Molecular analysis of photic inhibition of blood-feeding in Anopheles gambiae
© Das and Dimopoulos; licensee BioMed Central Ltd. 2008
Received: 25 June 2008
Accepted: 16 December 2008
Published: 16 December 2008
Anopheles gambiae mosquitoes exhibit an endophilic, nocturnal blood feeding behavior. Despite the importance of light as a regulator of malaria transmission, our knowledge on the molecular interactions between environmental cues, the circadian oscillators and the host seeking and feeding systems of the Anopheles mosquitoes is limited.
In the present study, we show that the blood feeding behavior of mosquitoes is under circadian control and can be modulated by light pulses, both in a clock dependent and in an independent manner. Short light pulses (~2–5 min) in the dark phase can inhibit the blood-feeding propensity of mosquitoes momentarily in a clock independent manner, while longer durations of light stimulation (~1–2 h) can induce a phase advance in blood-feeding propensity in a clock dependent manner. The temporary feeding inhibition after short light pulses may reflect a masking effect of light, an unknown mechanism which is known to superimpose on the true circadian regulation. Nonetheless, the shorter light pulses resulted in the differential regulation of a variety of genes including those implicated in the circadian control, suggesting that light induced masking effects also involve clock components. Light pulses (both short and long) also regulated genes implicated in feeding as well as different physiological processes like metabolism, transport, immunity and protease digestions. RNAi-mediated gene silencing assays of the light pulse regulated circadian factors timeless, cryptochrome and three takeout homologues significantly up-regulated the mosquito's blood-feeding propensity. In contrast, gene silencing of light pulse regulated olfactory factors down-regulated the mosquito's propensity to feed on blood.
Our study show that the mosquito's feeding behavior is under circadian control. Long and short light pulses can induce inhibition of blood-feeding through circadian and unknown mechanisms, respectively, that involve the chemosensory system.
The circadian clocks, or oscillators, that influence a variety of activities of an organism are regulated by a range of external environmental factors, such as light, temperature, humidity, food and social interactions [1–5]. Genetic and molecular analyses in a range of evolutionary distant organisms, including the fruit fly Drosophila melanogaster, have revealed a striking degree of conservation of these oscillators and their underlying molecular mechanisms, many of which involve transcriptional feedback loops [6, 7]. Timeless (tim) is one of the crucial factors that connects the endogenous clock with the external environment such as light . When light pulses are applied early in the subjective night (soon after the lights have gone off) a phase delay is produced. Conversely, when light pulses are applied just before lights would have come on, a phase advance is produced . Food is another potent clock-entraining cue, and the D. melanogaster takeout (to) gene has been shown to act as a molecular link between the circadian rhythm and feeding behavior [9, 10].
Despite the essential role of circadian clocks in the regulation of vector behaviors that enable disease transmission, little is known about the molecular control of the host-seeking and feeding behavior of mosquitoes. It has been shown that the A. gambiae flight activity peaks during the night time and during the twilight . Light has also been shown to inhibit A. gambiae flight activity and to re-set the rhythm by delaying it; however if the light phase begins early, it has a phase-advancing effect [12, 13]. The host-seeking and feeding behaviors of many hematophagous insects exhibit rhythmic biting activity ; reaching peak levels in mid-night and are largely controlled by several circadian and chemosensory systems [15, 16] involving a variety of odorant binding proteins (obp) and odorant receptors (or) that are linked to downstream signaling cascades [17, 18]. In D. melanogaster antennae, the chemosensory components have been shown to be regulated by peripheral circadian oscillators .
Here we have initiated a detailed dissection of the delicate interactions that occur between the mosquito's light-sensing system, the circadian oscillators and the host seeking and feeding systems. Several studies have reported the influence of light on the flight activity of mosquitoes [11–13, 20, 21], while there is no comprehensive information available on the regulation of the mosquito's blood-feeding behavior by light, at least at the molecular level. We show that light pulses can induce changes in A. gambiae blood-feeding behavior and in the mosquito's global transcriptome, including transcripts that are regulated by blood feeding. We have functionally characterized selected genes that respond to the light stimulus by RNAi mediated gene silencing and have shown that they can moderately influence the mosquitoes' blood feeding behavior. We show that this modulation of the mosquitoes' blood feeding behavior is dependent on light pulse duration and occurs both through clock entrainment mechanisms and by clock independent masking effects [22–24] that may occur in a phase dependent or independent manner . The light pulse stimulated alteration of mosquito feeding propensity implicates circadian factors that appear to down-regulate components of the chemosensory system.
Results and discussion
The blood feeding behavior of A. gambiae is under circadian control
Light pulse-induced alteration of A. gambiae blood-feeding behavior
A series of assays were performed to address the effect of long and short light pulses on the mosquito's blood feeding propensity. We investigated the effects of short (5 min) and long (2 h) light pulses, applied during the dark phase at 19.30 hours, on the blood-feeding pattern of mosquitoes in both LD and DD conditions (Figure 1B and 1C). A short light pulse of 5 min resulted in a transient inhibition (~30–50%) of blood feeding propensity in mosquitoes, in both LD and DD conditions (Figure 1B versus 1A). This observation suggests that the shorter light pulse did not evoke a behavioral phase shift, and the inhibition of feeding propensity is likely to reflect a masking effect . On the contrary, a longer 2 h light pulse resulted in an immediate inhibition (~50%) of the blood feeding propensity along with a phase-advance (by ~3–4 h) in mosquitoes that were maintained at both the LD and DD conditions (for 48 h), compared to the non-light pulse stimulated mosquitoes (Figure 1C versus 1A). Earlier studies have shown that an early initiation of the light phase results in a phase-advancing effect on mosquito flight activity .
Catalog of circadian (CIR) and chemosensory (CSR) genes that showed differential expression.
CK II BETA CHAIN
D7-RELATED 3 PRECURSOR
For these blood feeding studies we chose an experimental design involving an artificial membrane feeding system (Parafilm M) with commercial human blood because of the difficulty in standardization and replication of parameters associated with the use of a mouse or a human arm, as it will be subjective to different individuals. Similar artificial membrane-feeding assays were also performed with close proximity (1–3 cm) to human hands and continuous human exhalations. These results showed an apparent insensitivity of the mosquitoes to the applied light pulse; the mosquitoes' feeding propensity was largely unaffected by light pulse after this chemosensory over-stimulation (data not shown). However this does not mean that the mosquitoes' feeding behavior upon direct stimulation with a human is not regulated by circadian oscillators that can be manipulated through environmental cues. The exposure of mosquitoes to a human arm or hand and exhalations at a 1- to 3 cm distance is an extreme condition providing maximal concentrations of chemosensory and heat stimulants, while in nature mosquitoes have to sense the human host at a distance of few hundreds to a couple of yards, at conditions that are very likely to be better simulated by a membrane feeding system that only allows for limited chemosensory stimulation .
It has been shown that the taste receptors in mosquitoes can be induced by phagostimulants such as adenine nucleotides ; thus the blood feeding behavior in mosquitoes is a complex process that is regulated by a variety of factors. Host seeking, and hence the blood-feeding propensity of mosquitoes, has also been shown to be stimulated by various cues emitted by the host, such as CO2, lactic acid, 1 octen-3-ol, heat and color . In order to better understand the basis of the mosquitoes' attraction to the blood in the membrane feeding system, which may be regulated by the blood-specific odor, temperature and color, we designed a series of experiments that suggested that the mosquitoes were mainly attracted to a blood-specific chemosensory stimulant through the membrane feeding system (Additional file 3), and thereby supports the validity of our experimental design.
Relationships between the light pulse and blood feeding – induced transcriptomes
A light pulse stimulation for 2 min at 30 min prior to RNA harvest (arrays "b" and "c"; see Additional file 4), resulted in the regulation of 345 genes in the total mosquito (274 up-regulated and 71 down-regulated) and 374 genes in the head (124 up-regulated and 250 down-regulated) of females (Additional file 4). Mosquitoes exposed to continuous light for 2 h (array "a"; Additional file 5) showed regulation of 188 genes (146 up-regulated and 42 down-regulated) in whole female mosquitoes. The regulated genes represented a variety of functional classes, of which the most abundant were metabolism, immunity, oxidoreductive processes, and transporters (Figure 3A and Additional file 4). Of particular interest were the genes with predicted functions related to the mosquito's circadian clock, chemosensory and blood-digestive systems (Table 1). The robustness of the microarray data was validated by qRT-PCR (Additional file 6).
Circadian clock-related genes
Light pulse regulated six circadian genes in the head or total mosquito: a casein kinase and the tim genes were up-regulated in the total mosquito, while the transcript abundance of two putative to homologs and two circadian controlled precursor genes were down regulated in the head (Table 1 and Additional file 5). Casein kinase and tim have previously been shown to be involved in regulating the circadian rhythm in response to light pulse stimulation in various organisms . The mRNA abundance of the four major circadian genes: period (per), clock (clk), cycle (cyc) and cryptochrome 1 (cry1) was below threshold detection levels for microarray analyses (data not shown) at the assayed conditions (light on/off time-zone).
The to gene is involved in regulating the feeding behavior of D. melanogaster and has been shown to be up-regulated upon starvation . A similar study in A. gambiae with sugar feeding has shown up-regulation upon sugar starvation (see later section). The down-regulation of two putative to transcripts (AGAP004263 and AGAP012703) by light pulse stimulation, which may account for the decreased feeding propensity (Figure 2), is intriguing when related to the RNAi gene silencing of to genes that results in an increased feeding propensity (see later section). This is likely to reflect other functions of these genes, as has been found in D. melanogaster . A detailed phylogenetic study of the A. gambiae to genes with other homologues is discussed below. Two to transcripts (AGAP012703 and AGAP004262) were up-regulated in the carcass compartment after blood-feeding.
As many as twelve genes with putative chemosensory functions were exclusively regulated by light pulse stimulation; three were exclusively regulated by blood feeding, and three by both stimuli (Figure 3A and Additional file 5 ). Nine obps were down-regulated after light pulse stimulation in the female head; this pattern is likely to be related to the lower blood-feeding propensity of the mosquitoes after light pulse stimulation (Figure 2). The obp49 gene showed significant up-regulation in female mosquito head after blood feeding; this increased activity may indicate a specialized function for this gene, such as mate seeking or oviposition site finding. Another interesting feature was the significantly lower expression of several obps (15, 17, 19, 26, 4, 47 and 7) in light pulse-stimulated female versus male heads, while a direct comparison between the gene expression in non-stimulated female and male heads showed a higher expression of three obps (19, 22 and 26) in the male heads. Female obps play major role in host seeking and blood feeding, while the male obps are involved in pheromone binding for mating and nectar feeding and may not be regulated by light-entrainable host-seeking behavior in the same way as the female genes. A specific obp can play multiple roles in sensing different types of complex odors, since the odor sensing is dependent on several obps that are part of a specific odor code .
A D7 related 3 protein which is distantly related to obps, was up-regulated in the head by both light pulse stimulation and blood feeding, and was down-regulated in the carcass after blood feeding (Table 1, Additional file 5). Proteins belonging to the D7 family has been shown to be expressed in the salivary glands and were injected into the host with saliva during blood feeding . Among the other genes that showed regulation after light pulse stimulation includes a putative gustatory receptor, which was up-regulated in the female head when compared to the male head; and two pheromone binding proteins, of which one was up-regulated and the other was down-regulated in the female head (Additional file 5).
Blood digestive enzymes
As many as 23 genes involved in proteolytic digestion were regulated by blood feeding and 15 genes by light pulse stimulation, while 11 genes were regulated by both the stimuli (Figure 3A). The majority of these genes (17 out of 23) were up-regulated upon blood feeding; a finding that is consistent with the fact that this functional class plays a role in blood meal digestion. The carboxypeptidase genes were shown to follow a cyclical blood-inducible expression pattern with a peak every 3 h post blood meal , and in our assay two carboxypeptidase genes (ENSANGT00000016098 and AGAP011435) were up-regulated in the carcass after blood feeding, however AGAP011435 was down regulated in female head after a light pulse. Two chymotrypsin genes (chymotrypsin 1 and 2) have been shown to be induced in the midgut epithelium after blood feeding , and in our assay one chymotrypsin gene (AGAP010731) was similarly induced with feeding in female body, though it was down-regulated in female head after light pulse. In agreement with the down-regulation of two serine proteases in female body after blood feeding in our analysis, the gut-specific serine protease (AgChyL) has been earlier shown to be down-regulated during the peak hours of digestion (12 to 24 h) after a blood meal , although one of the serine protease (AGAP011477) showed up-regulation with light pulse in female head. An angiotensin converting enzyme and two aminopeptidase genes were down-regulated after blood feeding in the rest of the female body; however they were not regulated with light pulse. This finding is particularly interesting in light of a study that showed that A. gambiae aminopeptidase N (AgAPN1) was involved in blocking the development of Plasmodium ookinetes in several mosquito species .
Other functional gene groups
In D. melanogaster, a variety of physiological processes have been shown to be under circadian control and to be associated with periodic oscillations in gene expression patterns [39, 40]. A. gambiae genes regulated by light pulse and blood feeding which are involved in other functional groups like immunity, oxidative stress and redox, cytoskeletal and structural, metabolism and other housekeeping genes (Figure 3A and Additional file 5) are discussed below. A larger number of genes were regulated by light pulse: immunity (50 genes), metabolism (55 genes), cytoskeletal and structural (32 genes), redox/stress (53 genes), RTT (42 genes) and TRP (41) functional groups, compared to those regulated by blood feeding (36 genes, 43 genes, 6 genes, 29 genes 10 and 41 genes respectively). In the "diverse" functional category, there were 159 genes that were regulated exclusively by light pulse stimulation, 63 exclusively by blood feeding and 20 by both stimuli. Of the genes whose functions were unknown, 333 genes were regulated exclusively by a light pulse, 179 exclusively after blood feeding and 36 by both stimuli (Figure 3A). A significant number of light pulse- and blood feeding-regulated genes were most likely not identified in these assays because of limitations of the microarray methodology to detect mRNA of lower abundance. These major changes in gene expression are indicative of the highly significant impact that a light pulse can have on mosquito physiology, at least partly by modulating circadian oscillators.
As many as 50 genes with putative roles in the mosquito's innate immune system were found to be regulated by light pulse stimulation and 36 genes with blood-feeding exclusively. Thirty genes were regulated by both stimuli (Figure 3A) which adds up to 116 immune genes being regulated by the two stimuli (Additional file 4). These included genes encoding pattern recognition receptors (fibrinogen binding protein [FBN], gram negative bacteria binding protein [GNBP], thioester containing proteins [TEP], the peptidoglycan recognition protein family [PGRP], c type lectin [CTL], the gal lectin family [GAL-E], the scavenger receptor family, and the leucine rich repeat protein family [LRR]), serine protease cascade components (CLIP domain serine proteases and serpins), immune signaling pathway factors (cactus, rel1 and rel2), antimicrobial peptides (cecropin, gambicin, lysozyme, and defensin), and other functionally diverse proteins [41, 42]. It was recently shown in Drosophila that disruption of the circadian rhythm is often correlated with increased susceptibility to infection . The circadian regulation of the mosquito's innate immunity factors is particularly intriguing, since many of these molecules can modulate resistance to the malaria parasite Plasmodium . Interestingly, blood feeding alone up-regulated several immunity genes such as cecropin, fibrinogen immunolectins, leucine rich repeat protein, peroxidase precursor, thioester containing proteins as well as TOLL precursors and serpins. Bacterial proliferation occurs in the gut after a blood meal and this is likely to result in immune gene induction [35, 44]. Several immune genes were also down-regulated after blood-feeding and those includes among others; c-type lectin 8, fibrinogen immunolectin 38, a scavenger receptor protein and spaetzle 4 [35, 45].
Oxidative stress and redox genes
A variety of oxidoreductive and stress-related genes were found to be regulated upon light pulse stimulation (Figure 3A). Genes in this group included those encoding cytochromes, glutathione S-transferases (GST s), oxidoreductases, dehydrogenases and heat shock proteins. Most of the detoxification-related genes (18 genes); such as the cytochrome P450, GST and DNAJ genes, were down-regulated after light pulse stimulation in the female whole mosquito and head. These genes have been linked to insecticide resistance . Light pulse stimulation can change the redox state by generating several ions/molecules, resulting in up-regulation of several of these genes, including those for oxidoreductases, mitochondrial carrier proteins, heat shock proteins, and several dehydrogenases . It has been shown earlier that several detoxification enzymes are up-regulated after blood feeding, as a result of the release of reactive oxygen species during the digestion of heme ; a similar pattern observed in our assay, with most enzymes being up-regulated after blood feeding.
Cytoskeletal and structural genes
Thirty two genes encoding components of the cytoskeleton and those involved in structural maintenance, such as those encoding actin, myosin, tubulin, kinesin, tropomyosin, cuticle proteins and dynein, were mainly up-regulated (with few exceptions) by light pulse stimulation in both head and whole mosquitoes (Figure 3A). Six genes were regulated with blood-feeding exclusively and 4 genes were regulated by both stimuli (Figure 3A); hence 42 genes were regulated by both stimuli (Additional file 4). Many of these genes have previously been shown to be differentially expressed after blood feeding , and some play a role in the Plasmodium parasite's traversal of the midgut epithelial cells and the repair of the midgut epithelium after this invasion . Of particular interest are the peritrophin precursor gene, which is up-regulated in female head after both light pulse stimulation and blood feeding, and an annexin gene which was down-regulated in female head after light pulse stimulation. An annexin protein has been previously shown to be up-regulated by light pulse stimulation and serotonin in the eye circadian system of Aplysia species .
Metabolism and other housekeeping genes
As many as 55 genes encoding a variety of enzymes involved in a range of different metabolic pathways were differentially regulated upon light pulse stimulation; 33 were up-regulated and the remaining 22 were down-regulated in both whole female mosquitoes and in their heads (Figure 3A). A variety of replication-, transcription-, and translation-related genes (42 genes) were regulated by light pulse stimulation and to a lesser extent after blood feeding (10 genes), in both the head and whole mosquito Additional file 5. A variety of metabolic processes are tightly controlled by circadian oscillators, since these processes need to exhibit different activity levels during the day and night phases [39, 48]. Genes related to transport processes (41 genes) were also regulated (mainly up regulated in total mosquitoes) with light stimuli; this finding is consistent with reports that light entrainment is associated with adjustments in several photoreceptor molecules, and several ions, solute factors, and bio-molecules may need to be transported to other cellular compartments .
Circadian expression of the clock and chemosensory genes in A. gambiae
The regulation of several clock and chemosensory genes by a short light pulse stimulation (Figure 3A) which, however, was not capable of inducing a phase advance was intriguing (Figure 1B), and we pursued further characterization of selected genes (tim, per, cry 1, clk, putative to 1, obp4 and obp22) with regard to their mRNA abundance at successive time points over a LD 12:12 cycle to test for their circadian regulation (Figure 3B and 3C). This analysis showed some interesting features that may suggest that the circadian system of hematophagous insects share certain commonalities. All seven genes showed a higher expression pattern in female head at the night-dark phase (CT14), similarly to what has been observed in D. melanogaster . The expression pattern of these genes (except cry 1) showed a quite different expression pattern in the rest of the body where their mRNA abundance displayed the lowest level during the night-dark phase (CT14). A similar observation has been shown for the blood sucking sandfly Lutzomyia longipalpis, where several clock regulated genes were regulated in an opposite manner in female head and body .
Determination of RNAi knockdown efficiency and expression analysis of selected circadian and chemosensory genes
To identify putative components of the molecular interactions between the mosquito's light sensing system, light pulse-entrained circadian oscillators and its host-seeking and feeding systems, we selected 10 genes for RNAi gene silencing assays in adult female mosquitoes to assess their potential influence on the mosquito's blood-feeding behavior. Seven of these genes (tim, putative to1, to 2, to3, obp4, obp22, and obp26) were selected on the basis of their expression patterns (Additional file 5), and the remaining three (per, clk1 and cry) were included as representative of previously identified circadian clock factors. A series of assays were first carried out to determine the silencing efficiencies tissue specific expression specificities of the targeted genes.
The transcript abundance of these genes was assessed in the antennae (including proboscis and maxillary palps), the head, and the remaining body of 4-day-old female mosquitoes (Figure 4B). Since the circadian clock components have a rhythmic cyclical expression, RNA samples were collected at 4 hr intervals over a 24-h period (at CT0, 4, 8, 12, 16, 20 and 24) and then pooled for analysis of tissue-specific expression. The tim and per transcripts and all three putative to transcripts were detected at equal levels in all body parts, while cry 1 and clk showed slightly lower expression in the antennae than in the other body parts (Figure 4B). The three obps obp4, obp22, and obp26 were expressed only in the head and antennae and not in the rest of the body, consistent with previous reports [50, 51, 53].
Modulation of blood-feeding behavior through gene silencing of light-pulse regulated circadian and chemosensory factors
Phylogenetic analysis of the takeout homologs of A. gambiae and their regulation in response to starvation
Our study has established an important role for the light modulated circadian and chemosensory oscillators in the regulation of the mosquito's blood feeding behavior, and consequently its capacity to transmit blood-borne pathogens. While previous studies have described the A. gambiae flight activity during the night time and during the twilight [11, 12], here we provide insight into the molecular components and mechanisms that regulate the blood feeding behavior. We have shown that the mosquito's blood feeding behavior is under circadian control, with a peak during the late night (Figure 1). A short 2 minute light pulses can momentarily decrease the mosquito's blood-feeding propensity in a non-circadian fashion, while a longer light pulse of 2 hours can induce a phase advance phenomenon in the mosquito's feeding behavior, similarly to what has been shown for the circadian regulation on flight activity . The short 2-min light pulse can cause a profound change in the mosquito's transcriptome that reflected a modulation of a variety of physiological systems, including those that regulate the mosquito's vectorial capacity. We have mainly focused on transcripts that influence mosquito's feeding behavior; future studies are needed to more clearly define the specific ways in which light can contribute to the regulation of the mosquito's anti-Plasmodium defense system and its resistance to infection. The overlap between the light pulse- and blood feeding-induced transcriptomes encompassed a variety of genes, some of which represent molecular links between the mosquito's light sensing and feeding systems. The dosage dependent effect of the short 2-minute light pulse on feeding propensity (Figure 2) over time suggests it may represent a masking effect which however involves several clock factors (Figure 3A). Further detailed studies are required to better understand the impact of a masking mechanism on the mosquito's blood-feeding behavior, and whether this is a phase dependent or independent system. The circadian expression pattern and down-regulation of several obp transcripts after light pulse stimulation (Table 1), in conjunction with the gene-silencing phenotypes observed for these genes (Figure 5), establishes them as essential factors in the mosquito's host-seeking system and further supports an important role of the mosquito's chemosensory oscillators in the regulation of host seeking and feeding behavior [15, 16, 33]. Future work should focus on the characterization of these links since a detailed understanding of these interactions can lead to the development of novel malaria control strategies based on interference with the mosquito's host-seeking behavior.
Rearing of A. gambiae mosquitoes
A. gambiae Keele strain mosquitoes were raised at 27°C and 70% humidity, and adults were maintained on a 10% sucrose solution according to standard rearing condition . The insectary room was equipped with a Philips 25-watt T8 fluorescent lamp (3500K; daylight spectrum) having an intensity of ~800–1000 lux.
The blood feeding behavior of A. gambiae adult mosquitoes and the light pulse treatments
Each type of blood-feeding assay was repeated at least three independent times; with three replicas (of ~20 mosquitoes each) every time. In all assays, the 37°C blood meal was provided by standard artificial membrane feeding system (Parafilm M) for 10 min. The females were first entrained to the 12-h LD cycle of the insectary for 5–6 days before any assays were carried out.
For the time course study of mosquito blood feeding behavior; three mosquito cups (for 3 replicas) were given 37°C blood meal for 10 min, every 3 h (Figure 1). The set that was kept in constant DD, three cups for each set were kept in airtight plastic containers covered with aluminum foil for 48 h before the experiment was conducted. Every 3 h during the assay, one box was opened and the mosquitoes were given 37°C blood meal in dark for 10 min. For mosquitoes that were given light pulses (Figure 1B and 1C), mosquito cups for each set were kept in separate airtight transparent plastic container (3 replicas for each) and maintained in DD or LD conditions. At or before 19.30 hours, light pulses were given (for 5 min or 2 h), after which blood meal was provided for the next 24 hours, every 3 h. The DD or LD conditions were maintained even during blood-feeding time for all the assays.
For the light pulse assays in early morning hours (Figure 2); the night before the assay, each plastic container (containing 3 mosquito cups) was covered with aluminum foil just as the light went off for the night (light intensity in dark was ~1.5 lux). The following day prior to light phase initiation, light pulses were given at different time points and for different durations to each set (Figure 2A and 2B); by removing the aluminum foil and the cover of the respective boxes. After the light pulses, the boxes were covered with aluminum foil and returned to their original room, which was still in dark. The mosquitoes were given a 37°C blood meal for 10 min, when the light went on in the insectary (ZT 0). After feeding, the mosquitoes were sorted according to the amount of blood they had ingested: those that had not fed at all (non-fed), those with a fully stretched abdomen (fully fed) and those with a small quantity of blood in the abdomen (partially fed) (Figure 2 and Additional file 1). Statistical analysis Students T-tests were done and the p-values are shown in Additional file 2.
In all the assays, care was taken not to disturb the mosquitoes and to minimize their exposure to other external stimuli during the light pulse treatments: the experimenter wore a lab coat, double gloves, and a face mask. The light pulses were given to each set in a different room, so as to avoid the unnecessary exposure of any other experimental boxes to the light. In addition, all the different mosquito groups were always contained in their respective airtight plastic containers to reduce the exposure of other mosquito sets when working with one particular set.
RNA isolation and quantitative real-time PCR (qRT-PCR)
RNA was extracted and quantified (in triplicate) either from whole mosquitoes or separately from the antennae (also including the maxillary palps and proboscis), the head, and the rest of the body, using an RNeasy kit (Qiagen, Valencia, California, USA). The qRT-PCR assays were performed as previously described  and the ribosomal protein S7 gene was used for normalization of the cDNA templates. The fold differences in expression levels or gene silencing efficiency (see below) were calculated according to the standard EΔΔCt method .
RNAi gene-silencing assays in adult mosquitoes
RNA interference (RNAi) assays in adult female mosquitoes were performed using established RNAi methodology  injecting ~200 ngm dsRNA per mosquito. To silence the obp genes in head and antennae; at least 7–8 times more dsRNA (~1400–1600 ngm per mosquito) were injected to get a better silencing efficiency in these body parts, where diffusion of dsRNA through hemolymph would be less accessible. Three independent RNAi gene-silencing assays were performed for each gene with two replicas each time (~30 mosquitoes in each replica). The sequences of the primers used for making dsRNA are provided in Additional file 10. The gene silencing was verified by qRT-PCR, 4 days after injection of the dsRNA, and the A. gambiae ribosomal protein S7 gene was used for normalization of the cDNA templates . The primers used for silencing verification are presented in Additional file 11. To study the blood-feeding behavior of the mosquitoes after RNAi gene silencing; 4 days after dsRNA injections the mosquitoes were blood-fed at the time of light onset in the insectary (described earlier) and were scored for blood-fed mosquitoes versus unfed. For statistical analysis, Students T-tests and Mann Whitney tests were done and the p-values are shown in Additional file 8.
RNA was extracted (in triplicates) from total or head of the mosquitoes (as described earlier) of light pulsed and blood fed mosquitoes. The control samples were non-pulsed and unfed mosquitoes respectively. Sample preparations and microarray hybridizations were performed as previously described  with the experimental samples being labeled with Cy-3 and control samples being Cy-5 labeled. Scanning and data analyses were performed as previously described with a signal cut-off intensity of 100 to remove low-intensity/poorly hybridized spots from the analysis. Loc-Fit normalization (LOWESS) was performed independently for each data set through MIDAS (available online, TIGR). The TMEV software (available online, TIGR) was then used for SAM analysis with a 5% false discovery rate (FDR). Only transcripts that had signal values above or below the log2 cutoff value of ± 0.80 were used for further analysis as previously described. The expression data generated by the microarray were validated independently by real-time quantitative PCR (qRT-PCR) for some of the genes and plotted (Additional file 4). There was a high correlation (best-fit linear regression, R2 = 0.76; slope of the regression line, m = 0.91) between the log2-transformed values for the microarray and qRT-PCR. The microarray expression assays and their corresponding GEO accession numbers are:
GSM339808: non-light stimulated vs. 2 hr light stimulated rep 1GSM339819: non-light stimulated vs. 2 hr light stimulated rep 2GSM339907: non-light stimulated vs. 2 hr light stimulated rep 3GSM339909: non-light stimulated vs. 2 min light stimulated at -30 min rep 1GSM339910: non-light stimulated vs. 2 min light stimulated at -30 min rep 2GSM339911: non-light stimulated vs. 2 min light stimulated at -30 min rep 3GSM339912: non-light stimulated female head vs. 2 min light stimulated female head at -30 min rep 1GSM339913: non-light stimulated female head vs. 2 min light stimulated female head at -30 min rep 2GSM339914: non-light stimulated female head vs. 2 min light stimulated female head at -30 min rep 3GSM339915: 2 min light stimulated male head at -30 min vs. 2 min light stimulated female head at -30 min rep 1GSM339916: 2 min light stimulated male head at -30 min vs. 2 min light stimulated female head at -30 min rep 2GSM339917: 2 min light stimulated male head at -30 min vs. 2 min light stimulated female head at -30 min rep 3GSM339918: non-light stimulated male head vs. non-light stimulated female head rep 1GSM339943: non-light stimulated male head vs. non-light stimulated female head rep 2GSM339944: non-light stimulated male head vs. non-light stimulated female head rep 3GSM339945: non-light stimulated male head vs. 2 min light stimulated male head at -30 min rep 1GSM339946: non-light stimulated male head vs. 2 min light stimulated male head at -30 min rep 2GSM339947: non-light stimulated male head vs. 2 min light stimulated male head at -30 min rep 3GSM339949: non-bloodfed female head vs. bloodfed female head rep 1GSM339950: non-bloodfed female head vs. bloodfed female head rep 2GSM339951: non-bloodfed female head vs. bloodfed female head rep 3GSM339953: non-bloodfed female carcass vs. bloodfed female carcass rep 1GSM339954: non-bloodfed female carcass vs. bloodfed female carcass rep 2GSM339955: non-bloodfed female carcass vs. bloodfed female carcass rep 3
Green fluorescent protein
Odorant binding protein
Circadian clock controlled precursor
Pheromone binding protein/General Odorant binding protein
Quantative Real-Time PCR
We thank the microarray core and the insectary facility at the Johns Hopkins Malaria Research Institute. We thank Dr. Deborah McClellan at the Editing Referral Service, William H. Welch Medical Library, Johns' Hopkins University School of Medicine. We thank Dr. Larry Zwiebel for constructive discussions and feedback on this study. This work is supported by the Ellison Medical Foundation, the National Institutes of Health/National Institute for Allergy and Infectious Disease 1R01AI061576-01A1, United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases and Johns Hopkins School of Public Health.
- Emerson KJ, Letaw AD, Bradshaw WE, Holzapfel CM: Extrinsic light:dark cycles, rather than endogenous circadian cycles, affect the photoperiodic counter in the pitcher-plant mosquito, Wyeomyia smithii. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2008, 194: 611-615.PubMed CentralView ArticlePubMedGoogle Scholar
- Honma K, Honma S, Kohsaka M, Fukuda N: Seasonal variation in the human circadian rhythm: dissociation between sleep and temperature rhythm. Am J Physiol. 1992, 262: R885-R891.PubMedGoogle Scholar
- Ikeno T, Numata H, Goto SG: Molecular characterization of the circadian clock genes in the bean bug, Riptortus pedestris, and their expression patterns under long- and short-day conditions. Gene. 2008, 419: 56-61.View ArticlePubMedGoogle Scholar
- Sasagawa H, Narita R, Kitagawa Y, Kadowaki T: The expression of genes encoding visual components is regulated by a circadian clock, light environment and age in the honeybee (Apis mellifera. Eur J Neurosci. 2003, 17: 963-970.View ArticlePubMedGoogle Scholar
- Sidote D, Majercak J, Parikh V, Edery I: Differential effects of light and heat on the Drosophila circadian clock proteins PER and TIM. Mol Cell Biol. 1998, 18: 2004-2013.PubMed CentralView ArticlePubMedGoogle Scholar
- Hall JC: Genetics and molecular biology of rhythms in Drosophila and other insects. Adv Genet. 2003, 48: 1-280.View ArticlePubMedGoogle Scholar
- Hardin PE: The circadian timekeeping system of Drosophila. Curr Biol. 2005, 15: R714-R722.View ArticlePubMedGoogle Scholar
- Suri V, Qian Z, Hall JC, Rosbash M: Evidence that the TIM light response is relevant to light-induced phase shifts in Drosophila melanogaster. Neuron. 1998, 21: 225-234.View ArticlePubMedGoogle Scholar
- Sarov-Blat L, So WV, Liu L, Rosbash M: The Drosophila takeout gene is a novel molecular link between circadian rhythms and feeding behavior. Cell. 2000, 101: 647-656.View ArticlePubMedGoogle Scholar
- So WV, Sarov-Blat L, Kotarski CK, McDonald MJ, Allada R, Rosbash M: Takeout, a novel Drosophila gene under circadian clock transcriptional regulation. Mol Cell Biol. 2000, 20: 6935-6944.PubMed CentralView ArticlePubMedGoogle Scholar
- Jones MD, Ford MG, Gillett JD: Light-on and light-off effects on the circadian flight activity in the mosquito Anopheles gambiae. Nature. 1966, 211: 871-872.View ArticlePubMedGoogle Scholar
- Jones MD, Cubbin CM, Marsh D: The circadian rhythm of flight activity of the mosquito Anopheles gambiae: the light response rhythm. Journal of Experimental Biology. 1972, 57: 337-346.Google Scholar
- Jones MDR: Delayed effect of light on the mosquito clock. Nature. 1973, 245: 384-385.View ArticlePubMedGoogle Scholar
- Haddow AJ: Rhythmic biting activity of certain East African mosquitoes. Nature. 1956, 177: 531-532.View ArticlePubMedGoogle Scholar
- Klowden MJ, Blackmer JL, Chambers GM: Effects of larval nutrition on the host-seeking behavior of adult Aedes aegypti mosquitoes. J Am Mosq Control Assoc. 1988, 4: 73-75.PubMedGoogle Scholar
- Klowden MJ: Endogenous regulation of the attraction of Aedes aegypti mosquitoes. J Am Mosq Control Assoc. 1994, 10: 326-332.PubMedGoogle Scholar
- Zwiebel LJ, Takken W: Olfactory regulation of mosquito-host interactions. Insect Biochem Mol Biol. 2004, 34 (7): 645-652.PubMed CentralView ArticlePubMedGoogle Scholar
- Friend WG, Smith JJ: Factors affecting feeding by bloodsucking insects. Annu Rev Entomol. 1977, 22: 309-331.View ArticlePubMedGoogle Scholar
- Krishnan B, Dryer SE, Hardin PE: Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature. 1999, 400: 375-378.View ArticlePubMedGoogle Scholar
- Jones MD, Gubbins SJ: Modification of circadian flight activity in the mosquito Anopheles gambiae after insemination. Nature. 1977, 268: 731-732.View ArticlePubMedGoogle Scholar
- Jones MDR: Persistence in continuous light of a circadian rhythm in the mosquito Culex pipiens fatigans Wied. Nature. 1976, 261: 491-492.View ArticlePubMedGoogle Scholar
- Antle MC, Sterniczuk R, Smith VM, Hagel K: Non-photic modulation of phase shifts to long light pulses. Journal of Biological Rhythms. 2007, 22: 524-533.View ArticlePubMedGoogle Scholar
- Levy O, Dayan T, Kronfeld-Schor N: The relationship between the golden spiny mouse circadian system and its diurnal activity: an experimental field enclosures and laboratory study. Chronobiol Int. 2007, 24: 599-613.View ArticlePubMedGoogle Scholar
- Mrosovsky N, Thompson S: Negative and positive masking responses to light in retinal degenerate slow (rds/rds) mice during aging. Vision Res. 2008, 48: 1270-1273.View ArticlePubMedGoogle Scholar
- Aschoff J, von GC: Masking of circadian activity rhythms in hamsters by darkness. J Comp Physiol [A]. 1988, 162: 559-562.View ArticleGoogle Scholar
- Aschoff J: Advances in the Biosciences. Trends in Chronobiology. Edited by: Hekkens WT. 1988, Pergamon: Oxford, 149-161.Google Scholar
- Mrosovsky N: Masking: history, definitions, and measurement. Chronobiol Int. 1999, 16: 415-429.View ArticlePubMedGoogle Scholar
- Mboera LEG, Knols BGJ, Takken W, Torre A: The response of Anopheles gambiae s.l. and An. funestus (Diptera: Culicidae) to tents baited with human odour or carbon dioxide in Tanzania. Bull Entomol Res. 1997, 87: 173-178.View ArticleGoogle Scholar
- Galun R, vi-Dor Y, Bar-Zeev M: Feeding Response in Aedes aegypti: Stimulation by Adenosine Triphosphate. Science. 1963, 142: 1674-1675.View ArticlePubMedGoogle Scholar
- Williams CR, Ritchie SA, Russell RC, Eiras AE, Kline DL, Geier M: Geographic variation in attraction to human odor compounds by Aedes aegypti mosquitoes (Diptera: Culicidae): a laboratory study. J Chem Ecol. 2006, 32: 1625-1634.View ArticlePubMedGoogle Scholar
- Takken W, van Loon JJ, Adam W: Inhibition of host-seeking response and olfactory responsiveness in Anopheles gambiae following blood feeding. J Insect Physiol. 2001, 47: 303-310.View ArticlePubMedGoogle Scholar
- Dauwalder B, Tsujimoto S, Moss J, Mattox W: The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior. Genes Dev. 2002, 16: 2879-2892.PubMed CentralView ArticlePubMedGoogle Scholar
- Kwon HW, Lu T, Rutzler M, Zwiebel LJ: Olfactory responses in a gustatory organ of the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci USA. 2006, 103: 13526-13531.PubMed CentralView ArticlePubMedGoogle Scholar
- Calvo E, Mans BJ, Andersen JF, Ribeiro JM: Function and evolution of a mosquito salivary protein family. J Biol Chem. 2006, 281: 1935-1942.View ArticlePubMedGoogle Scholar
- Nirmala X, Marinotti O, James AA: The accumulation of specific mRNAs following multiple blood meals in Anopheles gambiae. Insect Mol Biol. 2005, 14: 95-103.View ArticlePubMedGoogle Scholar
- Vizioli J, Catteruccia F, della TA, Reckmann I, Muller HM: Blood digestion in the malaria mosquito Anopheles gambiae: molecular cloning and biochemical characterization of two inducible chymotrypsins. Eur J Biochem. 2001, 268: 4027-4035.View ArticlePubMedGoogle Scholar
- Shen Z, Edwards MJ, Jacobs-Lorena M: A gut-specific serine protease from the malaria vector Anopheles gambiae is downregulated after blood ingestion. Insect Mol Biol. 2000, 9: 223-229.View ArticlePubMedGoogle Scholar
- Dinglasan RR, Kalume DE, Kanzok SM, Ghosh AK, Muratova O, Pandey A, Jacobs-Lorena M: Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen. Proc Natl Acad Sci USA. 2007, 104: 13461-13466.PubMed CentralView ArticlePubMedGoogle Scholar
- McDonald MJ, Rosbash M: Microarray analysis and organization of circadian gene expression in Drosophila. Cell. 2001, 107: 567-578.View ArticlePubMedGoogle Scholar
- Ueda HR, Matsumoto A, Kawamura M, Iino M, Tanimura T, Hashimoto S: Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J Biol Chem. 2002, 277: 14048-14052.View ArticlePubMedGoogle Scholar
- Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G: Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog. 2006, 2: e52-PubMed CentralView ArticlePubMedGoogle Scholar
- Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC: Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol. 2005, 15: 1185-1195.View ArticlePubMedGoogle Scholar
- Shirasu-Hiza MM, Dionne MS, Pham LN, Ayres JS, Schneider DS: Interactions between circadian rhythm and immunity in Drosophila melanogaster. Curr Biol. 2007, 17: R353-R355.View ArticlePubMedGoogle Scholar
- Azambuja P, Garcia ES, Ratcliffe NA: Gut microbiota and parasite transmission by insect vectors. Trends Parasitol. 2005, 21: 568-572.View ArticlePubMedGoogle Scholar
- Marinotti O, Nguyen QK, Calvo E, James AA, Ribeiro JM: Microarray analysis of genes showing variable expression following a blood meal in Anopheles gambiae. Insect Mol Biol. 2005, 14: 365-373.View ArticlePubMedGoogle Scholar
- David JP, Strode C, Vontas J, Nikou D, Vaughan A, Pignatelli PM, Louis C, Hemingway J, Ranson H: The Anopheles gambiae detoxification chip: a highly specific microarray to study metabolic-based insecticide resistance in malaria vectors. Proc Natl Acad Sci USA. 2005, 102: 4080-4084.PubMed CentralView ArticlePubMedGoogle Scholar
- Raju U, Nunez-Regueiro M, Cook R, Kaetzel MA, Yeung SC, Eskin A: Identification of an annexin-like protein and its possible role in the Aplysia eye circadian system. J Neurochem. 1993, 61: 1236-1245.View ArticlePubMedGoogle Scholar
- Oishi K, Amagai N, Shirai H, Kadota K, Ohkura N, Ishida N: Genome-wide expression analysis reveals 100 adrenal gland-dependent circadian genes in the mouse liver. DNA Res. 2005, 12: 191-202.View ArticlePubMedGoogle Scholar
- Meireles-Filho AC, da S, Rivas GB, Gesto JS, Machado RC, Britto C, de Souza NA, Peixoto AA: The biological clock of an hematophagous insect: locomotor activity rhythms, circadian expression and downregulation after a blood meal. FEBS Lett. 2006, 580: 2-8.View ArticlePubMedGoogle Scholar
- Bohbot J, Pitts RJ, Kwon HW, Rutzler M, Robertson HM, Zwiebel LJ: Molecular characterization of the Aedes aegypti odorant receptor gene family. Insect Mol Biol. 2007, 16: 525-537.PubMed CentralPubMedGoogle Scholar
- Fox AN, Pitts RJ, Robertson HM, Carlson JR, Zwiebel LJ: Candidate odorant receptors from the malaria vector mosquito Anopheles gambiae and evidence of down-regulation in response to blood feeding. Proc Natl Acad Sci USA. 2001, 98: 14693-14697.PubMed CentralView ArticlePubMedGoogle Scholar
- Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA: Boosting NF-κB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity. 2006, 25: 677-685.View ArticlePubMedGoogle Scholar
- Xu PX, Zwiebel LJ, Smith DP: Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect Mol Biol. 2003, 12: 549-560.View ArticlePubMedGoogle Scholar
- Warr E, Das S, Dong Y, Dimopoulos G: The Gram-negative bacteria-binding protein gene family: its role in the innate immune system of Anopheles gambiae and in anti-Plasmodium defence. Insect Mol Biol. 2008, 17: 39-51.View ArticlePubMedGoogle Scholar
- Justice RW, Dimitratos S, Walter MF, Woods DF, Biessmann H: Sexual dimorphic expression of putative antennal carrier protein genes in the malaria vector Anopheles gambiae. Insect Mol Biol. 2003, 12: 581-594.View ArticlePubMedGoogle Scholar
- Bohbot J, Vogt RG: Antennal expressed genes of the yellow fever mosquito (Aedes aegypti L).; characterization of odorant-binding protein 10 and takeout. Insect Biochem Mol Biol. 2005, 35: 961-979.View ArticlePubMedGoogle Scholar
- Benedict MQ: Care and maintenance of Anopheline mosquitoes. The molecular biology of disease vectors: A methods manual. Edited by: Crampton JM. 1997, London: Champman & Hall, 3-12.View ArticleGoogle Scholar
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29: e45-PubMed CentralView ArticlePubMedGoogle Scholar
- Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC, Levashina EA: Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene. EMBO Rep. 2002, 3: 852-856.PubMed CentralView ArticlePubMedGoogle Scholar
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