A study of residual bio-efficacy of Inesfly 5AIGR and Inesfly 5AIGRNG insecticidal paints on Adult Anopheles gambiae complex mosquitoes on treated wall surfaces in Nigeria Communities: Masaka and Gidan-Zakara CURRENT STATUS: UNDER

Background Since insecticides are commonly used in agriculture/cultivation and in vector control, they are frequently found in agricultural water bodies, where mosquito larvae are exposed. Although their concentration is often so low as not to kill the larvae, they affect the development of the mosquitoes. In particular, their effects may be transmitted to adults to influence their characteristics of life-history and their vectorial competence for arboviruses infection and malaria. Such effects on vectorial competence of sub-lethal doses of insecticides are likely to be related to their impact on immune response. Insecticide exposure influences the immune response of insects in several ways. Organophosphates and organochlorines affect the number of hemocytes (for phagocytosis) However, it is not known whether larvae exposure will impact the immune response of adults. Method Assessment of the bio-efficacy and residual activities of insecticides sprayed, wall surface using CDC wall cone bioassay test. Entomological indices with Anopheles species collection was measured to determined vectorial involvement and residual bio-efficacy of of Inesfly 5AIGR/ Inesfly 5AIGRNG insecticidal paints on Adult Anopheles gambiae complex mosquitoes on treated wall surfaces in at both communities (Masaka and Gidan Zakara) from January to April and May through June, 2018. A bio-assay cone test was carried out at various wall surfaces/height treated with above named insecticidal paints at parameters of 0.5meter, 1meter, and 1.5meter respectively. The study investigated and evaluated the efficacy of insecticide paint using wall cone bioassay test from January through April, 2018 at Masaka and Gidan Zakara. The results showed a 100% efficacy of the Inesfly 5AIGR in reference to the wall parameter at 0.5m, l.0m, and 1.5m cone bioassay applied in houses sampled during the study period Anopheles were the most


Conclusion
The malaria vectorial competence of Anopheles species were 98% -100% susceptible to residual '1NESFLY' paints (5AIGR and 5AIGRNG) at various wall parameters of 0.5meters, 1meter and 1.5meters in the studied communities in central Nigetia. It is also imperative to state that the trial paints were in compliance with animals and human tolerance levels/standards during the during the trial periods, and fulfils the WHO criteria of insecticidal bio-efficacy (mortality >80%).

Background
Mosquito vectors ' immune system underlies their resistance to infections, and thus their capacity to spread pathogens to humans [1,2]. Although their immune response intensity has a strong genetic component [3], it's also affected by the climate. The male Anopheles gambiae if undernourished as larvae, have a less successful melanisation response [4], correspondingly, environmental effects on the ability to transmit pathogens. The vectorial competence of mosquitoes is influenced by the bacterial microbiota the mosquitoes acquire as larvae [5], and that the susceptibility of Aedes mosquitoes to arboviruses and of Anopheles mosquitoes to malaria parasites depend on the temperature and food conditions during larval development [6,7,8] The important aspect of the ecosystem is the use of insecticides. Since insecticides are commonly used in agriculture/cultivation and in vector control, they are frequently found in agricultural water bodies [9], where mosquito larvae are exposed. Although their concentration is often so low as not to kill the larvae, they affect the development of the mosquitoes. In particular, their effects may be transmitted to adults to influence their characteristics of life-history [10,11,12,13] and their vector competence for arboviruses infection [14,15] and malaria [16,17].
Such effects on vectorial competence of sub-lethal doses of insecticides are likely to be related to their impact on immune response [18]. Insecticide exposure influences the immune response of insects in several ways [18]. Organophosphates and organochlorines [19] affect the number of hemocytes (for phagocytosis) However, it is not known whether larvae exposure will impact the immune response of adults [20].
Malaria remains one of the most important tropical diseases [21,22]. It is estimated that 352 to 450 million people still lived in malaria endemic areas where there is little or no control of the disease [23]. Malaria has been eradicated from the USA, northern Australia, Israel, Cyrus and few other tropical Islands [24]. It was however, noticed in 1969 by the world health organization that the eradication was unrealistic [25].
The world health organization (WHO) declared that the main objective should be to control malaria and possibly reducing it to a point where it no longer constitute a major public health problem [26].
Malaria is widespread and common in sub-Sahara Africa. From the forgoing, it has become very imperative to implement control strategies that are effective in controlling the disease. In this study we evaluating the performance of wall cone bioassay code name Inesfly 5AIGR/ Inesfly 5AIGRNG insecticide paints for residual treatment against Anopheles mosquitoes infested (Malaria Endemic communities) areas in some selected local government area of Nasarawa State, Nigeria.

Study area
The trial of wall cone bioassay was carried out at two different locations, Masaka and Gidan Zakara communities of Keffi and Karu local government area of Nasarawa State in the north-central, Nigeria.
Keffi is about 53km away from the Federal Capital Territory, Abuja and is located on longitude 8050' (South) and latitude 7050' (North) and is 630m above sea level.

Sampling of Houses
Ten (10) houses were randomly selected at Masaka and Gidan Zakara for the trials, the houses were painted with lnesfly 5AIGR (Chlorpyrifos 1.5%, dsiazinon 1.5% and Pyriproxyfen 0.063%) and Inesfly 5AIGRNG (Alphacypermethrin 0.7%, d-allethrin 1.0%, and Pyriproxyfen 0.063%) paints on the plastered walls. Pyrethrum spray collection (PSC) of mosquitoes was conducted randomly before the paints were applied at both locations for a period of six (6) months. The procedure was conducted in order to determine the residual efficacy of the insecticide embedded admixture against the wild mosquitoes.

Mosquitoes Larval Sourcing
Mosquito larvae were sampled out every three (3) days within a 6 months period at both locations [Masaka and Gidan Zakara]. Anopheles gambiae mosquitoes larvae were reared to adult stage in the insectary laboratory of Nasarawa State University, Keffi.
Two (2) to three (3) days old non-blood fed female mosquitoes were used in the field for the cone bioassay testing on the painted walls.

Procedure
The twenty (20) houses were randomly selected in each sentinel site, PSC method was applied as described by the WHO (1975) to sample indoor-resting mosquitoes. The houses were sampled by two people, one inside, and the other one outside the confinement of the room using an aero 01 insecticide (Raid) containing the active ingredients of 0.250% Allethrin, 0-150% tetramethrin, 0.015% deltamethrin, and 99.5 85% inert ingredients.
Two sprayers sprayed the room in opposite directions with all the doors closed for 15 minutes; the technicians later entered and collected the mosquitoes that were knocked down from on a white piece of cloth which was spread on the floor prior to spraying.
Mosquitoes were collected using a pair of forceps and they were then placed in petri dishes containing damp filter paper. The anopheles mosquitoes were then kept on an absorbent paper in a cool box. The mosquitoe species were later identified and counted according to (Gilles and Medellin, B. 1968, Gilles and Coetzee, 1987., Kent, 2006).

Residual Activity of Insecticide
The evaluation assessment of the bio-efficacy and residual activities of 'Inesfly' insecticide paint on the walls were carried out from January through June, 2018 to ascertain the mortality of the exposed wild mosquitoes from the breeding sites within a time frame. The bio-efficacy as well as the decay rate of the insecticide paints on walls was measured using standard WHO cone test in the 20 randomly selected houses. The unpainted houses were used as controls. The cone test commences 24 hours post painting and it was carried out continually on a monthly bases for a period of six (6) months. The monitoring and evaluation of the insecticide paint residual efficacy was then determined.

Wall Cone Bioassay Test
Three cones were fixed using masking tapes on the interior walls at varied parameters, the lower point of 0.5m, middle point at 1.0m and the upper point at 1.5m. Three to five days-old unfed females of An. gambiae s.l. from the wild were used for the test as ten mosquitoes were gently transferred into each cone by an aspirator and exposed for 30 minutes and then observed for a period of 60 minutes. At the end of exposure time, the mosquitoes were transferred into insecticide free holding paper cups for further observation of 60 minutes and 24hrs holding period. The mosquitoes were fed with 10% sugar solution at the insectary laboratory.
The paper cups were kept in a cooling box and covered by damp towel to create favorable temperature and humidity in the insectary laboratory. Mortality was observed 24 hours post exposure and mosquitoes classified as dead if they are immobile or unable to stand or fly in coordinated manner or direction.

Results
Residual efficacy using cone bioassay test on Inesfly 5AIGR and Inesfly 5AIGRNG paints.
The residual efficacy of inesfly paints using wall cone bioassay was carried out to affirm the durability and efficacy of the paint applied to houses at Masaka and Gidan Zakara (Communities) both in keffi and Karu local government areas of Nasarawa state, Nigeria.
Monthly, the cone bioassay test were carried out on the wall parameters, mounted on the wall (0.5meters; 1.0meters; and 1 .5meters) respectively. At 0.5m 1.0m and 1 .5meters the cone bioassay test knockdown (KD) mortality status [For January] after 24hours showed a residual efficacy of 100% in the three households (Table 1).
In February through April, 2018, a 100% knockdown/mortality of mosquitoes after 24hours was comparably observed across same parameters heights. However in May, 2018, 0.5meters had 90% knockdown in household I, follow by household II (97%) mortality rate and household III 98% mortality rate after 24hours. In June, 0.5meters in household 1, showed a 90% knockdown while household II and household III showed a knockdown mortality of 97% and (96%) respectively. Household I and II at I.0meters showed a knockdown morality of 94% as against house III where 99% knockdown mortality of mosquitoes was chronicled. In a Similar manner, at 1.5meters a knockdown mortality rate of 98% was observed in household I and II, while household III showed a 93% mortality on the malaria vector (Table 1, Figure 1).  Figure 2).

Residual efficacy of Inesfly 5AIGR and Inesfly 5AIGRNG insecticide paint
The study investigated and evaluated the efficacy of insecticide paint using wall cone bioassay test from January through April, 2018 at Masaka and Gidan Zakara. The results showed a 100% efficacy of the Inesfly 5AIGR in reference to the wall parameter at 0.5m, l.0m, and 1.5m cone bioassay applied in houses sampled during the study period (Table 1 and Figure 1). Anopheles gambiae s.l were the most observed species as well as the most collected indoors compared to other species of mosquitoes this is in consonance with the results of Killeen et al., (2001) who reported similar cases of An. gambiae s.l that are typically endophagic, these trait appears to vary between locations. It was also observed during the study that An. gambiaense were more endophagic from the January periods through April (dry-season). This is in contrast to the wet season periods of May and June, 2018, where An. gambiae s.l. enter homes/houses in the early hours and exit in the morning hours.
[ Table 1]. The above findings shows the behavioral pattern of the female mosquitoes with respect to feeding activities that

Conclusion
The malaria vectorial competence of Anopheles species were 98% -100% susceptible to residual '1NESFLY' paints (5AIGR and 5AIGRNG) at various wall parameters of 0.5meters, 1meter and 1.5meters in the studied communities in central Nigetia. It is also imperative to state that the trial paints were in compliance with animals and human tolerance levels/standards during the during the trial periods, and fulfils the WHO criteria of insecticidal bio-efficacy (mortality >80%). The lack of malaria vector control and elimination(s) is recumbent in hands of various governments of the world as different vectors strains are emerging and re-emerging as a result of climate change. Human activities are contributing enormously to this change and if these trends are not scrutinized most especially in Sub-Saharan Africa, then the disputation and swedge against the control and elimination(s) of malaria will remain a mirage.

Ethical Approval and Consent to participate
Not applicable.

Consent for publication
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Availability of supporting data
Data is not restricted   Estimated frequency of the susceptibility and residual bio-efficacy of Inesfly 5IAGR &5IAGRNG insecticidal paints at Gidan-Zakara community against the malaria vector.