Analysis of cycle Gene Expression in Aedes aegypti Brains by In Situ Hybridization

Samira Chahad-Ehlers,Carla Gentile,Rafaela Vieira Bruno,José Bento Pereira Lima,Alexandre Afranio Peixoto,Nicholas S Foulkes

doi:10.1371/journal.pone.0052559

Abstract

Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc) gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR). Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on) compared to ZT11 (one before light-off), which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.

Highlights

The daily rhythms in behavior and metabolism of mosquitoes and other insects are controlled by endogenous circadian clocks [1], [2]
The Drosophila melanogaster central clock controlling activity behavior is composed by two main groups of neurons located dorsally and laterally (subdivided in dorsal lateral neurons (LNd), ventral large lateral neurons (l-LNv), posterior lateral neurons (LPN) and ventral small lateral neurons (s-LNv) clusters), while in mammals the clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus [3], [4]
Circadian activity rhythms are controlled by the central clock that lies in the central nervous system

Summary

Introduction

The daily rhythms in behavior and metabolism of mosquitoes and other insects are controlled by endogenous circadian clocks [1], [2]. When isolated from temporal cues, organisms exhibit rhythms that persist under constant conditions, with a free-running period that is closed to 24-hours (circadian rhythms) and exhibit temperature-compensation, maintaining a similar period over a broad range of temperature. These rhythms are synchronized (entrained) to the environment by external stimuli such as light-dark cycles [2]. In D. melanogaster, the clock is controlled by regulatory loops involving the core genes period (per), timeless (tim), Clock (Clk), cycle (cyc), vrille (vri), Par domain protein 1 (Pdp1) and clockwork orange (cwo) [5]. It is worth noticing that, in Drosophila, cyc expression does not oscillate with any detectable amplitude at the RNA or protein level [11]

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Conclusion