Phenotypic Physiognomies and Malaria Vectors KDT to Certain Insecticides in Some Populations of Anambra State, Southeast Nigeria

Emmanuel Okwudili Ogbuei¹ , Dennis Nnanna Aribodor¹ , David Ekene Nwele ^2,3 , Chibumma I. Nzeukwu¹ , Uzochukwu C. Uzochukwu ¹ , Ifeanyi Emmanuel Obiefule ¹ , Obiamaka Vivian Emma-Ogbuei^4,5

¹Department of Parasitology and Entomology, Faculty of Biosciences, Nnamdi Azikiwe University Awka, Anambra State, Nigeria

²Department of Applied Biology, Ebonyi State University AbakilikiEbonyi State, Nigeria

³Department of Biology/Biotechnology, David Umahi Federal University of Health Sciences Uburu Ebonyi State, Nigeria

⁴Department of Medical Services, NnamdiAzikiwe University Awka, Anambra State Nigeria

⁵Department of Family Medicine, Nnamdi Azikiwe University Teaching Hospital Nnewi, Anambra State Nigeria

Corresponding Author Email: oe.ogbuefi@unizik.edu.ng

DOI : https://doi.org/10.51470/ABF.2025.4.1.01

Abstract

Keywords

Download this article as:

Introduction:

The malaria issues in Nigeria particularly and Africa at large has triggeredterrific health dares to the general public where their vectors transmits diseases to humans especially pregnant women and children which are the most susceptible group because of their little or no resistance to the parasitic disease [1].In the combat to both manage and except malaria and its resultantvectors,some groups of insecticides such as organochloride, organophosphate, carbamate, and pyrethroidhave been ratified for Indoor Residual Spray (IRS),nonetheless only pyrethroids are currentlycertified in Insecticide Treated Nets (ITNs) usage. Thereason for this was because of their squat mammalian poisonousness and extraordinary insecticidal effectivenessas reported by [2]. Over time, this presence of resistance has triggered series of insecticide changes, ranging from DDT to pirimiphosmethyl, lambda-cyhalothrin, dieldrin, malathion, propoxurand deltamethrin to the current time [3]. It is on record that malaria vector-human contact has been reduced by the availability and usage ofITNs and IRS [4], nevertheless, insects have development both behavioral change and physiological resistanceleading to selection pressure [5]. In all of these Anopheles gambiaes.l. and Anopheles funestuss.l. which are the major two African malaria vector groups most effectively embattled by ITNs have equallywidespread insecticide resistance. Insecticide resistanceincorporateseither physiological, biochemical, molecular and behavioral mechanismsas reported by [6]. Physiological resistance and other multiple mechanisms like the one which are caused by knockdown resistance (kdr)mutations in the para-type sodium channel gene are ways through which mosquitoes evade pyrethroids and other insecticides[7]. Development of resistance to both DDT and pyrethroids have been established in Nigeriaand other Afro-tropical countries of the world owing to that reason,this would definitely have substantialconsequences for the success of vector survilliance, monitoring and other interventionsthat is currently ongoing[8]. Hence, the stout need that field populations of Anopheline mosquitoes be monitored by the development of suitableapparatuses. This strategy will definitely benefit the target populaceby eliminating malaria in the environment. Regretably, the control of malaria vectors will always be dragged behind by resistance to insecticides and there is urgent need to improve understanding of the underlying mechanisms of resistance to manage and curtail the situation [9]. The fight to ascertain resistance genes and DNA makers  have been subdued by target-site resistance andbehavioral changes or thickening of the cuticle. Whereas candidate gene and quantitative trait locus studies to solve this problem is not only anticipated but achiavable. AlthoughDDT and pyrethroids which are insecticides commonly used for malaria controlshare a common target site [10],but then again knockdown resistance (kdr)mutations in this para voltage-gated sodium channel can consequently confer cross-resistance to both pyrethroids, DDT and any other insecticides on that particular groupas observed by [11 and 12].The enhancement of monitoring and programmatic policymaking in Anopheles mosquito studies can make a positive swing toward the potential identification of resistance markers [13].Regardless of various malaria interpositionswhich include but not limited to the following;IRS, distributions of LLINs, and prompt and active treatment with antimalarialswhich have been rolledout by the World Health Organization in its functioningregions, malaria still remains a leading challenge in the health sector which requires uninterrupted studies in the struggle to both manage and eradicate the disease and its concurrent vectors. This is urgent and critical especially now that we are close to the pre-elimination stage of the disease.

Materials and methods

Study area:

Malaria vectors investigation was undertaken inthree local government areas of Central Senatorial Zone of Anambra State, Southeast Nigeria namely: Akamanator and Amaezikecommunities of Mgbakwu in Awka North L.G.A., Umuanumand Ezeawulu communities of Nibo in Awka South L.G.A., and then Ifiteand UmuAgidicommunities of EnugwuAgidi in Njikoka L.G.A. The State is located between Latitude 6 ̊ 12’45.68’’N to Latitude 6 ̊ 22’45.68’’N and Longitude 7° 04’19.16’’E to Longitude 7° 15’19.16’’E of Greenwich. It has an average daily temperature of 26.8 °C / 80.2 °F as recorded by[14]. Still,AwkaSouth LGA is located between Latitutude 6 ̊ 10’ North to Latatitude 6 ̊ 23’ N of the equator and Longitude 7° 04’ E to 7° 44’ E of Greenwich, Awka North is located between Latitude 6° 15’ N to 6° 35’ N of equator and Longitude 7°10’ E to 7°30’ E of Greenwich while Njikoka LGA is lying between Latitude 06° 20’ 58 ’’ N to 06° 21’ 00’’ N and Longitude 06° 12’ 55’’E to 06° 52’ 55’’E of Greenwich. The predictable population of 2020 forAnambra State is 6,182,924 as postulated by [15].Anambra State experiences a rainy season which falls between (April and October) and the dry season which cascadesbetween (November and March) as itsseasonal climatic condition. The areas experience a petite spell of harmattan between November and January when the atmosphere is generally mistywhich is a period of cold weather [16].The State has 11,450mm and above as its total annual rainfall for the six to seven months of the rainy season although the mean annual temperature ranges between 30.0–36.0 °Cas reported by [14]. Even though the English language is widely enunciated throughout the State as a secondary language, the indigenous Anambra State people are Igbo-speaking folks as their official language[14]. They cultivateyams, cassava, corn (maize), palm oil and kernelsand other agricultural productswhich are both consumed andtraded. This is possible because the soil in the State with its good vegetation sustains agricultural activities as reported by [14]. The current research covered the two main seasons of the year and was undertaken between September 2021 to August 2022 in Anambra State Southeast Nigeria.

Study design:

Thestudy design used for the current research was a completely randomized design (CRD) which comprised four treatments each of which was replicated four times with a control treatment on the laboratory bench. Each replicate was tagged as T1, T2, T3, and T4 whereas the control was tagged C1 and C2.

Mosquito larvae collection:

The collection of Larval were implementedon a monthly basis for three successive days from 9:00am to 12:00pm for twelve months of the year, it covered both rainy and dry seasons. Diverse water bodies were critically examined likecatchment pits, gutters, puddles,tyres, streams and river bodies, tyre tracks, containers, crab holes,excavations, hoof printsand rice fields formalaria vectors. To ensure that each site in the areas were explored in the course of sourcing for mosquito larvae, ladles and pipettes were used during the study.  Uneven debris that was collected alongside the larvae during the survey like sticks and plant leaves were carefully handpicked and thrown away. Amesh size sieve of 0.55mm was engaged during the study to distinguish the larvae from other debris. The reason for this usage was to ensure a comprehensive collection of mosquito populations of all possible vectors breeding in the study areas. This was achieved by embarking on wide-ranging larval sampling at the course of the investigation as described by[17].

Rearing of mosquito larvae:

A labelled container provided was used to keep all mosquito larvae collected. This was retained in cork plastic containersand labeled according to the site of collection. The time anddate of the collection were also noted before they were reared to adult stages. A provision of yeast in 500 ml larval bowls was made which was covered with a transparent netwasused to fedthe larvae. When the adult emerged, an aspirator was used toassigned the mosquitoes to cages. These newly emerged adults were fed with a 10% sugar solution saturated in cotton wool which was dropped on top of the mosquitoes cage.

World Health Organization (WHO) tube insecticide resistance bioassay tests:

Insecticide susceptibilityresistance bioassay tests were carried out using WHOimpregnated/control papers test-kits and standard techniques that were delivered by UniversitiSains Malaysia (USM), Penang, Malaysia. As described by[18], four (4) replicates of 3-5 day-old 25 non-blood-fed adult female mosquitoes were used for the study. The assays were carried out in consignments by exposing the adults of An. gambiaesfrom all sites in the study areas to impregnated papers which were achieved with a recommended concentration of a given insecticide in the assay tubes. Although for the control, paper without insecticide that was treated with paraffin oil was used. 0.75% Permethrin, 0.05% Deltamethrin, 0.1% Bendiocarb and 5% Malathion were the following insecticides that was used for the survey. Each replicate of 25 mosquitoes in each tube were exposed to the insecticide-impregnated filter paper for the total duration of time recommended. A total of 150 female non-blood-fed mosquitoes were exposed for each insecticide (using four replicates). (100 mosquitoes for the test tubes and 50 mosquitoes for the controls, using two replicates of 25 mosquitoes each). As the exposure period lasts, the number of mosquitoes knocked down were recorded after 10, 15, 20, 30, 40, 50, 60 minutes and 24 hours subsequently. After the experimental time lapses, the mosquitoes were subsequentlytransferred into the holding tube and fed with glucose solution via a pad of cotton wool soaked in 10% glucose solution. This solution is placed on the mesh-screen end of the holding tubes while the time taken to achieve 50%, 90%, and 95% of population knockdown (KDT_50, KDT₉₀ andKDT₉₅) were taken. Further evaluation was done using log-probit analysis. The mortality rate was determined after 24 hours post-exposure by counting the number of dead and alive mosquitoes on each tube. For an adult mosquito to be considered to be alive, it must be able to fly willy-nilly of the number of legs remaining. Whereas any otherknocked-down mosquito, whether or not it has lost a leg or wing, was considered and counted as dead. The condition for mosquitoes to be classified as either dead or knocked down was that they were immobile or even unable to stand or finally take off.

NB: Abbott’s formula was used in the study to precise the observed mortality when the mortality in the control is sandwiched between 5–20% as described by[19].

Analysis of data:

Recordsacquired from the survey were precised and scrutinized using comparative and descriptive statistics. Also to compare the differences between means, one-way analysis of variance (ANOVA) was usedwhereas the least significant difference (LSD) test wasused to compare the level of significance at 0.05% level of significance. Abbot Formula was used for the correction of mortality while Log-probit regression analysis was used to determinethe KDT_50, KDT₉₀ and KDT₉₅.

Agarose gel electrophoresis species identification for Anopheles gambiae s. l. complex.

(A):Lane 1 standsfor ladder of DNA whereas Lanes 18,12,8,3and 2 were products of An. coluzziiand then Lanes 17, 16, 15, 14, 11, 10, 9, 7, 6, 5 and 4 were products of An. gambiaewhereas Lanes 20, 19 and 13 were unamplified and thus unidentified.

(B): The Lane 1 stands for ladder ofDNA. Lanes 16, 11, 7and 5 were products of An. coluzziiwhile Lanes 15, 14, 13, 12, 10, 9and 8 were products of An. gambiaewhereas Lanes 20, 19, 18, 17, 6, 4, 3 and 2 were unamplified and thus unidentified as shown in Plate 1.

The Mean percentage knockdown time of An. gambiaes. l. that was exposed to different insecticides at varying time intervals in Awka South Local Government Areaof Anambra State

The Mean percentage knockdown time of An. gambiae s. l.that was exposed to different insecticides at varying time intervals in Awka North Local Government Areaof Anambra State

In Awka North LGA, the KDT₅₀time taken for 50% of the tested mosquitoes to be knockeddown,KDT₉₀time taken for 90% of the mosquitoes tested to be knockeddown, and KDT₉₅time taken for 95% of the mosquitoes tested to be knockeddown. Means of (4) Four Replicates (±s.e), df=3, Pv=0.002, Means of Eight (8) replicates (±s.e), df=7, Pv=0.106 as shown in

Mean percentage knockdown time of An. gambiae s. l. exposed to different insecticides at varying time intervals in NjikokaLocal Government Areaof Anambra State

In Njikoka LGA, the KDT₅₀time taken for 50% of the mosquitoes tested to be knockeddown,KDT₉₀time taken for 90% of the mosquitoes tested to be knockeddown, KDT₉₅time taken for 95% of the mosquitoes tested to be knockeddown were observed. Means of Four (4) Replicates (±s.e), df=3, Pv=0.001, Means of Eight (8) replicates (±s.e), df=7, Pv=0.142 as shown in Table 3.

Discussion:

Considering the species identification of malaria vectors inthree Local Government AreasofAnambra State, Southeast Nigeria under study, the total number of two thousand eight hundred and seventy (2,870) malaria vectors gathered together in the study was relatively low compared to the mosquitoestrappedby [20]in Papua-New Guinea wherefemale Anopheles mosquitoes of seven different speciestotallingthe number of 7,146which includes:Anopheleshinesorum, Anopheleskoliensis, Anopheleslongirostris, Anophelesbancroftii, Anophelesfarautis.s, Anopheles farautiand Anophelespunctulatuss.s were collected, including 2,611 (36.5%) blood-fed and 4,535 (63.5%) unfed mosquitoes. The high number of malaria vectors in the slated study could probably be because mosquitoes were sampled daily (consistent assessment) from dawn to dusk in five villages for four years (2012 to 2016)whereas the contemporary survey sampled only three communities within twelve calendar months. The seemingly low abundance of malaria vectors in the present study may be connected with the mean diurnal temperature range that increases the species sensitivity to probably changes in climates, leading to low presence of Anopheles species as also reported bythe studies done by [21 and 22].

Molecular identification of malaria vectorscollected from the three Local Government Areas comprised of 54.2% of Anopheles gambiaeand 45.8% of Anopheles coluzziieventhough there was no significant difference. The outcome was in agreement with previous studies done by [23, 24, 25 and 26] where the model results showed that An. gambiae, Anopheles coluzziiand An. arabiensisare widespread across all ecological zones in Nigeria including Anambra State where the current study was undertaken. Study findings also acclaimed that diverse malaria vectors breeds concurrently with each other in all the LGAs surveyed and these species co-exist in sympatric relationships as also reported by the survey of [27]. Coincidentally, this report is probably the second-time concurrent breeding for the two sibling species (Anopheles gambiaeand Anopheles coluzzii) has been reported in aenormousmeasuresynchronized study in the Eastern part of Nigeria after the research reported by [28].

The malaria vector knockdown times in the existing study showed that the mean percentage knockdown of An. gambiaes.l exposed to different insecticides at varying time intervals in Awka South LGA Anambra State as follows: delthamethrin (0.05%) was 62%, permethrin (0.75%) was 65%, whereas malathion (5.0%) and Bendiocarb (0.1%) both recorded 100% with KDT_50, KDT_90, and KDT₉₅ of 170 minutes, 396.5 minutes and 501 minutes respectively. Also the mean percentage knockdown of An. gambiae s. l. exposed to different insecticides at varying time intervals in Awka North LGA Anambra State recorded 54% for delthamethrin (0.05%), 22% for permethrin, whereas malathion (5.0%) and bendiocarb (0.1%) both recorded 100% with KDT_50, KDT₉₀ and KDT₉₅of 199minutes, 478minutes and 602minutes respectively. Whereas the mean percentage knockdown of An. gambiaes.l. exposed to different types of insecticides at varying time intervals in Njikoka LGA Anambra State were as follows: delthamethrin (0.05%) was 63%, permethrin was 42%, whereas malathion (5.0%) and Bendiocarb (0.1%) both recorded 100% with KDT_50, KDT₉₀and KDT₉₅of 186 minutes, 436.5 minutesand 562.3 minutes. In this study, the mean percentage knockdown of An. gambiaes.l.exposed to different insecticides at varying time intervals in all the LGAs showed that malaria vectors were fully susceptible to bendiocarb alongside with malathion. Outcomes of this current study substantiatedby means of the findings in Amansea, a community inAwka North LGA of Anambra Statewhere malaria vectors were only susceptible tobendiocarb which falls into the group of carbamates as reported by [29]. It’s recorded with substantiated data that between the year (2014 and 2015),the populations of malaria vecors showed the greatest resistance against DDT and later pyrethroids[30] andby the next year 2016, evidence-based data on resistance to at least one insecticide was reported from more than 80% of malaria-endemic countries which Nigeria is included. This is a serious threat to the continued efficacy and potency of key malaria management strategies. This probably may be due to excessive use of the pyrethroid group of insecticide before now as reported by [31]. Also widely distribution of LLINs in South-East Nigeria for more than ten yearsnow couple with the information that the pyrethroidsgroup of insecticides are the only group of insecticides used in LLINs may possibly to have goaded the condition. Transversely the three LGAs, Malathion (5%) and Bendiocarb (0.1%) performed better than other insecticides used in the present survey. This possibly will not be contemplative of the globalpresentation of the diverse insecticides in the pyrethroid class since Deltamethrinexistence as a type II pyrethroidcomprises an alpha-cyano group permitting it to wield anenhancedconsequence on insects as also postulated by[32].Nonetheless, the comparativeimpression of dwindled mosquito vulnerability to pyrethroids on vectorialcapability remains unidentified in the study areas largely due to a paucity of field data which other further studies have to address.

Conclusion and recommendations:

AlthoughextensiveAnopheles species resistance to pyrethroid has been reported in this study, there is unquestionably a foremostneed for timely management of mosquitoes vectors particularly as it edges the challengereadily accessible to accomplish the objective of scheming the diseases mosquitoes cause. The results on the insecticide resistance across these three LGAs revealed that An. gambiaes. l. populations were fully susceptible to malathion which is an organophosphate and bendiocarb which is a carbamate but resistant to the pyrethroids and permethrin and this may thwart the impact and continued effectiveness of pyrethroids insecticide in malaria vector control as time progresses.Also, the annotations in the present study showed anextensive resistance to one or more of the classes of insecticides used in public health in the LGAs in Anambra State, Nigeria. It is thereby observed that insecticide resistance will likely have weighty operational impact in the LGAs if no pre-emptive action is taken in Anambra Statepredominantly and Nigeria at large.

Whereas the worldwideobligation to eradicate malaria by 2030 necessitates immediate hard work that includes but not limited tothe establishment of development of a combination ofoperativeand effectual vector control strategies and interpositions in parasite control. Henceforward, a crucial necessity for unceasing and stretchedsusceptibility/insecticide resistance monitoring in the 3 LGAs particularly is not only immenent but urgent. This will assist ingaining a larger and purerimpression of the condition, and how to tackle it. It is of great importance that new insecticides with extraordinary susceptibility status be made to assist in the management and reduction of the bearing of resistance in malaria vectors as observed in the current study.

Author contributions:EOO, DNA and DENwrote the first draft and edited the manuscript, EOO, OVEand CINdeveloped the protocol, UCU and IEOoversaw study implementation and monitoring. EOO and DENdid the experimental design and molecular characterization of the malaria vector samples.

Acknowledgments: The writersremainvery appreciative and honestlythanked the nominatedcivicfront-runnersand research assistants for their assistance during the sample collection.

Funding:The researchers appreciate the Royal Society of Tropical Medicine and Hygiene (RSTMH) in collaboration with the National Institute for Health Research (NIHR)based in London, United Kingdom for the research grant that funded this study.

Competing interest: The writersassert that no struggles of interest exist amongst them.

Ethical approval and informed consent

We approve that all procedures used in the study were undertaken in harmony with the appropriateprocedures, protocols and regulations as accepted by the committee on ethics and human research.Research informed consent was sought and gottenwhile ethical approval was also acquired from the office of the Director, NnamdiAzikiwe University that is in charge of Human Research Ethics Committee withstudy Research Reference Number: NAU/HREC/2S/02/12/2023/05. The studypartakers were properly organized and study objectivesexplained to them by a sensitization rally before the commencement of the research. Participationin the survey was voluntary and participants had the liberty to withdraw from the study.

Data availability:The data used for this study are available upon reasonable request.

References:

1. World Health Organisation (2016). The Malaria Mannual: Guidelines for the rapid assessment of economic and cultural aspects of malaria. TGR/SER/MSR/95/. Geneva: 2016;170-9.
2. Goudet, J., Raymond, M., de Meeüs, T., Rousset, F. (1996). Testing differentiation in diploid populations Genetics. 1996: 144:1933–40.
3. Eze, N. C., Ezihe, E. K., and Chukwu, M. C. (2018). Larval abundance, distribution and species composition of mosquitoes in Obio-Akpor LGA, Rivers State, Nigeria. International Journal of Entomology Research2018:3(2)85-90.
4. Egwu, O., Ohaeri, C. C., Amaechi, E. C. and Ehisianya, C. N. (2018). Distribution and abundance of mosquito larvae in Ohafia, Abia State, Nigeria. Cuadernos de Investigación UNED2018;10(2): 379-85.
5. Okonkwo, N. J., Obiechina, I. O., Ugha, C. N., Irikannu, K. C., (2014). Mosquito species composition in Oba, Idemili South Local Government Area of Anambra State.  Researcher, 2014:6(8)51–6.
6. Afolabi, O. J., Simon-Oke, I. A. and Osomo, B. O. (2013). Distribution, abundance and diversity of mosquitoes in Akure, Ondo State, Nigeria. Journal of Parasitology and Vector Biology2013: 5(10) 132-6.
7. Abdulrasheed, D., Aliyu, A. O. and Hafsa, B. (2016). Surveillance of Mosquito Species Abundance and Composition in Azare, Katagum Local Government of Bauchi State, Nigeria. Journal of Pharmacy and Biological Sciences2016:11(6)105-9.
8. Aribodor, D. N., Nwaorgu, O. C., Ozumba, N. A. and Etega, H. (2011). Intermittent Preventive Treatment of Malaria in Pregnancy in Nigeria: Need for Improvement in Drug Administration during Antenatal Care. Journal of US-China Medical Science. 2011:74(8): 46-50.
9. Onyido, A. E., Agbata, V. O., Umeanaeto, P. U., and Obiukwu, M. O. (2011). Ecology of malaria vectors in a rainforest suburban community of Nigeria, African Research Review 2011:5(2):293-8.
10. Egwu, O., Ohaeri, C. C., Amaechi, E. C. and Ehisianya, C. N. (2018). Distribution and abundance of mosquito larvae in Ohafia, Abia State, Nigeria. Cuadernos de Investigación UNED2018;10(2): 379-85.
11. Goselle, O. N., Amobi, L. O., Ojile, J. O., David, A.(2017). Abundance of mosquito larvae in various microhabitats and the concern for invasion of human community. International Journal of Mosquito Research 2017;4(4): 119-25.
12. Mbanugo, J. I. and Okpalaononuju, C. N. (2003). Surveillance of mosquito vectors in some habitats Of Awka Metropolis, Anambra State Nigeria. The Nigerian Journal of Parasitology, 2003:24: 185-90.
13. Onyido, A. E., Ugha, C. N., Eneanya, O. A., Umeanaeto, P. U. (2014). Malaria vector bionomics in Abagana community of Anambra State, Southeastern Nigeria. Journal of American Science,2014:10(2):23-9
14. Microsoft Encarta College Dictionary and Encyclopedia. New York: St. Martin’s Press, 2009:276-9. [Accessed 23 November, 2024].
15. National Bureau of Statistics. National Population Commission 2006 Nigeria Census, 2006;1-9
16. Enete, I. C. (2008). Managing Aviation Operations during Harmattan in Nigeria for maximum operations. Inpritx Lagos State, Nigeria. 2008: 12-23.
17. Enete, I. C. (2004). A Study of Enugu Rainfall Patterns from the Viewpoint of Precipitation Dynamics. Nigeria Journal of Research and Production. 2004:5(4):98-108.
18. World Health Organization (2018). Report of the WHO Informal Consultation: Test Procedures for Insecticide Resistance Monitoring in Malaria Vectors, Bio-Efficacy and Persistence of Insecticides on Treated Surfaces. WHO/CDS/CPC?MALARIA 2018:98.12. Geneva, Switzerland.
19. Abbott, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 1925: 18:265-267.
20. Kevan, J. B., Katusele, M., Vinit, R., Koimbu, G., Vincent, N., Thomsen, E. K., Karl, S., Reimer, L. J.and Walker, E. D. (2019). Species abundance, composition, and nocturnal activity of femaleAnopheles (Diptera: Culicidae) in malaria-endemic villages of Papua New Guinea: assessment withbarrier screen sampling. Malaria Journal 18(96): 1-12.
21. Onyabe, D. Y., Vajime, C. G., Nock, I. H., Ndams, I. S., Akpa, A. U., Alaribe, A. A. (2009). The distribution of M and S molecular forms of Anopheles gambiaein Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene. 97:605-608.
22. Briga, M. and Verhulst, S. (2015). Large diurnal temperature range increases bird sensitivity to climate             change. Scientific Report. 2015: 5:16600. https://doi.org/10.1038/srep16600
23. Bruce-Chwatt, L. J. (1951). Malaria in Nigeria. Bull World Health Organization 4:301-327.
24. Onyabe, D. Y. and Conn, J. E. (2001). The Distribution of Two Major Malaria Vectors, Anopheles          gambiaeandAnopheles arabiensis, in Nigeria. MemInst Oswaldo Cruz, Rio de Janeiro.      96(8):1081-1084.
25. Okorie, P. N., McKenzie, F. E., Ademowo, O. G., Bockarie, M., Kelly-Hope, L. (2011). Nigeria Anopheles Vector Database: An Overview of 100 Years’ Research. PLoS ONE. 6(12):21-23.
26. Okorie Patricia, N., Popoola, Olayemi M. Awobifa, Kolade T. Ibrahim, George O. and Ademowo, K. O. K. (2014). Species composition and temporal distribution of mosquito populations inIbadan, Southwestern Nigeria. Journal of Entomology and Zoological Studies. 2014 August: 2(4): 164–169.
27. National Malaria Control Programme, (2013). World Health Organization and the INFORM Project. A description of the epidemiology of malaria to guide the planning of control in Nigeria. A report       prepared for the Federal Ministry of Health, Nigeria, the Roll Back Malaria Partnership and the Department for International Development, United Kingdom. November, 2013.
28. OkechukwuChukwuekezie, Emmanuel Nwosu, UdokaNwangwu, Festus Dogunro, CosmasOnwude, NnekaAgashi, EbukaEzihe, ClementinaAnioke, Stephen Anokwu, Emelda Eloy, Peter Attah, Francis Orizu, Sylvester Ewo, Angela Okoronkwo, Anumba Joseph, IjeomaIkeakor, Sylvester Haruna and VirgileGnanguenon. (2020). Resistance status of  Anopheles gambiae (s.l.) to four commonly used insecticides for malaria vector control in South-East Nigeria Parasites and Vectors 2020 volume 13: 152:1-12.
29. Nwankwo, E. N., Okorie, P. N., Acha, C. T., Okonkwo, O. E.(2017). Insecticide resistance in Anopheles gambiaes.l. mosquitoes in Awka, Anambra State, Southeast Nigeria. Journal of Mosquito Research. 2017;7:32–7.
30. Okorie, P. N., Ademowo, O. G., Irving, H., Kelly-Hope, L. A. (2015). Insecticide susceptibility of Anopheles coluzzii and Anopheles gambiae mosquitoes in Ibadan, Southwest Nigeria. Medical and Veterinary Entomology. 2015;29:44–50.
31. World health organization, (2017). Yellow fever in Nigeria. www.who.int/csr/don/22. December. Accessed on 11^th February 2024.
32. World Health Organization. (2013). Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. World Health Organization, Geneva, 2013;1-9.