Age-dependance of pteridines in the malaria vector, Anopheles stephensi

Introduction

Awareness of the epidemiology of vectors and vector borne diseases is necessary to develop integrated control measures needed to reduce the impact of vector-borne diseases [1]. To estimate the probability of vectorial capacity of a malaria vector, Anopheline mosquito surviving 1 day (p) is an essential element to find the daily rate of a potentially infective contact [2]. Generally, the main goal of any control operation is downsizing the vector population, which in turn reduces the transmission rate. Parasite transmission rate is directly dependent on the age of a female mosquito, therefore age determination is of great value to any control operation [3].

Determination of the physiological age of a female can be determined by the number of blood meal a female mosquito has taken. This blood meal is necessary to mature a batch of eggs. Changing the ovarian structure takes place during ovulation, which is the most common method for age determination of female mosquitoes. These methods can be categorized into two types: change in the tracheal system of ovary and development of basal plates in each ovariole [4]. The second method is based on the number of gonotrophic cycles [5].

In addition, a number of biochemical techniques have also been used to calculate the age of individual insects [6], [7], [8], [9]. Pteridine is a group of fluorescent chemicals derived from a pyrimidine-pyrazine ring structure, and changes in the level of these molecules have been utilized to estimate age in several dipterans [10]. By ageing the population of some dipteran, the concentration of pteridine molecules increased [11], [12], [13]. By contrast, total fluorescence decreased with age of the mosquito [9]. Pteridine molecules can be detected and characterized in the small quantities by fluorescence spectroscopy; therefore, this technique is very useful for age determination of field population of insects [9], [14].

Pteridines have been used to determine the chronological age of various Diptera, e.g. the new world screwworm Cochliomyia hominivorax [15]; the Simulium damnosum complex [16]; the Mexican fruit fly Anastrepha ludens [17]; the screwworms Chrysomia bezziana Villeneuve [18] and Cochliomyia hominivorax Coquerel [15]; tsetse flies Glossina spp. [11], Anopheles albimanus [8], Anopheles gambiae and Anopheles stephensi [9]; and stable fly Stomoxys calcitrans [19].

Pteridine concentrations were also used to measure the age determination of A. gambiae Giles and A. stephensi Liston mosquitoes in laboratory condition [9]. Dissimilarity of the pteridines pattern between reared and field collected mosquitoes was demonstrated in A. albimanus. It is stated that this variation may be due to uncontrollable variables in the field condition such exposure to the light, which affected the pteridines metabolism of individual mosquito [8].

In the current study, we used A. stephensi because it is one of the most potent vector of malaria in southern Iran [20]. However, this study investigates the correlation between concentration and pattern of pteridine molecules and age of the mosquito. The results showed that the association between pteridine concentrations and age is linear, and this correlation can be used as an index to measure age structure of a population. Additionally, blood or sugar diet did not affect on pattern of pteridine molecules.

Materials and methods

Results

Comparing with standards purchased pteridine molecules, four pteridines were detected in all three extracted components, including isoxanthopterin, xanthopterin, 6-biopterin and pterin-6-carboxylic acid, but no D-neopterin compound was found (Table 1). Among them, the fluorescent intensity of xanthopterin was highest while very low intensity of isoxanthopterin was observed (Figure 1).

Figure 1:

Detection of pteridines from whole body extraction of Anopheles stephensi.

Four pteridines were detected, including pterin-6-carboxylic acid (PCA), biopterin (BIO), xanthopterin (XAN) and isoxanthopterin (ISO).

The alteration of pteridines compounds of whole body, head and thorax was measured to 30 days post-emergence. The linear reduction in fluorescent of head, thorax and whole body was observed (Figure 2). Pteridine levels were decreased with age in a linear manner for all heads (R²=0.82), thorax (R²=0.72) and whole body (R²=0.88). Nearly eight-fold decrease in total pteridines was noted during the 30-day experimental period in head, thorax and whole body (Figure 2). No significant difference was observed in alteration of total pteridine between blood and sugar fed mosquitoes (Figure 3). The ratio of fluorescent intensity of xanthopterin, biopterin and pterin-6-carboxylic acid (comparing with Figure 1) deceased similarly throughout 30 days by ageing of mosquitoes (Figure 4).

Figure 2:

Comparison of the alteration of pteridines compounds of the whole body, head and thorax of Anopheles staphensi females during 30 days.

Pools of 10 specimens were extracted in 600 μL of buffer. Pteridine levels were decreased with age in a linear manner for all heads (R²=0.82), thorax (R²=0.72) and whole body (R²=0.88); n=10.

Figure 3:

Alteration of pteridines compounds of the whole body of Anopheles staphensi females fed on blood and sugar during 30 days.

The regression analysis for the blood-fed females for the period 1–30 days is Y=111.5X+741.7; p<0.001; r²=0.921. The regression analysis for the sugar-fed females for the period 1–30 days is Y=114.1X+773.7; p<0.001; r²=0.895; n=10.

Figure 4:

HPLC chromatograms of pteridines extracted from whole body of adult Anopheles stephensi of different age postemergence. The ratio of fluorescent intensity of xanthopterin, biopterin and pterin-6-carboxylic acid deceased similarly throughout 30 days by ageing of mosquitoes. Ten female mosquitoes in each age group were homogenized 100 mL of buffer containing 0.1 M NaOH and 0.15 M glycine with a pH of 7.2, centrifuged and after 0.2μm filtration loaded on 250×4.6 mm column of 5mm Spherisorb ODS C 18 column.

Discussion

Age determination of mosquitoes is critical in the transmission cycles of mosquito-borne pathogens [21]. The ovarian dissection technique, which is usually used for determination of the chronological age of mosquitoes, has some technical limitation such as the proportion of diagnostic ovarioles in the ovary declines with age and/or too few diagnostic ovarioles may occur in some mosquito populations [22].

The current study was conducted to determine whether pteridine compounds were suitable for age estimation of A. stephensi. We demonstrated a negative linear correlation between pteridine levels and chronological age of A. stephensi (Figure 2). Presence of isoxanthopterin, xanthopterin, biopterin and pterin-6-carboxylic acid was previously reported by Wu and Lehane [9] in An. gambiae and A. stephensi. We comparatively found xanthopterin at a relatively high level in whole body extraction (Figures 1 and 3) and, in contrast to the results of Wu and Lehane [9] showed biopterin as the most common pteridine molecule in their colony of A. stephensi. This dissimilarity in results may be due to different geographical population of A. stephensi we used. Change in molecular structure may occur in geographical populations of some insects [23]. As in previous study, we found variation in molecular receptors on midgut epithelial cells of the geographical populations of A. stephensi [24]. However, existence of dissimilarity of pteridines molecules either among species or geographical population of mosquitoes is inevitable. Thus, it is important to assemble the calibration curve for each area, and if possible, specimens used in assembling should be from mark-release-recaptured experiments.

However, total concentration of pteridines in body part would seem a more promising indicator of age determination as previously used for A. albimanus [8]. More recently, 6-biopterin as a standard was used to estimate pteridine concentration in honey bee, Apis mellifera [25]. However, their results did not show a variation among different population of honey bee.

Furthermore, the survival rate of mosquitoes depend on the environmental and seasonal conditions of the area. Hugo et al. [26] used transcriptional profiling to estimate age of Aedes aegypti in semifield condition during three seasons in a tropical area where Dengue fever transmission occurs periodically [26]. The authors demonstrated that the survival rate of the A. aegypti is dependent on seasons by estimating age of individual mosquito.

However, in a comparative field trial, if the technique becomes standardized for each environment, the quantification of fluorescence levels as simple, accurate and rapid technique can provide useful information on age structures of mosquito population. In conclusion, it is important to assemble the calibration curve on the same machine that will be used for determining the age of unknown mosquitoes. In this study, variation in level of different pteridine molecules was observed in different ages. Therefore, analyzing the total fluorescence level of whole body could be an appropriate estimation for age determination of mosquitoes.