The main vectors of malaria in Brazil are Anopheles darlingi, An. aquasalis, and some species of the An. albitarsis complex, whereas others have questionable importance with regard to the disease transmission. To identify these vectors in the State of Pará, Brazil, in a high-prevalence P. falciparum area, 565 anophelines were captured and identified while the seasonal variation and daily biting activity were determined. Of the seven anopheline species (An. strodei, An. albitarsis s.l., An. rondoni, An. darlingi, An. triannulatus, An. oswaldoi, and An. nuneztovari), the plasmodia circumsporozoite protein (CSP) was detected in three of them, with a total infection rate of 6.2%. An. darlingi was the most prevalent species (22.4%), followed by An. albitarsis (5.2%) and An. rondoni (3.6%). An. rondoni was found to be infected for the first time, which was also confirmed through PCR. This result possibly represents a new malaria vector based on its highest frequency, biting and seasonal activities in the peak of malaria transmission.
The insertion of foreign genes into the genome of an organism is an important tool to address the expression of a particular gene, to study parasite-vector interactions and also for practical uses. The first stable transformation of a mosquito was achieved by Coates et al. (1998) and by Jasinskiene et al. (1998) who expressed a species-specific eye colour gene, in Aedes aegypti (Linnaeus). A great achievement was obtained with the use of the green fluorescent protein (GFP), as a selective marker, because it has the advantage to express in a variety of species, including insects (Pinkerton et al. 2000, Horn & Wimmer 2000, Catteruccia et al. 2000, Kokoza et al. 2001, and on early developmental stages. Furthermore, with the discovery that the transposon piggyBac was able to work in different organisms [insects (Handler & Harrell 1999, Handler & MacCombs 2000, Grossman et al. 2001, Peloquin et al. 2000, Tamura et al. 2000, Kim et al. 2004, Franz et al. 2006) and even in mouse (Ding et al. 2005), planaria (Gonzalez-Estevez et al. 2003), and Plasmodium falciparum (Balu et al. 2005)], a wide horizon has opened on the transformation of insects of both agricultural and medical/veterinary importance.Although several mosquito species have been transformed so far, there are differences related to each species and consequently, adaptation of the transformation technique is required.Aedes fluviatilis (Lutz) is a zoophilic and anthropophilic species, with a geographical distribution comprehending the Southern part of Mexico to the Northern part of Argentina, east of Andes. This species has being used Here we report the first successful stable transformation of Ae. fluviatilis, by using the piggyBac transposable element, being the first report of genetically manipulation of mosquitoes in Latin America. MATERIALS AND METHODSAe. fluviatilis mosquitoes were reared at 27°C and 80% humidity under a 12 h light/dark cycle. For adults, 10% sucrose solution was offered ad libitum and females were fed on mouse blood. Larvae were fed on fish food (Goldfish Colour, Alcon).The transformation plasmids [piggyBac 3xP3-EGFP (Horn & Wimmer 2000) and phsp-Helper plasmid] were purified by using a Plasmid Maxi-Prep kit (Qiagen). Plasmids were mixed to a final concentration of 0.3 µg/µl (piggyBac) and 0.2 µg/µl (Helper plasmid) in injection buffer (5 mM KCl, 0.1 mM Na 2 HPO 4 , pH 6.8) plus 5% (vol/ vol) of green food colour to help visualization of injection mixture.Three days old females were fed on mouse blood and two days after, eggs were collected by forced egg laying. Thirty to 45 min embryos were lined up with their posterior ends towards the same side onto a square of filter paper soaked with isotonic buffer (150 mM NaCl, 5 mM KCl, 10 mM HEPES, 2.5 mM CaCl 2 , pH 7.2), the filter paper was dried-up and the embryos were transferred to a glass slide containing double-sided tape and covered with halocarbon oil 27 (Sigma-Aldrich).Microinjections were performed by using a Femtojet injector (Eppendorf) and an inverted microscope (TS100, Nikon) att...
We studied the involvement of the α8 subunit of nicotinic acetylcholine receptors (nAChRs) in olfactory learning and memory in Apis mellifera. We have previously shown, by injecting different nicotinic antagonists into the bee brain, that pharmacologically different subtypes of nAChRs are important for honeybee memory -α-bungarotoxin-sensitive receptors are necessary for memory consolidation and mecamylamine-sensitive receptors are involved in retrieval processes. Here, we took advantage of the honeybee genome sequencing and the development of a small interfering RNA (siRNA) tool to focus on the role of the α8 subunit, which has been shown to be expressed in brain areas important for olfactory learning, such as the antennal lobes and mushroom bodies. We first demonstrated the efficacy of the siRNA tool by showing a decrease of the α8 protein level at 6 h after brain injection of α8 siRNA. We then tested the general role of this subunit in olfactory conditioning, using brain systemic or localized siRNA injections in the antennal lobes or the calyces and vertical lobes of the mushroom bodies. These injections were performed at either 6 h before the learning acquisition or 6 h before the memory test. The most prominent result was that 6-h pre-test injection of siRNA in the mushroom body vertical lobes impaired memory retrieval at 24 and 48 h post-training. This indicated the importance of cholinergic extrinsic neurons and nAChRs containing the α8 subunit for this process.
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