Sporozoite Infection Rate and Identification of the Infective and Refractory Species of Anopheles gambiae (Giles) Complex

The ability of Anopheles gambiae complex mosquitoes to transmit Plasmodium infection is known to be variable within sibling species of the complex with strains that cannot transmit the parasite. High sporozoite infection rate recorded showed that A. gambiae mosquitoes are potent malaria vectors in southwestern Nigeria. The aim of this study was to identify the infective and refractory strains of A. gambiae mosquitoes and to determine the sporozoite infection rate in this area. The infective strains were A. gambiae (sensu stricto) and A. arabiensis, while the refractory strains were A. gambiae (sensu stricto). However, ovarian polytene chromosome banding patterns could not be used to distinguish between the infective and refractory strains of A. gambiae (sensu stricto). This study showed that the refractory strains of Anopheles gambiae complex are present, but in low frequencies, in southwestern Nigeria, and that the sibling species of Anopheles gambiae (A. gambiae s.s. and A. arabiensis) are potent malaria vectors.


Introduction
Malaria is one of the most deadly diseases affecting many people worldwide, particularly in the tropical and sub-tropical areas. It remains a major burden to human health in these regions (WHO, 1993(WHO, , 2009. Malaria that affects humans is caused by Plasmodium parasites that are adapted to propagating alternately in two different hosts, i.e. the primary hosts, Anopheles mosquitoes (vectors) and the secondary hosts, human beings. The four species of Plasmodium that infect humans and cause human malaria are P. falciparum, P. vivax, P. malariae and P. ovale, among which P. falciparum is the most infectious (Hoffman et al., 1996). Awolola et al. (2005) reported that ninety percent of infections are caused by P. falciparium in Nigeria. It has been reported that, in Nigeria alone, more than 200 million people have died from malaria (Kiszewski et al., 2004).
It has been reported that infective capacity varies among the sibling species of these complex, with strains of A. gambiae being poor vectors of P. falciparum, as the parasites do not develop well (or not at all) within them (Cirimotich et al., 2011;Collins et al., 1986). The strains that lack the capacity to transmit the parasites are said to be refractory to infection by the parasite. The ability of a mosquito to transmit malaria parasites depends on the ability of the parasite to complete its life cycle inside the mosquitoes' organism, i.e. reaching the infective (sporozoite) stage in the salivary glands of the host (Sanofi, 2013). The life cycle of P. falciparum may be incomplete in the refractory strains of A. gambiae due to two factors, namely (i) the vector's innate immune system, through lysis, melanization and hemocyte-mediated phagocytosis (Collins et al., 1986;Hurd et al., 2005;Vernick et al., 1995) and (ii) bacterial competition with the parasite in the midgut of the mosquitoes (Cirimotich et al, 2011). Macdonald (1952) defined sporozoite rate as the proportion of mosquitoes with sporozoites in their salivary glands. The parameter has been used as an epidemiological index to determine the infectivity of the species of Anopheles (Beier et al., 1990). Sporozoite rate can be determined by dissection and ELISA (Adungo et al., 1991;Beier et al., 1990). Determination of sporozoite rate by dissection has been found to be more accurate and sensitive than the ELISA method. ELISA is limited by overestimation (Adungo et al., 1991;Beier et al., 1990;Sanofi, 2013) and it cannot distinguish between infected and infectious mosquitoes.
There is paucity of information on the infective capacity and refractoriness of Anopheles gambiae complex in southwestern Nigeria; in addition, the refractory strains and the infective strains are yet to be identified. However, there is need for identifying these strains in order to know the effective malaria vectors among the sibling species of A. gambiae complex as it would contribute to the discovery of effective ways of controlling the malaria parasite and vector, because the effective vector will be targeted.

Materials and methods
Collection and processing of the samples Samples of mosquitoes larvae and pupae were collected from five towns of south-western Nigeria, Ado-Ekiti (7°37'16"N 5°13'17"E), Ekiti State; Ilesa (4 o 44'0" E 7 o 37'0" N) and Ile-Ife (7°28′N 4°34′E), Osun State; Ibadan (7°23′ 47″N 3°55′0″E), Oyo State and Akure (7°15′0″N 5°11′42″E), Ondo State. The mosquitoes larvae and pupae were collected by a dipper, made of a big plastic spoon, from temporary breeding places of rain pools in residential areas of the towns mentioned above. They were transported in bottles with mosquito net covers containing water from their natural habitats, to the laboratory in the Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria. The Anopheles larvae were selected within the samples using the resting position of the larvae in water and the diagnostic feature, lack of a respiratory siphon but spiracles on the 8 th abdominal segment.

Rearing of the Anopheles larvae
The Anopheles larvae that were selected from the samples were transferred into new trays with water from their natural habitats and reared in wooden cages (32 x 32 x 32 cm) in the laboratory, under fluctuating temperature between 26 °C and 32 °C; they were fed with finely ground dry biscuit powder lightly sprinkled on the water surface daily. The biscuit was chosen because its powder floats and forms suspension in water and in the same time it does not form colloids that could entangle the larvae and kill them. The larvae were maintained on this diet until they all emerged as adults. The trays that contained the larvae were transferred into new cages two days after the first emergent adults were observed. The transfer of the trays into new cages continued every two days, until all the larvae emerged as adults. This was vital for obtaining adults of the same age groups. The cages were labelled according to the last days of emergence and the locations of collection. The emerging adult mosquitoes were fed with 10% sugar solution. Cotton wool was inserted into a small bottle of sugar solution and the mosquitoes were feeding from the soaked part of the cotton wool that protruded out of the bottle.

Breeding F 1 generations of Anopheles mosquitoes from the emerged adults
Breeding Anopheles mosquito involved giving the adult mosquitoes blood meal so that they might lay eggs and afterwards rearing the laid eggs in order to obtain F1 adult mosquitoes.
According to Clements (1992) male mosquitoes become sexually mature within 24 hours (one day), while females become sexually mature between 48 hours (two days) and 72 hours (three days) after emergence. Female mosquitoes become receptive to males, usually prior to blood feeding, between two and three days after emergence (Marks et al., 2007). Shute (1936) reported that maximum mating occurs between three and five days after emergence. Therefore, the emergent female Anopheles mosquitoes from each location were blood-fed for 15 minutes on the fifth day after emergence in order to lay eggs thare are to be collected (Marks et al., 2007). This was done by allowing the mosquitoes to feed on an anaesthetized and immobilized mouse and this continued at intervals of three days. The mouse was anaesthetized following to the ethical guidelines of Canadian Council of Animal Care (CCAC, 2005) and International Society for Applied Ethology (ISAE, 2012). The mouse was restrained by tying its legs. The belly of the mouse was shaved prior to blood-feeding the mosquitoes in order to make the skin easily accessible to the mosquitoes.
A cup of water was then placed in the cage on the third day of blood-feeding the mosquitoes (8 th day after emergence) for egg collection, since the duration of the gonotrophic cycle (i.e the period between the blood meal, the egg maturation and the subsequent oviposition, is within three days) (Bruce-Chwatt, 1985). The sides of the cups were lined with filter paper and placed in the cages that contained the blood-fed mosquitoes for three days. The mosquitoes laid eggs on the moist filter paper at the sides of the cup.
The filter paper, on which the Anopheles mosquitoes laid eggs, was removed from the cups and the eggs on the filter paper were carefully washed with boiled and cooled water into hatching trays that also contained boiled and cooled water. The trays were then placed in the wooden rearing cages 32 x 32 x 32 cm, where they hatched to larvae under the fluctuating laboratory temperature between 26 °C and 32 °C; they were daily fed with finely ground dry biscuit powder lightly sprinkled on the water surface.
In order to ensure the true breed of the emerged F1 Anopheles larvae, species authentication was carried out by using their resting position in water, absence of breathing siphon and presence of palmate hairs on the eighth and fourth abdominal segments respectively (WHO, 1997). These diagnostic features were observed under a light microscope according to the method of Mark et al. (2007). The authenticated F1 Anopheles larvae were selected from the stock larvae and then transferred into new rearing trays.
The F1 Anopheles larvae were maintained on the biscuit powder diet until they pupated. The pupae were separated from the larvae by picking them with a picker and transferred into new trays that contained boiled and cooled water. The trays that contained the pupae were then transferred into new cages. They were kept in the cages until they emerged as adults. The emerged F1 adult mosquitoes were fed with 10% sugar solution.
Gender grouping of the F 1 adult mosquitoes On the third day of emergence, test tubes were used to collect the emergent F1 adult Anopheles mosquitoes from the cages and they grouped upon the gender, based on the structures of their antennae and palps (Service, 1980;WHO, 1997). Female mosquitoes were transferred into new cages, while the males were killed by starvation.

Selection for female Anopheles gambiae from the stock
On the fourth day after emergence, test tubes were used to collect the female Anopheles mosquitoes from their cages and the open ends of the test tubes were clogged with cotton balls. The mosquito in the test tube was then placed in a killing jar containing chloroform and anaesthetized for 45 to 60 seconds. The anaesthetized mosquitoes were removed from the killing jar and their morphological characteristics were observed under a stereomicroscope in order to identify and select A. gambiae females from the stock. The identification key developed by Mark et al. (2007) was first used for this purpose. The species of the female A. gambiae mosquitoes were then authenticated by using the Identification Key compiled from those published by Evans (1938), De Meillon (1947 and Hamon and Adam (1963). The identified female A. gambiae mosquitoes were then transferred into separate rearing cages and used for the subsequent experiments.

Infection of the F 1 A. gambiae females with Plasmodium falciparum
For the infection experiment, the female mosquitoes were starved for 18 to 20 hours prior to feeding them with the infectious blood meal (Gnémé et al., 2013). On the 5 th day of emergence, the identified A. gambiae females were infected by allowing them to feed on an individual who had been diagnosed positive for infection by P. Falciparum, following the regulations of Department of Health and Human Services (HHS, 2009). The infected individual dipped his hand through the sleeve into the cage that contained the female A. gambiae for 15 minutes, so that the mosquitoes obtained the infected blood meal from the individual and became engorged. The mosquitoes became infected by the parasite P. falciparum from the blood meal they took from the infected individual.
Mosquitoes that were engorged were identified by the colour and nature of their abdomen; they were removed from the cages and transferred into new cages. Female A. gambiae mosquitoes that took the blood meal and were engorged were identified by their dark red and swollen abdomen. These infected mosquitoes were then maintained on 10% sugar solution in new cages in the laboratory with fluctuating temperature between 26 °C and 32°C.

Dissection of the infected mosquito ovaries and salivary glands
In order to the determine the infective capacity of the mosquitoes and to identify the mosquito sibling species, the salivary glands and the ovaries of the mosquitoes were dissected under a dissecting microscope 16 and 18 days after infection, i.e. the time required by P. falciparum to reach salivary glands of the mosquitoes (WHO, 1975). The infected mosquitoes were blood fed a day before the dissection, after being starved for 18 to 20 hours. The dissection of ovaries and the salivary glands of the mosquitoes were carried out at the same.
Half gravid females (with ovaries at Christopher III stage) were selected from the mosquitoes 20-24 hours after blood feeding. Half gravid females are identified when the ovaries take up to 3/5 of the abdomen (Clements, 1992). The half gravid female mosquitoes were anaesthetized for 30 to 50 seconds with ethyl ether in an anesthetizing chamber. The anaesthetized mosquito was then placed on a clean microscope slide and dissected under a dissecting microscope. The wings and legs of the mosquito were first removed. The mosquito was then held down with a dissecting pin at the base (8 th /9 th segment) of the abdomen and simultaneously, the last two abdominal segments (genitalia) of the mosquito were gently pulled out with a forceps in order to extract the ovaries. The ovaries were immediately placed in a vial containing modified Carnoy's solution (Cornel, 2007). The ovaries were fixed and preserved in the modified Carnoy's solution in a refrigerator for 24-30 hours at 4 °C.
The mosquito, after extraction of the ovaries, was then placed on its side on a clean microscope slide and gentle pressure was applied on the thorax towards the mesonatum end in order to squeeze the salivary glands into the neck. While gently pressing the thorax, a dissecting needle was used to gently pull the head of the mosquito in such a way that the salivary glands were pulled out of the thorax with it. The salivary glands were gently detached from the head. The remains of the head and the thorax/abdomen were then removed from the slide and a drop of physiological solution (Hayes, 1953) was placed on the salivary gland. The physiological solution was vital for keeping the salivary glands from drying out immediately and to maintain the tissues in a reasonably normal state for observation under a microscope. A cover slip was then placed on the glands and gentle pressure was applied on the glands which helped to rupture the tissues and freed the sporozoites from the salivary glands of infected mosquitoes into the physiological solution.
The salivary glands were stained with Giemsa using the method of Marks et al. (2007). The stained salivary glands were inspected for infection by P. falciparum under x40 objective of the microscope. The pictures of the salivary glands were taken by an Acusscope LCD Digital Microscope DMS012 (Acusscope Inc., 2011). The number of the infective mosquitoes that had the parasite in their salivary glands and the refractory mosquitoes that did not have the parasite in their salivary glands were recorded.

Estimation of the sporozoite infection rate
The sporozoite infection rate (SIR) of each study area was determined (Macdonald, 1952;Kakkilaya, 2006).
The sporozoite infection rate was used as an indicator to determine the potential infectivity of the A. gambiae mosquitoes, and hence the infective capacity, if they successfully gained access into the natural environment.

Mosquito sibling species identification
The sibling species of the mosquitoes were identified by developing the cytogenetic maps of the polytene chromosomes prepared from the ovaries of the mosquitoes.
The polytene chromosome spread preparation and staining was done from the fixed ovary (Cornel, 2007). The slide that contained the chromosome spreads was then scanned at 100X magnification and the spreads that showed suitable levels of polytenization were than imaged by the digital imaging system of the Accuscope 3000 LED phasecontrast microscope (Accuscope Inc., 2011) under oil immersion at 1,000X magnification. The images of the chromosomes were processed using the Macormedia Fireworks image editing software version 8.0 (Macromedia Inc., 2005) on a personal computer. The software was used to increase the quality and the resolution of the polytene chromosome images. The cytogenetic maps of the chromosome images that showed well spread arms and suitable levels of polytenization were then prepared. The arms of the polytene chromosomes were recognized using the arm recognition landmarks of George et al. (2010). The cytogenetic map of the 2R arm of the mosquito polytene chromosomes was developed and the localization of each inversion breakpoint or landmark was determined with reference to the A. gambiae polytene chromosome maps published by Coluzzi et al. (2002) and George et al. (2010).
With reference to the data and information from Coluzzi et al. (2002) and George et al. (2010), a simple identification key was developed. The identification key, consisting of five inversion polymorphisms (2Rj, 2Rb, 2Rc, 2Ru and 2Rd) of 2R arm of the mosquito ovarian polytene chromosomes (Tab. 1), was employed in identifying the sibling species of the mosquitoes. susceptible to infection by the parasite (Fig. 1). Sporozoites were not observed in the salivary glands of the unsusceptible (refractory) mosquitoes. The infective strains, as well as refractory strains, of A. gambiae complex mosquitoes were recorded in all the locations.

Proportions of the infective and the refractory strains
The proportions of the infective (I) and the refractory (R) strains of female A. gambiae mosquitoes from each study area are shown in Tab. 2. There was a significant difference between the proportion of infective and the proportion of refractory strains in each study area (P<0.05), but the proportion of refractory strains as well as the proportion of infective strains between the study areas were not significantly different for P>0.05. However, the highest proportion of refractory strains of A. gambiae (0.205) was recorded in Ibadan, while the highest proportion of infective strains (0.826) was recorded in Ado-Ekiti.

Sporozoite infection rate
Tab. 3 shows the sporozoite infection rates of the mosquitoes from the study areas. The sporozoite rates did not significantly differ among the study areas (P > 0.05). A highest sporozoite index of 82.61% was obtained from Ado-Ekiti, while the least sporozoite rate of 79.55% was recorded in Ibadan. 410 Tab. 1. Simple identification key for identifying the sibling species of A. gambiae complex using common polymorphic inversions. (Tab. derived from Coluzzi et al., 2002 andGeorge et al., 2010) Inversion

Statistical methods
The data collected were subjected to statistical analyses. The proportions of the refractory and the infective mosquitoes and the sporozoite infection rate were estimated by using the Microsoft Office Excel 2007 software. Statistical tests were also carried out by using one-way analysis of variance (ANOVA) using the computerized database SPSS (Version 16.0) for Windows to determine the differences between proportions of the refractory and infective mosquitoes within each location and between the locations. Treatments were considered significant at P<0.05.

Results
Sporozoite stages of P. falciparum were observed in the dissected salivary glands of A. gambiae mosquitoes that were

Sibling Species Identification
Two sibling species of the A. gambiae complex, viz. A. gambiae (sensu stricto) and A. arabiensis were identified. The infective mosquitoes were A. gambiae (sensu stricto) and A. arabiensis, while the refractory mosquitoes were A. gambiae (sensu stricto).
The polytene chromosomes of the infective and refractory strains of A. gambiae (sensu stricto) mosquitoes had the same banding patterns.

Discussion
There are several reports on the species abundance and composition, sporozoite and infectivity rates, as well as vector competence of Anopheles gambiae in Lagos State, Nigeria (Awolola et al., 2002;Okwa et al., 2006;Okwa et al., 2007) and also in Igbo-Ora, Oyo State, Nigeria (Noutcha and Anumdu, 2009), but the available information on the sporozoite rate of Anopheles mosquitoes in southwestern Nigeria is scanty (Awolola et al., 2002;Awolola et al., 2005) and no direct evidence regarding the refractoriness of A. gambiae to infection by P. falciparum in southwestern Nigeria has been published. In addition, no reports of the identification of the refractory and infective strains of A. gambiae complex have been published. Hence, this study was carried out in areas where less or none of these pieces of information are available.
The significantly high proportions of infective strains of A. gambiae recorded showed that the species of A. gambiae complex are highly susceptible to P. falciparum infection. Similar reports have also been recorded showing that A. gambiae mosquitoes are highly susceptible to P. falciparum, (Molina-Cruz et al., 2012;Collins et al., 1986). It also corroborates the idea that despite the immune response of mosquitoes against Plasmodium infection, the parasites are capable of using some molecules produced by the mosquito to evade the mosquito innate immune response (Osta et al., 2004), which makes the mosquito susceptible.
The lack of significant differences among the sporozoite infection rates between the study areas might probably be due to the fact that the parasite and the vector originated from the same geographical origin (United States, 2004). This suggests a geographic scale of adaptation and sympatric association between the parasite and the vector. Similar reports of geographic scale of adaptation (Haris et al., 2012) and sympatric association (Gnémé et al., 2013) have been posited. This is also in agreement with the reports of Collins et al. (1986), Molina-Cruz et al., (2012) that showed the survival of P. falciparum in A. gambiae mosquitoes seemed to correlate with their geographical origin. It also shows the importance of sympatric association in vector-parasite association, and hence the ability of the parasite to evade the mosquito immune response and survive in the vector, which in turn determines the infective capacity and vector competence to transmit the parasite. Collins et al. (1986) and Molina-Cruz et al. (2012) also reported that the evasion of the A. gambiae immune system by P. falciparum may be the result of the parasite adaptation to the sympatric mosquito. However, the fact that some of the mosquitoes were refractory corroborates the reports of Cirimotich et al. Collins et al. (1986), Dimopoulos et al. (2001), Julián (2010) that state the fact that the mosquito's innate defence surveillance system is also capable of triggering defence reactions, which in some cases can terminate the development of all the parasites, leading to total refractoriness.
The high sporozoite infection rates in all the study areas, which ranged between 79.55% in Ibadan and 82.61% in Ado-Ekiti, showed that the species of A. gambiae complex are effective vectors of P. falciparum in southwestern Nigeria. The sporozoite infection rates were significantly higher than the P. falciparum sporozoite infection rates for A. gambiae reported in some other studies. For instance, the P. falciparum sporozoite infection rates for A. gambiae that were recorded at Igbo-Ora in 2001 and 2002, a rural community of Oyo State in south-west Nigeria, were 6.70% and 6.30% respectively (Noutcha and Anumdu, 2009). Along Badagry Axis of Lagos Lagoon, Lagos, Nigeria, P. falciparum sporozoite infection rates of 4.8% and 6.5% for A. gambiae s.s and A. melas respectively were reported (Oyewole et al., 2010). Oduola et al. (2012) reported that P. falciparum sporozoite infection rates of A. gambiae s.s. in six rural communities of Oyo State, Southwestern Nigeria, varied between 1.9% and 3.1%. Also in Western Kenya and in the Kiruhura District (formerly part of Mbarara District) in southwest of Uganda, low P. falciparum sporozoite infection rates of 6.3% and 0.84 -5.26% for A. gambiae were recorded respectively (Echodu et al., 2010). However, high P. falciparum sporozoite rates for A. gambiae were similarly reported in some other studies. Okwa et al. (2006) reported P. falciparum sporozoite rates of 62.9% for A. gambiae in Badagry area of Lagos, Nigeria. In another study, Okwa et al. (2007) reported P. falciparum sporozoite rates of 50%, 51.2%, 53.3%, 79.4% and 95.5% for A. gambiae in Amuro Odofin, Mushin, Ajeromi, Ojo and Alimosho areas of Lagos State, Nigeria respectively. In a similar study in which A. gambiae mosquitoes were infected with P. falciparum, using direct membrane feeding, in laboratory, 83.52% sporozoite infection rate was recorded (Ndiath et al., 2011) virtually equal to the sporozoite infection rates obtained in this study. Hence, the high P. falciparum sporozoite infection rates for A. gambiae recorded in all the study areas in this study justify the reports from other studies that A. gambiae is highly infective in southwestern Nigeria (Okwa et al., 2007) and it is a prominent malaria vector in Nigeria (Annon, 2003;Gilles and Coetzee, 1987). It has also been reported that A. gambiae is the most prominent malaria vector of P. falciparum, the mosquito species with the highest sporozoite rate and the most infected mosquitoes in the rain forest zone, south-west, of Nigeria (Okwa et al., 2008). The fact that the infective and refractory strains of A. gambiae (sensu stricto) had the same polytene chromosome banding patterns showed that the infective and refractory strains cannot be distinguished using their polytene chromosome banding patterns/photomaps.

Conclusions
This study showed that the refractory strains of Anopheles gambiae complex are present, but in low frequencies, in southwestern Nigeria, and that the sibling species of Anopheles gambiae (A. gambiae s.s. and A. arabiensis) are potent malaria vectors in the rain forest region of Nigeria. Even so, no conclusion could be drawn that refractoriness is species-specific, i.e. it might not be specific to A. gambiae (sensu stricto); hence, further studies are recommended.