A Simple Key for Identifying the Sibling Species of the Malaria Vector Anopheles gambiae ( Giles ) Complex by Polytene Chromosome Cytogenetics

It has been established that Anopheles gambiae complex sibling species are the major Plasmodium malaria vectors in Africa; however, not all the sibling species transmit the infection. Easier molecular methods, PCR-based assays, have been developed to distinguish the several members of the A. gambiae complex. However, malaria vector research in less developed countries, particularly sub-Saharan Africa, is being hampered by the lack of PCR facilities in laboratories and the cost of carrying out the assay within lack of funding. Hence, the present study was designed to develop a simple identification key, based on an affordable method of polytene chromosome cytotaxonomy, for identifying the major P. falciparum vectors. The Identification Key was successfully used to identify two members of the A. gambiae complex, A. gambiae sensu stricto and A. arabiensis, which are the most potent malaria vectors in Africa; even so, it could not be used to establish the infective and the refractory strains.


Introduction
Malaria remains a major burden to human health in the sub-tropical and tropical regions of the world, where Plamodium malaria is the most prevalent and infectious (WHO, 1993;Hoffman et al., 1996;Davidson, 2000;WHO, 2009).Plasmodium consists of four species, P. falciparum, P. vivax, P. malariae and P. ovale that cause human malaria (Nonstrand, 1978).Among the four species, P. falciparum is the most deadly (Hoffman et al., 1996;Miller and Marley, 1999).Sub-Saharan Africa, however, suffers from the highest global malaria transmission levels, and hence, high mortality rate due to malaria (Fontenille and Simard, 2004;Hay et al., 2005 and2009); in Nigeria alone, ninety percent of malaria infections are caused by P. falciparum.P. falciparum is adapted to propagating alternately in Anopheles mosquitoes and human beings (Wikipedia, 2013).
The A. gambiae complex members are the dominant vectors of malaria in sub-Saharan Africa (Sinka et al., 2010;Gregory and Yoosook, 2013).A. gambiae complex consists of eight sibling species, which include A. gambiae sensu stricto Giles; A. bwambae White; A. merus Dönitz; A. arabiensis Patton; A. quadriannulatus Theobald; A. amharicus Hunt, Coetzee and Fettene; A. melas Theobald; A. comorensis Brunhes, le Goff and Geoffroy (Gillies and DeMeillon, 1968;White, 1974 and1985;Hunt et al., 1998;Harbach, 2003;Coetzee et al., 2013).Amongst these sibling species, A. gambiae and A. Arabiensis are the two major malaria vectors in Africa (Gillies and De Meillon 1968), particularly in Nigeria (Boreham et al., 1979;Onyabe and Conn, 2001;Obembe and Awopetu, 2014).There are strains of A. gambiae complex sibling species in which the parasite cannot complete its life cycle; such strains are said to be refractory (Collins et al., 1986;Chris et al., 2011;Obembe and Awopetu, 2014).Members of A. gambiae complex are morphologically identical, but genetically distinctive (Coluzzi et al., 1979).Due to the genetic distinction, the sibling species can be distinguished by their polytene chromosome cytotaxonomy and molecular methods.The polytene chromosome cytotaxonomy is based on chromosomal inversions of the ovarian nurse cell polytene chromosomes (Coluzzi et al., 1979;George et al., 2010).Ten fixed inversions that can be used to differentiate the members of A. gambiae complex have been identified (Coluzzi et al., 1979;Coluzzi et al., 2002).In addition, drawings that depict the banding Separation of female Anopheles gambiae from the stock The F1 female Anopheles mosquitoes were anaesthetized on the fourth day and A. gambiae females were selected from the stock using a standard identification key (Mark et al., 2007).Further species authentications were carried out by using the collated Identification Key from Hamon and Adam (1963), De Meillon (1947) and Evans (1938).The female A. gambiae mosquitoes that were identified were infected with P. falciparum.

Female A. gambiae infection
In preparation for the infection experiment, the F1 female mosquitoes were starved for 18 to 20 hours.On the day-5, after emergence, the female mosquitoes were allowed to feed on a pre-diagnosed P. falciparum infected individual for about 15 minutes such that the mosquitoes became engorged.Mosquitoes that did not feed on the infected individual, as well as the ones that were not engorged, were sacrificed in chloroform.The infected mosquito colonies were kept on 10% sugar solution regimen in the laboratory with ambient temperature, between 26 °C and 32 °C.They were maintained in the laboratory for 16 to 18 days after infection, such that P. falciparum would have reached their salivary glands (WHO, 1975).

Identification of the A. gambiae sibling species
Sibling species identification was carried out by using a simple Identification Key developed from Coluzzi et al. (2002) and George et al. (2010) (Table 1).The infected mosquitoes were deprived of food for 18 to 20 hours after which they were fed with blood a day before the dissection.Half gravid females (Clements, 1992) were dissected between 20 -24 hours after blood feeding for ovarian extraction.Stained polytene chromosome spreads were prepared from fixed ovaries (Cornel, 2007).Images of the polytene chromosome spreads that showed suitable levels of polytenization were obtained by using the digital imaging system of the Accuscope 3000 LED phase contrast microscope (Accuscope Inc., 2011).The chromosome images were processed by the Macormedia Fireworks image editing software version 8.0 (Macromedia Inc., 2005).Chromosome arm recognition was carried out by using standard arm recognition landmarks (George et al., 2010).With reference to Coluzzi et al. (2002) and George et al. (2010), 2R arm cytogenetic maps of the mosquito polytene chromosomes were developed and the inversion (2Rj, 2Rb, 2Rc, 2Ru and 2Rd) breakpoints were characterized.The Identification Key in Table 1 was successfully used for identifying the sibling species of the mosquitoes.patterns of A. gambiae s.l.polytene chromosomes are now available for characterizing the divisions and subdivisions of the species, as well as the locations of the inversions (Coluzzi et al., 2002;Holt et al., 2002).The molecular method is PCR-based and refers to species-specific single nucleotide polymorphisms (SNPs) in the intergenic spacer region (IGS) of the A. gambiae DNA (Scott et al., 1993;Fettene and Temu, 2003;Besansky et al., 2006;Wilkins et al., 2006).
The polytene chromosome cyto-taxonomic method is affordable, but it can only differentiate semi-gravid females, which constitute a small proportion of mosquito population in the wild; molecular method is easier and it can identify all stages and genders in the population.Nevertheless, in sub-Saharan Africa, particularly in Nigeria, the use of molecular method in identifying the members of malaria vector species complexes is restricted by lack of the PCR facilities in laboratories and the cost of carrying out the assay coupled with lack of research funding; consequently, malaria vector research is restricted.Hence, there is need for a simple identification key based on the affordable polytene chromosome cytotaxonomy so as to encourage malaria vector research in Africa.

Study area
Two selected areas in Osun State, Ilesa (4 o 44' E, 7 o 37' N) and Ile-Ife (7°28′N, 4°34′E), in South-Western Nigeria were sampled.Anopheles larvae were collected from stagnant pools of water in these locations and transported to the laboratory at Obafemi Awolowo University, Ile-Ife, Nigeria.The Anopheles larvae were identified by using their resting position in water, as well as a diagnostic feature, lack of a respiratory siphon but spiracles on the 8th abdominal segment.

Rearing F1 generations of Anopheles mosquitoes
The Anopheles larvae collected from the study areas were used to breed F1 colonies of Anopheles larvae in the laboratory.The F1 Anopheles larvae species were validated by observing the diagnostic features, resting position of the larvae in water, absence of breathing siphon and presence of palmate hairs on the eighth and fourth abdominal segments respectively, under a dissecting microscope (WHO, 1997;Mark et al., 2007).The validated F1 Anopheles larvae were fed with biscuit diet until they emerged as adults.Gender grouping of the F1 adult mosquitoes was carried out on the third day of emergence based on their diagnostic features, antennae and palps structures (Service, 1980;WHO, 1997) The infective and the refractory strains The mosquito salivary glands were dissected immediately after ovary extraction.A drop of physiological solution (Hayes, 1953) and a cover slip were then placed on the salivary glands.They were broken by a gentle pressure applied to them in order to free the sporozoites into the physiological solution.The glands were then inspected under x40 objective of microscope for infection by P. falciparum.

Results
P. falciparum sporozoites were observed in the salivary glands of the mosquitoes, which indicated that the mosquitoes were susceptible to P. falciparum infection, and hence, they were infective.However, the salivary glands of some of the mosquitoes did not bear sporozoites, indicating that those mosquitoes were refractory to P. falciparum infection.Infective and the refractory strains were recorded in both locations.

Proportions of the infective and the refractory strains
Table 2 shows the proportions of the infective and the refractory strains of the A. gambiae mosquitoes from the two study areas.There was a significant between the proportions of infective and refractory strains within each study area (P < 0.05); contrarily, the proportions of refractory strains with the proportions of infective strains were not significantly different between the two study areas (P > 0.05).

Sporozoite rate
Sporozoite rates of the mosquitoes from both study areas are shown in Table 3.The sporozoite rates between the study areas were not significantly different (P > 0.05).

Sibling species identification
The Identification Key (Table 1) was successfully used to identify two sibling species, A. gambiae sensu stricto and A. arabiensis, of the A. complex.The observed infective strains include the two members of the complex, A. gambiae sensu stricto and A. arabiensis.It was noted that A. gambiae sensu stricto was the only refractory strain recorded.The infective strain and the refractory strain of A. gambiae sensu stricto have the same banding patterns, and hence, they could not be distinguished by the Identification Key.

Discussion
There is insufficient information on the identification of the sibling species of malaria vectors and their infective and refractory strains in south-western Nigeria (Obembe and Awopetu, 2014), perhaps, due to lack of facilities and funding for carrying out the molecular, PCR-based assay, method of identification.Hence, the current study was designed to promote malaria vector research, not only in Nigeria, but also in other less developed countries of the world, particularly in sub-Saharan Africa.
The proportions of A. gambiae complex recorded in the study areas were high, which shows that the members of A. gambiae complex are greatly susceptible to P. falciparum infection.In a similar research by Obembe and Awopetu (2014) in the study area, virtually, the same results were obtained.Similar reports were also recorded by Collins et al. (1986) and Alvaro et al. (2012).This corroborates the idea that the parasites are capable of evading the mosquito immune responses (Osta et al., 2004).However, the fact the parasite was not successful in reaching the salivary glands of some of the mosquito's supports the reports by Julián (2010) and Chris et al. (2011) that the mosquito inherent defence system is capable of terminating the development of all the parasites before they reach the salivary glands leading to total refractoriness.It also confirms the report by Obembe and Awopetu (2014) that refractory strains of A. gambiae are present in South-Western Nigeria, though at a very low frequency.
However, the sporozoite infection rates that were obtained were significantly higher than the ones previously reported, 6.70% and 6.30% in 2001 and 2002 respectively, at Igbo-Ora, Oyo State in South-Western Nigeria (Noutcha and Anumdu, 2009), as well as at Badagry Axis of Lagos Lagoon, Lagos State, Nigeria (Oyewole et al., 2010).However, Obembe and Awopetu (2014) has reported similarly high P. falciparum sporozoite rates in the study areas.Okwa et al. (2006) also reported a high sporozoite rate (62.9%) for A. gambiae in Badagry area of Lagos, Nigeria.In a related study, 83.52% P. falciparum sporozoite infection rate was recorded for A. gambiae (Ndiath et al., 2011).The high P. falciparum sporozoite rates shows that the A. gambiae complex members are prominent and potent P. falciparum vectors in South-Western Nigeria and similar report was given by Gilles and Coetzee (1987), Annon (2003), Okwa et al. (2007) and Okwa et al. (2008).
The identified members of the A. gambiae complex, A. gambiae sensu stricto and A. arabiensis, as similarly reported by Okwa et al. (2008) and Obembe and Awopetu (2014), are the major P. falciparum vectors in South-Western Nigeria.

Conclusions
The present study showed that A. gambiae sensu stricto and A. arabiensis are potent malaria vectors in southwestern, Nigeria.In addition, they can be identified by the Identification Key based on the affordable ovarian polytene chromosome cytotaxonomy.However, the method could not be used to characterize the refractory and infective strains of the A. gambiae sensu stricto.

. 98 Table 1 .
Simple identification key for identifying the sibling species of A. gambiae complex using common polymorphic inversions

Table 2 .
Proportions of refractory (R) and infective (I) A. gambiae mosquitoes from the study areas

Table 3 .
Sporozoite infection rates of A. gambiae mosquitoes from the study areas