Age-Dependance of Pteridines in the Malaria Vector, Anopheles Stephensi
Total Page：16
File Type：pdf, Size：1020Kb
Pteridines 2017; 28(3–4): 157–161 Hamideh Edalat, Mohammad Akhoundi and Hamidreza Basseri* Age-dependance of pteridines in the malaria vector, Anopheles stephensi DOI 10.1515/pterid-2017-0009 Received May 28, 2017; accepted May 29, 2017; previously published Introduction online July 29, 2017 Awareness of the epidemiology of vectors and vector Abstract: Determining the accurate age of malaria vectors borne diseases is necessary to develop integrated control is crucial to measure the risk of malaria transmission. measures needed to reduce the impact of vector-borne A group of fluorescent chemicals derived from a pyrim- diseases [1]. To estimate the probability of vectorial capac- idine-pyrazine ring structure known as pteridines from ity of a malaria vector, Anopheline mosquito surviving 1 the head, thorax and whole body of adult female Anoph- day (p) is an essential element to find the daily rate of a eles stephensi were identified and evaluated as a tool for potentially infective contact [2]. Generally, the main goal chronological and physiological age determination of of any control operation is downsizing the vector popula- malaria vectors. The female mosquitoes were collected tion, which in turn reduces the transmission rate. Para- from an insectary colony at an interval of every 5 days, site transmission rate is directly dependent on the age of up to 30 days, and the pteridines of head, thorax and a female mosquito, therefore age determination is of great whole body were detected fluorometrically by high-pres- value to any control operation [3]. sure liquid chromatography (HPLC) using excitation and Determination of the physiological age of a female can emission wavelengths of 365 and 455 nm, respectively. be determined by the number of blood meal a female mos- In addition, alteration of the pteridines compounds was quito has taken. This blood meal is necessary to mature a compared between blood and sugar fed mosquito groups. batch of eggs. Changing the ovarian structure takes place Although four pteridines including pterin-6-carboxylic during ovulation, which is the most common method for acid, biopterin, xanthopterin and isoxanthopterin were age determination of female mosquitoes. These methods detected, some of them were absent in the head or tho- can be categorized into two types: change in the tracheal rax of mosquitoes. Levels of all four pteridines were simi- system of ovary and development of basal plates in each larly decreased in a linear manner throughout 30 days. ovariole [4]. The second method is based on the number of No significant difference in alteration of pteridine com- gonotrophic cycles [5]. pounds was observed between the two groups of blood In addition, a number of biochemical techniques or sugar fed mosquitoes. This result indicates that diet have also been used to calculate the age of individual has a little effect on pteridines alteration. Age determi- insects [6–9]. Pteridine is a group of fluorescent chemi- nation based on pteridines, as an age-grading technique, cals derived from a pyrimidine-pyrazine ring structure, could be used for field collected mosquitoes, which have and changes in the level of these molecules have been uti- either sugar or blood meal. In addition, analyzing total lized to estimate age in several dipterans [10]. By ageing pteridine fluorescence from only whole body could be a the population of some dipteran, the concentration of convenient method to estimate the age. pteridine molecules increased [11–13]. By contrast, total fluorescence decreased with age of the mosquito [9]. Pteri- Keywords: age determination; Anopheles stephensi; dine molecules can be detected and characterized in the malaria; pteridine; Southern Iran. 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 chron- *Corresponding author: Hamidreza Basseri, Department of Medical ological age of various Diptera, e.g. the new world Entomology and Vector Control, School of Public Health, Tehran screwworm Cochliomyia hominivorax [15]; the Simulium University of Medical Sciences, Tehran, Iran, damnosum complex [16]; the Mexican fruit fly Anastre- E-mail: [email protected] Hamideh Edalat and Mohammad Akhoundi: Department of Medical pha ludens [17]; the screwworms Chrysomia bezziana Entomology and Vector Control, School of Public Health, Tehran Villeneuve [18] and Cochliomyia hominivorax Coquerel University of Medical Sciences, Tehran, Iran [15]; tsetse flies Glossina spp. [11], Anopheles albimanus 158 Edalat et al.: Age-dependance of pteridines and Anopheles stephensi [8], Anopheles gambiae and Anopheles stephensi [9]; and separately were extracted in groups of 10 specimens. Extraction was stable fly Stomoxys calcitrans [19]. performed by homogenizing the specimens in Eppendorff tube con- taining 0.1 M NaOH and 0.15 M glycine with a pH of 7.2, adjusted with Pteridine concentrations were also used to measure acetic acid. The pooled thorax and head capsules were extracted in the age determination of A. gambiae Giles and A. stephensi 100 μL of homogenizer buffer (0.1 M NaOH and 0.15 M glycine). Pools Liston mosquitoes in laboratory condition [9]. Dissimilar- of 10 specimens were extracted in 600 μL of buffer. All precipitated ity of the pteridines pattern between reared and field col- proteins were removed by centrifugation (20,000 × g, 4 °C, 20 min), lected mosquitoes was demonstrated in A. albimanus. It and supernatants were twice filtered through 0.2 μm syringe filters is stated that this variation may be due to uncontrollable containing PVDF membrane (Whatman, UK). Filtrates were adjusted to pH 6.5 with acetic acid before injection into the HPLC system. All variables in the field condition such exposure to the light, procedures described for the preparation of pteridine were carried which affected the pteridines metabolism of individual out under red light. mosquito [8]. In the current study, we used A. stephensi because it is one of the most potent vector of malaria in southern Analysis of pteridines by HPLC Iran [20]. However, this study investigates the correlation between concentration and pattern of pteridine molecules The extracts were separated using a 250 × 4.6-mm column of 5 mm and age of the mosquito. The results showed that the asso- Spherisorb ODS C18 column. Pteridines were separated and eluted + ciation between pteridine concentrations and age is linear, using an isocratic mobile phase of 20 mM (Na ) phosphate pH 6.5, 4% (v/v) methanol at a flow rate of 1 mL/min. Pteridines were detected and this correlation can be used as an index to measure age fluorometrically using excitation and emission wavelengths of 365 structure of a population. Additionally, blood or sugar diet and 455 nm, respectively. As standards, the purchased pteridines did not affect on pattern of pteridine molecules. (Sigma Chemical Co.) were used as follow: isoxanthopterin, xan- thopteridin, biopteridin, pterin-6-carboxylic acid and D-neopterin. Relative fluorescent intensities were measured by Chromatogate soft- ware, and the data were inserted in MS Excel sheet to analyze data Materials and methods and obtain correlations. Mosquito populations A biological form of A. stephensi, the intermediate form was reared under uniform conditions for larval and adult nutrition, tempera- Results ture (28 ± 2 °C), humidity (70 ± 10%) and photoperiod (12 h light-dark cycle). Larvae were white trays in bowls at a density of 300 larvae per Comparing with standards purchased pteridine mole- 500 mL of distilled water with 0.01% table salt and fed on fish food. cules, four pteridines were detected in all three extracted Females were fed on 10% fructose and blood three times per week components, including isoxanthopterin, xanthopterin, from the shaved flank of an anaesthetized guinea-pig. The head and 6-biopterin and pterin-6-carboxylic acid, but no D-neop- thorax were dissected from pools of 10 female mosquitoes at 1, 5, 10, 15, 20, 25 and 30 days posteclosion. In addition, pools of 10 whole terin compound was found (Table 1). Among them, the flu- bodies of mosquitoes at similar age groups were subjected for pteri- orescent intensity of xanthopterin was highest while very dine extraction. All samples were centrifuged and then stored dry in low intensity of isoxanthopterin was observed (Figure 1). the dark at −20 °C until use for HPLC analysis. The alteration of pteridines compounds of whole body, head and thorax was measured to 30 days post- Sugar and blood fed female mosquitoes emergence. The linear reduction in fluorescent of head, thorax and whole body was observed (Figure 2). Pteri- In order to find the effect of diet on the alteration of pteridines com- dine levels were decreased with age in a linear manner pounds, a group of mosquitoes who had access to only sugar for a month was compared with other group which fed only on blood meal three times per week for 30 days. Pools of 10 whole bodies of mosqui- Table 1: Concentration of different pteridines molecules detected in toes from each group, at age of 1, 5, 10, 15, 20, 25 and 30 days old, were thorax, head and whole body of Anopheles stephensi (n = 10). subjected for pteridine extraction and then analyzed fluorometrically by HPLC. Thorax, ng Head, ng Whole body, ng Isoxanthopteridin 0.23 ± 0.2 – 0.65 ± 0.2 Pteridine extraction Xanthopteridin – 3.76 ± 0.4 7.04 ± 0.6 Biopteridin 2.11 ± 0.4 2.54 ± 0.5 6.06 ± 0.5 Pteridin-6-carboxilic acid 2.05 ± 0.4 2.87 ± 0.3 4.98 ± 0.5 The pteridines were prepared as discussed by Wu and Lehane [9]. D-neopterin – – – The whole body, thorax and head capsule of adult female mosquitoes Edalat et al.: Age-dependance of pteridines and Anopheles stephensi 159 1000 XAN Retention time 900 750 800 , mV 700 600 500 BIO 500 Blood fed olts V 400 PCA Sugar fed 250 e fluorescent intensity 300 200 Relativ ISO 100 0 0 1 5 10 15 20 25 30 Days 01234 Time, min Figure 3: Alteration of pteridines compounds of the whole body of Anopheles staphensi females fed on blood and sugar during 30 days.