Evolutionary Genomics at the Human-Environment Interface in Africa.

We report on the first meeting of SMBE in Africa. SMBE Malawi was initiated to bring together African and international researchers who use genetics or genomics to study natural systems impacted by human activities. The goals of this conference were 1) to reach a world-class standard of science with a large number of contributions from Africa, 2) to initiate exchange between African and international researchers, and 3) to identify challenges and opportunities for evolutionary genomics research in Africa. As repored, we think that we have achieved these goals and make suggestions on the way forward for African evolutionary genomics research.

Coexistence of honeybees with distinct mitochondrial haplotypes and hybridised nuclear genomes on the Comoros Islands.

The honeybee, Apis mellifera, is a globally distributed species that has spread both naturally and by humans across the globe resulting in many natural and secondary contact zones. The geographic isolation of honeybees is likely to contribute to genetic differentiation. Secondary contact has resulted in hybridization at the nuclear genome, but replacement of mitochondrial. Here, we used a mitochondrial marker and 19 microsatellite markers to test for the variations in the mitochondrial and nuclear genomes of honeybee populations on the Comoros islands. We used samples of 160 workers for mtDNA analysis and 288 workers from 16 colonies spread across the three islands for microsatellite analyses. Our results showed that the wild honeybee populations of the Comoros Islands consist of coexisting mitochondrial haplotypes. One belongs to the typical African A-lineage, and the other, the newly described L-lineage, is closely related to Apis koschevnikovi, a honeybee species native to Southeast Asia. The nuclear genomes show complete hybridization, high genetic diversity, and strong differentiation according to the island of origin. Based on our results, we hypothesise that the Asian honeybee could have been transported from Southeast Asia to Madagascar and Comoros via the human migrations that occurred 6000 years ago, and has hybridised with African honeybees at the nuclear genome, but maternal ancestry still can be traced using the mtDNA markers. We conclude that mtDNA plays a pivotal role in adaptation to the local environment, with both haplotypes of the honeybees of Comoros contributing significantly to the mito-nuclear coadaptation resulting in maintenance at almost equal frequency.

Population abundance of Varroa destructor and its effects on Apis mellifera scutellata colonies in Kenya.

The devastating effects of Varroa destructor Anderson and Trueman on Western honeybee colonies (Apis mellifera L.) have been well documented. Not only do these mites cause physical damage to parasitized individuals when they feed on them, but they also transmit viruses and other pathogens, weaken colonies and ultimately may cause their death. Unlike the subspecies of European origin, the honeybees of African origin suffer less from mite infestations. Absconding is one of the factors contributing to low V. destructor population in honeybee colonies as it creates a brood-free period. For a long time, researchers hypothesized that absconding was the main mechanism to control the parasite. The effects of V. destructor are well documented under temperate climatic conditions with a break during winter. Therefore, our study aimed at investigating the impact of V. destructor population growth on colony size, absconding and productivity under natural infestation levels of a tropical/subtropical climate with continuous brood production. We measured several characteristics related to the mite populations, the bee colonies and the resources of the bee colonies for a period of 8 months. The seven colonies that absconded during the study period were not influenced by densities of V. destructor. Absconding of the colonies occurred as a result of low numbers of capped brood. Mite densities were generally low throughout the study period (ranged between 26.9 and 59.8 mites per month) but were positively associated with adult bee densities. The amount of open and capped brood was positively associated with densities of V. destructor in the brood and negatively associated with denisities of V. destructor on screen boards, which appeared as extremely important factors that should be monitored regularly alongside colony stores and availability of pollen.

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens.

BACKGROUND: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. RESULTS: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. CONCLUSIONS: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

Genetic underpinnings of division of labor in the honeybee (Apis mellifera).

Honeybees have been studied for centuries, starting with Aristotle, who wrote the first book about bee breeding. More than 2000 years later, the honeybee entered the genomic era as the first social insect whose genome was sequenced, leading to significant insight into the molecular mechanisms underlying social behavior. In addition, gene expression studies and knockdown using RNAi have extended the understanding of social interactions. Much of the work has focused on caste determination - the mechanism that results in reproductive division of labor, division of labor within the worker caste, and worker reproduction - an essential process underlying eusociality. Here we review the molecular factors involved in caste determination and the differential regulation of caste-specific genes. Recent findings suggest that division of labor is influenced by a small number of loci showing high levels of pleiotropy, suggesting that changes in a small number of genes lead to large changes in the phenotype.

Insects' essential role in understanding and broadening animal medication.

Like humans, animals use plants and other materials as medication against parasites. Recent decades have shown that the study of insects can greatly advance our understanding of medication behaviors. The ease of rearing insects under laboratory conditions has enabled controlled experiments to test critical hypotheses, while their spectrum of reproductive strategies and living arrangements - ranging from solitary to eusocial communities - has revealed that medication behaviors can evolve to maximize inclusive fitness through both direct and indirect fitness benefits. Studying insects has also demonstrated in some cases that medication can act through modulation of the host's innate immune system and microbiome. We highlight outstanding questions, focusing on costs and benefits in the context of inclusive host fitness.

Correction: Synergies of integrated pest and pollinator management in avocado farming in East Africa: An ex-ante economic analysis.

[This corrects the article DOI: 10.1371/journal.pone.0271241.].

Social scent marks do not improve avoidance of parasites in foraging bumblebees.

Foraging is a result of innate and acquired mechanisms, and is optimized in order to increase fitness. During foraging, an animal faces many threats, such as predation and infection. The uptake of parasites and diseases while foraging is common and an individual should be adapted to detect and avoid such threats, using cues from either the abiotic environment or the parasite. Social animals possess an additional cue to detect such contaminated food sources: information provided by conspecifics. Bumblebees avoid contaminated flowers, but the cues used by the bees to distinguish contamination remain unknown. Under controlled laboratory conditions, we tested the use of scent marks derived from other foragers in choosing between a contaminated (by Crithidia bombi) and an uncontaminated flower. As a positive control we tested the bee's choice between two flowers, one scented with geraniol and containing a highly rewarding sugar solution, and the other not scented and containing a poorer reward. The bees mainly chose the uncontaminated and the rewarding scented flowers. Scent marks did not increase the efficiency of the bumblebees in choosing the better flower. The bees from both experiments behaved similarly, showing that the main and most relevant cue used to choose the uncontaminated flower is the odour from the parasite itself. The adaptation of bumblebees to avoid flowers contaminated by C. bombi arose from the long-term host-parasite interaction between these species. This strong adaptation results in an innate behaviour of bees and a detection and aversion of the odour of contaminated flower nectar.

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.

Interactions between integrated pest management, pollinator supplementation, and normalized difference vegetation index in pumpkin, Cucurbita maxima (Cucurbitales: Cucurbitaceae), production.

Sustainable production of pumpkin (Cucurbita maxima Duchesne) partly relies on integrated pest management (IPM) and pollination services. A farmer-managed field study was carried out in Yatta and Masinga Sub-Counties of Machakos County, Kenya, to determine the effectiveness of a recommended IPM package and its interaction with stingless bee colonies (Hypotrigona sp.) for pollinator supplementation (PS). The IPM package comprised Lynfield traps with cuelure laced with the organophosphate malathion, sprays of Metarhizium anisopliae (Mechnikoff) Sorokin isolate ICIPE 69, the most widely used fungal biopesticide in sub-Saharan Africa, and protein baits incorporating spinosad. Four treatments-IPM, PS, integrated pest and pollinator management (which combined IPM and PS), and control-were replicated 4 times. The experiment was conducted in 600 m2 farms in 2 normalized difference vegetation index (NDVI) classes during 2 growing seasons (October 2019-March 2020 and March-July 2020). Fruits showing signs of infestation were incubated for emergence, fruit fly trap catches were counted weekly, and physiologically mature fruits were harvested. There was no effect of IPM, PS, and NDVI on yield across seasons. This study revealed no synergistic effect between IPM and PS in suppressing Tephritid fruit fly population densities and damage. Hypotrigona sp. is not an efficient pollinator of pumpkin. Therefore, we recommend testing other African stingless bees in pumpkin production systems for better pollination services and improved yields.

Reproductive biology of the Cape honeybee: a critique of Beekman et al.

Laying workers of the Cape honeybee parthenogenetically produce female offspring, whereas queens typically produce males. Beekman et al. confirm this observation, which has repeatedly been reported over the last 100 years including the notion that natural selection should favor asexual reproduction in Apis mellifera capensis. They attempt to support their arguments with an exceptionally surprising finding that A. m. capensis queens can parthenogenetically produce diploid homozygous queen offspring (homozygous diploid individuals develop into diploid males in the honeybee). Beekman et al. suggest that these homozygous queens are not viable because they did not find any homozygous individuals beyond the third larval instar. Even if this were true, such a lethal trait should be quickly eliminated by natural selection. The identification of sex (both with molecular and morphological markers) is possible but notoriously difficult in honeybees at the early larval stages. Ploidy is however a reliable indicator, and we therefore suggest that these "homozygous" larvae found in queen cells are actually drones reared from unfertilized eggs, a phenomenon well known by honeybee queen breeders.

Comparative analysis of detection limits and specificity of molecular diagnostic markers for three pathogens (Microsporidia, Nosema spp.) in the key pollinators Apis mellifera and Bombus terrestris.

Global pollinator decline has recently been discussed in the context of honey and bumble bee infections from various pathogens including viruses, bacteria, microsporidia and mites. The microsporidian pathogens Nosema apis, Nosema ceranae and Nosema bombi may in fact be major candidates contributing to this decline. Different molecular and non-molecular detection methods have been developed; however, a comparison, especially of the highly sensitive PCR based methods, is currently lacking. Here, we present the first comparative quantitative real-time PCR study of nine Nosema spp. primers within the framework of primer specificity and sensitivity. With the help of dilution series of defined numbers of spores, we reveal six primer pairs amplifying N. apis, six for N. bombi and four for N. ceranae. All appropriate primer pairs detected an amount of at least 10(4) spores, the majority of which were even as sensitive to detect such low amounts as 10(3) to ten spores. Species specificity of primers was observed for N. apis and N. bombi, but not for N. ceranae. Additionally, we did not find any significant correlation for the amplified fragments with PCR efficiency or the limit of detection. We discuss our findings on the background of false positive and negative results using quantitative real-time PCR. On the basis of these results, future research might be based on appropriate primer selection depending on the experimental needs. Primers may be selected on the basis of specificity or sensitivity. Pathogen species and load may be determined with higher precision enhancing all kinds of diagnostic studies.

Phytochemical composition and bio-functional properties of Apis mellifera propolis from Kenya.

There is an increased demand for natural products like propolis, yet little information is available about the chemical composition of African propolis and its bio-functional properties. Therefore, in this study, we aimed to quantify the phytochemicals and determine the antioxidant and antimicrobial properties of Apis mellifera propolis (n = 59) sourced from various regions in Kenya. Principal component analysis (PCA) showed that the sampling region had a remarkable impact on the propolis's composition and bio-functional properties. Generally, the propolis contained high amounts of phytochemicals, particularly alkaloids (5.76 g CE/100 g) and phenols (2.24 g GAE/100 g). Furthermore, analysis of propolis by gas chromatography-mass spectrometry (GC-MS) revealed various compounds with varying bio-functional activities. These compounds included triterpenoids alpha- and beta-amyrin, oleanen-3-yl-acetate, urs-12-en-24-oic acid, lanosta-8,24-dien-3-one, and hydrocarbons tricosane and nondecane, which have been reported to have either antimicrobial or antioxidant activities. The propolis samples collected from hotter climatic conditions contained a higher composition of phytochemicals, and additionally, they displayed higher antioxidant and antimicrobial activities than those obtained from cooler climatic conditions. Key findings of this study demonstrate the occurrence of relatively high phytochemical content in Kenya's propolis, which has antioxidant and antimicrobial properties; hence this potential could be harnessed for disease control.

Susceptibility of the Western Honey Bee Apis mellifera and the African Stingless Bee Meliponula ferruginea (Hymenoptera: Apidae) to the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana.

This study assessed the nontarget effect of entomopathogenic fungi on the Western honey bee Apis mellifera L. and the African stingless bee Meliponula ferruginea Cockrell (Hymenoptera: Apidae). Pathogenicity of five Metarhizium anisopliae (ICIPE 7, ICIPE 20, ICIPE 62, ICIPE 69, and ICIPE 78) (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) and one of Beauveria bassiana (ICIPE 284) (Balsamo) Vuillemin (Hypocreales: Cordicipitaceae) isolates were evaluated on bees at 108 conidia/ml. Conidial acquisition was evaluated immediately after exposure. Apis mellifera acquired more conidia (2.8 × 104-1.3 × 105 conidia per bee) compared to M. ferruginea (1.1 × 104-2.3 × 104 conidia per bee). In the bioassay with A. mellifera, ICIPE 7, ICIPE 20, and ICIPE 69 moderately reduced the survival by 16.9, 17.4, 15.3%, with lethal times LT10 = 7.4, 7.6, 8.1 d and LT25 = 8.7, 10.0, 9.9 d, respectively. The three isolates caused A. mellifera mycosis of 11.6-18.5%. None of the isolates had a significant effect on M. ferruginea. The tested isolates are nontoxic to bees according to the International Organization of Biological Control (IOBC) classification. However, the effect of ICIPE 7, ICIPE 20, and ICIPE 69 merits further studies on bee colonies, especially those of A. mellifera, under field conditions.

A quantitative in vitro cultivation technique to determine cell number and growth rates in strains of Crithidia bombi (Trypanosomatidae), a parasite of bumblebees.

The protozoan parasite Crithidia bombi and its host, the bumblebee Bombus terrestris, are used as a model system for the study of the evolutionary ecology of host-parasite interactions. In order to study these interactions we established a method for in vitro cultivation of single parasite strains. Additionally, a high-throughput method is developed for the determination of cell numbers in cultures by means of optical density (OD) measurements. The protocol for in vitro cultivation allowed for growing different strains on agar plates as well as in culture medium. A calibration curve for the relationship between cell number and OD has been developed. Subsequently, growth rates for different genotypes of C. bombi have been recorded. Significant differences in the growth rates and generation times between these genotypes were demonstrated. As this might be related to the virulence of the parasite, this relationship may be confirmed by in vivo growth rate determination. In comparison with conventional cell counting, the application of OD measurements allows for high-throughput experiments as the time taken to record each sample is reduced by a factor of 30. The in vitro cultivation method allows for controlled infection experiments in order to study host-parasite interactions.

A global-scale expert assessment of drivers and risks associated with pollinator decline.

Pollinator decline has attracted global attention and substantial efforts are underway to respond through national pollinator strategies and action plans. These policy responses require clarity on what is driving pollinator decline and what risks it generates for society in different parts of the world. Using a formal expert elicitation process, we evaluated the relative regional and global importance of eight drivers of pollinator decline and ten consequent risks to human well-being. Our results indicate that global policy responses should focus on reducing pressure from changes in land cover and configuration, land management and pesticides, as these were considered very important drivers in most regions. We quantify how the importance of drivers and risks from pollinator decline, differ among regions. For example, losing access to managed pollinators was considered a serious risk only for people in North America, whereas yield instability in pollinator-dependent crops was classed as a serious or high risk in four regions but only a moderate risk in Europe and North America. Overall, perceived risks were substantially higher in the Global South. Despite extensive research on pollinator decline, our analysis reveals considerable scientific uncertainty about what this means for human society.

Tissue Specificity in Social Context-Dependent lysozyme Expression in Bumblebees.

Group living at high densities may result in the enhanced transmission of pathogens. Social insects are obligate group-living species, which often also exhibit high relatedness and frequent social interactions amongst individuals, resulting in a high risk of disease spread. Social species seem to exhibit immune systems that provide colonies of social insects with a certain level of flexibility for adjustment of immune activity according to the risk of disease spread. In bumblebees, Bombus terrestris, it was demonstrated that in group-kept individuals, immune component activity and immune gene expression is increased, potentially as a prophylactic adaptation. Here, I tested whether social environment influences the gene expression pattern of two lysozyme genes, which are components of the antimicrobial response of the bumblebee. In addition, I tested gene expression activation in different tissues (gut, fat body). The analysis revealed that the gene, the density of individuals, the tissue, and the interaction of the latter are the main factors that influence the expression of lysozyme genes. This is the first report of a tissue-specific response towards the social environment. This has implications for gene regulation, which must be responsive to social context-dependent information.

Role of conspecifics and personal experience on behavioral avoidance of contaminated flowers by bumblebees.

Pollinators use multiple cues whilst foraging including direct cues from flowers and indirect cues from other pollinators. The use of indirect social cues is common in social insects, such as honeybees and bumblebees, where a social environment facilitates the ability to use such cues. Bumblebees use cues to forage on flowers according to previous foraging experiences. Flowers are an essential food source for pollinators but also pose a high risk of parasite infection through the shared use of flowers leading to parasite spillover. Nevertheless, bumblebees have evolved behavioral defense mechanisms to limit parasite infection by avoiding contaminated flowers. Mechanisms underlying the avoidance of contaminated flowers by bumblebees are poorly understood. Bumblebees were recorded having the choice to forage on non-contaminated flowers and flowers contaminated by a trypan osome gut parasite, Crithidia bombi. The use of different treatments with presence or absence of conspecifics on both contaminated and non-contaminated flowers allowed to investigate the role of social visual cues on their pathogen avoidance behavior. Bumblebees are expected to use social visual cues to avoid contaminated flowers. Our study reveals that the presence of a conspecific on flowers either contaminated or not does not help bumblebee foragers avoiding contaminated flowers. Nevertheless, bumblebees whereas gaining experience tend to avoid their conspecific when placed on contaminated flower and copy it when on the non-contaminated flower. Our experiment suggests a detrimental impact of floral scent on disease avoidance behavior.

The Invasion of the Dwarf Honeybee, Apis florea, along the River Nile in Sudan.

The spread of the dwarf honeybee, Apis florea, in Sudan along the river Nile in a linear fashion provides a good model for studying the population dynamics and genetic effects of an invasion by a honeybee species. We use microsatellite DNA analyses to assess the population structure of both invasive A. florea and native Apis mellifera along the river Nile. The invasive A. florea had significantly higher population densities than the wild, native A. mellifera. Nevertheless, we found no indication of competitive displacement, suggesting that although A. florea had a high invasive potential, it coexisted with the native A. mellifera along the river Nile. The genetic data indicated that the invasion of A. florea was established by a single colony.