for both subspecies, the quality and quantity of the individual pheromone components vary with age, and that from the onset, A. m. capensis parasites make use of gene pathways typically upregulated in queens in achieving reproductive dominance. Due to the high production of 9-hydroxy-decenoic acid (9-HDA) the precursor to the queen substance 9-oxo-decenoic acid (9-ODA) in newly emerged capensis clones, we argue that clones are primed for parasitism upon emergence and develop into fully fledged parasites depending on the colony's social environment.