Species Composition, Phenotypic and Genotypic Resistance Levels in Major Malaria Vectors in Teso North and Teso South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Knockdown resistance (kdr) is strongly linked to pyrethroid insecticide resistance in Anopheles gambiae in Africa, which may have vital significance to the current increased use of pyrethroid-treated bed net programmes. The study is aimed at determining species composition, levels of insecticide resistance, and knockdown patterns in Anopheles gambiae sensu lato in areas with and areas without insecticide resistance in Teso North and Teso South subcounties, Western Kenya. MATERIALS AND METHODS: For WHO vulnerability tests, mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes (4944 at 100 mosquitoes per insecticide) which were exposed to 0.75% permethrin, 0.05% deltamethrin, and 0.1% bendiocarb using the WHO tube assay method. Species identification and kdr East gene PCRs were also performed on randomly selected mosquitoes from the collections; including adult mosquitoes (3448) sampled using standard collection methods. RESULTS: Anopheles gambiae sensu stricto were the majority in terms of species composition at 78.9%. Bendiocarb caused 100% mortality while deltamethrin had higher insecticidal effects (77%) on female mosquitoes than permethrin (71%). Susceptible Kengatunyi cluster had higher proportion of An. arabiensis (20.9%) than resistant Rwatama (10.7%). Kengatunyi mosquitoes exposed to deltamethrin had the highest KDT50 R of 8.2. Both Anopheles gambiae sensu stricto and Anopheles arabiensis had equal S allelic frequency of 0.84. Indoor resting mosquitoes had 100% mortality rate after 24 h since exposure. Overall SS genotypic frequency in Teso North and Teso South subcounties was 79.4% against 13.7% homozygous LL genotype and 6.9% heterozygous LS genotype. There was a significant difference (ρ < 0.05) in S allele frequencies between Kengatunyi (0.61) and Rwatama (0.95). Mosquito samples collected in 2013 had the highest S allelic frequency of 0.87. Discussion. Most likely, the higher the selection pressure exerted indoors by insecticidal nets, the higher were the resistance alleles. Use of pyrethroid impregnated nets and agrochemicals may have caused female mosquitoes to select for pyrethroid resistance. Different modes of action and chemical properties in different types of pyrethroids aggravated by a variety of edaphic and climatic factors may have caused different levels of susceptibility in both indoor and outdoor vectors to pyrethroids and carbamate. Species composition and populations in each collection method may have been influenced by insecticide resistance capacity in different species. Conclusions and Recommendations. Both phenotypic and genotypic insecticide resistance levels have been confirmed in Teso North and Teso South subcounties in Western Kenya. Insecticide resistance management practices in Kenya should be fast tracked and harmonized with agricultural sector agrochemical-based activities and legislation, and possibly switch to carbamate use in order to ease selection pressure on pyrethroids which are useable in insecticidal nets and indoor residual spray due to their low human toxicity. The implication of such high resistance levels in mosquitoes collected in Teso subcounties is that resistance is likely to persist and or even increase if monomolecules of permethrin and deltamethrin or both continue to be used in all net- and nonnet-based mosquito control purposes. Usage of mutually reinforcing piperonyl butoxide (PBO) that prohibits particular enzymes vital in metabolic activities inside mosquito systems and has been integrated into pyrethroid-LLINs to create pyrethroid-PBO nets is an extremely viable option.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. METHODS: Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. RESULTS: The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.

Vulnerable Habitats Alter African Meliponine Bee's (Hymenoptera: Apidae) Assemblages in an Eastern Afromontane Biodiversity Hotspot.

Habitat degradation has over time formed synergy with other factors to contribute to dwindling populations of both fauna and flora by altering their habitats. The disturbance of natural habitats affects the diversity of both vertebrates and invertebrates by altering both feeding and nesting sites for which organisms are known to depend on for survival. Little is known of the extent to which vulnerable habitats could shape the diversity of most indigent pollinators such as African meliponine bee species in tropical ecosystems. This study was conducted to determine how disturbance could shape the natural occurrence of African meliponine bee species in different ecological habitats of Taita Hills, leading to changes in their diversity. A total of four species depicted by the Renyi diversity profile was recorded in five of the six main habitat types surveyed, and a further extrapolation with Shannon index (EH) also predicted the highest species richness of 4.24 in a deciduous habitat type. These meliponine bee species (Hypotrigona gribodoi, Hypotrigona ruspolii, Meliponula ferruginea (black), and Plebeina hildebrandti) were observed to be unevenly distributed across all habitats, further indicating that mixed deciduous habitat was more diverse than acacia-dominated bush lands, grasslands, and exotic forest patches. Geometric morphometrics categorized all four meliponine bee species into two major clusters-cluster 1 (H gribodoi, H ruspolii, M ferruginea (black)) and cluster 2 (P hildebrandti)-and further discriminated populations against the 4 potential habitats they are likely to persist or survive in. Each habitat appeared to consist of a cluster of subpopulations and may possibly reveal ecotypes within the four meliponine populations. This has revealed that unprecedented conversions of natural habitats to agroecosystems are a key driving factor causing increased habitat isolation and vulnerability in this Afromontane region which may potentially distort local assemblages of native pollinators, such as meliponine bee species.

The Biology and Control of the Greater Wax Moth, Galleria mellonella.

The greater wax moth, Galleria mellonella Linnaeus, is a ubiquitous pest of the honeybee, Apis mellifera Linnaeus, and Apis cerana Fabricius. The greater wax moth larvae burrow into the edge of unsealed cells with pollen, bee brood, and honey through to the midrib of honeybee comb. Burrowing larvae leave behind masses of webs which causes galleriasis and later absconding of colonies. The damage caused by G. mellonella larvae is severe in tropical and sub-tropical regions, and is believed to be one of the contributing factors to the decline in both feral and wild honeybee populations. Previously, the pest was considered a nuisance in honeybee colonies, therefore, most studies have focused on the pest as a model for in vivo studies of toxicology and pathogenicity. It is currently widespread, especially in Africa, and the potential of transmitting honeybee viruses has raised legitimate concern, thus, there is need for more studies to find sustainable integrated management strategies. However, our knowledge of this pest is limited. This review provides an overview of the current knowledge on the biology, distribution, economic damage, and management options. In addition, we provide prospects that need consideration for better understanding and management of the pest.

Temperature-dependent phenology of Plutella xylostella (Lepidoptera: Plutellidae): Simulation and visualization of current and future distributions along the Eastern Afromontane.

There is a scarcity of laboratory and field-based results showing the movement of the diamondback moth (DBM) Plutella xylostella (L.) across a spatial scale. We studied the population growth of the diamondback moth (DBM) Plutella xylostella (L.) under six constant temperatures, to understand and predict population changes along altitudinal gradients and under climate change scenarios. Non-linear functions were fitted to continuously model DBM development, mortality, longevity and oviposition. We compiled the best-fitted functions for each life stage to yield a phenology model, which we stochastically simulated to estimate the life table parameters. Three temperature-dependent indices (establishment, generation and activity) were derived from a logistic population growth model and then coupled to collected current (2013) and downscaled temperature data from AFRICLIM (2055) for geospatial mapping. To measure and predict the impacts of temperature change on the pest's biology, we mapped the indices along the altitudinal gradients of Mt. Kilimanjaro (Tanzania) and Taita Hills (Kenya) and assessed the differences between 2013 and 2055 climate scenarios. The optimal temperatures for development of DBM were 32.5, 33.5 and 33°C for eggs, larvae and pupae, respectively. Mortality rates increased due to extreme temperatures to 53.3, 70.0 and 52.4% for egg, larvae and pupae, respectively. The net reproduction rate reached a peak of 87.4 female offspring/female/generation at 20°C. Spatial simulations indicated that survival and establishment of DBM increased with a decrease in temperature, from low to high altitude. However, we observed a higher number of DBM generations at low altitude. The model predicted DBM population growth reduction in the low and medium altitudes by 2055. At higher altitude, it predicted an increase in the level of suitability for establishment with a decrease in the number of generations per year. If climate change occurs as per the selected scenario, DBM infestation may reduce in the selected region. The study highlights the need to validate these predictions with other interacting factors such as cropping practices, host plants and natural enemies.

Rearing Method and Developmental Biology of the African Coffee White Stem Borer, Monochamus leuconotus (Coleoptera: Cerambycidae).

Although the African coffee white stem borer, Monochamus leuconotus (Pascoe) (Coleoptera: Cerambycidae), is a major insect pest of coffee in Africa, there is much to learn about its biology. The present paper describes an original rearing method for M. leuconotus and provides unpublished biological data for the pest. The coffee white stem borer colony was started with larvae extracted from infested coffee stems collected in the field. Larvae were reared on artificial diet while the adults were kept in reproduction cages with coffee sticks as feeding and oviposition substrate. Under our rearing conditions, mean adult longevity was 89.5 d for females and 81.8 d for males. Fecundity was 40.7 eggs per female on average, with an oviposition period of 53 d (one female laid 151 eggs for a period of 145 d). Egg viability was low (39%) and the incubation period was 26.6 d. Larval and pupal survival was high (80.9 and 88.5%, respectively). Larval development duration was variable, with a mean of 251.4 d, whereas pupal development was less variable, with a mean of 26.6 d. The rearing method described here is easily replicable and allowed a colony of coffee white stem borer to be continuously maintained in the laboratory for three years. Useful data on coffee white stem borer's life history, including reproductive and feeding behaviors, are reported here and compared with previous reports in order to fill the knowledge gaps of this important but neglected pest of coffee.

Chemical communication in the honey bee scarab pest Oplostomus haroldi: role of (Z)-9-pentacosene.

Oplostomus haroldi Witte belongs to a unique genus of afro-tropical scarabs that have associations with honey bee colonies, from which they derive vital nutrients. Although the attributes of the honey bee nest impose barriers to communication among nest invaders, this beetle still is able to detect conspecific mates for reproduction. Here, we show, through behavioral studies, that cuticular lipids serve as mate discrimination cues in this beetle. We observed five steps during mating: arrestment, alignment, mounting, and copulation, and a post-copulatory stage, lasting ~40-70 % of the total mating duration, that suggested mate guarding. Chemical analysis identified the same nine straight-chain alkanes (C(23)-C(31)), six methyl-branched alkanes (6), and five mono-unsaturated alkenes in the cuticular lipids of both sexes. Methyl alkanes constituted the major component (46 %) of male cuticular lipids, while mono-unsaturated alkenes were most abundant (53 %) in females. (Z)-9-Pentacosene was twice as abundant in females than in males, and ~20 fold more concentrated in beetles than in worker bees. In mating assays, (Z)-9-pentacosene elicited arrestment, alignment, and mounting, but not copulation, by male beetles. These results represent the first evidence of a contact sex pheromone in a scarab beetle. Such contact pheromones may be an essential, cryptic mechanism for arthropods associated with eusocial insects.

Pathogenicity of Metarhizium anisopliae (Metch) Sorok and Beauveria bassiana (Bals) Vuill to adult Phlebotomus duboscqi (Neveu-Lemaire) in the laboratory.

BACKGROUND & OBJECTIVES: Biological control of sandflies using entomopathogenic fungi is a possible alternative to the expensive synthetic chemical control. It is potentially sustainable, less hazardous, and relatively inexpensive and merits further investigations. The objective of this study was to identify the most pathogenic fungal isolate(s) to sandflies in the laboratory. METHODS: Isolates of entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were screened for their pathogenicity against Phlebotomus duboscqi. Adult flies were contaminated using the technique described by Migiro et al (2010). Briefly, flies were exposed to 0.1 g of dry conidia evenly spread on a cotton velvet cloth covering the inner side of a cylindrical plastic tube (95 mm long × 48 mm diam). In all 25 sandflies were transferred into the cylindrical tube and allowed to walk on the velvet for one minute, after which they were transferred from the velvet into the cages in Perplex. Insects in the control treatments were exposed to fungusfree velvet cloth before being transferred into similar cages. The treatments were maintained at 25 ± 2°C, 60-70% RH and 12L: 12D photoperiod. The experiment was replicated 5 times. The most pathogenic isolates were selected for further studies. RESULTS: A total of 19 isolates were screened against adult sandflies in the laboratory. Mortality in the controls was approximately 16.8 ± 1.7 %. All the isolates were found to be pathogenic to P. duboscqi. Mortality ranged between 76.8 and 100% on all the fungal isolates tested. The lethal time taken to 50% (LT50) and 90% (LT90) mortality ranged from 3.0-7.8 days and from 5.3-16.2 days, respectively. The virulent isolates, causing mortalities of 97.5-100%, were selected for further studies. INTERPRETATION & CONCLUSION: The high susceptibility of sandflies to entomopathogenic fungi suggests that fungi are potential alternatives to chemical control methods. We conclude that application of entomopathogenic fungi could result in acute mortalities of sandflies and reduction of parasite transmission and subsequently, reduction of leishmaniasis risk. This method of biological control has great potential as a new strategy for leishmaniasis control.

Comparative performance of the Mbita trap, CDC light trap and the human landing catch in the sampling of Anopheles arabiensis, An. funestus and culicine species in a rice irrigation in western Kenya.

BACKGROUND: Mosquitoes sampling is an important component in malaria control. However, most of the methods used have several shortcomings and hence there is a need to develop and calibrate new methods. The Mbita trap for capturing host-seeking mosquitoes was recently developed and successfully tested in Kenya. However, the Mbita trap is less effective at catching outdoor-biting Anopheles funestus and Anopheles arabiensis in Madagascar and, thus, there is need to further evaluate this trap in diverse epidemiological settings. This study reports a field evaluation of the Mbita trap in a rice irrigation scheme in Kenya METHODS: The mosquito sampling efficiency of the Mbita trap was compared to that of the CDC light trap and the human landing catch in western Kenya. Data was analysed by Bayesian regression of linear and non-linear models. RESULTS: The Mbita trap caught about 17%, 60%, and 20% of the number of An. arabiensis, An. funestus, and culicine species caught in the human landing collections respectively. There was consistency in sampling proportionality between the Mbita trap and the human landing catch for both An. arabiensis and the culicine species. For An. funestus, the Mbita trap portrayed some density-dependent sampling efficiency that suggested lowered sampling efficiency of human landing catch at low densities. The CDC light trap caught about 60%, 120%, and 552% of the number of An. arabiensis, An. funestus, and culicine species caught in the human landing collections respectively. There was consistency in the sampling proportionality between the CDC light trap and the human landing catch for both An. arabiensis and An. funestus, whereas for the culicines, there was no simple relationship between the two methods. CONCLUSIONS: The Mbita trap is less sensitive than either the human landing catch or the CDC light trap. However, for a given investment of time and money, it is likely to catch more mosquitoes over a longer (and hence more representative) period. This trap can therefore be recommended for use by community members for passive mosquito surveillance. Nonetheless, there is still a need to develop new sampling methods for some epidemiological settings. The human landing catch should be maintained as the standard reference method for use in calibrating new methods for sampling the human biting population of mosquitoes.

Comparative field evaluation of the Mbita trap, the Centers for Disease Control light trap, and the human landing catch for sampling of malaria vectors in western Kenya.

The mosquito sampling efficiency of a new bed net trap (the Mbita trap) was compared with that of the Centers for Disease Control miniature light trap (hung adjacent to an occupied bed net) and the human landing catch in western Kenya. Overall, the Mbita trap caught 48.7 +/- 4.8% (mean +/- SEM) the number of Anopheles gambiae Giles sensu lato caught in the human landing catch and 27.4 +/- 8.2% of the number caught by the light trap. The corresponding figures for Anopheles funestus Giles were 74.6 +/- 1.3% and 39.2 +/- 1.9%, respectively. Despite the clear differences in the numbers of mosquitoes caught by each method, both the Mbita trap and light trap catches were directly proportional to human landing catches regardless of mosquito density. No significant differences in parity or sporozoite incidence were observed between mosquitoes caught by the three methods for either An. gambiae s.l. or An. funestus. Identification of the sibling species of the An. gambiae complex by a polymerase chain reaction indicated that the ratio of An. gambiae Giles sensu stricto to An. arabiensis Patton did not vary according to the sampling method used. It is concluded that the Mbita trap is a promising tool for sampling malaria vector populations since its catch can be readily converted into equivalent human biting catch, it can be applied more intensively, it requires neither expensive equipment nor skilled personnel, and it samples mosquitoes in an exposure-free manner. Such intensive sampling capability will allow cost-effective surveillance of malaria transmission at much finer spatial and temporal resolution than has been previously possible.