Alphaviruses Detected in Mosquitoes in the North-Eastern Regions of South Africa, 2014 to 2018.

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa.

Malaria Vector Surveillance and Control in an Elimination Setting in South Africa.

South Africa's malaria elimination plans are aligned to the World Health Organization's aim for a malaria-free world and include specific objectives within a specified time frame. These are proving difficult to achieve owing to the sporadic nature of locally acquired malaria in some affected districts, while other districts that were endemic for the disease are either malaria-free or very close to that goal. The WHO also specifies that continued measures to prevent the re-establishment of transmission are required in areas where elimination has been achieved. These measures include routine malaria vector surveillance in endemic districts that are free of malaria to assess receptivity and risk of reintroduction, which may prove difficult to justify in the face of competing public health priorities and limited resources. These issues are discussed here within the framework of vector surveillance and control and include recommendations on how they can be addressed going forward.

The effect of blood feeding on insecticide resistance intensity and adult longevity in the major malaria vector Anopheles funestus (Diptera: Culicidae).

Insecticide-based vector control is key to the reduction and elimination of malaria. Although insecticide resistance is common in malaria vector populations, the operational implications are often unclear. High intensity pyrethroid resistance in the major malaria vector Anopheles funestus has been linked to control failure in Southern Africa. The aim of this study was to assess linkages between mosquito age, blood feeding and the intensity of pyrethroid resistance in two An. funestus laboratory strains that originate from southern Mozambique, namely the moderately pyrethroid resistant FUMOZ and the highly resistant FUMOZ-R. Resistance tended to decline with age. This effect was significantly mitigated by blood feeding and was most apparent in cohorts that received multiple blood meals. In the absence of insecticide exposure, blood feeding tended to increase longevity of An. funestus females and, following insecticide exposure, enhanced their levels of deltamethrin resistance, even in older age groups. These effects were more marked in FUMOZ-R compared to FUMOZ. In terms of programmatic decision-making, these data suggest that it would be useful to assess the level and intensity of resistance in older female cohorts wherever possible, notwithstanding the standard protocols for resistance testing using age-standardised samples.

Characterisation of the epigenetic architecture of the major malaria vector Anopheles arabiensis (Diptera: Culicidae) after treatment with epigenetic modulators and heavy metals.

Anopheles arabiensis (a member of the An. gambiae species complex) is a major vector of malaria in sub-Saharan Africa. Despite its disease vector status, there is currently a paucity of epigenetic information for this species. The aim this study was therefore to analyse global epigenetic markers and their response to metal exposure in insecticide susceptible and resistant laboratory strains of An. arabiensis. This was done using commercially available epigenetic marker quantification kits. In order to validate the efficacy of the kits, several kits were assessed to determine whether changes induced by known epigenetic modulators were detectable using these platforms. The efficacy of the dosages used were determined by examining the effect of the dosages used on insecticide resistant phenotypes. Upon confirmation that the dosages used were sufficient to induce a phenotypic change, the effect on epigenetic markers was assessed. Commercial kits were used to quantify 5-methylcysteine (5-mC) and 5-hydroxymethylcysteine (5-hmC) methylation in DNA, m6A methylation in mRNA as well as Histone Acetyl Transferase (HAT) activity. There was a marked difference in the phenotypic response in adult mosquitoes of the insecticide susceptible strain compared to that of its' resistant counterpart. For males and females of the resistant strain, exposure to nucleic acid modifying drugs typically increased their tolerance to insecticides. The patterns of changes in 5-mC methylation by epigenetic modulators was congruent with previous studies which quantified by mass spectrometry. The two strains differed in methylation patterns under control conditions and responded differentially to larval metal exposure. In the resistant strain, which previously was demonstrated to show increased detoxification enzyme activity and insecticide tolerance after the same treatment, the potential increase in transcriptional activity appeared to be modulated by reduced methylation and increased HAT activity. This study suggests that the commercial epigenetic quantification kits can be used to characterise phenotypic changes in An. arabiensis, and also shows that epigenetic regulation of the response to metal exposure is regulated at the DNA as opposed to the RNA level.

Detection of Insect-Specific Flaviviruses in Mosquitoes (Diptera: Culicidae) in Northeastern Regions of South Africa.

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

Potential Mosquito Vectors for Shuni Virus, South Africa, 2014-2018.

Shuni virus is associated with neurologic and febrile illness in animals and humans. To determine potential vectors, we collected mosquitoes in South Africa and detected the virus in species of the genera Mansonia, Culex, Aedes, and Anopheles. These mosquitoes may be associated with Shuni virus outbreaks in Africa and emergence in other regions.

Estimates of the population size and dispersal range of Anopheles arabiensis in Northern KwaZulu-Natal, South Africa: implications for a planned pilot programme to release sterile male mosquitoes.

BACKGROUND: Anopheles arabiensis is a major malaria vector, recently implicated as contributing to ongoing residual malaria transmission in South Africa, which feeds and rests both indoors and outdoors. This species is, therefore, not effectively targeted using core malaria vector control interventions alone. Additionally, increasing resistance to available insecticides necessitates investigations into complementary non-insecticide-based vector control methods for outdoor-resting mosquitoes. The feasibility of the sterile insect technique (SIT) as a complementary vector control intervention is being investigated in South Africa. Successful implementation of an SIT programme largely depends on inundating a target insect population with sterilized laboratory-bred males. Therefore, knowledge of the native population size and dispersal ability of released sterile laboratory-reared males is critical. In this study, we estimated the male An. arabiensis population size and the dispersal of released males in an area targeted for a pilot sterile male release programme. METHODS: Three separate releases were performed within a 2-year period. Approximately 5000-15,000 laboratory-reared male An. arabiensis (KWAG) were produced and marked for mark-release-recapture experiments. To recapture released mosquitoes, cloth tubes were deployed in widening concentric circles. The average dispersal distance of released males was calculated and the wild male An. arabiensis population size was estimated using two Lincoln index formulae. The natural population was sampled concurrently and Anopheles species diversity examined. RESULTS: The Anopheles gambiae complex and An. funestus group species made up the majority of wild collections along with other anophelines. The An. arabiensis population size was estimated to be between 550 and 9500 males per hectare depending on time of year, weather conditions and method used. Average dispersal distance of marked males ranged from 58 to 86 m. Marked males were found in swarms with wild males, indicating that laboratory-reared males are able to locate and participate in mating swarms. CONCLUSIONS: It was logistically feasible to conduct mark-release-recapture studies at the current scale. The population size estimates obtained may provide a guideline for the initial number of males to use for a pending SIT pilot trial. It is promising for future SIT trials that laboratory-reared marked males participated in natural swarms, appearing at the right place at the right time.

Use of alternative bioassays to explore the impact of pyrethroid resistance on LLIN efficacy.

BACKGROUND: There is substantial concern that the spread of insecticide resistance will render long-lasting insecticide-treated nets (LLINs) ineffective. However, there is limited evidence supporting a clear association between insecticide resistance and malaria incidence or prevalence in the field. We suggest that one reason for this disconnect is that the standard WHO assays used in surveillance to classify mosquito populations as resistant are not designed to determine how resistance might impact LLIN efficacy. The standard assays expose young, unfed female mosquitoes to a diagnostic insecticide dose in a single, forced exposure, whereas in the field, mosquitoes vary in their age, blood-feeding status, and the frequency or intensity of LLIN exposure. These more realistic conditions could ultimately impact the capacity of "resistant" mosquitoes to transmit malaria. METHODS: Here, we test this hypothesis using two different assays that allow female mosquitoes to contact a LLIN as they host-seek and blood-feed. We quantified mortality after both single and multiple exposures, using seven different strains of Anopheles ranging in pyrethroid resistance intensity. RESULTS: We found that strains classified as 1×-resistant to the pyrethroid insecticide deltamethrin in the standard WHO assay exhibited > 90% mortality over 24 h following more realistic LLIN contact. Mosquitoes that were able to blood-feed had increased survival compared to their unfed counterparts, but none of the 1×-resistant strains survived for 12 days post-exposure (the typical period for malaria parasite development within the mosquito). Mosquitoes that were 5×- and 10×-resistant (i.e. moderate or high intensity resistance based on the WHO assays) survived a single LLIN exposure well. However, only about 2-3% of these mosquitoes survived multiple exposures over the course of 12 days and successfully blood-fed during the last exposure. CONCLUSIONS: These results suggest that the standard assays provide limited insight into how resistance might impact LLIN efficacy. In our laboratory setting, there appears little functional consequence of 1×-resistance and even mosquitoes with moderate (5×) or high (10×) intensity resistance can suffer substantial reduction in transmission potential. Monitoring efforts should focus on better characterizing intensity of resistance to inform resistance management strategies and prioritize deployment of next generation vector control products.

Member species of the Anopheles gambiae complex can be misidentified as Anopheles leesoni.

BACKGROUND: Accurate Anopheles species identification is key for effective malaria vector control. Identification primarily depends on morphological analysis of field samples as well as molecular species-specific identifications. During an intra-laboratory assessment (proficiency testing) of the Anopheles funestus group multiplex PCR assay, it was noted that Anopheles arabiensis can be misidentified as Anopheles leesoni, a zoophilic member of the An. funestus group. The aim of this project was, therefore, to ascertain whether other members of the Anopheles gambiae complex can also be misidentified as An. leesoni when using the standard An. funestus multiplex PCR. METHODS: The An. funestus multiplex PCR was used to amplify DNA from An. gambiae complex specimens. These included specimens from the laboratory colonies and field samples from the Democratic Republic of Congo. Amplified DNA from these specimens, using the universal (UV) and An. leesoni species-specific primers (LEES), were sequence analysed. Additionally, An. leesoni DNA was processed through the diagnostic An. gambiae multiplex PCR to determine if this species can be misidentified as a member of the An. gambiae complex. RESULTS: Laboratory-colonized as well as field-collected samples of An. arabiensis, An. gambiae, Anopheles merus, Anopheles quadriannulatus, Anopheles coluzzii as well as Anopheles moucheti produced an amplicon of similar size to that of An. leesoni when using an An. funestus multiplex PCR. Sequence analysis confirmed that the UV and LEES primers amplify a segment of the ITS2 region of members of the An. gambiae complex and An. moucheti. The reverse was not true, i.e. the An. gambiae multiplex PCR does not amplify DNA from An. leesoni. CONCLUSION: This investigation shows that An. arabiensis, An. gambiae, An. merus, An. quadriannulatus, An. coluzzii and An. moucheti can be misidentified as An. leesoni when using An. funestus multiplex PCR. This shows the importance of identifying specimens using standard morphological dichotomous keys as far as possible prior to the use of appropriate PCR-based identification methods. Should there be doubt concerning field-collected specimens molecularly identified as An. leesoni, the An. gambiae multiplex PCR and sequencing of the internal transcribed spacer 2 (ITS2) can be used to eliminate false identifications.

Second generation effects of larval metal pollutant exposure on reproduction, longevity and insecticide tolerance in the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

BACKGROUND: Members of the Anopheles gambiae complex breed in clean, sunlit temporary bodies of water. Anthropogenic pollution is, however, altering the breeding sites of the vectors with numerous biological effects. Although the effects of larval metal pollution have previously been examined, this study aims to assess the transgenerational effects of larval metal pollution on the major malaria vector An. arabiensis. METHODS: Two laboratory strains of An. arabiensis, SENN (insecticide-susceptible) and SENN-DDT (insecticide-resistant), were used in this study. After being bred in water polluted with either cadmium chloride, copper nitrate or lead nitrate, several life history characteristics that can have epidemiological implications (fertility, apoptotic damage to reproductive structures, adult longevity and insecticide tolerance) were examined in the adults and compared to those of adults bred in clean water. RESULTS: All metal treatments reduced fecundity in SENN, but only lead treatment reduced fertility in SENN-DDT. Cadmium chloride exposure resulted in apoptosis and deformation of the testes in both strains. After breeding generation F0 in polluted water, F1 larvae bred in clean water showed an increase in longevity in SENN-DDT adult females. In contrast, after breeding the F0 generation in polluted water, longevity was reduced after cadmium and copper exposure in the F1 generation. Larval metal exposure resulted in an increase in insecticide tolerance in adults of the SENN strain, with SENN-DDT adults gaining the greatest fold increase in insecticide tolerance. CONCLUSIONS: This study demonstrates that a single exposure to metal pollution can have transgenerational effects that are not negated by subsequent breeding in clean water.

Effects of inorganic fertilizer on larval development, adult longevity and insecticide susceptibility in the malaria vector Anopheles arabiensis (Diptera: Culicidae).

BACKGROUND: Exposure to inorganic fertilizer is common for the major malaria vector Anopheles arabiensis, which is closely associated with agricultural activities. The aim of this study was to understand if insecticide susceptible and resistant individuals respond to fertilizer exposure in the same manner. Two laboratory strains, SENN, an insecticide susceptible strain, and SENN-DDT, an insecticide resistant strain selected strain selected from SENN, were used in this study. Both strains were exposed to one of three concentrations of a combination nitrogen-phosphorus-potassium (NPK) inorganic fertilizer, as well as nitrogenous (urea), phosphorus (superphosphate) and kaelic (potassium chloride, KCl) elemental fertilizer. The time to pupation was monitored, adult longevity was assessed and the insecticide tolerance of adults was determined. The effect of elemental fertilizers on ovipositioning site choice was also assessed. RESULTS: For both strains, urea increased the number of eggs laid, while superphosphate resulted in a significant decrease in egg laying. Larval NPK exposure decreased the time to pupation in the SENN strain but not in SENN-DDT. Urea exposure increased the time to pupation in both strains, while KCl decreased the time to pupation in both strains. Larval NPK exposure only affected adult male longevity at high concentrations. Larval exposure to NPK and KCl resulted in increased insecticide tolerance in both strains, with variable efficacy from strain to strain. CONCLUSION: Exposure to inorganic fertilizers has a greater effect on insecticide susceptible An. arabiensis as compared to resistant strains, where the primary advantage is increased insecticide tolerance. These data also demonstrate that larval fertilizer exposure can affect fecundity and fertility, and alter the life histories of adult An. arabiensis. © 2019 Society of Chemical Industry.

Metabolic rate does not vary with seasonal change in Anopheles arabiensis adults in South Africa.

An important component of South Africa's malaria elimination agenda is identifying the entomological drivers of residual transmission, especially those that present opportunities for enhanced vector control. Seasonal mosquito density correlates directly with malaria transmission in South Africa. Transmission is highest during the warm rainy season and lowest but not entirely absent during the cooler dry season. The factors that sustain dry-season mosquito survival remain unknown. The aim of this project was therefore to investigate seasonal change in metabolic rate to determine the presence or absence of winter dormancy in malaria vector mosquitoes. Metabolic rate, determined by CO2 production during closed-system respirometry, was measured from wild anophelines collected from KwaZulu-Natal Province, South Africa. Monthly sampling spanned all four seasons (summer, autumn, winter, and spring) in 2017. Anopheles arabiensis and An. parensis specimens formed the majority of the total 437 identified specimens (n = 216 and n = 162, respectively). Metabolic rate data from wild-caught mosquitoes showed no significant seasonal disparities for An. arabiensis and An. parensis males and females. Further laboratory experiments assessed the effect of manipulated photoperiod, representing seasonal day-length changes, on the metabolic rate of colonized An. arabiensis mosquitoes. Simulations of midwinter (10 h:14 h light dark) and midsummer (14 h:10 h) day-length showed no significant effect on the metabolic rate of these mosquitoes. Age (in days) had a significant effect on the metabolic rate of both male and female colonized adult An. arabiensis mosquitoes which may be linked to developmental factors during maturation of adults. These data suggest that the South African populations of the malaria vector species An. arabiensis and An. parensis do not curtail their breeding and foraging activities during the colder and drier winter months. Overwintering by diapause does not appear to be triggered in the adult mosquito stage in An. arabiensis. However, their respective population densities do decrease considerably during winter leading to reduced malaria transmission and the opportunity for control by winter larviciding of known breeding sites.

The contribution of gut bacteria to insecticide resistance and the life histories of the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

The gut microbiota of mosquitoes is a crucial determinant of their fitness. As such, the biology of the gut microbiota of Anopheles arabiensis, a major malaria vector of Southern Africa, was investigated. Two laboratory strains of An. arabiensis were used; SENN, an insecticide susceptible strain, and SENN-DDT, a resistant strain. The strains were supplemented with either non-commensal bacteria or antibiotics via a sucrose source to sterilize the gut. The strains were fed the broad-spectrum bactericidal antibiotic gentamicin, or a preferentially gram-positive bactericidal (vancomycin), gram-negative bactericidal (streptomycin) or broad-spectrum bacteriostatic (erythromycin), either by sugar supplementation or by artificially-spiked blood-meal. The effects on adult mosquito longevity and insecticide resistance phenotype were assessed. Bacteria from the midgut of both strains were characterised by MALDI-TOF mass spectroscopy. Bactericidal antibiotics increased longevity in SENN-DDT. Bacterial supplementation increased insecticide tolerance. Antibiotic supplementation via sugar decreased tolerance to the insecticides deltamethrin and malathion. Blood-supplemented vancomycin decreased insecticide resistance, while gentamicin and streptomycin increased resistance. SENN showed a greater gut bacterial diversity than SENN-DDT, with both strains dominated by Gram-negative bacteria. This study suggests a crucial role for bacteria in An. arabiensis life history, and that gut microflora play variable roles in insecticide resistant and susceptible mosquitoes.

The effects of larval organic fertiliser exposure on the larval development, adult longevity and insecticide tolerance of zoophilic members of the Anopheles gambiae complex (Diptera: Culicidae).

Zoophilic members of the Anopheles gambiae complex are often associated with cattle. As such, it is likely that the immature aquatic stages will be exposed to cattle faeces as a pollutant. This study aimed to examine the effect of cattle manure on members of the An. gambiae complex found in South Africa. In this study, a commercial organic fertiliser originating from cattle manure was used as a proxy for cattle faeces. Laboratory strains of An. merus, An. quadriannulatus as well as four An. arabiensis strains (SENN and MBN: insecticide susceptible, MBN-DDT: insecticide resistant, unselected, SENN-DDT: insecticide resistant: selected for resistance) were used in this study. The effect of larval fertiliser exposure on larval development rate and adult longevity was assessed in all three species. The effect of larval fertiliser exposure on subsequent adult size, insecticide tolerance and detoxification enzyme activity of the four strains of the malaria vector An. arabiensis was also assessed. Following fertiliser treatment, all strains and species showed a significantly increased rate of larval development, with insecticide susceptible strains gaining the greatest advantage. The adult longevities of An. merus, An. quadriannulatus, insecticide susceptible and resistant An. arabiensis were significantly increased following fertiliser treatment. Insecticide susceptible and resistant An. arabiensis adults were significantly larger after larval organic fertiliser exposure. Larval fertiliser exposure also increased insecticide tolerance in adult An. arabiensis, particularly in the insecticide resistant, selected strain. This 4.7 fold increase in deltamethrin tolerance translated to an increase in pyrethroid resistance intensity, which could exert operational effects. In general, larval exposure to cattle faeces significantly affects the life histories of members of the An. gambiae complex.

The effect of commercial herbicide exposure on the life history and insecticide resistance phenotypes of the major malaria vector Anopheles arabiensis (Diptera: culicidae).

Herbicides, such as atrazine and glyphosate, are common agrochemicals known to pollute surface ground water. As such, aquatic invertebrates associated with agricultural activities can be exposed to varying doses of these xenobiotics. Anopheles arabiensis, a major malaria vector species in southern Africa, is often closely associated with agricultural activities. This study aimed to examine the effects of larval atrazine or glyphosate exposure on larval and adult life history traits on two laboratory strains of An. arabiensis; one insecticide susceptible (SENN), the other selected for resistance (SENN DDT). Atrazine delayed time to pupation in both strains, but markedly more so in SENN DDT. Glyphosate treatment reduced time to pupation in SENN DDT. Larval atrazine exposure decreased adult longevity in SENN, while both herbicide treatments significantly increased adult longevity in SENN DDT. Larval glyphosate exposure was the more potent enhancer of insecticide tolerance in adult mosquitoes. In SENN DDT, it reduced deltamethrin and malathion-induced mortality, and the LT50 s for these insecticides were increased in association with herbicide exposure. Glyphosate exposure also increased the LT50 s for malathion and deltamethrin in SENN. Exposure to both herbicides had contrasting effects on detoxification enzyme activities. Although both increased cytochrome P450 activity, they had opposite effects on those enzymes involved in reactive oxygen species detoxification. Glyphosate decreased glutathione S-transferase activity, but increased catalase activity with atrazine having the opposite effect. This study demonstrates that larval exposure to the herbicides atrazine and glyphosate can affect the insecticide susceptibilities and life history traits of epidemiological importance in An. arabiensis, with glyphosate being the more potent effector of insecticide resistance.

The effect of metal pollution on the life history and insecticide resistance phenotype of the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

Metal exposure is one of the commonest anthropogenic pollutants mosquito larvae are exposed to, both in agricultural and urban settings. As members of the Anopheles gambiae complex, which contains several major malaria vector species including An. arabiensis, are increasingly adapting to polluted environments, this study examined the effects of larval metal exposure on various life history traits of epidemiological importance. Two laboratory strains of An. arabiensis, SENN (insecticide susceptible) and SENN DDT (insecticide resistant), were reared in maximum acceptable toxicity concentrations, (MATC-the highest legally accepted concentration) of cadmium chloride, lead nitrate and copper nitrate. Following these exposures, time to pupation, adult size and longevity were determined. Larvae reared in double the MATC were assessed for changes in malathion and deltamethrin tolerance, measured by lethal time bottle bioassay, as well as changes in detoxification enzyme activity. As defence against oxidative stress has previously been demonstrated to affect the expression of insecticide resistance, catalase, glutathione peroxidase and superoxide dismutase activity was assessed. The relative metal toxicity to metal naïve larvae was also assessed. SENN DDT larvae were more tolerant of metal pollution than SENN larvae. Pupation in SENN larvae was significantly reduced by metal exposure, while adult longevity was not affected. SENN DDT showed decreased adult size after larval metal exposure. Adult insecticide tolerance was increased after larval metal exposure, and this effect appeared to be mediated by increased ß-esterase, cytochrome P450 and superoxide dismutase activity. These data suggest an enzyme-mediated positive link between tolerance to metal pollutants and insecticide resistance in adult mosquitoes. Furthermore, exposure of larvae to metal pollutants may have operational consequences under an insecticide-based vector control scenario by increasing the expression of insecticide resistance in adults.

Population Dynamics and Plasmodium falciparum (Haemosporida: Plasmodiidae) Infectivity Rates for the Malaria Vector Anopheles arabiensis (Diptera: Culicidae) at Mamfene, KwaZulu-Natal, South Africa.

Anopheles arabiensis (Patton; Diptera: Culicidae) is a major malaria vector in the southern African region. In South Africa, effective control of this species using indoor-based interventions is reduced owing to its tendency to rest outdoors. As South Africa moves towards malaria elimination there is a need for complementary vector control strategies. One of the methods under consideration is the use of the sterile insect technique (SIT). Key to the successful implementation of an SIT programme is prior knowledge of the size and spatial distribution of the target population. Understanding mosquito population dynamics for both males and females is critical for efficient programme implementation. It is thus necessary to use outdoor-based population monitoring tools capable of sampling both sexes of the target population. In this project mosquito surveillance and evaluation of tools capable of collecting both genders were carried out at Mamfene in northern KwaZulu-Natal Province, South Africa, during the period January 2014 to December 2015. Outdoor- and indoor-resting Anopheles mosquitoes were sampled in three sections of Mamfene over the 2-yr sampling period using modified plastic buckets, clay pots and window exit traps. Morphological and molecular techniques were used for species identifications of all samples. Wild-caught adult females were tested for Plasmodium falciparum (Welch; Haemosporida: Plasmodiidae) infectivity. Out of 1,705 mosquitoes collected, 1,259 (73.8%) and 255 (15%) were identified as members of either the Anopheles gambiae complex or Anopheles funestus group respectively. An. arabiensis was the most abundant species contributing 78.8% of identified specimens. Mosquito density was highest in summer and lowest during winter. Clay pots yielded 16.3 mosquitoes per trap compared to 10.5 for modified plastic buckets over the 2-yr sampling period. P. falciparum infection rates for An. arabiensis were 0.7% and 0.5% for 2014 and 2015, respectively. Logistic regression analysis showed an association between An. arabiensis catches with Section and season of collection but not with sex and collection methods. These data confirmed the presence of a perennial An. arabiensis population at Mamfene and constitute the first records of P. falciparum infective An. arabiensis from South Africa, confirming this species as a major vector in the malaria endemic provinces of the country.

The effects of ingestion of hormonal host factors on the longevity and insecticide resistance phenotype of the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

Exogenous vertebrate-derived factors circulating in the blood have the capacity to modulate the biology of haematophagous insects. These include insulin, insulin growth factor 1 (IGF) and transforming growth factor ß1 (TGFß). The effects of the consumption of these three proteins were examined on laboratory strains of Anopheles arabiensis. SENN, an insecticide susceptible strain and SENN DDT, a resistant strain selected from SENN, were fed with host factor-supplemented sucrose. Adult longevity was measured and insecticide resistance phenotype over time was assessed by WHO bioassay. Detoxification and oxidative stress defence enzyme activity was assessed calorimetrically. Insulin supplementation augmented insecticide resistance in young adult mosquitoes. This effect was due to the hormonal nature of the protein, as heat-denatured insulin did not elicit the same response. In contrast, IGF and TGFß consumption generally reduced the expression of insecticide resistance. Insulin ingestion significantly reduced longevity in the insecticide susceptible strain. IGF elicited the same response in the susceptible strain, while TGF consumption had no effect on either strain. Consumption of all factors significantly decreased Glutathione S-transferase activity and increased cytochrome P450 and superoxide dismutase activity. This suggests that the altered detoxification phenotype is mediated primarily by cytochrome P450 activity, which would result in an increase in oxidative stress. The increased superoxide dismutase activity suggests that this enzyme class alleviates the oxidative stress as opposed to glutathione-based redox systems. Oxidative stress responses play a crucial role in insecticide resistance and longevity. These data show that ingested hormonal factors can affect mosquito longevity and insecticide susceptibility, both of which are important characteristics in terms of malaria transmission and control.

Benchmarking insecticide resistance intensity bioassays for Anopheles malaria vector species against resistance phenotypes of known epidemiological significance.

BACKGROUND: Insecticide use via indoor residual spraying (IRS) or treated nets is the primary method for controlling malaria vector populations. The incidence of insecticide resistance in vector populations is burgeoning globally making resistance management key to the design of effective malaria control and elimination strategies. Vector populations can be assessed for insecticide resistance using a binary (susceptible or resistant) classification based on the use of the standard WHO insecticide susceptibility assay for adult anopheline mosquitoes. However, the recent scaling up of vector control activities has necessitated a revision of the WHO bioassay protocol to include the production of information that not only diagnoses resistance but also gives information on the intensity of expression of resistance phenotypes detected. This revised protocol is expected to inform on the range of resistance phenotypes in a target vector population using discriminating/diagnostic insecticide concentrations (DC) as well as their potential operational significance using 5× DC and 10× DC assays. The aim of this project was to use the revised protocol to assess the intensity of pyrethroid resistance in a range of insecticide resistant Anopheles strains with known resistance mechanisms and for which there is evidence of operational significance in the field setting from which these colonies were derived. METHODS: Diagnostic concentration (DC) bioassays followed by 5× DC and 10× DC assays using the pyrethroid insecticides permethrin and deltamethrin were conducted according to the standard WHO bioassay method against pyrethroid resistant laboratory strains of Anopheles funestus, An. arabiensis and An. gambiae. RESULTS: Low to moderate resistance intensities were recorded for the An. arabiensis and An. gambiae strains while moderate to high intensities were recorded for the An. funestus strains. CONCLUSIONS: It is evident that resistance intensity assays can add predictive value to the decision making process in vector control settings, although more so in an IRS setting and especially when bench-marked against resistance phenotypes of known operational significance.

The effect of elevated temperatures on the life history and insecticide resistance phenotype of the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

BACKGROUND: Temperature plays a crucial role in the life history of insects. Recent climate change research has highlighted the importance of elevated temperature on malaria vector distribution. This study aims to examine the role of elevated temperatures on epidemiologically important life-history traits in the major malaria vector, Anopheles arabiensis. Specifically, the differential effects of temperature on insecticide-resistant and susceptible strains were examined. METHODS: Two laboratory strains of A. arabiensis, the insecticide-susceptible SENN and the insecticide-resistant SENN DDT strains, were used to examine the effect of elevated temperatures on larval development and adult longevity. The effects of various elevated temperatures on insecticide resistance phenotypes were also examined and the biochemical basis of the changes in insecticide resistance phenotype was assessed. RESULTS: SENN and SENN DDT larvae developed at similar rates at elevated temperatures. SENN DDT adult survivorship did not vary between control and elevated temperatures, while the longevity of SENN adults at constantly elevated temperatures was significantly reduced. SENN DDT adults lived significantly longer than SENN at constantly elevated temperatures. Elevated rearing temperatures, as well as a short-term exposure to 37 and 39 °C as adults, augmented pyrethroid resistance in adult SENN DDT, and increased pyrethroid tolerance in SENN. Detoxification enzyme activity was not implicated in this phenotypic effect. Quercertin-induced synergism of inducible heat shock proteins negated this temperature-mediated augmentation of pyrethroid resistance. CONCLUSION: Insecticide-resistant A. arabiensis live longer than their susceptible counterparts at elevated temperatures. Exposure to heat shock augments pyrethroid resistance in both resistant and susceptible strains. This response is potentially mediated by inducible heat shock proteins.