The use of entomopathogenic fungi against Anopheles funestus giles (Diptera: Culicidae)

Malaria vector control relies primarily on the application of chemical insecticides. The increasing incidence of insecticide resistance in target vector populations coupled with the threat of environmental contamination are of major concern in terms of this approach. The use of biological agents to complement existing insecticide based control strategies has been proposed, e.g. Metarhizium anisopliae and Beauveria bassiana. The efficacy of M. anisopliae (Metschnikoff) Sorokin strain ICIPE-30 and Beauveria bassiana (Balsamo) Vuillemin isolate I93-825 alone or in combination was assessed against laboratory strains of the major malaria vector Anopheles funestus. Samples of adult females from three laboratory strains of An. funestus were exposed to dry conidia of M. anisopliae and B. bassiana for 3, 6 or 24 hours. Of these An. funestus strains, Fang was fully susceptible to all insecticides, Fumoz was partly resistant to pyrethroid insecticides and Fumoz-R has been intensively selected for pyrethroid resistance. Following inoculations, the rate of mortality in all strains was approximately 6-fold higher in fungus infected cohorts compared to their corresponding uninfected control cohorts. Susceptibility to fungal infection in the colonies appeared to follow their pattern of susceptibility to insecticide whereby Fang showed higher rates of mortality following fungus infection than Fumoz and Fumoz- R. Further, Mosquitoes placed in forced proximity of fungal spores for only three hours showed significantly lower rates of mortality than those placed in similar circumstances for 24 hours, showing that the probability of acquiring an infection is a function of time and that a longer potential exposure time leads to the acquisition of greater numbers of infective spores. Approximately 99% of all fungus infected mosquitoes (infection confirmed by followup sporulation tests on cadavers) died within 14 days of acquiring their infection. Fourteen days is the maximum time required by malarial parasites to reach the infective sporozoite stage. Experiments were designed to quantify a possible interaction between susceptibility to the pyrethroid insecticide permethrin and susceptibility to B. bassiana or M. anisopliae infection, based on the hypothesis that intoxication or infection with one of these agents exerts a synergistic effect on susceptibility to the other. Further, the effect of a non insecticidal substance that inhibits the activity of monooxygenases - piperonyl butoxide (PBO) – on subsequent susceptibility to fungus infection in pyrethroid resistant An. funestus was tested. Fumoz-R infected with fungus proved significantly more susceptible to pyrethroid intoxication post fungus infection than uninfected samples from the same cohort. Pre-exposure to PBO did not affect subsequent susceptibility to fungus infection, suggesting that monooxygenases play a negligible role in protection against fungus infection. Experiments were designed to test for variation in fungus induced mortality rates between blood fed and unfed cohorts of female An. funestus as well as to test for differences in fecundity in response to fungal infection. Females blood fed post exposure to fungus showed a slightly higher rate of mortality compared to unfed fungus infected females. Females blood fed prior to fungus infection showed comparable rates of mortality with those of unfed fungus infected cohorts. These results suggest that blood-feeding may affect susceptibility to fungus infection, although the effect is slight at best and of no concern in terms of fungal pathogenicity. Fungus infected females produced significantly fewer eggs than uninfected females. The proportion of progeny from fungus infected females surviving to adulthood was also significantly reduced by comparison to the progeny of uninfected females. In order to test the effectiveness of entomopathogenic fungi in semi-field conditions it is important to first survey the local malaria vector species composition including their insecticide susceptibility status. Baseline mosquito surveillance was conducted in the Mamfene region of northern KwaZulu-Natal, South Africa, in order to assess the feasibility of using clay pots treated with dry conidia of entomopathogenic fungi as a delivery/infection system in a field environment. Anopheles arabiensis, An. parensis, An. funestus, An. merus and An. quadriannulatus were collected inside houses between February and September, 2005. A sample of An. parensis, a nonvector, was shown to present false positives for the presence of P. falciparum circumsporozoites using the standard ELISA method. This result highlighted the importance of accurate species identification and vector incrimination, and showed how over-reliance on standard methodologies without suitable quality assurance can lead to inaccurate information about malaria transmission dynamics in a given area. Resistance to permethrin (pyrethroid) was detected in An. arabiensis and An. parensis. Biochemical analysis and insecticide-synergist assays showed monooxygenase elevation leading to monooxygenase based permethrin detoxification in An. arabiensis. Preliminary laboratory tests revealed that clay pots treated with dry conidia of B. bassiana or M. anisopliae are suitable for spore delivery to anopheline mosquitoes resting inside them. However, the efficacy of treated pots, measured in terms of relative infectivity, decreased with increasing time lapse since treatment, so that by 3 months post treatment their efficacy was negligible under standard laboratory conditions. I conclude that dry conidia of B. bassiana and M. anisopliae are effective pathogens against An. funestus. Infective spores can be delivered using either an attractant such as sucrose or by treating the surfaces of preferred resting sites such as clay pots. Their pathogenicity is not significantly affected by monooxygenase based insecticide resistance, and in almost all cases fungus infected females die within 14 days of acquiring an infection, regardless of bloodfeeding status. Further, fungal infection significantly attenuates the expression of insecticide resistance, and also significantly reduces fecundity and fertility in infected An. funestus females. These factors enhance the potential of entomopathogenic fungi as biocontrol agents in areas where resistance to insecticide occurs in target vector populations

Gene flowanalysis of anopheles arabiensis (Diptera:culicidae) populations in southern africa using microsatellite DNA markers

0009014A Msc thesis Science Animal plant and environmental sciencesAnopheles arabiensis is considered an important vector of human malaria in the southern African region where the disease is the major cause of morbidity and mortality. Gene flow plays an important role in malaria control with the spread of insecticide resistance. The main objectives of this study were to (i) measure the genetic variability within and between five populations (Botswana, Namibia, South Africa, Swaziland and Zimbabwe) of wild An. arabiensis and (ii) estimate the level of gene flow between natural populations across the Southern-limits of An. arabiensis. A total of 1225 An. arabiensis specimens were identified out of 1300 mosquitoes collected from 2000-2003 with the sample sizes ranging from 180-292 per country. Variation at four microsatellite markers was investigated on non-denaturing polyacrylamide gels. The results showed fewer variations between populations (2.96%) than within populations (82.60%) suggesting considerable homogeneity. However, estimates of gene flow (Nm) calculated from mean FST and RST statistics were relatively low, 1.14 and 1.19 respectively, suggesting somewhat restricted gene flow between populations. The occurrence of gene flow within subpopulations of An. arabiensis in Zimbabwe but not in South Africa is interesting with regard to the spread of insecticide resistance in Zimbabwe. The results presented here are obviously subject to the limitations inherent in manual, silver staining method of analysing microsatellite DNA markers. It is possible that a different set of results would be obtained if an Automated Sequencing Analyzer were used. i

Mosquito community composition in South Africa and some neighboring countries

BACKGROUND: A century of studies have described particular aspects of relatively few mosquito species in southern Africa, mostly those species involved with disease transmission, specifically malaria and arboviruses. Patterns of community composition such as mosquito abundance and species diversity are often useful measures for medical entomologists to guide broader insights and projections regarding disease dynamics and potential introduction, spread or maintenance of globally spreading pathogens. However, little research has addressed these indicators in southern Africa. RESULTS: We collected 7882 mosquitoes from net and light traps at 11 localities comprising 66 species in 8 genera. We collected an additional 8 species using supplementary collection techniques such as larval sampling, sweep-netting and indoor pyrethrum knockdown catches. Highest diversity and species richness was found in the Okavango Delta of Botswana and in South Africa's Kruger National Park, while the lowest diversity and abundances were in the extreme southern tip of South Africa and in semi-desert Kalahari close to the South Africa border with Botswana. Species composition was more similar between proximal localities than distant ones (Linear model P-value = 0.005). Multiple arbovirus vector species were detected in all localities we surveyed (proportion of vector mosquito numbers were > 0.5 in all locations except Shingwedzi). Their proportions were highest (> 90%) in Vilankulo and Kogelberg. CONCLUSIONS: Multiple known arbovirus vector species were found in all study sites, whereas anopheline human malaria vector species in only some sites. The combination of net traps and light traps effectively sampled mosquito species attracted to carbon-dioxide or light, accounting for 89% of the 74 species collected. The 11% remaining species were collected using supplementary collection techniques mentioned above. The diversity of species weas highest in savanna type habitats, whereas low diversities were found in the drier Kalahari sands regions and the southern Cape fynbos regions.publishersversionpublishe

Indoor collections of the Anopheles funestus group (Diptera: Culicidae) in sprayed houses in northern KwaZulu-Natal, South Africa

BACKGROUND: Insecticide resistance in malaria vector mosquitoes presents a serious problem for those involved in control of this disease. South Africa experienced a severe malaria epidemic during 1999/2000 due to pyrethroid resistance in the major vector Anopheles funestus. Subsequent monitoring and surveillance of mosquito populations were conducted as part of the malaria vector control programme. METHODS: A sample of 269 Anopheles funestus s.l. was collected in Mamfene, northern KwaZulu-Natal, using exit window traps in pyrethroid sprayed houses between May and June 2005. Mosquitoes were identified to species level, assayed for insecticide susceptibility, analysed for Plasmodium falciparum infectivity and blood meal source. RESULTS: Of the 220 mosquitoes identified using the rDNA PCR method, two (0.9%) were An. funestus s.s. and 218 (99.1%) Anopheles parensis. Standard WHO insecticide susceptibility tests were performed on F1 progeny from wild caught An. parensis females and a significant survival 24 h post exposure was detected in 40% of families exposed to 0.05% deltamethrin. Biochemical analysis of F1 An. parensis showed no elevation in levels/activity of the detoxifying enzyme systems when compared with an insecticide susceptible An. funestus laboratory strain. Among the 149 female An. parensis tested for P. falciparum circumsporozoite infections, 13.4% were positive. All ELISA positive specimens (n = 20) were re-examined for P. falciparum infections using a PCR assay and none were found to be positive. Direct ELISA analysis of 169 blood meal positive specimens showed > 75% of blood meals were taken from animals. All blood fed, false positive mosquito samples for the detection of sporozoites of P. falciparum were zoophilic. CONCLUSION: The combination of pyrethroid resistance and P. falciparum false-positivity in An. parensis poses a problem for vector control. If accurate species identification had not been carried out, scarce resources would have been wasted in the unnecessary changing of control strategies to combat a non-vector species

Comparing efficacy of a sweep net and a dip method for collection of mosquito larvae in large bodies of water in South Africa [version 1; referees : 2 approved]

In this study we tested an alternative method for collecting mosquito larvae called the sweep net catch method and compared its efficiency to that of the traditional dip method. The two methods were compared in various water bodies within Kruger National Park and Lapalala Wilderness area, South Africa. The sweep net catch method performed 5 times better in the collection of Anopheles larvae and equally as well as the dip method in the collection of Culex larvae (p =8.58 x 10 ). Based on 15 replicates the collector’s experience level did not play a significant role in the relative numbers of larvae collected using either method. This simple and effective sweep net catch method will greatly improve the mosquito larval sampling capacity in the field setting.Supplementary material: Larval rearing methods.Both Cornel and Braack were beneficiaries of a Carnegie African Diaspora Fellowship Program grant (IIE Grantee ID: 15410201) which partly enabled this study.http://f1000.com/reportsam2017UP Centre for Sustainable Malaria Control (UP CSMC

Microporous polyolefin strands as controlled-release devices for mosquito repellents

The main vectors of malaria in Africa, i.e. An. arabiensis, An. gambiae s.s. and An. funestus, are attracted by human foot odour and they tend to bite victims in the ankle area. Hence, affordable mosquito-repellent polymer-foot bracelets with long lasting protection could reduce infective lower limb bites and therefore help to reduce the overall malaria transmission rate. This study investigated the possibility of increasing the duration of repellence activity by incorporating repellents into inexpensive thermoplastic polymers, namely poly(ethylene-co-vinyl acetate) (EVA) and linear low-density polyethylene (LLDPE). Volatile repellents need to be released into the surrounding air to be effective, i.e. they are continuously lost to the atmosphere. This means that the bracelet should also act as a reservoir for relatively large quantities of the active compound. Towards this goal, polymer strands containing mosquito repellent were prepared by twin-screw extrusion compounding. A co-continuous phase structure was achieved by rapid quenching in an ice bath of the homogeneous polymer-repellent melt mixture exiting the extruder. Phase separation occurred through spinodal decomposition that trapped the liquid repellent in the microporous polymer matrix. A skin-like membrane that covered the extruded polymer strands controlled the release rate. Strands that contained up to 30 wt-% of either DEET or Icaridin provided effective protection against mosquito bites even after 12 weeks of ageing at 50 °C.The Deutsche Forschungsgemeinschaft (DFG), Germany, under Grant AN 212/22-1.http://http://www.elsevier.com/locate/cej2020-03-15hj2019Chemical EngineeringMedical VirologyStatistic

Bio-sanitation treatment using essential oils against E. coli O157:H7 on fresh lettuce

The study was undertaken to develop an effective bio-sanitation treatment using essential oils for the highly perishable leafy vegetable lettuce. The combination of thyme oil and tea tree oil mixed together had a significantly higher total phenolic content as well as higher antioxidant scavenging activity than other single oils or oil mixtures. Lettuce inoculated with Escherichia coli O157:H7 showed significantly reduced population counts in the presence of the sanitation treatment of a combination of thyme + tea tree oil applied by dipping at 0.75% + 0.75% or 1.5% + 1.5% concentrations compared to commercial sodium hypochlorite (200 mg/L NaOCl) washing after 5 days shelf life at 10°C. These results indicate that thyme + tea tree oil dipping treatment could be used to control E. coli O157:H7 contamination in lettuce as an alternative to NaOCl

Bio-sanitation treatment using essential oils against E. coli O157:H7 on fresh lettuce

The study was undertaken to develop an effective bio-sanitation treatment using essential oils for the highly perishable leafy vegetable lettuce. The combination of thyme oil and tea tree oil mixed together had a significantly higher total phenolic content as well as higher antioxidant scavenging activity than other single oils or oil mixtures. Lettuce inoculated with Escherichia coli O157:H7 showed significantly reduced population counts in the presence of the sanitation treatment of a combination of thyme + tea tree oil applied by dipping at 0.75% + 0.75% or 1.5% + 1.5% concentrations compared to commercial sodium hypochlorite (200 mg/L NaOCl) washing after 5 days shelf life at 10°C. These results indicate that thyme + tea tree oil dipping treatment could be used to control E. coli O157:H7 contamination in lettuce as an alternative to NaOCl

Comparing efficacy of a sweep net and a dip method for collection of mosquito larvae in large bodies of water in South Africa

In this study we tested an alternative method for collecting mosquito larvae called the sweep net catch method and compared its efficiency to that of the traditional dip method. The two methods were compared in various water bodies within Kruger National Park and Lapalala Wilderness area, South Africa. The sweep net catch method performed 5 times better in the collection of Anopheles larvae and equally as well as the dip method in the collection of Culex larvae (p =8.58 x 10 -5). Based on 15 replicates the collector's experience level did not play a significant role in the relative numbers of larvae collected using either method. This simple and effective sweep net catch method will greatly improve the mosquito larval sampling capacity in the field setting