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.

Optimisation of laboratory-rearing parameters for Anopheles funestus larvae and adults.

Anopheles funestus is one of the major malaria vectors in Africa. As with the other main vectors, insecticide resistance in this species threatens existing vector control strategies. Unfortunately, scientific investigations, which could improve understanding of this vector species or lead to the development of new control strategies, are currently limited by difficulties in laboratory rearing of the species. In an attempt to optimise laboratory-rearing conditions for An. funestus, the effect of an artificial blood-feeding system for adults, different larval diet doses, and a range of other rearing conditions on the life history traits of an existing colony were investigated. Firstly, fecundity and fertility in An. funestus adult females fed on either live guinea pigs or bovine blood supplied through an artificial membrane feeding system were assessed. Secondly, a life-table approach was used to assess the impact of larval food dose (mg/larvae), larval density (larvae/cm2), and the depth of water used for larval rearing on life history traits. Fecundity was significantly higher when females were blood-fed on live anaesthetised guinea pigs than when fed on defibrinated bovine blood. However, the fertility of these eggs did not differ significantly between the two feeding methods or blood meal sources. Mosquitoes fed on defibrinated bovine blood using the artificial membrane feeding system showed an increase in egg production when the blood-feeding frequency was increased, but this difference was not statistically significant. The quantity of larval food influenced both time-to-pupation and pupal production. Increasing the larval densities resulted in reduced both time-to-pupation and pupal productivity. An optimal larval density of 0.48 larvae/cm2 was vital in preventing overcrowding. Increased water depth in the larval trays, was associated with significantly lower pupal production and reduced pupal weight. In conclusion, these results show that An. funestus can be reared using defibrinated bovine blood delivered via an artificial membrane feeding system. The quantity of larval food, optimal larval density, and depth of water used for larval rearing are critical factors influencing colony productivity. These findings can be used to improve current guidelines for rearing An. funestus under insectary conditions.

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.

Staggered larval time-to-hatch and insecticide resistance in the major malaria vector Anopheles gambiae S form.

BACKGROUND: Anopheles gambiae is a major vector of malaria in the West African region. Resistance to multiple insecticides has been recorded in An. gambiae S form in the Ahafo region of Ghana. A laboratory population (GAH) established using wild material from this locality has enabled a mechanistic characterization of each resistance phenotype as well as an analysis of another adaptive characteristic - staggered larval time-to-hatch. METHODS: Individual egg batches obtained from wild caught females collected from Ghana and the Republic of the Congo were monitored for staggered larval time-to-hatch. In addition, early and late larval time-to-hatch sub-colonies were selected from GAH. These selected sub-colonies were cross-mated and their hybrid progeny were subsequently intercrossed and back-crossed to the parental strains. The insecticide susceptibilities of the GAH base colony and the time-to-hatch selected sub-colonies were quantified for four insecticide classes using insecticide bioassays. Resistance phenotypes were mechanistically characterized using insecticide-synergist bioassays and diagnostic molecular assays for known reduced target-site sensitivity mutations. RESULTS: Anopheles gambiae GAH showed varying levels of resistance to all insecticide classes. Metabolic detoxification and reduced target-site sensitivity mechanisms were implicated. Most wild-caught families showed staggered larval time-to-hatch. However, some families were either exclusively early hatching or late hatching. Most GAH larvae hatched early but many egg batches contained a proportion of late hatching larvae. Crosses between the time-to-hatch selected sub-colonies yielded ambiguous results that did not fit any hypothetical models based on single-locus Mendelian inheritance. There was significant variation in the expression of insecticide resistance between the time-to-hatch phenotypes. CONCLUSIONS: An adaptive response to the presence of multiple insecticide classes necessarily involves the development of multiple resistance mechanisms whose effectiveness may be enhanced by intra-population variation in the expression of resistance phenotypes. The variation in the expression of insecticide resistance in association with selection for larval time-to-hatch may induce this kind of enhanced adaptive plasticity as a consequence of pleiotropy, whereby mosquitoes are able to complete their aquatic life stages in a variable breeding environment using staggered larval time-to-hatch, giving rise to an adult population with enhanced variation in the expression of insecticide resistance.

A standardised method of marking male mosquitoes with fluorescent dust.

BACKGROUND: Prior to a major release campaign of sterile insects, including the sterile insect technique, male mosquitoes must be marked and released (small scale) to determine key parameters including wild population abundance, dispersal and survival. Marking insects has been routinely carried out for over 100 years; however, there is no gold standard regarding the marking of specific disease-transmitting mosquitoes including Anopheles arabiensis, Aedes aegypti and Aedes albopictus. The research presented offers a novel dusting technique and optimal dust colour and quantities, suitable for small-scale releases, such as mark-release-recapture studies. METHODS: We sought to establish a suitable dust colour and quantity for batches of 100 male An. arabiensis, that was visible both by eye and under UV light, long-lasting and did not negatively impact longevity. A set of lower dust weights were selected to conduct longevity experiments with both Ae. aegypti and Ae. albopictus to underpin the optimal dust weight. A further study assessed the potential of marked male An. arabiensis to transfer their mark to undusted males and females. RESULTS: The longevity of male An. arabiensis marked with various dust colours was not significantly reduced when compared to unmarked controls. Furthermore, the chosen dust quantity (5 mg) did not negatively impact longevity (P = 0.717) and provided a long-lasting mark. Dust transfer was found to occur from marked An. arabiensis males to unmarked males and females when left in close proximity. However, this was only noticeable when examining individuals under a stereomicroscope and thus deemed negligible. Overall, male Ae. aegypti and Ae. albopictus displayed a greater sensitivity to dusting. Only the lowest dust weight (0.5 mg) did not significantly reduce longevity (P = 0.888) in Ae. aegypti, whilst the lowest two dust weights (0.5 and 0.75 mg) had no significant impact on longevity (P = 0.951 and 0.166, respectively) in Ae. albopictus. CONCLUSION: We have devised a fast, inexpensive and simple marking method and provided recommended dust quantities for several major species of disease-causing mosquitoes. The novel technique provides an evenly distributed, long-lasting mark which is non-detrimental. Our results will be useful for future MRR studies, prior to a major release campaign.

Malaria Vectors and Vector Surveillance in Limpopo Province (South Africa): 1927 to 2018.

Despite the annual implementation of a robust and extensive indoor residual spraying programme against malaria vectors in Limpopo Province (South Africa), significant transmission continues and is a serious impediment to South Africa's malaria elimination objectives. In order to gain a better understanding regarding possible causes of this residual malaria, we conducted a literature review of the historical species composition and abundance of malaria vector mosquitoes in the Limpopo River Valley region of the Vhembe District, northern Limpopo Province, the region with the highest remaining annual malaria cases in South Africa. In addition, mosquito surveys were carried out in the same region between October 2017 and October 2018. A total of 2225 adult mosquitoes were collected using CO2-baited tent and light traps, human landing catches and cow-baited traps. Of the 1443 Anopheles collected, 516 were members of the An. gambiae complex and 511 An. funestus group. In the malaria endemic rural areas outside the Kruger National Park, one specimen each of An. gambiae s.s. and An. funestus and only three of An. arabiensis were collected. The latter species was abundant at a remote hot spring in the neighboring Kruger National Park. Eighteen other species of Anopheles were collected. Our survey results support the historical findings that An. arabiensis, the species widely held to be the prime malaria vector in South Africa, is a rare species in the malaria endemic Limpopo River Valley. The implications of the mosquito surveys for malaria transmission, elimination and vector control in northern Limpopo Province and neighboring regions are discussed.

Characterization of chemosensitivity and resistance of human cancer cell lines to platinum(II) versus platinum(IV) anticancer agents.

Platinum (Pt)(IV) complexes are thought to function as prodrugs for anticancer Pt(II) drugs. We studied two pairs of Pt(II)/Pt(IV) complexes to explore whether there were differences in their cytotoxic activities, their abilities to cause acquired resistance and their gene expression profiles in the resistant lines. Microtiter methods were used to evaluate the antiproliferative activity of cisplatin, oxoplatin, [trans-d,l-(1,2-diaminocyclo-hexane)]dichloroplatinum(II) [DACH-Pt(II)] and cis,trans-[trans-d,l-(1,2-diaminocyclo-hexane)]-dichlorodihydroxoplatinum(IV) [DACH-Pt(IV)] in a panel of 14 human cancer cell lines. Cisplatin and oxoplatin showed significant similar spectra of cytotoxicity, whereas DACH-Pt(II) and DACH-Pt(IV) did not. DACH-Pt(IV) required more than 24 h to reach full potency, whereas the other three Pt complexes achieved maximal activity in less than 24 h. The SISO cervical cell line was made four- to six-fold resistant to the four Pt complexes by weekly exposure to the respective agent. Glutathione (GSH) levels increased in all resistant lines except for the DACH-Pt(IV) resistant line. The catalytic concentrations of various redox enzymes (GSH transferase, GSH peroxidase, GSH reductase, catalase) were all unchanged in the resistant lines relative to the native line. Multidrug resistance protein 2 expression was detected in the cisplatin-resistant and oxoplatin-resistant cell lines but not in the native line. The transcription of 29,000 genes in the SISO lines resistant to either cisplatin or oxoplatin was studied by DNA-microarray methods and compared with the native line. Overall changes in gene transcription were very different between the cisplatin-resistant and oxoplatin-resistant cell lines. Thus, Pt(IV) complexes seem to have biological actions that distinguish them from their Pt(II) counterparts, even when they show cross-resistance.

A review on the progress of sex-separation techniques for sterile insect technique applications against Anopheles arabiensis.

The feasibility of the sterile insect technique (SIT) as a malaria vector control strategy against Anopheles arabiensis has been under investigation over the past decade. One of the critical steps required for the application of this technique to mosquito control is the availability of an efficient and effective sex-separation system. Sex-separation systems eliminate female mosquitoes from the production line prior to irradiation and field release of sterile males. This is necessary because female mosquitoes can transmit pathogens such as malaria and, therefore, their release must be prevented. Sex separation also increases the efficiency of an SIT programme. Various sex-separation strategies have been explored including the exploitation of developmental and behavioural differences between male and female mosquitoes, and genetic approaches. Most of these are however species-specific and are not indicated for the major African malaria vectors such as An. arabiensis. As there is currently no reliable sex-separation method for An. arabiensis, various strategies were explored in an attempt to develop a robust system that can be applied on a mass-rearing scale. The progress and challenges faced during the development of a sexing system for future pilot and/or large-scale SIT release programmes against An. arabiensis are reviewed here. Three methods of sex separation were examined. The first is the use of pupal size for gender prediction. The second is the elimination of blood-feeding adult females through the addition of an endectocide to a blood meal source. The third is the establishment of a genetic sexing strain (GSS) carrying an insecticide resistance selectable marker (dieldrin-resistance rdl gene and/or other GABA receptor antagonists that can be used as alternative insecticides to dieldrin) or a temperature-sensitive lethal marker.

Embryonic development and rates of metabolic activity in early and late hatching eggs of the major malaria vector Anopheles gambiae.

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO2 output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.

Insecticide resistance in malaria vector mosquitoes at four localities in Ghana, West Africa.

BACKGROUND: Malaria vector control programmes that rely on insecticide-based interventions such as indoor house spraying with residual insecticides or insecticide treated bed nets, need to base their decision-making process on sound baseline data. More and more commercial entities in Africa, such as mining companies, are realising the value to staff productivity of controlling malaria transmission in their areas of operation.This paper presents baseline entomological data obtained during surveys conducted for four mining operations in Ghana, West Africa. RESULTS: The vast majority of the samples were identified as Anopheles gambiae S form with only a few M form specimens being identified from Tarkwa. Plasmodium falciparum infection rates ranged from 4.5 to 8.6% in An. gambiae and 1.81 to 8.06% in An. funestus. High survival rates on standard WHO bioassay tests were recorded for all insecticide classes except the organophosphates that showed reasonable mortality at all locations (i.e. >90%). The West African kdr mutation was detected and showed high frequencies in all populations. CONCLUSIONS: The data highlight the complexity of the situation prevailing in southern Ghana and the challenges facing the malaria vector control programmes in this region. Vector control programmes in Ghana need to carefully consider the resistance profiles of the local mosquito populations in order to base their resistance management strategies on sound scientific data.

Multiple insecticide resistance in Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo.

Successful implementation of an integrated vector control program will rely on availability of accurate vector information in the specific location. However, such information can be limited in some countries. The aim of this study was to obtain baseline vector information from Pointe Noire on the Congo coast (Republic of the Congo). Field sampling was conducted during April 2009 in the village of Boutoto and its surrounds, close to the city of Pointe Noire. Anopheles gambiae sensu lato mosquitoes were collected resting indoors. Samples were analyzed for insecticide susceptibility, species identification, and Plasmodium sporozoite infection. Molecular and biochemical assays were conducted to characterize insecticide resistance mechanisms. The malaria vector A. gambiae S-form was the only mosquito species identified, and it had a high Plasmodium falciparum infection rate (9.6%). Multiple insecticide resistance was detected in this population with full susceptibility to only one insecticide class, the organophosphates. Dieldrin and DDT resistance was mainly attributed to target-site resistance (the Rdl and L1014F/L1014S kdr mutations respectively), whereas pyrethroid resistance was mainly attributed to P450 metabolic enzyme-mediated detoxification in addition to kdr. The role of various insecticide resistance mechanisms revealed a complex association between metabolic detoxification and reduced target-site sensitivity.