Identification The Sibling Species Of Anopheles Gambiae S.L, And Determine The Insecticide Resistance Mechanism Present In The Population

CHAPTER ONE

• INTRODUCTION

1.1              Background of the Study

Malaria remains the world most important parasitic disease of public health importance (WHO, 2013). It is the major public health problem in Nigeria contributing a quarter of the malaria burden in Africa (WHO, 2008). The disease is the most important cause of human morbidity and mortality and with enormous medical, emotional and economic impact in the world (Boccolini, et al., 2012; Coetzee, et al., 2000). Nigeria is one of the six countries in the WHO African region with the highest burden of malaria. Nigeria contributes a quarter of malaria burden in Africa (World Malaria Report, 2012). In 2013, malaria killed an estimated 584 000 people with over 2 million cases (WHO, 2014). Nigeria is reported to have the unenviable record of contributing about 25% of the world malaria burden (WHO, 2012).

Anopheles mosquito transmits malaria. The most important vectors of malaria are members of Anopheles gambiae s.l. (complex), a group of morphologically identical yet genetically and behaviourally distinct species that differ markedly in their ability to transmit the diseases (Coluzzi, 1979). Members of the species complex include Anopheles gambiae s.s., An. arabiensis, An. merus, An. melas, An. bwambe, and An. quadriannulatus (Coetzee, et al., 2000).

The major control interventions against malaria vectors include Insecticide-treated mosquito nets (ITNs) and indoor residual spray (IRS) with insecticides. IRS has helped to

eliminate malaria from great parts of Asia, Russia, Europe, and Latin America. Long- lasting insecticidal nets (LLINs) are the preferred form of ITNs for public health distribution programmes. WHO recommends coverage for all at-risk persons, and the most cost effective way to achieve this is through provision of free LLINs for sleeping every night. (Pluess, et al., 2010; WHO, 2013).

However, these control efforts are threatened by several challenges which includes incorrect identification, biology distribution and insecticide resistance (Awolola, et al., 2002, 2005; Ranson, et al., 2011; Ibrahim, et al., 2014; Okorie, et al., 2015), and lack of surveillance research interventions (Slutsker, 2012).

To overcome these challenges from undermining control programs, correct identification, distribution of Anopheles vector must be understood and insecticide resistance management must reduce the current overreliance on pyrethroids. These compounds are used widely for indoor residual spraying and uniquely for insecticide- treated bed nets as it is the only class of insecticide of the four major classes of insecticide recommended for malaria control by World Health Organization.

A mutation at a single target site can result in mosquito resistance to DDT and pyrethroids or to organophosphates and carbamates. Furthermore, mosquitoes can express multiple insecticide-resistance mechanisms (Perera, 2008). For example, in several populations of the major malaria vector in Africa, Anopheles gambiae s.l. mosquitoes, mutations in the DDT/pyrethroid target site, known as knockdown resistance (kdr) alleles, have been found in conjunction with resistance alleles of the

acetylcholinesterase gene (Ace-1), the target site of organophosphates and carbamates (Yewhalaw, et al., 2011).

1.2              Statement of Problem

Information on species composition, distribution and susceptibility status of Anopheles to insecticides are important parameters to assess the effectiveness of malaria vector control strategies. Such information is currently unavailable to malaria control programmes in Kaltungo and Balanga Local Government Area of Gombe state.

• Significance of the Study

The identification of malaria vector species, distribution and susceptibility status to insecticides are vital to effective malaria control strategies. If meaningful control strategies are to be formulated against the malaria vectors in the Sahel, studies to confirm the predominant sibling species of An. gambiae sensu lato must be carried out (Gadzama, 1983). This study therefore, identifies An. gambiae to species level, molecular forms, distribution and resistance mechanism present in the resistant Anopheles population in Gombe state. This information will help in formulating appropriate control strategies in Kaltungo and Balanga Local Government Area of Gombe state.

• Aims and Objectives of the Study

The aim of this study is to identify the sibling species of Anopheles gambiae s.l, and determine the insecticide resistance mechanism present in the population.

1.4.1        Objectives

1. To identify the Anopheles mosquitoes collected using morphological keys.
2. To identify the sibling species of Anopheles gambiae complex using species- specific PCR assay.
3. To identify and determine the distribution of molecular forms of An. gambiae s.s. [ gambiae (S form) and An. coluzii (M form)] using Restriction Length Fragment Polymorphism (RFLP) technique.
4. To determine the distribution of molecular forms of Anopheles gambiae s.
5. To identify the involvement of Knockdown resistant (kdr) point mutation in the resistant population of gambiae complex in the study area.

CHAPTER TWO

• LITERATURE REVIEW

2.1              Malaria Burden

Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. Malaria is widespread in the tropics and also occurs in subtropical and temperate regions. According to the latest WHO estimates, released in December 2015, there were 214 million cases of malaria in 2015 and 438 000 deaths. Between 2000 and 2015, malaria incidence among populations at risk fell by 37% globally; during the same period, malaria mortality rates among populations at risk decreased by 60%. An estimated 6.2 million malaria deaths have been averted globally since 2001.

Some 15 countries, mainly in sub-Saharan Africa accounts for 80% of malaria cases and 78% deaths globally. Since 2000, the decline in malaria incidence in these 15 countries (32%) has lagged behind that of other countries globally (53%).

In 2015, 95 countries and territories had on-going malaria transmission, Malaria is preventable and curable, and increased efforts are dramatically reducing the malaria burden in many places. Between 2000 and 2015, malaria incidence among populations at risk (the rate of new cases) fell by 37% globally. In that same period, malaria death rates among populations at risk fell by 60% globally among all age groups and by 65% among children under 5 years of age. Sub-Saharan Africa carries a disproportionately high share

of the global malaria burden. In 2015, the region was home to 88% of malaria cases and 90% of malaria deaths (WHO, 2015).

2.1.1        Malaria in Nigeria

 In Nigeria, malaria is caused by three species of In Nigeria. According to WHO (2015), malaria estimated case ranges between 42 million to 78 million while the estimated recorded death ranges from 81,000 to 150,000 with 76% of population living in high transmission areas and 24% in low transmission areas. This report shows that Nigeria still remains an endemic country of malaria.

The parasite Plasmodium: P. falciparum (100%), P. vivax (0%) are the major species that transmits malaria infection in Nigeria. The principal method of malaria transmission is through the bite of infected female Anopheles mosquito (WHO, 2015).

• The Anopheles Vector

 The biology of the main African malaria vectors has been part of literature for over 50 years. The vectors have been variously described and identified as sub-species, forms, varieties, races, etc. These have been carried out in terms of morphological differences, distribution, biology, ecology, behaviour among others. In West and Central Africa, five different species are considered major malaria vectors: An. gambiae, An. arabiensis, An. funestus, An. nili and An. moucheti. At least 4 or 5 other species are considered secondary or locally (Dixit, et al., 2010) important vectors, e.g. An. paludis, An. hancocki, An. melas among others.Of the seven sibling species of the complex, An. gambiae s.s. and An. arabiensis is the most widely distributed and efficient vectors of malaria in sub-Saharan African (Coetzee, 2004; Edillo, et al., 2004). These two species are broadly sympatric but there are may be areas where only one or the other may be found (Edillo, et al., 2004).

• Anopheles gambiae Complex

 The Anopheles gambiae sensu lato (s.l.) species complex contains the most important mosquito vectors of malaria in sub-Saharan Africa. It comprises seven morphologically indistinguishable sibling species up to four of which may be sympatric (Coetzee, 2004). The principal malaria vectors in the complex are Anopheles gambiae sensu stricto (s.s.) and Anopheles arabiensis. Of the remaining members, Anopheles quadriannulatus species A, which is widespread in southern Africa, and Anopheles quadriannulatus species B, found in Ethiopia, are considered to be zoophilic non-malaria vectors (Coetzee, 2004; Coetzee, et al., 2000). Anopheles melas and An. merus are salt water breeding and consequently only important vectors in coastal region (Moreno, et al., 2004).

• Molecular Forms of gambiae s.s.

In West Africa, An. gambiae s.s. has been divided into five chromosomal forms

designated with a non-Linean nomenclature: bamako, mopti, savanna, forest, and bissau

(Coluzzi, et al., 1985). Anopheles gambiae s.s. exists throughout most of Sub-Saharan Africa, but there are many polymorphisms including chromosomal inversions that appear to be involved in the adaptation of subpopulations to different environments (Coluzzi, et al., 2002). Anopheles gambiae s.s. is undergoing speciation, being split into two “molecular forms”, currently named An. coluzzi (M form) and An. gambiae (S form). Speciation is the main process promoting biological diversity and in the context of public health it increases epidemiological complexity and these molecular forms (M and S) have been identified to be reproductively isolated (della Torre, et al., 2001). The S form (now An. gambiae) is distributed widely throughout the An. gambiae species range, whereas the M form (now An. coluzzi) is common but restricted to western parts of Africa, and hybridization between them is rare in most areas of sympatry (della Torre, et al., 2005). It is vital to understand such differentiation and genetic subdivision and its importance in vector evolution, as this complexity can affect malaria control, including resistance to insecticides (Lynd, et al., 2010) and susceptibility to malaria parasites and other infections (White, et al., 2011).

Differences in the adult mosquito transcriptome between the M and S molecular forms appear to be minimal but evidence is accumulating that the larval stages are differentially adapted to particular features of breeding sites, with the M form (now An. coluzzi) being generally more common in large areas of irrigation for crop cultivation (Diabate, et al., 2008).