Evaluation of the Long-Lasting Insecticidal Net Interceptor LN: Laboratory and Experimental Hut Studies against Anopheline and Culicine Mosquitoes in Northeastern Tanzania

Long lasting insecticidal nets (LN) are a primary method of malaria prevention. Before new types of LN are approved they need to meet quality and efficacy standards set by the WHO Pesticide Evaluation Scheme. The process of evaluation has three phases. In Phase I the candidate LN must meet threshold bioassay criteria after 20 standardized washes. In Phase II washed and unwashed LNs are evaluated in experimental huts against wild, free flying anopheline mosquitoes. In Phase III the LN are distributed to households in malaria endemic areas, sampled over three years of use and tested for continuing insecticidal efficacy. Interceptor® LN (BASF Corporation, Germany) is made of polyester netting coated with a wash resistant formulation of alpha-cypermethrin. Interceptor LN was subjected to bioassay evaluation and then to experimental hut trial against pyrethroid-susceptible Anopheles gambiae and An. funestus and resistant Culex quinquefasciatus. Mosquito mortality, blood feeding inhibition and personal protection were compared between untreated nets, conventional alpha-cypermethrin treated nets (CTN) washed 20 times and LNs washed 0, 20 and 30 times. In Phase I Interceptor LN demonstrated superior wash resistance and efficacy to the CTN. In the Phase II hut trial the LN killed 92% of female An. gambiae when unwashed and 76% when washed 20 times; the CTN washed 20 times killed 44%. The LN out-performed the CTN in personal protection and blood-feeding inhibition. The trend for An. funestus was similar to An. gambiae for all outcomes. Few pyrethroid-resistant Cx. quinquefasciatus were killed and yet the level of personal protection (75-90%) against Culex was similar to that of susceptible An. gambiae (76-80%) even after 20 washes. This protection is relevant because Cx. quinquefasciatus is a vector of lymphatic filariasis in East Africa. After 20 washes and 60 nights’ use the LN retained 27% of its initial insecticide dose. Interceptor LN meets the approval criteria set by WHO and is recommended for use in disease control against East African vectors of malaria and filariasis. Some constraints associated with the phase II evaluation criteria, in particular the washing procedure, are critically reviewed

Chemical Mediation of Oviposition by Anopheles Mosquitoes : a Push-Pull System Driven by Volatiles Associated with Larval Stages

The oviposition behavior of mosquitoes is mediated by chemical cues. In the malaria mosquito Anopheles gambiae, conspecific larvae produce infochemicals that affect this behavior. Emanations from first instar larvae proved strongly attractive to gravid females, while those from fourth instars caused oviposition deterrence, suggesting that larval developmental stage affected the oviposition choice of the female mosquito. We examined the nature of these chemicals by headspace collection of emanations of water in which larvae of different stages were developing. Four chemicals with putative effects on oviposition behavior were identified: dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) were identified in emanations from water containing fourth instars; nonane and 2,4-pentanedione (2,4-PD) were identified in emanations from water containing both first and fourth instars. Dual-choice oviposition studies with these compounds were done in the laboratory and in semi-field experiments in Tanzania. In the laboratory, DMDS and DMTS were associated with oviposition-deterrent effects, while results with nonane and 2,4-PD were inconclusive. In further studies DMDS and DMTS evoked egg retention, while with nonane and 2,4-PD 88% and 100% of female mosquitoes, respectively, laid eggs. In dual-choice semi-field trials DMDS and DMTS caused oviposition deterrence, while nonane and 2,4-PD evoked attraction, inducing females to lay more eggs in bowls containing these compounds compared to the controls. We conclude that oviposition of An. gambiae is mediated by these four infochemicals associated with conspecific larvae, eliciting either attraction or deterrence. High levels of egg retention occurred when females were exposed to chemicals associated with fourth instar larvae.</p

Aedes albopictus invasion across Africa: the time is now for cross-country collaboration and control

The distribution of Aedes albopictus across west Africa is well documented. However, little has been done to synthesise data and establish the current distribution of this invasive vector in central and east Africa. In this Viewpoint, we show that A albopictus is establishing across Africa, how this is potentially related to urbanisation, and how establishment poses risks of near-term increases in arbovirus transmission. We then use existing species distribution maps for A albopictus and Aedes aegypti to produce consensus estimates of suitability and make these estimates accessible. Although urban development and increased trade have economic and other societal gains, the resulting potential changes in Aedes-borne virus epidemiology require a discussion of how cross-country collaboration and mitigation could be facilitated. Failure to respond to species invasion could result in increased transmission of Aedes-associated pathogens, including dengue, chikungunya, and Rift Valley fever viruses

An experimental hut evaluation of Olyset® nets against anopheline mosquitoes after seven years use in Tanzanian villages

BACKGROUND: Long-lasting insecticidal nets (LLINs) are advocated by WHO for protection against malaria. Of the three brands of LLINs currently approved by WHO, Olyset(R) is the only one currently granted full recommendation. With this type of LLIN, the insecticide (permethrin) is incorporated into the polyethylene fibre during manufacture and diffuses from the core to the surface, thereby maintaining surface concentrations. It has not been determined for how long Olyset nets remain protective against mosquitoes in household use. METHODS: Examples of Olyset nets, which had been in use in Tanzanian villages for seven years, were tested in experimental huts against naturally entering Anopheles gambiae and Anopheles funestus mosquitoes. Performance was compared with new Olyset nets, conventionally treated ITNs (either newly treated with alphacypermethrin or taken from local villages after 1.5 years of use) and untreated nets. All nets were artificially holed except for the seven-year Olyset nets, which had developed holes during prolonged domestic use. RESULTS: Anopheles funestus and An. gambiae in NE Tanzania are susceptible to pyrethroids. The new Olyset nets caused high mortality against An. funestus (73.9%) and An. gambiae (62.7%) in experimental huts. The seven-year Olyset nets caused 58.9% mortality against An. funestus and 40.0% mortality against An. gambiae. The freshly treated alphacypermethrin nets also caused high mortality against An. funestus (70.6%) and An. gambiae (72.0%); this decreased to 58.4% and 69.6% respectively after 1.5 years of use. The new Olyset nets inhibited blood-feeding by 40-50%. The 7 year Olyset nets showed no feeding inhibition over that shown by the untreated nets. The alphacypermethrin treated nets failed to inhibit blood-feeding after 1.5 years of use. However iHhhdn laboratory tunnel tests samples of all types of treated net including the 7 year Olyset inhibited blood-feeding by more than 95%. CONCLUSION: After seven years of use Olyset nets were still strongly insecticidal. Mosquito mortality decreased by only 20-35% over this period. However, Olyset would not provide personal protection after seven years unless it was in good condition and all holes fully repaired

Tropical Data: Approach and Methodology as Applied to Trachoma Prevalence Surveys

PURPOSE: Population-based prevalence surveys are essential for decision-making on interventions to achieve trachoma elimination as a public health problem. This paper outlines the methodologies of Tropical Data, which supports work to undertake those surveys. METHODS: Tropical Data is a consortium of partners that supports health ministries worldwide to conduct globally standardised prevalence surveys that conform to World Health Organization recommendations. Founding principles are health ministry ownership, partnership and collaboration, and quality assurance and quality control at every step of the survey process. Support covers survey planning, survey design, training, electronic data collection and fieldwork, and data management, analysis and dissemination. Methods are adapted to meet local context and needs. Customisations, operational research and integration of other diseases into routine trachoma surveys have also been supported. RESULTS: Between 29th February 2016 and 24th April 2023, 3373 trachoma surveys across 50 countries have been supported, resulting in 10,818,502 people being examined for trachoma. CONCLUSION: This health ministry-led, standardised approach, with support from the start to the end of the survey process, has helped all trachoma elimination stakeholders to know where interventions are needed, where interventions can be stopped, and when elimination as a public health problem has been achieved. Flexibility to meet specific country contexts, adaptation to changes in global guidance and adjustments in response to user feedback have facilitated innovation in evidence-based methodologies, and supported health ministries to strive for global disease control targets

Olfactory mediation of oviposition in Anopheles mosquitoes : A new strategy for malaria vector control

Mosquito-borne diseases are among the most serious infectious diseases in the world caused by parasites and pathogens. The African mosquito Anopheles gambiae transmits malaria parasites as well as filarial worms. The mosquitoes feed on humans inside and outside houses, which complicates control strategies. Current control strategies focus mostly on reducing contact with host-seeking mosquitoes through the use of long-lasting insecticide-impregnated nets (LLINs) and indoor residual spraying (IRS). Despite the fact that these tools have lowered the burden of malaria in many parts of the world, transmission is sustained by vectors that bite outdoors. This thesis proposes a new strategy for malaria vector control to be added to existing control tools. It goes beyond blood-feeding processes and it targets all mosquitoes which are feeding indoors and outdoors. The proposed strategy is based on manipulation of oviposition behaviour using chemical cues that mimic olfactory cues emitted by breeding sites. Mosquitoes are attracted to lay their eggs on selected water bodies with specific biotic and abiotic factors. Organic chemical cues, emitted by these sites, are presumed to act as infochemicals enabling these mosquitoes to find suitable breeding sites. Identification of the odours that mediate the site-seeking behaviour of mosquitoes can lead to the development of novel intervention methods for mosquito-borne disease control. My study marked an important progress in the development of mosquito surveillance and control strategies using infochemicals by: (1) generating knowledge on the biology and ecology of immature stages of Anopheles mosquitoes in the study area, (2) providing evidence that mosquitoes select breeding sites preferentially and lay eggs on suitable sites while avoiding unsuitable ones, (3) identifying and characterizing volatile organic chemicals that mediate oviposition behaviour in mosquitoes, (4) generating evidence that oviposition infochemicals can be used to divert oviposition activities to specific breeding sites, which can be targeted by an environmentally safe larvicide: a lure-and-kill method. The research aimed to understand the reasons that prevent early and late larval stages from sharing breeding sites, and also why breeding sites with late stage larvae are not immediately succeeded by early stage larvae (Chapter 5). Several studies have indicated a possible role of habitat materials in the production of volatiles chemicals which either attract or deter egg-laying mosquitoes. A review of recently published studies showed that oviposition attractants and deterrents are produced by biotic factors present in breeding sites (Chapter 2). In my study it was found that breeding sites which are occupied by larvae play an important role in the production of odours that mediate oviposition behaviour of mosquitoes (Chapter 3). These infochemicals can potentially be used for manipulation of mosquito behaviour by making protected resources unsuitable for mosquitoes, while luring them towards attractive sources. Such push and pull strategies using ovipostion infochemicals for other mosquitoes were reviewed in order to develop similar surveillance and control strategies for malaria vectors. Among other larviciding products reviewed, bacterial products such as Bacillus sphaericus (Bs) and Bacillus thuringiensis israelensis (Bti) emerged with the potential to be combined with attractive infochemicals in the development of a lure and kill strategy (Chapter 2). To understand the factors influencing oviposition behaviour of Anopheles mosquitoes, a series of dual-choice behavioural experiments were conducted in the laboratory using larval habitats materials. Results show that the presence of larvae in breeding sites mediates oviposition behaviour of Anopheles mosquitoes (Chapter 3). The presence of early-stage larvae in oviposition cups attracted conspecific gravid mosquitoes to lay eggs, whereas the presence of late-stage larvae in the cups deterred conspecific gravid mosquitoes to lay eggs. The response was density dependent: as larval density increased so did the response, until reaching a threshold. Moreover, this behaviour was manifested more profoundly in the presence of larvae than larval-conditioned water. To test whether larval habitats produce volatiles that mediate oviposition behaviour, the oviposition cups containing larvae were covered by a filter paper to prevent gravid females from seeing the larvae in the cups. Results show that both olfactory and visual cues are involved in mediating the oviposition behaviour of anopheline mosquitoes. To confirm the findings that larval habitats emit infochemicals that mediate oviposition behaviour of &nbsp;gravid mosquitoes, larval habitats containing early- or late-stage larvae were placed in containers in the laboratory. Headspace volatiles of the larval microcosm were entrained and analyzed by gas chromatography - mass spectrometry (GC-MS). Analysis of the volatiles emitted by larvae in-vitro led to the identification of 16 compounds, and four of them, when tested with An. gambiae, had an effect on oviposition behaviour (Chapter 4). Volatile chemicals extracted from headspace collections of larvae mediated similar behaviour; volatiles entrapped from young larvae attracted conspecific gravid mosquitoes, whereas those from late-stage larvae deterred conspecific gravid mosquitoes to lay eggs. Nonane and 2,4-pentenedione, which were isolated from headspace collection of larvae, enhanced the attractiveness of a synthetic solution and could be used to increase mosquito egg deposition for monitoring or vector control purposes. Dimethyldisulphide (DMDS) and dimethyltrisulphide (DMTS), which were isolated from headspace collection of late-stage larvae only, decreased mosquito egg deposition in a synthetic solution and may act as a spatial deterrent. Laboratory and semi-field experiments in Tanzania showed similar results and suggested that nonane and 2,4-pentanedione can be used as mid-range cues for oviposition. The study described in Chapter 5 provided ecological characteristics of the field site where observations on the behaviour and distribution pattern of larvae were done. This is the field site where initial observations on succession of larvae in breeding sites and the actual testing of infochemicals took place. The factors influencing the selection of breeding sites by gravid females and distribution patterns of mosquito larvae were explored. Human activities such as brick-making and rice production were found to influence oviposition site selection and hence constitute a risk factor for malaria and other mosquito-borne diseases. The presence of larvae of Anopheles mosquitoes in breeding sites was associated with vegetation, presence of culicine larvae, shallowness and sunlight. The association between anopheline and culicine larvae is a vital finding that suggests that gravid mosquitoes of the two species use similar cues in selecting suitable breeding sites. Moreover, results from the study suggested that productivity of anopheline larvae in the field can be reduced by introducing larval predators, water management, or water pollution with rotting vegetation. The findings obtained after careful observation of the field site helped to design surveillance and larval control strategies using synthetic infochemicals identified in Chapter 4. Field experiments in east-central Tanzania showed results similar to the laboratory and semi-field studies and suggested that larval habitat volatiles also attract other disease vectors (Chapter 6). This study showed that infochemicals from selected sites can induce an oviposition response in mosquitoes under natural conditions, and that soil from a breeding site and nonane both induced oviposition in An. gambiae and Cx. quinquefasciatus with a synergistic effect caused by nonane + soil in An. gambiae. Therefore, the study also revealed that the oviposition-site-selection process of gravid mosquitoes is mediated by a combination of infochemicals derived from natural breeding sites. This knowledge can be exploited to develop a control strategy based on the push-pull or attract-and-kill concept. Furthermore, the knowledge can be utilized to develop other surveillance tools for mosquito vectors of human and animal diseases. In order to explore the potential of using a push-pull system for mosquito control, the attractants were combined with environmentally friendly biolarvicides in order to achieve more effective control of their aquatic larval offspring (Chapter 7). As an attractant for gravid mosquitoes, nonane, which originated from An. gambiae larvae, was selected. As toxicant for mosquito larvae, Bacillus thuringiensis var israeliensis (Bti) and Temephos were selected. Mosquitoes were successfully attracted to lay eggs in water-filled clay pots treated with attractants and combined with larvicides. Therefore, a lure-and-kill system was successfully implemented; mosquitoes were attracted to lay eggs in intervention pots containing toxicants, consequently, all emerged larvae died before reaching the pupal stage. The results presented in this thesis create new opportunities for applied research, especially on the development of novel vector-borne disease control strategies using infochemicals (Chapter 8). The volatiles produced by the larval habitats play an important role in the site-selection behaviour of An. gambiae and Cx. quinquefasciatus mosquitoes. The attractiveness of the synthetic chemicals identified in this study may be improved by making blends of attractants or in combination with other infochemicals identified elsewhere. Identified compounds can be used in push-pull and/or lure-and-kill strategies for the manipulation of mosquitoes; thereby reducing the abundance of mosquitoes, the human-biting frequency, and the intensity of pathogen transmission. Research on the role of oviposition-based infochemicals in the site-seeking behaviour of mosquitoes may lead to a better understanding of vector behaviour and contribute to the fight against vector-borne diseases. The main conclusions from this thesis can be summarized as follows: Volatiles produced by larval habitats play an important role in the mediation of oviposition behaviour of An. gambiae and in the dispersal of mosquito species. The analysis of the headspace from larval microcosms led to the identification of 16 compounds. Out of these, four (DMDS, DMTS, nonane and 2,4-pentanedione) had behavioural effects on An. gambiae. Nonane acts as an oviposition cue for Cx. quinquefasciatus as well. Mosquitoes are governed by multiple cues in the selection of suitable breeding sites, thus further research on the role of combined infochemicals to optimize selection of breeding sites should be initiated. These infochemicals can be used in push-pull or attract&ndash;and-kill strategies for surveillance and control of malaria mosquito larvae. Thus, odour-based technologies can improve the surveillance, sampling and control strategies for disease vectors. In this way the burden resulting from mosquito-borne diseases such as morbidity, mortality and economic losses will be decreased

Synergism between nonane and emanations from soil as cues in oviposition‐site selection of natural populations of Anopheles gambiae and Culex quinquefasciatus

Background: Olfactory cues have been shown to have an important role in guiding gravid mosquito females to selected sites for egg laying. The objective of this study was to determine the influence of emanations from soil from a breeding site and the putative oviposition pheromone nonane on oviposition-site selection of natural populations of Anopheles gambiae sensu lato (s.l.) and Culex quinquefasciatus. Methods: This field-based study was conducted in Mvomero District in East-central Tanzania. In a dual-choice experimental set up, clay bowls were dug into the ground and filled with one of the following treatments: (i) distilled water + autoclaved soil (control), (ii) distilled water + soil from a natural mosquito breeding site, (iii) distilled water + nonane and (iv) distilled water + nonane + soil from a natural breeding site. Soil was dried and autoclaved or dried only before use. After five days of incubation, larvae were collected daily for 10 days. The median number of larvae per bowl per day was used as outcome measure. Results: Autoclaved soil had a significant attractive effect on oviposition behaviour of Cx. quinquefasciatus (median values ± s.e: 8.0 ± 1.1; P < 0.005) but no effect on An. gambiae (median value ± s.e: 0.0 ± 0.2; P = 0.18). Nonane and emanations from untreated soil significantly and positively influenced the selection of oviposition sites by both An. gambiae s.l. (median values ± s.e.: 12.0 ± 2.0 and 4.5 ± 1.5, respectively; P < 0.0001) and Cx. quinquefasciatus (median values ± s.e.: 19.0 ± 1.3 and 17.0 ± 2.0, respectively; P < 0.0001). A mixture of nonane and untreated soil caused a synergistic effect on oviposition behaviour in An. gambiae s.l. (median value ± s.e.: 23.5 ± 2.5; P < 0.0001) compared to either nonane (median values ± s.e.: 12.0 ± 2.0; P < 0.0001) or untreated soil alone (median value ± s.e.: 4.5 ± 1.5; P < 0.0001). A synergistic effect of nonane mixed with untreated soil was also found in Cx. quinquefasciatus (median value ± s.e.: 41.0 ± 2.1; P < 0.0001) compared to either nonane (median value ± s.e. 19.0 ± 1.3; P < 0.0001) or untreated soil alone (median value ± s.e.: 17.0 ± 2.0; P < 0.0001). The oviposition activity index for An. gambiae was 0.56 (P < 0.001) and for Cx. quinquefasciatus 0.59 (P < 0.0001). Conclusions: The larval pheromone nonane and emanations from breeding-site soil both induced oviposition in wild An. gambiae s.l. and Cx. quinquefasciatus, with a synergistic effect when both stimuli were present simultaneously. This is the first study in which nonane is shown to cause oviposition under natural conditions, suggesting that this compound can potentially be exploited for the management of mosquito vectors

Exploiting the chemical ecology of mosquito oviposition behavior in mosquito surveillance and control: a review

Vector control is an important component of the interventions aimed at mosquito-borne disease control. Current and future mosquito control strategies are likely to rely largely on the understanding of the behavior of the vector, by exploiting mosquito biology and behavior, while using cost-effective, carefully timed larvicidal and high-impact, low-volume adulticidal applications. Here we review the knowledge on the ecology of mosquito oviposition behavior with emphasis on the potential role of infochemicals in surveillance and control of mosquito-borne diseases. A search of PubMed, Embase, Web of Science, Global Health Archive, and Google Scholar databases was conducted using the keywords mosquito, infochemical, pheromone, kairomone, allomone, synomone, apneumone, attractant, host-seeking, and oviposition. Articles in English from 1974 to 2019 were reviewed to gain comprehensive understanding of current knowledge on infochemicals in mosquito resource-searching behavior. Oviposition of many mosquito species is mediated by infochemicals that comprise pheromones, kairomones, synomones, allomones, and apneumones. The novel putative infochemicals that mediate oviposition in the mosquito subfamilies Anophelinae and Culicinae were identified. The role of infochemicals in surveillance and control of these and other mosquito tribes is discussed with respect to origin of the chemical cues and how these affect gravid mosquitoes. Oviposition attractants and deterrents can potentially be used for manipulation of mosquito behavior by making protected resources unsuitable for mosquitoes (push) while luring them towards attractive sources (pull). In this review, strategies of targeting breeding sites with environmentally friendly larvicides with the aim to develop appropriate trap-and-kill techniques are discussed

The Influence of Larval Stage and Density on Oviposition Site-Selection Behavior of the Afrotropical Malaria Mosquito Anopheles coluzzii (Diptera: Culicidae)

In the selection of oviposition sites female mosquitoes use various cues to assess site quality to optimize survival of progeny. The presence of conspecific larvae influences this process. Interactive effects of oviposition site selection were studied in the malaria mosquito Anopheles coluzzii Coetzee & Wilkerson in dual- and no-choice assays, by exposing single gravid mosquitoes to oviposition cups containing 1) larvae of different developmental stages, 2) larvae-conditioned water (LCW), and 3) cups where visual cues of conspecific larvae were absent. Early-stage conspecific larvae had a positive effect on the oviposition response. By contrast, late stages of conspecific larvae had a negative effect. Oviposition choice was dependent on larval density. Moreover, in oviposition cups where larvae were hidden from view, late-stage larvae had a significant negative effect on oviposition suggesting the involvement of olfactory cues. LCW had no effect on oviposition response, indicating involvement of chemicals produced by larvae in vivo. It is concluded that the presence of larvae in a breeding site affects the oviposition response depending on the development stage of the larvae. These responses appear to be mediated by olfactory cues emitted by the larval habitat containing live larvae, resulting in the enhanced reproductive fitness of the females