Larval salinity tolerance of two members of the Anopheles funestus group.

The Anopheles funestus group (Diptera: Culicidae) is one of the main species groups involved in malaria transmission in the Afrotropical regions. Basic research into this group has been limited because its members are eurygamic (they tend not to mate in confined spaces), which makes laboratory colonization difficult. Currently, only a few An. funestus Giles colonies are available and no colonies of other members of the group have been established. As information on the larval biology of members of the An. funestus group is limited, the present study aims to determine the effects of different salt concentrations on survival rates of the aquatic stages of two members of the An. funestus group, Anopheles funestus and Anopheles rivulorum Leeson. There were statistically significant negative trends in hatch rate and larval survival rate in An. funestus with increasing salt concentrations, with no larvae surviving to pupae at concentrations that included >15% seawater. Anopheles rivulorum, by contrast, showed no significant trends in hatch rate or larval survival with increasing salt concentrations. This is the first report on salinity tolerance in An. rivulorum. A basic understanding of these variations in salinity tolerance provides vital information on the biology, ecology and colony rearing of members of the An. funestus group.

Age-related pyrethroid resistance is not a function of P450 gene expression in the major African malaria vector, Anopheles funestus (Diptera: Culicidae).

Anopheles funestus is a major vector of malaria in most of the African region. Resistance to pyrethroid and carbamate insecticides has been recorded in populations of this species in South Africa and Mozambique. The P450 gene, CYP6P9, has been shown to be highly transcribed in a permethrin (pyrethroid)-resistant laboratory strain, FUMOZ-R, originating from southern Mozambique. We examined the relationship between pyrethroid resistance and gene transcription levels of two closely related genes, CYP6P9 and CYP6P13, in FUMOZ-R. Levels of resistance to 0.75% permethrin were determined based on standard WHO insecticide susceptibility assays using females and males of different ages, ranging from 3 to 30 days old. The transcription levels of the two genes were quantified using qPCR for each age cohort. In the WHO insecticide susceptibility assays, survival of both males and females significantly decreased as age increased. Quantitative analysis of the two genes CYP6P9 and CYP6P13 showed the highest levels of expression at 10 days of age. There was no correlation between expression of these two genes and pyrethroid survival by age. We conclude that the resistance of this mosquito strain to permethrin is not directly related to age-mediated differences in CYP6P9 and CYP6P13 expression.

Aedes species (Diptera: Culicidae) ecological and host feeding patterns in the north-eastern parts of South Africa, 2014-2018.

BACKGROUND: There is a paucity of recent data and knowledge on mosquito diversity and potential vectors of arboviruses in South Africa, with most of the available data dating back to the 1950s-1970s. Aedes and Culex species are the major vectors of some of the principal arboviruses which have emerged and re-emerged in the past few decades. METHODS: In this study we used entomological surveillance in selected areas in the north-eastern parts of South Africa from 2014 to 2018 to assess mosquito diversity, with special emphasis on the Aedes species. The impact of trap types and environmental conditions was also investigated. Identification of the blood meal sources of engorged females collected during the study period was carried out, and DNA barcodes were generated for selected species. RESULTS: Overall, 18.5% of the total Culicidae mosquitoes collected belonged to the genus Aedes, with 14 species recognised or suspected vectors of arboviruses. Species belonging to the Neomelaniconion subgenus were commonly collected in the Bushveld savanna at conservation areas, especially Aedes mcintoshi and Aedes circumluteolus. Aedes aegypti was present in all sites, albeit in low numbers. Temperature was a limiting factor for the Aedes population, and they were almost exclusively collected at temperatures between 18 °C and 27 °C. The cytochrome oxidase subunit I (COI) barcode fragment was amplified for 21 Aedes species, and for nine of these species it was the first sequence information uploaded on GenBank. CONCLUSION: This study provides a better understanding of the diversity and relative abundance of Aedes species in the north-east of South Africa. The information provided here will contribute to future arboviral research and implementation of efficient vector control and prevention strategies.

Evaluation of the pyrrole insecticide chlorfenapyr against pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae).

OBJECTIVE: To evaluate the pyrrole insecticide chlorfenapyr, which has a novel non-neurotoxic mode of action and is a promising alternative to conventional adulticides, against Anopheles funestus. METHOD: The toxicity of a range of concentrations of chlorfenapyr against pyrethroid resistant and susceptible laboratory reared southern African An. funestus was assessed using standard WHO protocols and analysed using probit analysis. RESULTS: The pyrethroid resistant strain showed consistently higher LD50 and LD95 values compared to the susceptible strain, but these differences were not statistically significant and the magnitude was twofold at most. The LD50 values recorded for An. funestus are approximately three-fold higher than those reported elsewhere for other species of anopheline. CONCLUSIONS: Monooxygenase based pyrethroid resistance in An. funestus does not influence the toxic effect of chlorfenapyr. It is unlikely that such a small decrease in susceptibility of An. funestus to chlorfenapyr relative to other anophelines would have any operational implications. Chlorfenapyr is an important addition to insecticides available for malaria vector control, and could be used as a resistance management tool to either circumvent or slow the development of resistance.

Implementing malaria control in South Africa, Eswatini and southern Mozambique during the COVID-19 pandemic.

The COVID-19 pandemic has strained healthcare delivery systems in a number of southern African countries. Despite this, it is imperative that malaria control and elimination activities continue, especially to reduce as far as possible the number and rate of hospitalisations caused by malaria. The implementation of enhanced malaria control/elimination activities in the context of COVID-19 requires measures to protect healthcare workers and the communities they serve. The aim of this review is therefore to present innovative ideas for the timely implementation of malaria control without increasing the risk of COVID-19 to healthcare workers and communities. Specific recommendations for parasite and vector surveillance, diagnosis, case management, mosquito vector control and community outreach and sensitisation are given.

Malaria vector composition and insecticide susceptibility status in Guinea Conakry, West Africa.

This study provides data on malaria vector species composition and insecticide susceptibility status from three localities in Guinea Conakry. A total of 497 mosquitoes were collected resting indoors and morphologically identified as belonging to the Anopheles gambiae complex. The majority of these were An. gambiae s.s. (99.6%), but a small percentage (0.4%) were identified as Anopheles arabiensis. Thirty-four Anopheles funestus s.s. were also collected. The molecular S form of An. gambiae s.s. was predominant over the M form in Siguiri (95%) and Boffa (97.4%), whereas at Mt Nimba the M form was more abundant (61.4%) than the S form (38.1%). One hybrid M/S specimen was recorded from Mt Nimba. Siguiri populations showed high levels of resistance to DDT, dieldrin and bendiocarb. Anopheles gambiae from Boffa were largely susceptible to the insecticides tested. At Mt Nimba, resistance to DDT and bendicocarb was detected. Biochemical enzyme analysis showed that an altered acetylcholinesterase is operating in the field at low levels. The frequency of the 1014F kdr allele in the An. gambiae S form was 0.24 at Siguiri and 0.14 at Mt Nimba. A single RR specimen was found in the M form. The heterogeneity in species composition and resistance profiles between sites requires vector control interventions to be tailored to each site based on the data collected from ongoing monitoring and surveillance.

Test for association between dieldrin resistance and 2La inversion polymorphism in Anopheles coluzzii from Lagos, Nigeria.

The assortment of paracentric chromosomal inversion 2La is associated with the maintenance of dieldrin resistance in laboratory colonies of the malaria vector Anopheles gambiae. This association has not been tested in field populations. The aim of this study was to test the association between inversion 2La and dieldrin resistance in a field population of An. coluzzii in Nigeria. Field collected immature stages of Anopheles were raised to adults and exposed to 4% dieldrin according to WHO criteria. Knockdown was recorded at 10 min intervals for 1 hour and final mortality was recorded 24 hours post exposure. Species and inversion 2La diagnostic PCR assays were conducted on the resistant and susceptible mosquitoes. The mosquitoes were highly resistant to 4% dieldrin (17.1% knock down and 25.7% final mortality; KDT50 and KDT95 calculated as 170 and 1, 514 minutes respectively). Frequencies of 2La in both the resistant and susceptible cohorts assorted within HardyWeinberg estimates (χ2=1.32, p=0.8 for dead/susceptible mosquitoes and χ2=2.54, p=0.5 for survivors or resistant mosquitoes). However, a higher number of heterozygous mosquitoes were observed in the resistant cohort compared to the susceptible, with significant variation in karyotype frequencies (χ2=11.08, DF=2, p<0.05) and a significantly higher frequency of the 2La inversion arrangement in the resistant cohort (Pearson's χ2 = 4.58, p = 0.03.). These data are the first to associate paracentric chromosome inversion 2La and dieldrin resistance in field population of An. coluzzii. Dieldrin resistance shows a weak but significant association with 2La whose assortment is affected by positive heterosis. Variation in the assortment of 2La inversion arrangements between resistant and susceptible cohorts of this An. coluzzii population suggests that dieldrin resistance is at least partially linked to inversion 2La which may explain the persistence of dieldrin resistance in this population despite a significant absence of selection for resistance to this insecticide.

Over expression of a cytochrome P450 (CYP6P9) in a major African malaria vector, Anopheles Funestus, resistant to pyrethroids.

Anopheles funestus Giles is one of the major African malaria vectors. It has previously been implicated in a major outbreak of malaria in KwaZulu/Natal, South Africa, during the period 1996 to 2000. The re-emergence of this vector was associated with monooxygenase-based resistance to pyrethroid insecticides. We have identified a gene from the monooxygenase CYP6 family, CYP6P9, which is over expressed in a pyrethroid resistant strain originating from Mozambique. Quantitative Real-Time PCR shows that this gene is highly over expressed in the egg and adult stages of the resistant strain relative to the susceptible strain but the larval stages showed almost no difference in expression between strains. This gene is genetically linked to a major locus associated with pyrethroid resistance in this A. funestus population.

Stable chromosomal inversion polymorphisms and insecticide resistance in the malaria vector mosquito Anopheles gambiae (Diptera: Culicidae).

Anopheles gambiae Giles has been implicated as a major vector of malaria in Africa. A number of paracentric chromosomal inversions have been observed as polymorphisms in wild and laboratory populations of this species. These polymorphisms have been used to demonstrate the existence of five reproductive units in West African populations that are currently described as incipient species. They have also been correlated with various behavioral characteristics such as adaptation to aridity and feeding preference and have been associated with insecticide resistance. Two paracentric inversions namely 2La and 2Rb are highly ubiquitous in the wild and laboratory populations sampled. Both inversions are easily conserved during laboratory colonization of wild material and one shows significant positive heterosis with respect to Hardy-Weinberg proportions. Inversion 2La has previously been associated with dieldrin resistance and inversion 2Rb shows an association with DDT resistance based on this study. The stability and maintenance of these inversions as polymorphisms provides an explanation for the transmission and continued presence of DDT and dieldrin resistance in a laboratory strain of An. gambiae in the absence of insecticide selection pressure. This effect may also be operational in wild populations. Stable inversion polymorphism also provides a possible mechanism for the continual inheritance of suitable genetic factors that otherwise compromise the fitness of genetically modified malaria vector mosquitoes.

Evaluation of a polymerase chain reaction assay for detection of pyrethroid insecticide resistance in the malaria vector species of the Anopheles gambiae complex.

A published polymerase chain reaction assay proved reliable for detecting nerve-insensitivity (kdr-type) resistant and susceptible alleles to the pyrethroid insecticide permethrin in the malaria vectors Anopheles gambiae s.s. and An. arabiensis and the nonvector species An. quadriannulatus. The assay detects pyrethroid susceptible and resistance alleles mediated by a mutation in region II of the para-type sodium channel gene, showing that resistance is conferred by pyrethroid target site insensitivity. Analysis of results of the assay suggests that more than 1 mechanism conferring pyrethroid resistance is operating in an An. gambiae s.s. strain from Côte d'Ivoire. The value of the assay as a tool for monitoring this mode of insecticide resistance in wild populations is discussed.

Relative developmental and reproductive fitness associated with pyrethroid resistance in the major southern African malaria vector, Anopheles funestus.

The effect of pyrethroid resistance on the fitness of a laboratory strain of Anopheles funestus originating from southern Mozambique was evaluated by comparing the developmental and reproductive characteristics of a pyrethroid resistant strain with an insecticide susceptible strain. Fitness was evaluated in terms of fecundity, fertility, egg production, developmental time and life stage progression and survival. Of the eggs laid by females of the resistant strain, 81.5% hatched while only 66.9% were recorded in the susceptible strain. The time from egg hatch to adult emergence was longer for the resistant strain (15.9 days) than the susceptible strain (15.2 days). A significantly higher proportion of eggs from the resistant strain (61.6%) survived to adulthood compared with those of the susceptible strain (49%). Fecundity and larval and pupal survival did not differ significantly between strains. Of spermathecae dissected from females of the resistant strain, 56.8% were fertilized compared to 52.6% from the susceptible strain. The proportion of females that successfully produced eggs was 43.3% and 23.3% for the resistant and susceptible strains respectively. Complete failure of larval hatch was recorded in 28.6% of susceptible strain families compared to 7.7% of resistant families. Our results show that pyrethroid resistance in southern African An. funestus does not incur any loss of fitness under laboratory conditions. These results suggest that the removal of pyrethroid insecticide selection pressure may not lead to a regression of resistance alleles in pyrethroid resistant An. funestus populations in southern Africa.

Insecticide resistance in the malarial mosquito Anopheles arabiensis and association with the kdr mutation.

A colony of Anopheles arabiensis Patton (Diptera: Culicidae) from the Sennar region of Sudan was selected for resistance to dichlorodiphenyltrichloroethane (DDT). Adults from the F-16 generation of the resistant strain were exposed to all four classes of insecticides approved for use in malaria vector control and showed high levels of resistance to them all (24-h mortalities: malathion, 16.7%; bendiocarb, 33.3%; DDT, 12.1%; dieldrin, 0%; deltamethrin, 24.0%; permethrin, 0%). Comparisons between the unselected base colony and the DDT-resistant strain showed elevated glutathione-S-transferase (P<0.05) in both sexes and elevated esterases (P<0.05) in males only. The Leu-Phe mutation in the sodium channel gene was detected by polymerase chain reaction and sequencing, but showed no correlation with the resistant phenotype. These results do not provide any explanation as to why this colony exhibits such widespread resistance and further studies are needed to determine the precise mechanisms involved. The implications for malaria vector control in central Sudan are serious and resistance management (e.g. through the rotational use of different classes of insecticides) is recommended.

Dieldrin resistance in the malaria vector Anopheles gambiae in Ghana.

Anopheles gambiae Giles s.s. (Diptera: Culicidae) is one of the principal vectors of malaria in the Ashanti region of central Ghana. High levels of resistance to dieldrin were recorded in a wild-caught sample from Obuasi (south of Kumasi) as well as a laboratory colony established using material from the wild population. Cytogenetic analysis of wild-caught and laboratory samples revealed chromosomal polymorphism for inversions 2La and 2Rb. Although inversion 2La has previously been shown to be associated with dieldrin resistance in certain other laboratory strains originating from West Africa, there was no obvious association between inversion karyotype assortment and the resistance phenotype in the Obuasi population. In addition, polymerase chain reaction analysis indicated the presence of the alanine296 to glycine mutation in the GABA (gamma amino-butyric acid) receptor (which has been mapped to a chromosomal position within inversion 2La). This mutation has previously been shown to be associated with dieldrin resistance in the same An. gambiae laboratory strains of West African origin. Our data show only a weak association between the dieldrin resistance phenotype and the presence of this mutation, suggesting that another dieldrin resistance mechanism is operational in the Obuasi population. Biochemical and synergist exposure assays suggest a metabolic component, probably mediated by monooxygenase P450 enzymes. We conclude that dieldrin resistance in the An. gambiae population of the Obuasi region occurs at a high level - most likely in the absence of selection - and that control of the resistance phenotype is polyfactorial and must include components other than mutations in the GABA receptor locus.

Laboratory selection for and characteristics of pyrethroid resistance in the malaria vector Anopheles funestus.

A laboratory colony of Anopheles funestus Giles (Diptera: Culicidae) was established in 2000 from material collected from southern Mozambique where pyrethroid resistance had been demonstrated in the wild population. A subsample of the colony was selected for pyrethroid resistance using 0.1% lambda-cyhalothrin. Bioassay susceptibility tests in subsequent generations F(2) to F(4) showed increased resistance with each successive generation. Survival of individual mosquitoes fed only on 10% sugar solution, increased with age up to 4 days, but by day 10 had decreased significantly. However, females that had been mated and given bloodmeals showed no such increase in mortality with age. Biochemical analysis of resistant and susceptible individuals showed increased monooxygenase and glutathione S-transferase activity but no significant correlation with age of the mosquitoes.

Independent mutations in the Rdl locus confer dieldrin resistance to Anopheles gambiae and An. arabiensis.

Substitutions of a conserved alanine residue in the Rdl locus coding for a gamma-aminobutyric acid (GABA) receptor subunit with serine or glycine confer resistance to dieldrin in various insect species. Here, we show that alanine to glycine substitution in the Rdl locus of the malaria vector, Anopheles gambiae, is genetically linked to resistance to dieldrin. An alanine to serine substitution developed independently in a dieldrin resistant strain of An. arabiensis. An allele-specific polymerase chain reaction (PCR) assay was able to differentiate dieldrin resistant and susceptible mosquitoes.

Resistance to dieldrin + fipronil assorts with chromosome inversion 2La in the malaria vector Anopheles gambiae.

Cyclodiene insecticide resistance in malaria vector mosquitoes of the Anopheles gambiae species complex (Diptera: Culicidae) has been reported previously from several parts of Africa. We report resistance to dieldrin, a cyclodiene, in two laboratory strains of An. gambiae Giles sensu stricto code-named Ian P20 from Nigeria (1979) and CIG from Cote d'Ivoire (1997). Dieldrin resistance levels were high in adult female mosquitoes (40-75% survived exposure to 4% dieldrin for 1 h) and was closely linked with chromosomal paracentric inversion 2La. This inversion did not occur in Hardy-Weinberg proportions, but showed an excess of heterozygotes in both strains, which may account for the high levels of resistance. This linkage also suggests that dieldrin resistance in Ian P20 is dominant. After subsamples of strain Ian P20 were exposed for 1 h to dieldrin 4% or fipronil 2% (discriminating concentrations), the resultant mortality-rates (61% and 65%) were not significantly different. Most survivors after fipronil treatment also survived subsequent exposure to dieldrin (46/50=92%). This apparent cross-resistance between insecticides of two classes (cyclodiene and phenyl pyrazole) has implications for the management of insecticide resistance in wild populations of the An. gambiae complex.

Absence of the kdr mutation in the molecular 'M' form suggests different pyrethroid resistance mechanisms in the malaria vector mosquito Anopheles gambiae s.s.

Field tests conducted on adult Anopheles mosquitoes using standard WHO procedures, diagnostic kits and test papers in south-western Nigeria showed pyrethroid (deltamethrin and permethrin) resistance in adult populations of Anopheles gambiae sensu stricto. The knock-down resistance (kdr) mutation involved in pyrethroid resistance was only found in the molecular S form of An. gambiae s.s. even in area where both molecular M and S forms occurred in sympatry. The absence of the kdr mutation in the M form suggests an additional pyrethroid resistance mechanism in An. gambiae s.s.

Anopheles arabiensis and An. quadriannulatus resistance to DDT in South Africa.

The malaria control programme of KwaZulu-Natal Province, South Africa, includes Mamfene and Mlambo communities. Western-type houses there are currently sprayed with deltamethrin, whereas traditional houses are sprayed with DDT for malaria control. In 2002, mosquitoes of the Anopheles gambiae complex (Diptera: Culicidae) were collected from DDT-sprayed houses, by window exit traps, and from man-baited nets outdoors. Larval collections were also carried out at Mzinweni Pan near Mlambo. Species of the An. gambiae complex were identified by rDNA polymerase chain reaction assay. The majority of samples collected by window trap and baited nets were identified as the malaria vector An. arabiensis Patton, with a few An. merus Dönitz and An. quadriannulatus (Theobald). The larval collections were predominantly An. quadriannulatus with a small number of An. arabiensis. Standard WHO insecticide susceptibility tests using 4% DDT and 0.05% deltamethrin were performed on both wild-caught females and laboratory-reared progeny from wild-caught females. Wild-caught An. arabiensis samples from window traps gave 63% and 100% mortality 24-h post-exposure to DDT or deltamethrin, respectively. Wild-caught An. arabiensis samples from man-baited net traps gave 81% mortality 24-h post-exposure to DDT. The F1 progeny from 22 An. arabiensis females showed average mortality of 86.5% 24-h post-exposure to DDT. Less than 80% mortality was recorded from five of these families. Biochemical analyses of samples from each of the families revealed comparatively high levels of glutathione-S-transferases and non-specific esterases in some families, but without significant correlation to bioassay results. Wild-caught An. quadriannulatus larvae were reared through to adults and assayed on 4% DDT, giving 47% (n = 36) mortality 24-h post-exposure. Finding DDT resistance in the vector An. arabiensis, close to the area where we previously reported pyrethroid-resistance in the vector An. funestus Giles, indicates an urgent need to develop a strategy of insecticide resistance management for the malaria control programmes of southern Africa.

Anopheles funestus resistant to pyrethroid insecticides in South Africa.

Northern Kwazulu/Natal (KZN) Province of South Africa borders on southern Mozambique, between Swaziland and the Indian Ocean. To control malaria vectors in KZN, houses were sprayed annually with residual DDT 2 g/ m2 until 1996 when the treatment changed to deltamethrin 20-25 mg/m2. At Ndumu (27 degrees 02'S, 32 degrees 19'E) the recorded malaria incidence increased more than six-fold between 1995 and 1999. Entomological surveys during late 1999 found mosquitoes of the Anopheles funestus group (Diptera: Culicidae) resting in sprayed houses in some sectors of Ndumu area. This very endophilic-vector of malaria had been eliminated from South Africa by DDT spraying in the 1950s, leaving the less endophilic An. arabiensis Patton as the only vector of known importance in KZN. Deltamethrin-sprayed houses at Ndumu were checked for insecticide efficacy by bioassay using susceptible An. arabiensis (laboratory-reared) that demonstrated 100% mortality. Members of the An. funestus group from Ndumu houses (29 males, 116 females) were identified by the rDNA PCR method and four species were found: 74 An. funestus Giles sensu stricto, 34 An. parensis Gillies, seven An. rivulorum Leeson and one An. leesoni Evans. Among An. funestus s.s. females, 5.4% (4/74) were positive for Plasmodium falciparum by ELISA and PCR tests. To test for pyrethroid resistance, mosquito adults were exposed to permethrin discriminating dosage and mortality scored 24h post-exposure: survival rates of wild-caught healthy males were 5/10 An. funestus, 1/9 An. rivulorum and 0/2 An. parensis; survival rates of laboratory-reared adult progeny from 19 An. funestus females averaged 14% (after 1h exposure to 1% permethrin 25:75cis:trans on papers in WHO test kits) and 27% (after 30 min in a bottle with 25 microg permethrin 40:60cis:trans). Anopheles funestus families showing >20% survival in these two resistance test procedures numbered 5/19 and 12/19, respectively. Progeny from 15 of the families were tested on 4% DDT impregnated papers and gave 100% mortality. Finding these proportions of pyrethroid-resistant An. funestus, associated with a malaria upsurge at Ndumu, has serious implications for malaria vector control operations in southern Africa.

Bioassay and biochemical analyses of insecticide resistance in southern African Anopheles funestus (Diptera: Culicidae).

Anopheles funestus Giles has been implicated as a major malaria vector in sub-Saharan Africa where pyrethroid insecticides are widely used in agriculture and public health. Samples of this species from northern Kwazulu/Natal in South Africa and the Beluluane region of southern Mozambique showed evidence of resistance to pyrethroid insecticides. Insecticide exposure, synergist and biochemical assays conducted on A. funestus suggested that elevated levels of mixed function oxidases were responsible for the detoxification of pyrethroids in resistant mosquitoes in these areas. The data suggested that this mechanism was also conferring cross-resistance to the carbamate insecticide propoxur.