Comparison of life table attributes from newly established colonies of Anopheles albimanus and Anopheles vestitipennis in northern Belize

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1 December, 2003 Journal of Vector Ecology 200 Comparison of life table attributes from newly established colonies of Anopheles albimanus and Anopheles vestitipennis in northern Belize John P. Grieco 1, Nicole L. Achee 1, Ireneo Briceno 2, Russell King 2, Richard Andre 1, Donald Roberts 1, and Eliska Rejmankova 3 1 Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, U.S.A. 2 Ministry of Health, Belize City, Belize, Central America 3 Department of Environmental Science and Policy, University of California, Davis, CA 95616, U.S.A. Received 2 February 2003; Accepted 5 May 2003 ABSTRACT: A life table study was conducted for recently established colonies of Anopheles albimanus and Anopheles vestitipennis in Belize, Central America. The colonies were reared in the northern Orange Walk District under uncontrolled environmental conditions (29-32 C, 87-90% RH, and 13:11 L:D photoperiod). The mean time of larval development for An. albimanus was 10.8 days for males and 11.7 days for females. Mean times for An. vestitipennis larval development were 11.3 days for males and 13.5 days for females. Anopheles albimanus exhibited a 92% survival rate from egg hatch to adult emergence, while that of An. vestitipennis was 82%. Neither species showed a sex ratio that was significantly different from 1:1. Adult male An. albimanus lived for an average of 13.6 days, while the females lived for an average of 21.2 days. The An. vestitipennis adult males lived for a mean of 14.8 days, while the females lived considerably longer with a mean of 25.6 days. The reproductive rate and age of mean cohort reproduction were calculated as 287 and 11.3 days for An. albimanus and 302 and 13.4 days for An. vestitipennis. The r/b and B/D ratios of 0.13 and 1.15, respectively, for An. albimanus and 0.1 and 1.1, respectively, for An. vestitipennis indicate that these species have a low potential for colonization. Journal of Vector Ecology 28 (2): Keyword Index: Anopheles, colony, life table, malaria, Belize. INTRODUCTION Two of the primary vectors of malaria in Belize, Central America, are Anopheles albimanus Wiedemann and An. vestitipennis Dyar and Knab. While An. albimanus is recognized as an important vector of malaria throughout much of Mexico, Central America, parts of northern South America, and Haiti (Rachou et al. 1965), it demonstrates only low levels of natural field infections to Plasmodium vivax (Ramsey et al. 1986). This finding has led many researchers to conclude that the transmission potential for this species occurs only when the population levels are high (Breeland 1972, Weidhaas et al. 1974). Anopheles vestitipennis, on the other hand, has demonstrated a number of characteristics that contribute to greater vector potential such as strong endophagic and anthropophagic behaviors (Grieco et al. 2000), as well as high minimum field infection rates (Achee et al. 2000). The potential of any anopheline species to be a competent vector of malaria, however, depends on a number of physical and physiological components associated with the life history of the mosquito. Various biological characteristics such as stage specific survivorship, rate of larval development, fecundity, and larval and adult mortality are critical aspects of disease transmission potential and constitute information that could contribute to control strategies (Seawright et al. 1979). Age-specific horizontal life tables can be used to summarize the mortality and reproductive characteristics of a given species (Reisen et al. 1979, Reisen and Mahmood 1980). When all of the variables are controlled for at the optima for each instar, this information can provide characterizations about the population that represent the maximum expression of these attributes for a given population or species. The goals of this study were twofold: 1) to gain preliminary information on laboratory rearing of An. albimanus and An. vestitipennis for use in future studies

2 December, 2003 Journal of Vector Ecology 201 involving these species and, 2) to compare life table data on these two mosquito species to further the understanding of their potential as vectors of malaria within Belize, Central America. MATERIALS AND METHODS Field population An insectary was constructed at a field station located in Orange Walk, Belize (N W ). The insectary contained no artificial light source, external humidifier or temperature control to maintain environmental variables. These variables fluctuated with natural conditions via open windows adjacent to the insectary. Temperatures within the insectary ranged from 28 o C to 32 o C with an average temperature of 30 o C throughout the course of the study. Outdoor temperatures during the same time ranged from 26 o C to 34 o C while the average temperature remained the same, 30 o C. The relative humidity recorded within the cages ranged from 87%-90% with an average of 89%. The light:dark cycle was recorded as 13L:11D based on ambient light from natural sunrise and sunset as measured via windows adjacent to the insectary. Wild caught blood-fed females of An. albimanus and An. vestitipennis were used to establish colonies. Females were obtained from human baited collections conducted at the field station. Eggs from blood-engorged populations of An. vestitipennis and An. albimanus were set in 12 white enamel pans measuring 34.3 x 25.4 cm with a depth of 4.4 cm. Larvae were fed a diet of ground fish flakes and brewer s yeast mixed in a 3:1 (weight/ weight) ratio. The mixture was made into a suspension with regular tap water in approximately a 10% suspension (weight/volume). Three ml of this suspension were applied daily for the first 5 days after which time it was increased to 6 ml. Pans were checked daily for the presence of pupae. Once pupae were observed, they were removed from the rearing pans and placed into separate clear styrene 9-dram vials. An inverted 5-dram clear styrene vial inserted in the top of the larger tube formed a sealed rearing chamber. The vial was labeled with information from the pan from which the pupae had been picked. Upon emergence, adults were briefly placed in a freezer to knock the specimen down for species identification. Adults were placed into a 30 cm 2 screened rearing cage labeled with the date of first emergence. Adults that had emerged within three days of each other were placed into the same cage to insure that all members of the population were within a three-day age range.vials containing cotton wicks soaked in a 10% sugar solution were placed in each adult cage to provide a source of carbohydrate. Sugar solution and cotton wicks were replaced every three days to minimize the growth of mold and bacteria. Beginning 24 h after adult emergence, mosquitoes were offered a blood meal from the researcher. Small plastic cups lined with filter paper were filled with tap water and placed in the cages to provide oviposition sites for the mosquitoes. Cups were checked on a daily basis. When eggs were observed, the egg laden filter papers were removed and placed in Ziploc bags containing moist paper towels for 24 h. After 24 h, the eggs were counted and transferred to pans filled with rainwater and pans were labeled with the date of oviposition and generation number. Life table analysis The colony was allowed to reach the F 4 generation prior to beginning life table observations. Upon reaching the F 4 generation, 6 pans were set up with 20 An. albimanus 1 st instar larvae (all larvae were within 12 h of hatch) per pan. An additional 6 pans were set up with 20 An. vestitipennis 1 st instar larvae. Each pan was provided with a standard amount of food, and pans were continuously examined for any signs of contamination. Once the immatures reached the 4 th instar, pans were checked twice daily for pupae. Pupae were picked and placed into separate clear styrene 9-dram vials as described above. Each vial contained approximately 5 ml of water from the pan of origin for each pupa. Vials were labeled with the date and time of pupation. Each pupa was designated with a number from 1 to 20, and the time of pupation was recorded on a data sheet next to the corresponding pupa. Upon emergence, the sex of the mosquitoes was determined and recorded along with the time of adult emergence on the data sheet next to the corresponding pupal number. Mosquitoes that died in the process of emerging were also recorded. This experiment was replicated and resulted in observations on a total of 600 An. albimanus and 600 An. vestitipennis larvae. Adult lifespan After determining the sex, the adult mosquitoes were promptly placed in adult rearing cages (50 females and 50 males). Four cages of 100 anophelines were set up to quantify adult longevity. Cages had white paper placed on the bottom of the cage to facilitate identification and removal of dead specimens. The cages were examined daily in order to locate, record, and promptly remove dead mosquitoes. For every dead mosquito, information regarding cage number, sex, and date of death was recorded. If a mosquito was located on the bottom but did not appear dead, a light puff of air from a mouth

3 202 Journal of Vector Ecology December, 2003 aspirator was blown onto it. If no movement was observed within a three-h period, it was considered to be dead. Adult feeding followed the same procedure as was used for the regular colony. Both a 10% sugar solution and a bloodmeal were offered to the mosquitoes every day. This procedure was repeated until all dead mosquitoes had been removed from the cage. Data analysis Standard life table analysis was performed on the colony data (Mahmood 1997). The median times for pupation and eclosion were calculated by fitting a regression line to the pupation and emergence data as a Probit equation in the form of (p) = a + blnx, where p was the cumulative proportion either pupating or emerging on each day and x being equal to the age in days. The median values were obtained by solving the equation for 50% pupation or 50% emergence. These values were obtained for each pan and for each trial to check for variations between pans. Larval survival was determined by counting the number of larvae reaching the pupal stage and dividing that value by the total number of 1 st instar larvae placed in the pan (i.e., 20 larvae). The same procedure was used for those 1 st instar larvae that emerged as adults. The sex ratio was calculated by dividing the number of males by the total number of adults that emerged. Age specific survivorship was calculated as l x =y x / y 0, where y x was the number of mosquitoes that were alive on day x and y 0 was the starting number of mosquitoes in the population. This provided an estimate of the daily survivorship of the species. Age specific life expectancy was calculated as e x = T x /l x, where T x = w x = 0 L x = (l x + l x + 1) / 2 with w being equal to the final day a mosquito was still alive in the cage. For example, the value of e 1 represents the total number of days a mosquito would be expected to live after it emerges as an adult on day 1. The true measure of a population s fecundity is the number of females that are produced by an individual. For this reason, it is critical to know the number of females produced by each member of a specific cohort or generation. This was accomplished by solving for R o in w R 0 = a x = 0 L x, and where l x m x In this equation, a equals the proportion of the population which survives from egg to adult. The value m x can be calculated using m x = E x p. In this equation, E x equals the mean number of larvae that are produced per female per time interval (i.e., the number of days after adult emergence, as designated by x). The value p equals the proportion of the emerging adult population that will be female (i.e., this value is equal to the female sex ratio). Another critical statistic is instantaneous rate of increase per female and is designated as r. The equation used in this determination is a modified version of the Eueler-Lotka equation (Mahmood 1997), w.0 = a x = 0 1 L x m x e -r(x) where e is the natural logarithm. To calculate the instantaneous birthrate (B), the equation used was B = ln(1+b) where w /b = x = 0 1 L x e -r(x) The death rate (D) was then calculated as D = B-r. RESULTS The larvae of An. albimanus began pupating eight days after eclosion and finished by day 12. Anopheles vestitipennis took slightly longer with pupation beginning 9 days after eclosion and completing by day 14. The results of the life table analysis for An. albimanus and An. vestitipennis are presented in Table 1. The male/ female sex ratios for both species were not significantly different from 1:1 (P<0.01) with 45.8% An. albimanus being male and 49.7% An. vestitipennis being male. The overall mean survival rate from eclosion to pupation was 93% for An. albimanus and 87% for An. vestitipennis and from eclosion to adult was 92% and 82%, respectively. On average, it required 11.9 days for male An. albimanus to go from eclosion to adult emergence while the females took 12.4 days (Table 1). It took 12.2 days for male An. vestitipennis to go from eclosion to adult emergence and the females took slightly more than two wks (14.2 days). In all instances, the males demonstrated a shorter adult life span than the females. Adult females of both species began taking a blood meal approximately 36 h after emerging with the first egg batch being laid 5 days after adult emergence for An. albimanus and after 7 days for An. vestitipennis. The requirement of a partial pregravid blood meal was not observed. A plot of the number of female offspring produced by a particular

4 December, 2003 Journal of Vector Ecology 203 Table 1. Life table attributes of An. albimanus and An. vestitipennis in colony from northern Belize, Central America conducted from January to October, Attribute An. albimanus An. vestitipennis Days to Median Pupation a Males Females Days to Median Emergence a Males Females Mean Survival from Hatching to Pupation 93% 87% Mean Survival from Hatching to Adult Emergence 92% 82% Mean Adult Survival (Days) Males Females Sex Ratio males/total 45.8% 49.7% a Measured as median number of days for completion of development. Figure 1. Age specific survivorship (l x ) for colony adult An. albimanus and An. vestitipennis from Belize, Central America. l x Age (days) An. albimanus (males) An. albimanus (females) An. vestitipennis (males) An. vestitipennis (females)

5 204 Journal of Vector Ecology December, An. albimanus An. vestitipennis Expectation of Life (e x ) l x m x Age (days) Figure 2. Number of female offspring produced per female per generation of An. albimanus and An. vestitipennis from newly established colonies from Belize, Central America. An. albimanus An. vestitipennis Age(days) Figure 3. Comparison of expectation of life for newly established colonies of An. albimanus and An. vestitipennis as a function of age. female of age x (L x m x ) for both An. albimanus and An. vestitipennis showed a curve peaking within the first 2 wk of life and then declining dramatically as the age of the females increased (Figure 2). The area under the curve for each species represents the reproductive rate (R 0 ) of that species. This is representative of the mean number of female offspring produced by a single female from a cohort during the course of her life span. Calculating the area under the curve resulted in a net reproductive rate for An. albimanus of 287 females and for An. vestitipennis of 302 females. The expectation of life (Figure 3) for females after emergence was 19.6 days for An. albimanus and 24.2 days for An. vestitipennis. The age of the mean Cohort reproduction (T 0 ) was 11.3 days for An. albimanus and 13.4 days for An. vestitipennis. The mean generation time for An. albimanus was 16.6 days and for An. vestitipennis was 21.2 days. The r-value (r being the rate of instantaneous female growth per female per day of life) was calculated to be 0.34 for An. albimanus. The instantaneous birth and death rates were 2.54 and 2.2, respectively. This resulted in an r/b ratio of 0.13 and a B/D ratio of For An. vestitipennis the r-value was calculated to be The instantaneous birth and death rates were 3.01 and 2.74, respectively. The calculated r/b ratio was 0.1 and the

6 December, 2003 Journal of Vector Ecology 205 B/D ratio was 1.1. DISCUSSION Findings presented in this paper represent observations on newly established colonies of An. albimanus and An. vestitipennis reared under uncontrolled conditions. While these environmental variables may not represent optimal conditions for one or both of the species involved, they do closely mimic the natural setting in which the field population was collected. The longevity of a population is an extremely important facet of vector potential. The malaria parasite requires a certain amount of time under specific temperature regimes to penetrate the gut lining of the mosquito, develop into oocysts, and finally have the sporozoites rupture out of the mature oocysts and infect the salivary glands of the mosquito. If the life span of the mosquito is not sufficiently long, the parasite will be unable to complete its sporogonic life cycle and transmission cannot take place. When the temperature is held at a constant 24 o C, the process of sporogony takes 9 days for P. vivax and 11 days for P. falciparum to complete (Sandosham and Thomas 1983). With most mosquitoes requiring 24 h after emergence to take their first blood meal, an anopheline would have to survive at least 10 days to become infective under this temperature regime. In the present study, the colonization of An. vestitipennis and An. albimanus was successfully accomplished using field-collected material from Belize. The conditions under which these mosquitoes were maintained are similar to those experienced by indoor resting females. Field-collected adults produced large numbers of eggs after receiving a single blood meal. The first egg batch generally occurred between 5 days to 1 wk with the majority of egg production for both species taking place during the second wk of adult life. The hatch rate from eggs produced from the study populations for these two species was 89% for An. albimanus and 72% for An. vestitipennis. The survival rate from hatch to adult was higher for An. albimanus than for An. vestitipennis, 91.7% and 82.2%, respectively. The life table attributes of An. albimanus in this study do not differ dramatically from the previous studies conducted on this species in Belize (Grieco 2000), but they are a departure from data presented in previous studies conducted on this species (Mahmood 1997, Haile and Weidhaas 1977). Previous studies show developmental times that are considerably shorter than those presented here. In the present study, the mean time of female larval development was 11.7 days, only slightly longer than the 10.9 days previously observed from colony attempts made in the southern Toledo District of Belize. These differences are due in part to differences in environmental conditions (i.e., temperature) for larval development. One consistent finding, however, was that males developed more quickly than females. The life table attributes for An. vestitipennis have not been previously reported. The developmental time for this species, as compared to An. albimanus, shows a greater disparity between males and females with the mean male development time more than 2 days shorter than for the females. On average An. vestitipennis takes longer to develop than does An. albimanus by about 4 days. The low hatch rate, higher larval mortality, and longer development time for An. vestitipennis may contribute to the low larval densities found under natural conditions (Grieco 2000). Despite high levels of egg production, insufficient hatch rates and low survival rates of immature stages might result in slow population growth rates. Low hatch rates, however, must be considered carefully. These rates may be a reflection of a recently established colony and not indicative of naturally occurring rates. The length of time spent in the larval stage would also contribute to increased exposure to numerous predators found in the aquatic environment and lead to decreased larval densities. A longer time spent in the larval stage also would make this species more subject to drastic environmental fluctuations such as lowering water levels due to dry conditions. Based on the number of females produced per female per day, indications are that the first two weeks of life for an adult female An. albimanus constitutes more than 60% of her egg producing potential. Almost 70% of An. vestitipennis egg production occurs within the first two weeks of the adult female s life span. With female An. albimanus living an average 21.2 days, the last week of life is not as critical to sustaining population density as the first two weeks. This is more pronounced with An. vestitipennis, in which the final two weeks of adult life contribute little to the overall population density. When applied to a natural setting, it is understandable that there will be a reduction in the length of the adult life span due to predation and other adverse environmental factors. Therefore, high reproductive activity during the first week of life is necessary for these species to be successful. For multivoltine species with overlapping generations, the r-value is a much better reflection of the growth potential per unit of time (Mahmood 1997). The r-value is more representative of this growth because it takes into account both the number of progeny

7 206 Journal of Vector Ecology December, 2003 produced and the times at which these progeny were produced (Hacker 1972). The r-value for An. albimanus was higher than in previous studies due to a longer adult life span. However, as with other studies (Mahmood 1997) this r-value indicates An. albimanus to be an r- strategist. The equation for calculating the r-value places more weight on those eggs laid earlier in the life of a female. Although An. vestitipennis has a longer lifespan, it begins laying eggs later in life. For this reason, the r- value of An. vestitipennis is slightly lower than that calculated for An. albimanus. The adult populations of these two species show similar trends with regard to aging males succumbing before females, as reported for other species (Reisen and Mahmood 1980). The males in this study lived for an average of 15.2 days and the females lived for an average of 19.4 days. The r/b and B/D ratios of 0.13 and 1.15 are low but are considerably higher than those calculated for An. vestitipennis, 0.09 and 1.10, respectively. Based on these values, both An. albimanus and An. vestitipennis show low potential for colonization. These values indicate that the populations are subject to very slow growth and that minor increases in mortality due to a variety of problems could severely impact a colony to the point of complete elimination. The varying age composition of the female members of a species as well as the length of the adult survivorship play important roles in the vectorial capacity of a particular species. In nature, each blood meal represents an additional chance for a female mosquito to become infected with malaria parasites. Therefore, the vectorial importance of each age grouping becomes greater in relation to the physiological age of an individual mosquito (Detinova 1962). The relationship between age and probability of being infected is countered by the cumulative increase in adult mortality that is associated with an individual mosquito reaching the upper limits of its expected life span. Factors that play a role in this cumulative mortality are predation, environmental conditions, and increased physiological age. Therefore, the earlier that a mosquito acquires malaria parasites in a blood meal, the greater the likelihood that transmission can take place. Using 10 days as the shortest period of time for a species to take its first infected bloodmeal and then become infective, we can use the calculation of expectation of life to determine the remaining number of days in which the mosquito could possibly feed and transmit the parasite. For example, An. albimanus can be expected to survive for an additional 13 days under laboratory conditions while An. vestitipennis can be expected to survive an additional 19.2 days. The gonotrophic cycle of An. albimanus has been recorded at between 2 and 3 days (Rodriguez et al. 1992; Weidhaas et al. 1974). Taking all available data into consideration, An. albimanus would feed an additional five to six times, thus increasing the chance of malaria transmission if the female took an infectious blood meal soon after emerging. Not much is known of the gonotrophic cycle of An. vestitipennis, but from this study the female lifespan is substantially long enough to assume multiple feeds within the 19.2 days of life after 10 days of emergence. This would indicate that this species will continue to blood feed multiple times, thus increasing its potential for the transmission of the malaria parasite. Although these studies were conducted on laboratory colonies under uncontrolled conditions, the results obtained give some indication of what can be expected of a natural population. The data obtained from these analyses indicate the potential for An. albimanus to quickly reach high population densities in the wild. Anopheles vestitipennis, while also producing large numbers of eggs, are exposed to a number of constraints that may limit rapid increases in population density, namely its low hatch rate, low larval survival, and lengthy larval development. Although An. vestitipennis population density is subject to slow growth, it is a longlived species (i.e. on average more than four days longer than An. albimanus). When examining the raw data, the lifespan of the longest lived female of An. albimanus was 11 days shorter than the longest lived female of An. vestitipennis. This would provide many more opportunities for this species to become infective and subsequently transmit the parasite given the right environmental conditions. Additional studies are presently underway to gain more insight into the gonotrophic cycle of An. vestitipennis so that a better understanding of the number of feeds and length between feeds can be incorporated into the analyses presented in this paper. Further studies also are required to determine the life table attributes for the other species found in Belize, specifically An. darlingi. When side-by-side comparisons can be made between these three vector species, we will better understand their respective roles in malaria transmission in Belize, Central America. Acknowledgments We are grateful for the administrative and logistical support provided by Dr. Errol Vanzie and Dr. Jorge Polanco (Ministry of Health, Belize). This research was supported by the NIH-NSF Ecology of Infectious Diseases program, Grant # R01 AI49726, Environmental Determinants of Malaria in Belize. Disclaimer: The opinions and assertions contained in this

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