A Study of the Susceptibility of Anopheles species to Pyrethroids on Long Lasting Insecticide Treated Nets (LLINs) and Ownership and Usage of LLINs in Some Communities in Obi Local Government Area the Susceptibility of Anopheles speciesto Pyrethroids on Long Lasting Insecticide Treated and

The use of Long-Lasting Insecticide-treated Nets (LLINs) for Malaria vector control depends entirely on the continued susceptibility of Anopheles mosquitoes to pyrethroid Insecticides. This research is aimed at comparing the susceptibility of Anopheles species to pyrethroids on Long Lasting Insecticide-treated Nets in Okpokwu, Adiko and Obarike communities of Obi Benue State, Nigeria. LLINs of ages 0 (control nets), 1, 2, 3, 4, years were collected from each of the three communities. Anopheles larvae were also collected from the three communities, reared to adulthood from April-June 2015. Emerged adult female mosquitoes were tested for susceptibility to pyrethroids on each of the net ages collected. The mosquito in total were susceptible to pyrethroids on LLINs, with a mortality rate of 98.5%. The highest mortality, 100% was recorded in Adiko community, 98% mortality in Obarike community and the lowest, 97.6% mortality in Okpokwu community. The relationship between the mortality of mosquitoes and the different study communities was not significant. Mosquito samples were susceptible to net ages 0, 1 and 2 years with mortality rates of 100% each; 98% mortality with net age 3 years and 94% mortality with net age 4 years. The relationship between the mortality of mosquitoes and the different net ages was significant (P < 0.05). Out of 316 individuals that responded to questionnaires, a total of 279 (88.3%) owned LLINs out of which only 177 (63.4%) used LLINs previous night before this study. There was a significant relationship between the ownership and utilisation of LLINs with respect to the communities (r = 0.99), occupation (r = 0.94), age (r = 0.99) and gender (r = 1) of the respondents. This study concluded that mosquito populations from Obi LGA are susceptible to pyrethroids on LLINs indicating that LLINs are veritable tools for malaria control.

97% of Nigeria's population. The remaining 3% of the population live in the malaria free highlands [3]. There are an estimated 100 million malaria cases with over 300,000 deaths per year in Nigeria.
Malaria is the commonest cause of outpatient attendance across all age groups with about 66% of clinic attendance thus constituting a huge burden on the economy of Nigeria [4]. Nigeria accounted for 27% of malaria cases and 24% of malaria deaths globally in 2016 [5]. Anopheles gambiae is the primary mosquito vector responsible for transmission of malaria in most of sub-Saharan Africa [6,7].
Malaria vector control programmes in Africa rely heavily on the use of pesticides for insecticide-treated nets (ITNs)/long-lasting insecticide-treated nets (LLINs) and for indoor residual spraying (IRS) [8]. The use of these strategies is known to contribute to the reduction in malaria transmission [9,10]. The effectiveness of the current vector control depends much on the susceptibility of the local malaria vectors to insecticides used [8]. The emergence of resistance of these vectors to this insecticides have become one of the greatest challenges to these strategies and to the vision of future eradication of malaria infection as well as a threat to the global health of populations especially in tropical and subtropical regions of the world.

Study Area
The study was carried out in Obi Local Government Area (LGA) of Benue State between the months of April to June 2015. Obi LGA is located between latitude 6 0 56" and 7 0 10" N and longitude 8 0 10" and 8 0 20" E, covering an expanse area of about 516km 2 and situated at the South-Central part of Benue State. By the year 2015, the population of Obi is estimated to be 128075 with 3% growth rate [11]. Adiko, Okpowu-ito and Obarike were the communities in Obi L.G.A. that were used for the study. Obi Local Government Area lies in the Southern Guinea Savannah biome which is a transitional zone between the Northern Sudan Savannah and the forest belt of the South with its characteristic coarse grasses and numerous species of scattered trees widely developed as park-land.
The climate of the area falls within the tropical wet and dry climatic belt designated "AW". The climate can be described as tropical sub-humid type with two distinct seasons: wet and dry seasons. Rainfall averages 8 months annually between April and November with totals ranging between 1200-2000mm, though subject to seasonal variation which may lead to either earlier or later rains, in 2015 rain began in the month of February. The annual dry season in the area lasts four to five months. Temperature is relatively high over the area throughout the year averaging 28 0 C -32 0 C with an occasional peak at 37 0 C between March and April. This is combined with an average relative humidity (RH) of over 80% in the morning and falls to between 50% and 70% in the afternoon.
The people of Obi L.G.A. speak Igede and are predominantly farmers with maize, cassava, rice, groundnuts, and yams the main farm produce (Figures 1,2).

Ethical Consent
A letter of introduction was obtained from the Department of Biological Sciences, Benue State University, Makurdi for the purpose of the research. Permission was obtained from the Community Heads of the three communities where the study was carried out.
Volunteered participants were briefed on the relevance of the research, after which they orally indicated their consent.

Collection of LLINs
A total of 15 LLINs were used in this study. Five LLINs of different ages 0, 1yr, 2yrs 3yrs and 4yrs were randomly collected from different households in each of the three different communities.
Age 0 nets were unused nets that served as the control nets while the rest of the nets were being used in the communities. These nets were replaced by new ones for each one that was collected.

Net Cages
Three cages were built (16 x 16 inches). One for vector samples from each of the communities.

Larval Collection and Rearing of Anopheles Species
Field Anopheles larval collection was done in natural larval breeding sites in the three selected communities between the months of April and June 2015. Anopheles larvae were identified based on their position on water which was parallel to water surface while the pupae are comma-shaped when viewed from the side. Collection was done using a collecting spoon. Care was taken in other not to disturb the larvae. The larvae and pupae were collected with breeding site water and transferred into plastic containers that were well labelled with the names of the three different communities. The plastic containers were uncovered to allow air so that the larvae and pupae can breathe while they were being transported to the laboratory. Yeast/liver powder which served as feed to the larvae and pupae was spread on the surface of the plastic containers. This was done moderately to avoid pollution of water and mortality. The water in the container was monitored regularly and as soon as it is noticed to be polluted it was changed to avoid high mortality. Pieces of cotton wools soaked in sugar solution and gently squeezed to drain out excess solution was placed in the cage at various location the sugar solution served as feed to the adult stage that emerged [12][13][14].

Sorting of Mosquitoes
Sorting out of Adult female Anopheles species from the males was done by simple observation using major differentiating morphological characteristics according to Gilles and Coetzee [6] such as the palps not swollen at the end and the antennae nonplumose for the females while the for the males the palps are swollen and the antennae plumose in nature. The females were sucked out using an aspirator.

Bioassay
Cone-test: The knock down (KD) effects of pyrethroids on the nets collected were tested using test kits (WHO cone) and standard procedures as recommended by the World Health Organization/ World Health Organization Pesticide Evaluation Schemes [12][13][14]. Pieces of nets 25cm by 25cm were carefully cut out from the old nets of different ages and firmly placed on the wall in the laboratory. The cone was placed on the nets to be tested; adhesive sponge tapes were used to fix the cones to the wall. Ten batches each of 2-3 days old non-blood fed laboratory bred Adult female Anopheles gambiae mosquitoes were aspirated and transferred into cones. After exposing the mosquitoes for 3 minutes, they were carefully taken out of the cones and transferred to empty cage for observation. The knock down effect of pyrethroids on each of the net age was recorded at 10 minutes interval over a One-hour exposure period. The final mortality was recorded after 24 hours.
The experiment was replicated five times for each of the net age collected from households using the same number of mosquitoes.
Controls included batches of mosquitoes from each site exposed to unused nets (net age 0). The Bioassays was carried out at room temperature. The average knockdown, percentage knockdown and percentage mortality were calculated after the experiment.

Knock-down/Mortality
Integrated vector management [13] manual and video was used in estimating the knock-down and mortality of the Anopheles mosquitoes.

Statistical Analysis
Difference in average mortality between the selected communities, across ages of nets [14,15] and criteria for efficacy of at least >80% mortality or >90% knockdown used to evaluate the resistance/susceptibility status of the mosquitoes tested. Chisquare test and Analysis of Variance were used in analysing the result while Correlation was used in the analysis of questionnaire findings.

Mosquito Population
Anopheles gambiae larvae and pupae were found in large populations in the three communities. They were collected more from pools, temporary streams, rice fields and abandoned wells.
The mosquitoes were reared in the laboratory between the months of April and June 2015 using Insect cages (16 x 16) inches and Plastic containers. Table 1 shows the overall knockdown of mosquitoes across the communities to be 671 (89.5%). Adiko community had the highest knockdown of 232 (92.8%) while Okpokwu community had the lowest knockdown of 208 (83.2%). There was a significant relationship between locality and knockdown (χ 2 calculated = 15.648, df = 2, P < 0.05). Table 2 shows that the total number of mortality (%) of mosquitoes recorded across the different communities was 739 (98.5%). The highest mortality of (100%) was recorded in Adiko community, while the lowest (97.6%) was at Okpokwu community. The relationship between the mortality of mosquitoes and communities was not significant (χ 2 calculated = 5.720, df = 2, P > 0.05). Table 3 shows the knockdown of mosquitoes across the different net ages. The highest (100%) knockdown was recorded with age 0 nets, while the least (68.7%) knockdown was recorded with age 4 years. There was a significant relationship between the knockdown of mosquitoes and the different ages of the nets that were used for the study (χ 2 calculated = 105.180, df = 4, P < 0.05). Table 4. shows the mortality (%) of mosquitoes across the different net ages. for net ages 0. 1 and 2 years 100% mortality was recorded while 98.7% and 94.0% mortality were recorded for net age 3 and 4 years, respectively. The relationship between mortality of mosquitoes and net ages used for the study was significant (χ 2 calculated 28.048, df = 4, P < 0.05).

Practices of LLINs
Findings on the ownership, utilisation and handling practices of LLINs of 316 respondents to administered questionnaires.
The overall ownership and utilisation of LLINs across different communities as recorded in Table 5 Table 6 shows that a higher ownership and utilisation of LLINs    Table 8. There was a significant relationship between the ownership and utilisation of LLINs with respect to the age of the respondents (r = 0.99, n = 7, P = 0.001). Table 9 shows       (χ2 calculated = 45.319, df = 10, P < 0.05) Figure 3 shows that majority of the respondents that own LLINs got them from the free distribution campaign number 303 (96%) while only 13 (4%) purchased theirs. Information on Figure   4 shows that the major reason why some respondents did not own LLINs was that they sought for nets but were not given, while lack of knowledge of LLINs was not a reason for non-ownership of nets as all the respondents knew about LLINs. Figure 5 shows that the major reasons why the respondents owned nets but did not use them were because of the feeling of heat and discomfort while a few 2 (2.0) and 3 (2.9) complained of suffocation and ineffectiveness.

Discussion
World Health Organisation [15] recommendations for testing susceptibility/resistance states that 98-100% mosquito mortality indicates susceptibility, 80-97% suggest potential resistance that needs to be confirmed, and < 80% mortality suggests resistance.
Based on these criteria, the present study overall, showed a full susceptibility of Anopheles gambiae to pyrethroids on LLINs, (98.50%) which was being used in Obi LGA Area of Benue State. This is like some earlier reports from other parts of the country [16]. However, this study contrasts with studies which reported widespread resistance to pyrethroids in the African malaria vectors [17][18][19][20][21]. The differences in the levels of insecticide susceptibility/ resistance have been explained as probably a reflection of differential selection pressure exerted on field mosquito populations. The primary source of this selection pressure is still unclear although evidence is accumulating to incriminate the agricultural use of insecticides in selection for resistance in malaria vectors [22]. Owing to this explanation the susceptibility but not resistance reported in this study could be attributed to the fact that agricultural activities in Obi LGA are subsistent, hence the use of insecticide have not been extensive enough to cause resistance in malaria vectors.
In this study the community-based report showed a slightly reduced susceptibility of mosquitoes to pyrethroids on LLINs in With respect to the occupation of the respondents the higher percentage of ownership recorded among farmers could be attributed to the fact that farmers earn less income than civil servants and hence may take more advantage of the free distribution campaign to save cost: Also the higher percentage of utilisation reported amongst civil servants than farmers could be explained as a reason that civil servants that have resorted to using LLINs may be more aware of the importance of using LLINs for malaria control, while the farmers who recorded the lowest percentage of utilisation may out of negligence or other behaviours like forgetfulness not use their Nets. There was a significant relationship between the ownership and utilisation of LLINs with respect to the community, Gender, Occupation and Age of respondents (P < 0.05).
The present study established that majority of the respondents 166 (59.5%) that owned LLINs have nets that have lasted 1 > 2 years and also majority of the respondents that owned LLINs have never washed them 151 (54.1%). These facts could account for the reason why mosquitoes exposed to them were highly susceptible, and this complies with of World Health Organization in 2007 [26] recommendations that LLINs should only be washed for at least 20 times during its lasting periods.

Conclusion
Reliability on LLINs, as effective control methods for malaria infection, depends on the continued susceptibility of Anopheles gambiae to pyrethroids. This study reveals that mosquito samples from Obi LGA are susceptible to pyrethroids on LLINs, despite the use of insecticides for pest control in agricultural activities.
This study also reveals that LLINs are the major tools used for the control of malaria in the study area for pest control in agricultural activities. This study also reveals that LLINs are the major tools used for the control of malaria in the study area.