The w MelPop strain of Wolbachia interferes with dopamine levels in Aedes aegypti
© Moreira et al; licensee BioMed Central Ltd. 2011
Received: 22 November 2010
Accepted: 28 February 2011
Published: 28 February 2011
Wolbachia is an intracellular bacterium that has been stably transinfected into the mosquito vector of dengue, Aedes aegypti. This inherited infection causes a range of metabolic and phenotypic alterations in the mosquito, which might be related to neuronal abnormalities. In order to determine if these alterations were caused by the manipulation of neuroamines by this bacterium, we studied the expression of genes involved in the dopamine biosynthetic pathway and also measured the amount of dopamine in infected and uninfected mosquitoes of different ages. Wolbachia-infected mosquitoes exhibit greater expression of some genes related to the melanization pathway, but not for those directly linked to dopamine production. Although dopamine levels were higher in Wolbachia-positive mosquitoes this was not consistent across all insect ages nor was it related to the previously described Wolbachia induced "bendy" and "shaky" phenotypes.
Aedes aegypti is the main vector of dengue, one of the leading arboviral diseases of humans throughout the tropics of the world . In an attempt to develop a new biocontrol approach to reduce dengue transmission, A. aegypti mosquitoes have been transinfected with the common inherited bacterial symbiont of insects, Wolbachia. The wMelPop-CLA strain of Wolbachia when present in A. aegypti induces a range of effects including reductions in adult lifespan and blockage of vector competence for a range of human pathogens [2–5]. In addition some other effects have also been reported. Infected mosquitoes exhibit increases in both locomotor activity and metabolic rate . As Wolbachia-infected A. aegypti females age, they obtain fewer and smaller blood meals and show increasing difficulty in completing the process of blood feeding [7, 8]. Old female mosquitoes (>15 days) also display behavioral defects described as a "bendy" proboscis or jittering of the body termed "shaky", suggesting that their neuronal function is somehow impaired.
The biogenic amines dopamine, serotonin and octopamine act as signaling molecules in many diverse physiological contexts, including behavior, fertility, and reproduction, and in the development of neuronal and non-neuronal tissues . In insects, dopamine is involved in several other biological processes, that span from cuticle formation , to egg chorion hardening , to gonadotropic regulation  and to the immune response [13, 14]. Dopamine is associated with locomotor defects in Drosophila  and mammals  and, in humans its deficiency is the cause of motor defects such as Parkinson disease .
To examine a possible relationship between dopamine production and the previously described behavioral defects in mosquitoes associated with Wolbachia infection, we characterized transcription of a number of genes involved in the dopamine biosynthetic pathway  (Figure 1A) in mosquito heads at 3 different adult ages. A. aegypti mosquitoes, w MelPop-CLA infected and tetracycline cured strains , were reared in a controlled environment insectary at 26°C and approx. 80% humidity. All experiments were carried out on sucrose fed individuals. Thirty day-old Wolbachia-infected females were sorted into two different phenotypic categories - normal and abnormal (containing "shaky" or "bendy") by placing insects into a cage and observing their probing behavior on a human hand .
For gene expression studies pools containing 20 female mosquitoes were dissected in ice-cold PBS, pooled, snap frozen in liquid nitrogen and extracted for total RNA using Trizol (Invitrogen Corp., Carlsbad, CA). Total RNA was treated with 2 μL of DNase I (Roche) for 30 minutes at 37°C in a 20 μL reaction to eliminate genomic DNA. Approximately 0.5 μg of total RNA was reverse transcribed using random primers and SuperScript III reverse transcriptase (Invitrogen) according to manufacturer's protocols.
Primers used for quantitative PCR analysis of genes related to dopamine metabolism
Expression of all genes reduces with age, regardless of infection status (Figure 1; see Table S1; Additional file 1). However expression is significantly higher in Wolbachia infected mosquitoes for the three-prophenoloxidase (ppo) genes, but not for DDC or Ebony (Figure 1B-F). The increased expression of genes related to the melanization pathway in Wolbachia infected insects is consistent with the finding that several immunity genes (including AAEL011764) are also up-regulated by the presence of this bacterium in A. aegypti[3, 4] and more recently that Wolbachia-infected mosquitoes exhibit higher levels of melanization in their hemolymph .
Dopamine levels were also determined in mosquitoes of three ages (5, 15, and 30 days). Our hypothesis was that Wolbachia-infected mosquitoes, would exhibit lower levels of dopamine, resembling the situation in Parkinson's Disease in humans . Mosquitoes were anesthetized with CO2 and then decapitated. Preliminary analysis indicated pooling 5 heads was sufficient to achieve a robust dopamine signal and hence all reported sample sizes represent pools of 5 heads. Heads were snap frozen on dry ice and kept at -80°C for up to two weeks. One hundred microliters of 0.1 M perchloric acid containing 5 ng deoxyepinephrine (as an internal standard) was added to each tube. Heads were sonicated using a probe setting of 60% amp/20 Khz for 5 sec, followed by 10 sec rest and another 5 sec sonication (on ice). Tubes were centrifuged for 5 min at 13,000 rpm. Supernatant was filtered through a 4 mm/0.22 syringe filter (PM Separations, Australia) and injected into a HPLC system that consisted of a degasser, autosampler and an isocratic pump (Model 1100, Agilent Technologies, Inc. CA), a Sunfire C18 column, 4.6 mm × 150 mm, 5 um; (Waters Corporation, MA) and a Coulochem III (ESA Laboratories, Inc. MA) electrochemical detector. The mobile phase was a 12% acetonitrile/75 mM potassium dihydrogen phosphate buffer containing 1 mM EDTA and 1.4 mM octane sulfonic acid adjusted to pH 4.13 with phosphoric acid. Flow rate was 1.2 ml/min. The conditioning cell (Model 5020, ESA Laboratories, Inc. MA) operated at +350 mV with the first and second electrode of the analytical cell (Model 5014B, ESA Laboratories, Inc. MA) potentials set to -150 mV and +250 mV respectively. Data were quantified by calculating peak-height ratios for dopamine relative to deoxyepinephrine, and calibrated using a standard curve. Data were then processed with Chemstation software (Rev B.01.03, Agilent Technologies, Inc. CA). Samples were corrected for dilution and expressed as pg/mosquito head. Dopamine levels were analysed using standard t-tests comparing the effect of line (+Wolb or -Wolb) at each of three adult ages (5,15 or 30 days) using Statistica 8.0 (StatSoft, Inc.). In each case comparisons were only made between mosquitoes collected on the same day and analysed in the same batch.
Taken together, our findings indicate that Wolbachia-infected mosquitoes have higher dopamine levels in their heads, although the effect was not present at all ages examined. These higher dopamine levels are consistent with previous observation that Wolbachia-infected mosquitoes exhibit higher activity . There may be other behaviours governed by dopamine that are also affected by Wolbachia infection however there is no obvious correlation between higher dopamine levels found here and the abnormal behavioral phenotypes "shaky" and "bendy" we examined. The positive correlation between Wolbachia densities and prevalence of behavioral phenotypes suggests that bacterial densities are predictive of virulence.
We are grateful to Nichola Kenny and Jenny Gough for assistance with mosquito rearing. This research was supported by a grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative of the Bill and Melinda Gates Foundation and the National Health and Medical Research Council, Australia.
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