Genome-wide patterns of gene expression during aging in the African malaria vector Anopheles gambiae

Abstract

The primary means of reducing malaria transmission is through reduction in longevity in days of the adult female stage of the Anopheles vector. However, assessing chronological age is limited to crude physiologic methods which categorize the females binomially as either very young (nulliparous) or not very young (parous). Yet the epidemiologically relevant reduction in life span falls within the latter category. Age-grading methods that delineate chronological age, using accurate molecular surrogates based upon gene expression profiles, will allow quantification of the longevity-reducing effects of vector control tools aimed at the adult, female mosquito. In this study, microarray analyses of gene expression profiles in the African malaria vector Anopheles gambiae were conducted during natural senescence of females in laboratory conditions. Results showed that detoxification-related and stress-responsive genes were up-regulated as mosquitoes aged. A total of 276 transcripts had age-dependent expression, independently of blood feeding and egg laying events. Expression of 112 (40.6%) of these transcripts increased or decreased monotonically with increasing chronologic age. Seven candidate genes for practical age assessment were tested by quantitative gene amplification in the An. gambiae G3 strain in a laboratory experiment and the Mbita strain in field enclosures set up in western Kenya under conditions closely resembling natural ones. Results were similar between experiments, indicating that senescence is marked by changes in gene expression and that chronological age can be gauged accurately and repeatedly with this method. These results indicate that the method may be suitable for accurate gauging of the age in days of field-caught, female An. gambiae.

Figure 1. Hierarchical clustering analysis of gene expression in Anopheles gambiae adult female mosquitoes of different age groups maintained with access to plant sugar or sugar and rabbit blood.

Graphic heat maps represent transcripts expressed differentially among all the age groups (n = 276, p values <0.001). Each row represents data for one probe and each column corresponds to an age group, referred as chronological age (days post adult emergence, pupal-adult ecdysis).

Figure 2. Flowchart of data analysis and identification of age-related transcript accumulation.

The numbers in parentheses represent the number of gene-specific transcripts in each category. The ‘⇑’ symbol indicates gene expression increasing with age; ‘⇓’ indicates expression decreasing with age; ‘⇑,⇑’ expression increasing with age and up-regulated by blood feeding; ‘⇓,⇓’ expression decreasing with age and down-regulated by blood feeding; ‘⇑,⇓’ expression increasing with age and down-regulated by blood feeding; and ‘⇓,⇑’ expression decreasing with age and up-regulated by blood feeding. Genes in the ‘⇑’ and ‘⇓’ categories are considered candidates for the development of a gene expression-based mosquito age-grading methodology.

Figure 3. Distribution of the Anopheles gambiae genes with expression modulated by aging according to their putative functions.

Gene ontology functional class were derived and displayed separately for genes with decreasing (in green) or increasing expression (in red) as the mosquitoes age.

Figure 4. Semi qRT-PCR validation of gene expression patterns of four selected genes reveal consistent gene expression variations between two strains maintained in distinct environments.

Anopheles gambiae G3 strain mosquitoes raised in a local insectary, at UCI, CA, USA (Lab colony) and Mbita strain mosquitoes maintained in the MalariaSphere in western Kenya (MalariaSphere populations) were used as the source of RNA. RNA extraction and qRT-PCR conditions were identical to all samples. Age refers to chronological days post pupal emergence in adult females.

Figure 5. Validation of variations in gene expression by qRT-PCR.

Regression analyses were performed correlating chronological ages and transcript abundance for 4 of the age-grading candidate genes. Mosquito chronological age is the days post pupal emergence.