Bio-functional properties and phytochemical composition of selected Apis mellifera honey from Africa.

Globally, the demand for natural remedies such as honey to manage ailments has increased. Yet, the health benefits and chemical composition of African honeys are not well understood. Therefore, this study aimed to characterise the bio-functional properties and the phytochemical composition of 18 Apis mellifera honeys from Kenya, Uganda, and Cameroon in comparison to the popular and commercially available Manuka 5+ honey from New Zealand. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH-RSA) was used to determine the antioxidant property, whilst the agar well diffusion and broth dilution (Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)) assays were used to determine antimicrobial property. Further, colorimetric methods were used for phytochemical analysis. Our results showed that honeys collected from Rift Valley region of Kenya (e.g. Poi, Salabani and Mbechot) and Western region of Cameron (e.g. Bangoulap) had the highest antioxidant (DPPH RSA of 41.52-43.81%) and antimicrobial (MIC (3.125-6.25% w/v) and MBC (6.25-12.5% w/v)) activities. Additionally, the total flavonoid (770-970 mg QE/100 g), phenol (944.79-1047.53 mg GAE/100 g), terpenoid (239.78-320.89 mg LE/100 g) and alkaloid (119.40-266.57 mg CE/100 g) contents reached the highest levels in these bioactive African honeys, which significantly and positively correlated with their bio-functional properties. The functional and phytochemical composition of these bioactive African honeys were similar to or higher than those of the Manuka 5+ honey. Furthermore, gas chromatography-mass spectrometry analysis of African honeys revealed 10 most prominent volatile organic compounds that contribute to their geographical distinction: triacontane, heptacosane, (Z)-9-tricosene, tetracosane, 6-propyl-2,3-dihydropyran-2,4-dione, octacosane, 1,2,4-trimethylcyclohexane, 1,3-bis(1,1-dimethylethyl) benzene, 2-methylheptane and phytol. Overall, our findings suggest that some of the tested African honeys are natural sources of antimicrobial and antioxidant therapies that can be exploited upon further research and commercialized as high value honey.

Effects of Niemann-Pick type C2 (NPC2) gene transcripts silencing on behavior of Varroa destructor and molecular changes in the putative olfactory gene networks.

To understand the role of two Niemann-Pick type C2 (NPC2) transcripts, Vd40090 (NP1) and Vd74517 (NP5), in the chemosensing pathway of Varroa destructor, we evaluated the impact of NP5 silencing on mites behavior and compared the effect of silencing of either transcripts on the interaction between chemosensory transcripts. In contrast to silencing NP1, which reduced feeding and reproduction by the mite (Nganso et al., 2021), silencing of NP5 reduced significantly the host reaching ability, but it did not affect the feeding on nurse bee. However, silencing of either transcript changed dramatically the co-expression patterns among the putative chemosensory genes, binding proteins and receptors. The results suggest the role of gustatory receptors in the detection of long-range chemical cues in the chemosensory cascade of the Varroa mite.

Insights into gene manipulation techniques for Acari functional genomics.

Functional genomics is an essential tool for elucidating the structure and function of genes in any living organism. Here, we review the use of different gene manipulation techniques in functional genomics of Acari (mites and ticks). Some of these Acari species inflict severe economic losses to managed crops and health problems to humans, wild and domestic animals, but many also provide important ecosystem services worldwide. Currently, RNA interference (RNAi) is the leading gene expression manipulation tool followed by gene editing via the bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 system (CRISPR-Cas9). Whilst RNAi, via siRNA, does not always lead to expected outcomes, the exploitations of the CRISPR systems in Acari are still in their infancy and are limited only to CRISP/Cas9 to date. In this review, we discuss the advantages and disadvantages of RNAi and CRISPR-Cas9 and the technical challenges associated with their exploitations. We also compare the biochemical machinery of RNAi and CRISPR-Cas9 technologies. We highlight some potential solutions for experimental optimization of each mechanism in gene function studies. The potential benefits of adopting various CRISPR-Cas9 systems for expanding on functional genomics experiments in Acari are also discussed.

The effects of crude propolis, its volatiles and ethanolic extracts on the ecto-parasitic mite, Varroa destructor and health of the African savannah honey bee, Apis mellifera scutellata.

Propolis is a hive product composed of biologically active plant resins, and has been shown to enhance individual honey bee (Apis mellifera L.) health. Propolis has also been demonstrated to mitigate, in part, the negative effects caused by the ecto-parasitic mite Varroa destructor and its associated viruses on the health of managed European honey bee colonies. However, its effect on the health status of African honey bees remains largely unknown. Here, we found that the African savannah honey bees, A. m. scutellata in Kenya, deposited approximately two and half-fold more propolis in their colonies during periods of increased than reduced worker brood rearing. This finding suggested that A. m. scutellata may use high quantities of propolis prophylactically to protect their young brood; yet, we observed no significant correlation between the quantity of propolis and the amount of worker brood or mite-infestation level on adult workers. Furthermore, whereas propolis volatiles or propolis placed in direct contact with the mites had no effect on mite survival under laboratory conditions, the ethanolic extract of propolis significantly reduced mite survival when compared with untreated control. These results suggest the presence of mite deterrent compounds in the ethanolic extract of the African honey bee propolis.

A genome-wide screening for RNAi pathway proteins in Acari.

BACKGROUND: RNA interference (RNAi) is a highly conserved, sequence-specific gene silencing mechanism present in Eukaryotes. Three RNAi pathways are known, namely micro-RNA (miRNA), piwi-interacting RNA (piRNA) and short interfering RNA (siRNA). However, little knowledge exists about the proteins involved in these pathways in Acari. Moreover, variable successes has been obtained in gene knockdown via siRNA pathway in their functional genomics and management. We hypothesized that the clue may be in the variability of the composition and the efficacy of siRNA machinery among Acari. RESULTS: Both comparative genomic analyses and domain annotation suggest that all the analyzed species have homologs of putative core proteins that mediate cleaving of targeted genes via the three RNAi pathways. We identified putative homologs of Caenorhabditis elegans RNA-dependent RNA polymerase (RdRP) protein in all species though no secondary Argonaute homologs that operate with this protein in siRNA amplification mechanism were found, suggesting that the siRNA amplification mechanism present in Acari may be distinct from that described in C. elegans. Moreover, the genomes of these species do not encode homologs of C. elegans systemic RNAi defective-1 (Sid-1) protein that mediate silencing of the mRNA target throughout the treated organisms suggesting that the phenomena of systemic RNAi that has been reported in some Acari species probably occur through a different mechanism. However, homologs of putative RNAi spreading defective-3 (Rsd-3) protein and scavenger receptors namely Eater and SR-CI that mediate endocytosis cellular update of dsRNA in C. elegans and Drosophila melanogaster were found in Acari genomes. This result suggests that cellular dsRNA uptake in Acari is endocytosis-dependent. Detailed phylogenetic analyses of core RNAi pathway proteins in the studied species revealed that their evolution is compatible with the proposed monophyletic evolution of this group. CONCLUSIONS: Our analyses have revealed the potential activity of all three pathways in Acari. Still, much experimental work remains to be done to confirm the mechanisms behind these pathways in particular those that govern systemic/parental RNAi and siRNA amplification in Acari. Disclosure of these mechanisms will facilitate the development of new and specific management tools for the harmful species and enrichment of the beneficial species.

How Crucial is the Functional Pit Organ for the Varroa Mite?

Olfaction as well as gustation, are essential for animal survival, allowing behavioral modulation according to environmental input. We focused our study on an obligate ecto-parasitic mite of honey bees, the Varroa destructor Anderson and Trueman (Parasitiformes, Mesostigmata, Varroidae). By mechanically blocking the main olfactory organ on Varroa forelegs by varnishing with nail polish, we were able to show that other sensory organs cannot significantly compensate chemosensory abilities required for mite's host selection, identification as well as reproduction. In fact, we found that mites with blocked forelegs had a significantly lower ability to reach a host bee than those with varnished idiosoma and unvarnished control. Furthermore, fewer foreleg blocked mites were feeding on the nurse bees and their reproduction in the brood cells was significantly impaired. The inhibition of reproduction was also reflected in altered expression levels of vitellogenin and vitellogenin receptor genes in foreleg-blocked mites.

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.

Hygienic and grooming behaviors in African and European honeybees-New damage categories in Varroa destructor.

Varroa destructor is an ectoparasitic pest of honeybees, and a threat to the survival of the apiculture industry. Several studies have shown that unlike European honeybees, African honeybee populations appear to be minimally affected when attacked by this mite. However, little is known about the underlying drivers contributing to survival of African honeybee populations against the mite. We hypothesized that resistant behavioral defenses are responsible for the survival of African honeybees against the ectoparasite. We tested this hypothesis by comparing grooming and hygienic behaviors in the African savannah honeybee Apis mellifera scutellata in Kenya and A. mellifera hybrids of European origin in Florida, USA against the mite. Grooming behavior was assessed by determining adult mite infestation levels, daily mite fall per colony and percentage mite damage (as an indicator of adult grooming rate), while hygienic behavior was assessed by determining the brood removal rate after freeze killing a section of the brood. Our results identified two additional undescribed damaged mite categories along with the six previously known damage categories associated with the grooming behavior of both honeybee subspecies. Adult mite infestation level was approximately three-fold higher in A. mellifera hybrids of European origin than in A. m. scutellata, however, brood removal rate, adult grooming rate and daily natural mite fall were similar in both honeybee subspecies. Unlike A. mellifera hybrids of European origin, adult grooming rate and brood removal rate did not correlate with mite infestation levels on adult worker honeybee of A. m. scutellata though they were more aggressive towards the mites than their European counterparts. Our results provide valuable insights into the tolerance mechanisms that contribute to the survival of A. m. scutellata against the mite.