Pteridine concentrations differ between ... - Wiley Online Library

Medical and Veterinary Entomology (2002) 16, 225±234

Pteridine concentrations differ between insectary-reared and field-collected Anopheles albimanus mosquitoes of the same physiological age R . P . P E N I L L A * , M . H . R O D R IÂ G U E Z y, A . D . L OÂ P E Z * , J . M . V I A D E R - S A L V A D OÂ z and C . N . S AÂ N C H E Z z *

Centro de InvestigacioÂn de Paludismo, Tapachula, Chiapas, yCentro de Investigaciones sobre Enfermedades Infecciosas, Cuernavaca, Morelos, and zDepartamento de BioquõÂ mica, Facultad de Medicina, Universidad AutoÂnoma de Nuevo LeoÂn, Mexico

Abstract. Biopterin, isoxanthopterin and 6-pterincarboxylic acid were identified in the head of the malaria vector mosquito Anopheles albimanus Weidemann (Diptera: Culicidae) by HPLC. Total pteridine concentrations (TPC) were estimated in heads, body parts (BP: abdomen, legs and wings) and whole bodies of insectary-reared and field-collected females, by spectrofluorometry, to investigate whether they could be used for age determination. Pteridine concentrations diminished with age in both mosquito groups. TPC correlated with chronological age in insectary-reared sugar-fed females (heads: r2 0.35, BP: r2 0.34, P < 0.001), but lower correlation occurred in blood-fed females (heads: r2 0.22, BP: r2 0.27). TPC differed among females of the same age fed with blood at different times (P < 0.05), indicating that bloodmeals modify the diminution rate of pteridines with age. Nevertheless, a polynomial significant correlation was documented for TPC and the number of ovipositions (heads: r2 0.24, BP: r2 0.27, whole body: r2 0.52, P < 0.001) in insectary-reared mosquitoes. This correlation was lower in field-collected mosquitoes (heads: r2 0.14, BP: r2 0.10, P < 0.05), which showed a remarkable pteridine increase in one-parous females. The correlation of TPC in whole body with physiological age was much less (r2 0.03). These observations indicate that TPC determination by spectrofluorometry is not a reliable method to estimate the age of An. albimanus females from the feral population. Key words. Anopheles albimanus, age determination, feral mosquitoes, fluorescence, HPLC, insectary, malaria vector, mosquito age, physiological age, pteridines, spectrofluorometry, Mexico.

Introduction The age structure of mosquito populations is an important parameter for the estimation of their vectorial capacity (Garrett-Jones, 1964), and could be used to monitor fluctuations in population size and dispersal rate (Tyndale-

Correspondence: Dr R. P. Penilla, Centro de InvestigacioÂn de Paludismo, Instituto Nacional de Salud PuÂblica, Apartado postal 537 Tapachula, Chiapas 30700, Mexico. E-mail: [email protected] #

2002 The Royal Entomological Society

Biscoe, 1984). These estimations are frequently used to predict or monitor vector populations response to control programmes (Detinova, 1968), as in the case of malaria control using insecticides aimed at reducing the possibility of mosquitoes surviving to sporogony (MacDonald, 1957; Bown et al., 1991). The most popular method used to determine anopheline physiological age is based on the observation of morphological changes occurring in the ovaries after each oviposition (Detinova, 1962). However, this timeconsuming method requires special training, which limits its application. There is therefore a need to develop new easily 225

226 R. P. Penilla et al. performed methods, that could be automated, in order to handle bigger sample sizes. Pteridines are purin metabolic products (fluorescent pigments) synthesized in the fat body and transported to other body parts, where they accumulate and function as light filters in eyes, enzyme cofactors or sexual coloration pigments (Ziegler & Harmsen, 1969). Pteridines have been used to determine the chronological age of various Diptera, e.g. the Mexican fruit fly Anastrepha ludens Loew (TomicCarruthers et al., 1996), the screw-worms Chrysomia bezziana Villeneuve (Wall et al., 1990) and Cochliomyia hominivorax Coquerel (Thomas & Chen, 1989), tsetse flies Glossina spp. (Lehane & Mail, 1985; Langley et al., 1988; Msangi & Lehane, 1991), the horn-fly Haematobia irritans (L.) (Krafsur et al., 1992), blowfly Lucilia sericata Meigen (Wall et al., 1991), face-fly Musca autumnalis De Geer (Krafsur et al., 1995), housefly Musca domestica (L.) (McIntyre & Gooding, 1994), stable fly Stomoxys calcitrans (L.) (Mail et al., 1983; Lehane et al., 1986) and the blackfly Simulium damnosum Theobald s.l. (Cheke et al., 1990; Millest et al., 1992). In all these species, pteridine concentrations correlated linearly with age, their measurement being more reliably indicative than the follicular relic method of age-grading for S. calcitrans (Mail et al., 1983). Wu & Lehane (1999) demonstrated that pteridine concentrations in laboratory Anopheles gambiae Giles and An. stephensi Liston mosquitoes were inversely proportional to their age. This observation suggested that pteridine quantification could be used for age determination of mosquito populations. However, as environmental conditions and exposure to light differ between insectary-reared and field-collected mosquitoes, a test using field-collected mosquito populations was necessary to explore this possibility. The objective of this study was to identify and measure pteridines in body parts and whole bodies of insectary-reared and field-collected An. albimanus females, one of the main malaria vectors in Mexico and Central America (RodrõÂ guez & Loyola, 1989), and to investigate their relationship to chronological and physiological age.

Materials and methods Mosquito populations Females of Anopheles albimanus were obtained from field collections near Tapachula and from an insectary colony (white-striped strain) maintained at the Centro de InvestigacioÂn de Paludismo (RodrõÂ guez et al., 1992). Insectary-reared mosquitoes were maintained at 25±27 C and relative humidity 60±70% on a diet of 10% sucrose solution, with LD 12 : 12 h photoperiod. Also, An. albimanus females were captured during the evening, resting on vegetation and in cattle corrals in villages of the coastal lowlands of southern Chiapas State, Mexico. They were transported to laboratory and processed the next morning. #

Pteridine identification For pool analysis of five Anopheles albimanus females, the heads were removed and homogenized with 300 mL of chloroform : methanol (2 : 1) in an Eppendorf tube. Five hundred mL of glycine (11.5 g/L, pH 10) were added, stirred for 2 min and centrifuged at 2040 g for 4 min. Ten mL of HCl 12 N were added to the supernatant and filtered through 0.45 mm Durapore filters (Millipore, CA., U.S.A.). Pteridine identification in supernatants was carried out in the Facultad de Medicina, Universidad AutoÂnoma de Nuevo LeoÂn by high performance liquid chromatography (HPLC, System Gold, Beckman,) in ion-pair mode in an analytic Ultrasphere-ODS (5 mm, 150 4.6 nm, Beckman) reverse-phase column, and fluorescence detection (System Gold, Beckman) (excitation filter 305±395 nm, emission filter 420±650 nm). A gradient elution with solution A (sodium heptanosulphonate 10 mM, 1% AcOH in deionized water) and solution B (sodium heptanosulphonate 10 mM, 1% AcOH in methanol : deionized water 70 : 30) from 0% B to 35% B by concave curve at 20 min, 35% B for 3 min and from 35% B to 0% B at 5 min, a flow rate of 1 mL/min was used. Eluents were filtered through 0.45 mm Durapore filters and degassed before using. The chromatogram obtained from the extract of heads was compared with chromatograms of commercial standard pteridines [isoxanthopterin, D-()-neopterin, leucopterin, xanthopterin, 6-pterincarboxylic acid, biopterin, pterine, 6,7-dimethylpterine, aminopterin (Sigma Chemicals, St Louis, MO, U.S.A.) and folic acid (ICN Biochemicals Cleveland, OH, U.S.A.)] and a mixture of all of them. The peaks were correlated by retention times and retention times relative to that of biopterin.

Pteridine concentration measurement in laboratory and field-collected mosquitoes Total pteridine concentration (TPC) in heads, body parts (BP: abdomen, legs and wings) and whole body of individual An. albimanus females was measured using the method of Lehane & Mail (1985). Heads, BP or whole body of individual mosquitoes were separated and homogenized in a mortar with 500 mL of chloroform : methanol (2 : 1 v/v) and 150 mL of 0.1 M NaOH adjusted with glycine to pH 10. The mortar was washed three times with 200 mL of 0.1 M NaOH. Homogenized samples were sonicated to 16 amplitude microns for 20 s and centrifuged at 1500 g for 3 min. The aqueous phase of each sample was added to 0.1 M NaOH to produce a final 2 mL solution. Solutions were filtered (0.45 mm nucleopore, Costar, CA, U.S.A.) and their fluorescence was measured using a Sequoia-Turner Model 450 Fluorometer, CA, U.S.A. (gain 50, excitation monochromator set at 360 nm and emission monochromator set at 430 nm). Samples were read against a control solution of 0.1 M NaOH pH 10. Pteridine concentrations were calculated using a standard calibration curve made

2002 The Royal Entomological Society, Medical and Veterinary Entomology, 16, 225±234

Pteridine variation in Anopheles females

6.5 8.6 10.6 10.8 12.0 13.2 14.6 15.5 22.4 23.2

Results Identification of pteridines Only isoxanthopterin, 6-pterincarboxylic acid and biopterin were identified in mosquito head extracts by HPLC. No other pteridines corresponding to other standards retention times were detected, but other peaks, which #

9.6 0.0 6.4 53.6 58.9

rt (min)

RRT (min)

0.16 0.53 82.50 17.64 0.48

6.8 ± ± ± 12.2

54.1 ± ± ± 17.6

115.00 0.59 0.83 0.22 636.50

± 14.8 ± ± ±

± 0.0 ± ± ±

0.2000 0.1000

21.05

14.77 1.70 2.67

12.22 6.76

0.0000

The physiological age of field-collected An. albimanus females was determined by ovarian dissection using the method suggested by Detinova (1962). TPC was determined in heads, BP (wings and legs) and whole body of these specimens. The size of mosquitoes tested (heads and BP) was also estimated by measuring the wing length. Correlations between TPC and mosquito size and between TPC and physiological age were investigated using a linear or polynomial regression analysis. A t-test was used to compare TPC between different physiological stages and TPC and size between insectary-reared and fieldcollected mosquitoes (Zar, 1984).

55.5 41.1 27.4 26.0 17.8

MDC (ng/head)

0.0000

Effect of body size and physiological age on pteridine concentrations in field-collected mosquitoes

RRT (min)

7.07

Isoxanthopterin D-()-Neopterin Leucopterin Xanthopterin 6-Pterincarboxylic acid Folic acid Biopterin Pterine 6,7-Dimethylpterine Aminopterin

2.08

Insectary-reared An. albimanus were kept in groups of 1000 in either of the following conditions: (1) maintained on a diet of 10% sucrose only; (2) fed on a rabbit on day 2 after emergence and allowed to oviposit and fed on a rabbit after each oviposition. These mosquitoes were maintained on 10% sucrose between bloodmeals. Batches of mosquitoes from the last group were separated after every oviposition and maintained on a sucrose diet afterwards. Individual samples of mosquitoes were taken every day for pteridine determinations in heads and BP (abdomens, legs and wings). These experiments were repeated three times, each one from different mosquito batches. The size of all mosquitoes tested was estimated by measuring the wing length (both extremes) using a stereoscopic ocular micrometer. Pteridine content was also analysed in whole bodies of nulliparous mosquitoes and samples of mosquitoes taken immediately after every one of four ovipositions. Correlations between TPC and mosquito size and between TPC and the chronological and physiological age were investigated using a linear or polynomial regression analysis. A one factor ANOVA or t-test were used to compare TPC within and between physiological stages, within chronological age (Zar, 1984).

rt (min)

Compound

0.2000

Effect of body size, age, blood-feeding and oviposition on pteridine concentrations in insectary-reared mosquitoes

Table 1. Pteridines detected in the head of Anopheles albimanus. Chromatographic retention time (rt), relative retention time to biopterin (RRT) and minimal detectable concentration (MDC) of the 10 standard compounds and mosquito head extract.

0.1000

from a standard solution of 6-pterincarboxylic acid (Sigma Chemicals) in 0.1 M NaOH, pH 10.

227

0.00

10.00

20.00

28.00

Fig. 1. HPLC chromatogram of head extract of five Anopheles albimanus females, showing the characteristic retention time (min) and fluorescence for isoxanthopterin (6.76), 6-pterincarboxylic ac. (12.22) and biopterin (14.77).

did not correspond to any of the pteridine standards, were observed (Table 1, Fig. 1). Effect of size, age, blood-feeding and oviposition on pteridine concentrations in insectary-reared mosquitoes. TPC was determined in 705 heads, 752 BP and 480 whole bodies of insectary-reared mosquitoes. The range of TPC was 2.18±20.10 ng in heads, 10.10±60.56 ng in BP and 0±88.54 ng in whole bodies. A significant linear correlation was found between mosquito size and TPC of BP (r2 0.16, P < 0.001), but no correlation existed with TPC of heads (r2 < 0.01, P > 0.6). The correlation between mosquito size and TPC of BP was more evident in sugar-fed mosquitoes (r2 0.21, P < 0.001; Fig. 2). There was a significant negative linear correlation (P < 0.001) between TPC in heads (r2 0.16) and


228 R. P. Penilla et al.

Fig. 2. Effect of mosquito size on total pteridine concentration (TPC) in body parts (abdomens, wings and legs) of insectary-reared Anopheles albimanus females: fed on sucrose and blood (a) and sucrose-fed only (b).

Fig. 3. Effect of the chronological age on pteridine concentration in insectary-reared Anopheles albimanus females: individual head (a,c); body parts (b,d); blood-fed (a,b); sucrose-fed nulliparous (c,d). #


Pteridine variation in Anopheles females BP (r2 0.23) and the chronological age of insectary-reared mosquitoes (Fig. 3), data fitted in a cubic or quadratic curve improved this correlation (P < 0.001, r2 0.22 and r2 0.27, respectively). Correlations were slightly better when only data from sucrose-fed nulliparous mosquitoes were plotted (heads: linear, r2 0.22 or quadratic, r2 0.35 and BP: linear, r2 0.31 or cubic, r2 0.34; Fig. 3). There was a significant negative quadratic correlation (P < 0.001) between the number of ovipositions and the TPC in heads (r2 0.24), BP (r2 0.27) and whole body (r2 0.52) (Fig. 4) in blood-fed mosquitoes. TPCs in head extracts of all physiological age groups were statistically lower (P < 0.01) than that of nulliparous females (mean SE in nulliparous, 11.11 0.4 ng, n 57; with one oviposition, 8.10 0.5 ng, n 31; with two ovipositiones, 6.47 0.4 ng, n 49; with three ovipositions, 7.17 0.4 ng, n 39; with four ovipositions 9.11 0.5 ng, n 56).

229

TPCs in BP of females with four, three and two ovipositions were statistically lower (P < 0.01) than that of nulliparous and one oviposition females (mean SE in nulliparous, 39.69 1.1 ng, n 58; with one oviposition, 37.12 1.0 ng, n 59; with two ovipositions, 31.99 1.1 ng, n 49; with three ovipositions, 28.04 1.2 ng, n 39; with four ovipositions, 27.40 1.1 ng, n 60). TPC in BP of females with four ovipositions was also statistically lower than that of females with two ovipositions (P < 0.01). TPC in whole bodies of females of all physiological ages were statistically lower than that of females within each previous oviposition group (P < 0.001, n 96 for each oviposition group), except between females of the groups with three and four ovipositions (mean SE in nulliparous, 56.22 1.5 ng; with one oviposition, 48.45 1.1 ng; with two ovipositions, 34.31 1.3 ng; with three ovipositions, 20.10 1.7 ng; with four ovipositions 18.05 1.6 ng).

Fig. 4. Effect of physiological age on pteridine concentration in insectary-reared Anopheles albimanus females: individual head (a); body parts (b) and whole body (c). Only females that had ingested another bloodmeal after each oviposition were included. #


230 R. P. Penilla et al. Table 2. Effect of bloodmeals on pteridine concentration in heads of insectary-reared females of the same chronological age (mean SE, n in parentheses), according t-test or one factor ANOVA with multiple comparisons. Bloodmeals number Age day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

1

11.36 0.68 (20) 12.42 1.17 (9) 11.67 0.99 (8) 10.04 0.77 (20) 7.39 0.62 (8) 7.85 0.41 (19) 7.61 1.08 (9) 6.58 0.46 (8) 7.16 0.46 (18) 5.18 0.39 (9)

2

3

4

7.84 0.80 (10) 8.58 0.89 (12) 7.77 0.55 (9) 6.01 0.27 (20) 8.23 0.47 (20)

6.70 0.36 (19) 6.73 0.73 (9) 7.02 0.36 (18) 7.95 1.13 (7) 10.19 0.79 (4) 5.67 0.62 (3)

7.96 0.41 (19) 6.81 0.28 (8) 5.33 0.50 (15) 6.51 0.39 (10) 5.81 0.22 (10) 7.00 0.28 (10)

7.59 0.76 (19) 6.09 0.91 (11) 6.79 0.65 (9) 6.56 0.66 (17)

6.49 0.39 (20)

5.68 0.35 (10) 5.83 0.24 (9) 8.09 0.39 (18)** 5.72 0.42 (10) 5.85 0.33 (17) 5.25 0.28 (10)

10.15 0.89 (27)*

5.18 0.16 (10)

6.49 0.30 (18) 4.53 0.26 (10)

6.30 0.35 (30)*

5.72 0.24 (27) 6.66 0.38 (20)

19 20

6.22 0.16 (9)

21

11.30 0.31 (10)***

5.74 0.38 (14) 4.09 0.20 (10)

22 23

12.34 0.66 (8)*** 7.42 0.67 (10)*

4.60 0.18 (10) 4.23 0.21 (10)

Bold numbers indicate values significantly higher than the previous or subsequent bloodmeal group. *P < 0.05, **P < 0.01 and ***P < 0.001.

In mosquitoes of the same chronological age, a significant increase in TPC in BP, but not in head extracts, was detected in females with one or two bloodmeals, compared to that of nulliparous females (P < 0.05, Tables 2 and 3). However, significant increases in TPC in head and BP extracts were observed after the third and #

fourth bloodmeals in females from 11 to 22 days old, compared to that of females with one or two bloodmeals (P < 0.05). In these mosquitoes, TPCs were significantly higher in BP than in heads (mean SE in heads, 8.09 0.2 ng, in BP (abdomen, wings and legs), 30.86 0.7 ng, P < 0.001).


Pteridine variation in Anopheles females

231

Table 3. Effect of bloodmeals on pteridine concentration in BP of insectary-reared females of the same chronological age (mean SE, n in parentheses), according t-test or one factor ANOVA with multiple comparisons. Bloodmeals number Age day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0 35.97 2.02 (20) 45.52 1.94 (10) 38.61 1.58 (18) 43.28 2.86 (10) 32.0 1.62 (20) 41.98 3.72 (10) 21.26 0.69 (9) 29.12 1.45 (10) 27.12 2.12 (19) 23.09 1.64 (10) 21.75 1.36 (10) 28.05 1.48 (19)

1

2

3

4

41.49 1.76 (20)*** 32.77 1.30 (10) 35.26 2.06 (19)*** 36.26 1.37 (10)* 30.19 1.33 (19) 25.81 2.56 (10) 27.31 0.95 (19) 27.98 0.75 (10) 26.35 1.31 (10)

25.98 1.93 (10) 35.46 1.42 (19)* 32.19 2.97 (10)* 30.66 1.665 (19)** 23.51 2.01 (10) 24.70 1.21 (20)

32.23 2.62 (10)** 26.91 2.30 (10) 32.24 1.40 (9)* 24.82 2.89 (10) 27.75 1.97 (10)

24.81 1.45 (20)

20.72 1.18 (10) 27.12 1.69 (10)

25.07 1.44 (20)

21.18 0.99 (10) 30.39 1.73 (10)

25.49 1.11 (10)

19.75 1.63 (10) 22.68 2.23 (9)

18.37 2.07 (10) 24.01 0.91 (20)

26.88 1.10 (10)* 23.51 1.67 (10)

29.21 1.43 (20)*** 35.0 2.05 (10)***

23.33 1.60 (10)

18.39 1.39 (3) 25.74 1.92 (7)

17.12 1.94 (5) 24.23 1.90 (14)

22.58 1.09 (10) 28.88 1.67 (10)

22.72 1.63 (20) 24.92 1.49 (8)

25.74 1.59 (8) 19.98 2.31 (4)

25.21 1.55 (6) 26.77 1.38 (10)* 31.65 3.25 (10)

20 21

20.38 1.92 (9)

22

Bold numbers indicate values significantly higher than the previous or subsequent bloodmeal group. *P < 0.05, **P < 0.01 and ***P < 0.001.

Effect of size and physiological age on pteridine concentrations in field-collected specimens TPC was determined in heads and BP of 81 field-collected An. albimanus females, and in whole bodies of 960. The range of TPC was 2.42±10.56 ng in heads, 2.42±13.82 ng in BP, and 7.74±130.81 ng in whole bodies. No correlation was #

found between mosquito size and TPC in heads or BP (r2 < 1%, P > 0.3). The relationship between TPC and the number of ovary dilatations was fitted in a quadratic regression (P < 0.01) in heads (r2 0.14) and BP (r2 0.11), whereas correlation in whole body was lower with a lineal regression (r2 0.03) (Fig. 5). TPC in heads (mean SE 4.43 0.2 ng, n 3) and BP (mean SE 3.19 0.4 ng,


232 R. P. Penilla et al.

Fig. 5. Effect of physiological age on pteridine concentration in field-collected Anopheles albimanus females: individual head (a); body parts (b) and whole body (c).

n 3) of four ovary-dilatations females were significantly lower (P < 0.05) than all the groups of minor physiological age except those of three dilatations (mean SE in heads of nulliparous, 6.01 0.3 ng, n 28; with one dilatation, 7.12 0.2 ng, n 27; with two dilatations, 6.58 0.3 ng, n 18; with three dilatations, 5.9 0.6 ng, n 5 and BP in nulliparous, 6.74 0.4 ng, n 28; with one dilatation, 7.32 0.4 ng, n 27; with two dilatations, 7.31 0.5 ng, n 18; with three dilatations, 7.16 1.5 ng, n 5). TPC in whole bodies of females with one to three dilatations (mean SE in females with one dilatation, 35.87 0.9 ng, n 292; with two dilatations, 33.88 1.7 ng, n 65; with three dilatations, 29.91 5.2 ng, n 13) were significantly lower (P < 0.05) than that of nulliparous females (mean SE, 40.53 0.7 ng, n 590). Pteridine concentrations were statistically higher (P < 0.05) in BP (mean SE, 6.95 0.2 ng) than in heads #

(mean SE, 6.44 0.2 ng) of field-collected mosquitoes. The size of these mosquitoes was significantly higher than those of insectary-reared mosquitoes (mean SE wing length, 3.63 0.03 mm and 3.35 0.01 mm, respectively, P < 0.01). However, TPC in heads of insectary-reared nulliparous females were 1.85 times significantly higher than those of field-collected ones (mean SE, 11.11 0.43 ng (n 57) and 6.01 0.35 ng (n 28), respectively, P < 0.001), being similar between insectary-reared and field-collected mosquitoes when compared within each physiological age in parous females. Similar results were obtained in whole body TPC, where insectary-reared nulliparous females were 1.39 times significantly higher than those of field-collected mosquitoes (mean SE, 56.22 1.53 ng, n 96 and 40.53 0.65 ng, respectively, n 590, P < 0.001). However in whole body TPC, these differences were only detected in females with one oviposition, with 1.35 times significantly higher TPC


Pteridine variation in Anopheles females concentration in insectary-reared mosquitoes compared to field-collected mosquitoes (mean SE, 48.45 1.06 ng, n 96 and 35.87 0.86 ng, n 292, respectively, P < 0.001). Discussion The pteridines biopterin, isoxanthopterin and 6-pterincarboxylic acid, as identified in head extracts of An. albimanus, are among the most commonly distributed natural pteridines of insects (Chefurka, 1965). All of them were also identified in whole body extracts of An. gambiae and An. stephensi (Wu & Lehane, 1999), and biopterin was previously identified in adults of An. atroparvus Van Thiel, An. labranchiae Falleroni and An. stephensi (Cerioni et al., 1975). It is possible that the pteridines we identified are not the only ones present in An. albimanus, and that others were present at very low concentrations for their detection by the chromatographic conditions we used. However, Wu & Lehane (1999) estimated that these contribute only a small proportion (1±10%) of the total pteridine-derived fluorescence in anophelines. Total pteridine concentration in BP increased with body size in insectary-reared mosquitoes, but the linear regression analysis indicated that this correlation explains only 16% of the variability in the data, and when pteridine concentrations were analysed in relation to mosquito age, no differences were observed after adjusting for body size. This observations agree with those in Glossina, indicating that body size does not affect the accumulation rate of pteridines in these insects (Wall et al., 1991). By contrast, Wu & Lehane (1999), using reversed-phase HPLC to measure pteridines, demonstrated that introducing a correction for mosquito body size considerably improved the predictive value of pteridine-derived fluorescence for mosquito age. This indicates the importance of the sensitivity of the assay method; possibly the less sensitive method (spectrofluorometry) used in our study precluded the detection of small variations in pteridines in heads of both insectaryreared and field-collected An. albimanus and in BP of feral mosquitoes. As for An. stephensi and An. gambiae (Wu & Lehane, 1999), we found no accumulation of pteridines with age in An. albimanus female. This may be associated with nocturnal activity. Pteridines accumulate in insects when stimulated by sunlight (Hanser, 1948) and function as eye pigments in day-active species (Ziegler & Harmsen, 1969). Like other insects that do not use pteridines as light filters, screening pigments or sexual dyes (Laudani & Lecis, 1970), mature anophelines apparently eliminate these compounds. The observed reduction of pteridine concentrations with age of Anopheles raised the possibility of their use as a simpler and faster technique than ovarian dissection for mosquito age determination (Wu & Lehane, 1999). Our results with insectary-reared An. albimanus coincide with those observed for An. gambiae and An. stephensi under laboratory conditions. However, pteridine concentrations in field-collected An. albimanus females did not follow the #

233

same pattern as for those reared in the insectary. This was probably due to uncontrollable variables in the field, such as frequency and type of bloodmeal sources and exposure to light. The effect of bloodmeals was observed in insectaryreared mosquitoes of the same chronological age, but different feeding history. Chronological age and pteridine concentrations correlated better in sugar-fed mosquitoes. Whereas pteridine levels significantly increased in heads after the third or fourth bloodmeal, and in BP after each bloodmeal, this occurred in a small proportion of mosquitoes and was not reflected in the correlation curve. In field-collected mosquitoes, pteridine levels increased in heads and BP after the first bloodmeal (represented by one ovarian dilatation), but tended to decrease with physiological age. The initial increase followed by a decrease resulted in an overlap between TPC in very young and the oldest female groups. When whole bodies were used, a very good correlation was observed in insectary-reared mosquitoes, with a decrease in pteridines as physiological age progressed (r2 0.52), but this correlation was very poor (r2 0.03) in field-collected mosquitoes. Although the spectrofluorometric method used in this work is less sensitive than the reversed-phase HPLC used by Wu & Lehane (1999), our findings of a decline in pteridine with age coincide with theirs for insectary-reared Anopheles. Hence, the variable pteridine concentration with age of feral mosquitoes may be attributed to factors affecting pteridine metabolism, rather than any imprecision of our assay method. Similarly, Lardeux et al. (2000) could not correlate mosquito age with TPC spectrofluorometrically for the diurnally active Aedes polynesiensis Marks and the nocturnally active Culex quinquefasciatus Say. Our findings for An. albimanus show more conclusively that quantification of pteridines cannot be used for age determination of individual mosquitoes from feral populations.

Acknowledgements We thank D.N. Bown and R.M. Chandler-Burns for their critical reading of the manuscript and A. D. RodrõÂ guez for helping in the statistical analysis. The help of M.C. RodrõÂ guez, C. Villarreal and J.I. Arredondo is also appreciated.

References Bown, D.N., RodrõÂ guez, M.H., Arredondo-JimeÂ nez, J.I., Loyola, E.G. & RodrõÂ guez, M.C. (1991) Age structure and abundance levels in the entomological evaluation of an insecticide used in the control of Anopheles albimanus in Southern Mexico. Journal of the American Mosquito Control Association, 7, 180±187. Cerioni, E., Contini, C. & Laudani, U. (1975) Occurrence and distribution of known pterins in some species of Diptera. Chemistry and Biology of Pteridines (ed. by W. Pfleiderer),


234 R. P. Penilla et al. pp. 851±860. Proceedings of the 5th International Symposium of the University of Konstanz, Switzerland. Chefurka, W. (1965) Metabolism of pterins. The Physiology of Insecta, Vol. 2. Academic Press, London, pp. 730±737. Cheke, R.A., Dutton, M., Avissey, H.S. & Lehane, M.J. (1990) Increase with age and fly size of pteridine concentrations in different members of the Simulium damnosum species complex. Acta Leidensia, 59, 307±14. Detinova, T.S. (1962) Age-grouping methods in Diptera of medical importance with special reference to some vectors of malaria. World Health Organization, Monograph Series, 47, 1±216. Detinova, T.S. (1968) Age structure of insect populations of medical importance. Annual Review of Entomology, 13, 427±450. Garrett-Jones, C. (1964) The human blood index of malaria vectors in relation to epidemiological assessment. Bulletin World Health Organization, 30, 241±261. Hanser, G. (1948) Uber die histogenese der augenpigmentgranula bei verschiedenen rassen von Ephestia kuhniella Z. und Ptychopoda seriata Schrk Zeitschrift fuÈr Vererblehre, 82, 74±97. Krafsur, E.S., Moon, R.D. & Kim, Y. (1995) Age structure and reproductive composition of summer Musca autumnalis (Diptera: Muscidae) populations estimated by pterin concentrations. Journal of Medical Entomology, 32, 685±96. Krafsur, E.S., Rosales, A.L., Robison-cox, J.F. & Turner, J.P. (1992) Age structure of Horn Fly (Diptera: Muscidae) populations estimated by pterin concentrations. Journal of Medical Entomology, 29, 678±686. Langley, P.A., Hall, H.J.R. & Felton, T. (1988) Determining the age of tsetse flies, Glossina spp (Diptera: Glossinidae) on appraisal of the pteridin fluorescence technique. Bulletin of Entomological Research, 78, 387±395. Lardeux, F., Ung, A. & Chebret, M. (2000) Spectrofluorometers are not adequate for aging Aedes and Culex (Diptera: Culicidae) using pteridine fluorescence. Journal of Medical Entomology, 37, 769±773. Laudani, U. & Lecis, A.R. (1970) n Le pterine di Anopheles atroparvus (Diptera Nematocera) e di un mutante per il colore degli occhi. Rivista di Biologia, 62, Fasc. 1, 43±49. Lehane, M.J., Chadwick, J., Hower, M.A. & Mail, T.S. (1986) Improvements in the pteridine method of determining age in adult Stomoxys calcitrans. Journal of Economic Entomology, 79, 1714±1719. Lehane, M.J. & Mail, T.S. (1985) Determining the age of adult male and female Glossina morsitans morsitans using a new technique. Ecological Entomology, 10, 219±224. MacDonald, G. (1957) The Epidemiology and Control of Malaria. Oxford University Press, London. Mail, T.S., Chadwick, J. & Lehane, M.J. (1983) Determining the age of adults of Stomoxys calcitrans (L.) (Diptera: Muscidae). Bulletin of Entomological Research, 73, 501±525.

#

McIntyre, G.S. & Gooding, R.H. (1994) Pteridine accumulation in Musca domestica. Journal of Insect Physiology, 41, 357±368. Millest, A.L., Cheke, R.A., Howe, M.A., Lehane, M.J. & Garms, R. (1992) Determining the ages of adult females of different members of the Simulium damnosum complex (Diptera: Simuliidae) by the pteridine accumulation method. Bulletin of Entomological Research, 82, 219±226. Msangi, A. & Lehane, M.J. (1991) A method for determining the age of very young tsetse flies (Diptera: Glossinidae) and an investigation of the factors determining head fluorescent levels in newly emerged adults. Bulletin of Entomological Research, 81, 185±188. RodrõÂ guez, M.H. & Loyola, E.G. (1989) SituacioÂn epidemioloÂgica actual y perspectivas de la investigacioÂn entomoloÂgica en MeÂxico. Memorias del IV Simposio Nacional de EntomologõÂa MeÂdica y Veterinaria. Sociedad Mexicana de EntomologõÂ a, Oaxtepec, Morelos, MeÂxico. RodrõÂ guez, M.H., Orozco, A., ChaÂvez, B. & MartõÂ nez-Palomo, A. (1992) Scanning electron microscopy of egg hatching of Anopheles albimanus (Diptera: Culicidae). Journal of Medical Entomology, 29, 887±890. Thomas, D.B. & Chen, A.C. (1989) Age determination in the adult screwworm (Diptera: Calliphoridae) by pteridine levels. Journal of Economic Entomology, 82, 1140±1144. Tomic-Carruthers, N., Robacker, D.C. & Mangan, R.L. (1996) Identification and age-dependance of pteridines in the head of adult Mexican fruit fly, Anastrepha ludens. Journal of Insect Physiology, 42, 359±366. Tyndale-Biscoe, M. (1984) Age-grading methods in adult insects: a review. Bulletin of Entomological Research, 74, 341±377. Wall, R., Langley, P.A., Morgan, K.L. (1991) Ovarian development and pteridine accumulation for age determination in the blowfly Lucilia sericata. Journal of Insect Physiology, 37, 863±868. Wall, R., Langley, P.A., Stevens, J. & Clarke, G.M. (1990) Age determination in the old-world screwworm fly Chrysomia bezziana by pteridine fluorescence. Journal of Insect Physiology, 36, 213±218. Wu, D. & Lehane, M.J. (1999) Pteridine fluorescence for age determination of Anopheles mosquitoes. Medical and Veterinary Entomology, 13, 48±52. Zar, J.H. (1984) Biostatistical Analysis, 2nd edn. Prentice Hall, Inc, Englewood Cliffs, N.J. Ziegler, I. & Harmsen, R. (1969) The biology of pteridines in insects. Advances in Insect Physiology, 6, 139±203.

Accepted 11 April 2002
