Salivary Gland Protein Repertoire from Aedes aegypti Mosquitoes

Abstract

Diseases caused by arthropod-borne viruses are a significant threat to the health of human and animal populations throughout the world. Better knowledge of the molecules synthesized in the salivary gland and saliva of hematophagous arthropods could be of use for improving the control of pathogen transmission. Recently, a sialome analysis of three Aedes aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) carried out in our laboratory allowed us to identify 44 saliva proteins. Of these secreted proteins, none was exclusively expressed in one colony, suggesting that expression of salivary proteins is highly conserved across populations. In another study, we reported that some of these salivary proteins could be used as the genus-specific markers for travelers' exposure to mosquito vectors. Here, comparison of salivary gland protein profiles between these same three Ae. aegypti colonies was performed using the one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis method. As observed at the saliva level, no significant differences were detected between these three colonies. The salivary gland protein repertoire from the Ae. aegypti mosquito was analyzed using a proteomic approach. One hundred and twenty proteins were identified in these salivary glands representing the largest description of the Ae. aegypti salivary gland protein catalog. We succeeded in identifying 15 secreted proteins, some of which have already been reported as being involved in blood feeding. A comparison of the proteins identified between the salivary glands and the sialome is discussed.

Introduction

M osquitoes from the Aedes genus (e.g., Aedes aegypti and Aedes albopictus) transmit arboviruses such as the Dengue, Yellow fever, or Chikungunya viruses to humans, causing significant morbidity and mortality throughout the world (Halstead 1988, Monath 1994, James 1996). Few vaccines or antiviral agents are available, and ways of controlling the transmission of these pathogens are needed. Pathogen transmission can occur in two ways during blood feeding: (i) when an adult female mosquito ingests infected blood from humans or (ii) via salivation from an infected mosquito to humans. This phenomenon requires interactions between virus and host salivary gland cells through cell receptors (Yazi Mendoza et al. 2002, Salas-Benito et al. 2007). Salivary glands are used as reservoirs for the viruses that are injected during blood meals (Beerntsen et al. 2000). The salivary glands of arthropods thus play a key function in virus transmission.

At the bite site, mosquitoes inject salivary proteins, which are produced in the salivary glands and facilitate blood meals. These salivary proteins counteract vertebrate hemostasis (platelet aggregation, blood clotting, and vasoconstriction), and may modulate the human immune response (Ribeiro 1995, Ribeiro and Francischetti 2003). The regurgitated proteins may also induce an antibody response against the salivary antigens of arthropods in people living in endemic areas (Gillespie et al. 2000, Remoue et al. 2006, 2007), or in travelers transiently exposed to vectors in tropical areas (Orlandi-Pradines et al. 2007). This antibody response could be used to distinguish Anopheles sp. from Aedes sp. exposure (Orlandi-Pradines et al. 2007). Mosquito saliva could be used as an epidemiological marker for evaluating individual human exposure to mosquito bites. Taken together, these data suggest that better knowledge of saliva and salivary gland components is an important issue for understanding the mechanisms of blood feeding and virus transmission, as well as for evaluating human exposure to vectors.

Recently, we compared the relative abundance of saliva proteins collected from three Ae. aegypti colonies (Almeras et al. 2008). We observed that protein profiles were superimposed, despite significant variations in the relative abundance of two protein bands. For the first time, based on a proteomic approach, 44 distinct proteins were identified in the saliva collected from Ae. aegypti mosquitoes (Orlandi-Pradines et al. 2007, Almeras et al. 2008). These observations will serve as a basis for future work to determine the possible role of these proteins in blood feeding success and viral transmission, or their use as antigenic epidemiological markers to measure human exposure to Aedes mosquitoes.

However, collecting mosquito saliva is extremely time consuming and labor intensive, so most research groups prefer to work on mosquito salivary glands. To investigate the mosquito salivary protein repertoire, the salivary-gland cDNA library from adult females was randomly sequenced, and analyzing the transcripts made it possible to predict the protein secreted. Sialotranscriptome from several mosquito species such as Anopheles gambiae (Arca et al. 1999, 2005, Francischetti et al. 2002, Calvo et al. 2006b), Anopheles stephensi (Valenzuela et al. 2003), Anopheles darlingi (Calvo et al. 2004), Anopheles funestus (Calvo et al. 2007), Anopheles dirus B (Diptera: Culicidae) (Jariyapan et al. 2006), Ae. aegypti (Valenzuela et al. 2002, Ribeiro et al. 2007), Ae. albopictus (Arca et al. 2007), or Culex pipiens quinquefasciatus (Ribeiro et al. 2004), and C. pipiens pallens (Chen et al. 2007) were performed. These descriptions of salivary gland transcripts play a part in improving our knowledge of the components of saliva and the discovery of new pharmacological agents. However, very few data are available regarding the proteins genuinely expressed in the salivary glands of Ae. aegypti mosquitoes. Despite the description of 614 transcripts in the salivary glands of Ae. aegypti, only 24 proteins have been identified by mass spectrometry (MS) (Ribeiro et al. 2007).

The objectives of the present study were (i) to compare the salivary gland protein profiles from three Ae. aegypti colonies (Rockefeller, PAEA, and Formosus) that are maintained under highly inbred laboratory rearing using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis (1D DIGE), and (ii) to investigate the salivary gland protein repertoire from these colonies by means of a proteomic approach. Finally, a comparison of the proteins identified between the salivary glands and saliva will be discussed.

Materials and Methods

Mosquitoes and salivary glands extraction

Three populations of 10-day-old uninfected Ae. aegypti mosquitoes, which differed in their origins and laboratory colonization histories, were used in this study (Table 1). Adult female Ae. aegypti from Formosus, Rockefeller, and PAEA colonies were reared at the Institut Pasteur (Paris) and maintained under strictly identical standard conditions: 26°C and 60% humidity. Mosquitoes were selected 2 days after their first blood feeding on rabbit blood maintained at 37°C. The salivary glands from adult mosquito females were dissected using a fine entomological needle under a stereomicroscope at 4 × magnification, in two independent experiments (at 2-month intervals), in which the mosquitoes from the three colonies were handled at the same time under strictly identical conditions. The salivary glands from each experiment were pooled by colony into a microcentrifuge tube on ice in phosphate-buffered saline and then stored frozen at −20°C until needed.

Table 1.

Characteristics of the Aedes aegypti Colonies

Ae. aegypti colonies	Origin	Collection date (length of time in laboratory, years)
Formosus	Senegal (Kedougou)	2002 (5)
Rockefeller	Caribbean	∼1950 (∼50)
PAEA	Tahiti (Papeete)	1987 (20)

CyDye labeling and SDS-PAGE

The salivary glands were disrupted by ultrasonication (Vibracell 72412; Bioblock Scientific, Illkirch, France) for 5 min on ice at maximum amplitude. Salivary gland homogenates (SGH) were centrifuged for 15 min at 16,100 g, and the protein concentration of the supernatant was determined in duplicate using the Lowry method (DC Protein assay Kit; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Salivary gland proteins were then concentrated by precipitation with acetone (Sigma, St. Louis, MI), and were suspended in a buffer containing 8 M urea (Sigma), 2 M thiourea (Sigma), 4% (w/v) CHAPS (Sigma), and 30 mM Tris (Sigma), adjusted to pH 8.5 to obtain a protein concentration adjusted to 2.5 μg/μL.

The salivary gland proteins were then minimally labeled with CyDye according to the manufacturer's recommended protocols. Briefly, proteins (25 μg) were labeled with 200 pmol of either cyanine 5 (Cy5) or Cy3 or Cy2 (GE Healthcare, Uppsala, Sweden), freshly dissolved in anhydrous DMF (Sigma), and incubated on ice for 30 min in the dark. The reaction was quenched with 1 μL of free lysine (10 nM; Sigma) by incubation of 10 min on ice. Cy5, Cy3, and Cy2 labeled samples were then pooled, and an equal volume of 2 × Tris buffer was added containing 5% (w/v) SDS (Sigma). For protein separation, 15 μg of pooled labeled samples reduced with 1% (w/v) dithiothreitol (Sigma) was loaded per lane on to a 10% SDS-PAGE.

Image analysis

After electrophoresis, the gels with CyDye-labeled proteins were scanned three times with a Typhoon™ Trio Image scanner (GE Healthcare), each time at different excitation wavelengths (Cy3, 580 BP 30/green [532 nm]; Cy5, 670 BP 30/red [633 nm]; Cy2, 520 BP 40/blue [488]). Prescans were performed to adjust the photomultiplier tube voltage to obtain images with a maximum intensity of 60,000 to 80,000 U. Images of salivary gland profiles were further analyzed using ImageQuant™ TL software (GE Healthcare). Background subtraction was performed and the densitometry profiles were normalized to take into account global differences. Relative abundance in proteins from each band was estimated by dividing the area under the curve of the peak corresponding to the band by the sum total of the areas under the curves for all the bands. The gels were then stained with Sypro Ruby (Bio-Rad) according to the manufacturer's protocol.

In-gel tryptic digest

Excised bands from the Sypro Ruby–stained gels were prepared as described previously by Almeras et al. (2008). Samples were then stored at −20°C before their analysis with MS.

MS analysis

The resulting peptides were extracted from the gel and analyzed by nanoscale capillary liquid chromatography-tandem MS (nano LC-MS/MS). Purification and analysis were performed on a C18 capillary column using a CapLC system (Waters, Milford, MA) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF Ultima; Waters). Chromatographic separations were conducted on a reversed-phased capillary column (Atlantis™ dC18, 3 μm, 75 μm × 150 mm Nano Ease™; Waters) with a 180–200 nL/min flow. The gradient profile consisted in a linear gradient from 95% A (H₂O, 0.1% HCOOH) to 60% B (80% acetonitrile, 0.1% HCOOH) in 60 min followed by a linear gradient to 95% B in 10 min. Mass data acquisitions were piloted by MassLynx 4.0 software using automatic switching between MS and MS/MS modes. The internal parameters of Q-TOF were set as follows. The electro-spray capillary voltage was set to 3.2 kV, the cone voltage was set to 30 V, and the source temperature was set to 80°C. The MS survey scan was m/z 400–1300 with a scan time of 1 s and an interscan time of 0.1 s. When the intensity of a peak rose above a threshold of 15 counts, tandem mass spectra were acquired. Normalized collision energies for peptide fragmentation were set using the charge-state recognition files for +2 and +3 peptide ions. The scan range for MS/MS acquisition was from m/z 50 to 1500 with a scan time of 1 s and an interscan time of 0.1 s. Fragmentation was performed using argon as the collision gas and with the collision energy profile optimized for various mass ranges and charge of precursor ions. Mass data collected during a nano LC-MS/MS analysis were processed using ProteinLynx Global Server 2.2 software (Waters) with the following parameters: no background subtraction, smooth 3/2 Savitzky Golay and no deisotoping to generate peak lists in the micromass pkl format. Pkl files were then fed into a local search engine Mascot Daemon v2.2.2 (Matrix Science, London, UK). The data were screened against the National Center for Biotechnology Information nonredundant protein database (January 10, 2008) with other Metazoa (273,686 sequences) as a taxonomy setup. Search parameters allowed for one missed tryptic cleavage site, the carbamidomethylation of cysteine, and the possible oxidation of methionine; precursor and product ion mass error tolerance was <0.2 Da. All identified proteins had a Mascot score greater than 67, corresponding to a statistically significant (p < 0.05) confident identification.

Statistical analysis

Differences in the relative abundance of each protein band between the colonies were analyzed taking into account the hypothetical differences between gels or experiments using two-way analysis of variance, with the colony as a factor and the gel or the experiment as a cofactor. To take into account that multiple tests performed, we considered significant p-values after the Bonferroni correction: p < 0.05/30 ≈0.0017. All statistical analyses were done with SAS software version 9.1 (SAS Institute, Cary, NC).

Results and Discussion

The most frequent strategy employed to study the molecules expressed in the salivary glands of mosquitoes encompasses either random sequencing of clones from salivary gland cDNA libraries or uses a signal trapping method for specific isolation of cDNA-encoding proteins with signal peptides (Arca et al. 2002, Francischetti et al. 2002, Lanfrancotti et al. 2002, Valenzuela et al. 2003, Calvo et al. 2004). Although emerging evidence suggests that transcriptome profiling is necessary, this approach seems to be insufficient for comprehensive delineation of biological systems. It is possible that some transcripts identified might not be expressed at the protein level (Mounsey et al. 2002). Conversely, if a protein is able to be identified inside a tissue, the corresponding transcript can be automatically designated as a protein-coding region. Therefore, in addition to monitoring gene expression at the transcriptional level, large-scale analysis of the proteome is also important for the understanding of the cellular, metabolic, and regulatory networks in living organisms or specific tissues.

In this study, we carried out a proteomic analysis of salivary gland proteins from three Ae. aegypti colonies, and compared protein identifications between the salivary glands and sialomes from these mosquitoes. Good reproducibility of salivary gland protein profiles was observed between colonies, and more than 100 distinct proteins were successfully identified.

Comparative analysis of salivary gland protein profiles from Ae. aegypti colonies

Recently, we performed a sialome (i.e., proteins present in saliva) protein profile comparison between three Ae. aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) reared under identical laboratory conditions (Almeras et al. 2008). Despite significant quantitative differences for only two protein bands, salivary profile analysis indicated that major proteins were detectable in the three colonies. These data suggested that Ae. aegypti colonies conserved their own species characteristics. In this study, we compared the salivary gland protein profiles from these same three Ae. aegypti colonies. A 1D DIGE method was chosen to increase the accuracy of this comparison, providing high sensitivity and reproducibility between experiments. DIGE technology, using sample multiplexing and the linear dynamic range of fluorescent protein labeling, allows small differences to be accurately detected and quantified with statistical confidence, rather than conventional one-sample-per-lane techniques (Chakravarti et al. 2005, Timms and Cramer 2008).

SGH from PAEA, Rockefeller, and Formosus colonies were thus, respectively, labeled with Cy5, Cy3, and Cy2, followed by 1D SDS-PAGE separation of the salivary glands. Protein profiles analyzed with Image Quant TL software made it possible to detect numerous bands with molecular weights ranging from about 7 to 225 kDa (Fig. 1). Thirty protein bands could be detected on the gel for each colony; however, these individual bands presented a wide dynamic concentration range (i.e., the bands differed in intensity; Fig. 1). For the three Ae. aegypti colonies, seven predominant bands were observed (band numbers 9, 10, 13, 15, 19, 21, and 22; Fig. 1).

FIG. 1.

Comparative salivary gland protein profiles of three Aedes aegypti colonies. Salivary gland proteins collected from Formosus, Rockefeller, and PAEA Ae. aegypti colonies were, respectively, labeled with cyanine 2 (Cy2), Cy3, and Cy5, before being mixed and separated on 10% SDS-PAGE gels. Salivary gland protein profiles from each Ae. aegypti colony, and the merged protein profiles are indicated at the top of the gel. The numbers on the right side of the gel correspond to the 30 bands excised for further analysis by mass spectrometry. Band identity is listed in Table 2. Standard molecular masses are indicated on the left side. MW, molecular weight.

Before comparing protein profiles between colonies, the variations associated with the sample collection (experimental effect) and with protein migration (gel effect) were assessed. Two gels were then performed under the same conditions as the gel presented in Figure 1. A pool of labeled samples collected at two time points (as described in the Materials and Methods section) were loaded on each gel (data not shown). A densitometric scan and normalization of the gels were performed, giving an accurate comparison of the salivary gland protein profiles between colonies. Statistical tests (analysis of variance) were performed to determine bands that differed in intensity between colonies, taking into account gel or experimental effects as described previously (Almeras et al. 2008). No statistically significant difference was detected between the three Ae. aegypti colonies. Several hypotheses could explain this result: first, as observed previously in the sialome (Almeras et al. 2008), the three colonies conserved their own species characteristics limiting protein expression variations between these mosquito colonies; second, despite the high sensitivity and reproducibility of CyDye protein labeling, the 1D DIGE technique is insufficient for detecting significant protein variations (perhaps additional experiments with a better protein separation method [such as 2D DIGE] could be performed to verify protein pattern reproducibility between these three colonies); third, as observed in a previous Ae. aegypti sialome analysis (Almeras et al. 2008), a long history of laboratory rearing could have limited environmental pressures and induced a homogenization of salivary gland protein repertoires. To determine if inbred rearing has induced a decrease in protein colony singularity, a comparison of salivary gland protein profiles between these colonies and their counterparts collected in the field would have to be performed. In summary, this analysis shows that despite variations in band intensity, no statistically significant differences were noted between the three colonies, suggesting low variability in protein expression as observed at the saliva level.

Identification of Ae. aegypti salivary gland proteins

Thirty bands numbered in Figure 1 were excised and submitted to trypsin digestion before an analysis of peptide mixture by MS (LC-MS/MS) for identification. Each protein band was analyzed twice. Only the protein band numbered 30 failed to be identified by MS. All the other bands excised allowed the identification of at least one protein, corresponding to 164 proteins identified (Table 2). As expected, several proteins could be identified in each excised band, such as bands 8 and 21, which contain 7 and 5 proteins, respectively (Table 2). Inversely, the same protein was also detected in several excised bands, such as malate dehydrogenase (gi|108875864, identified in bands 20, 21, and 22) or ADP, ATP carrier protein (gi|108872852, identified in bands 11, 22, 23, and 24). The 164 proteins identified effectively correspond to 120 distinct proteins according to their NCBI numbers (Table 2).

Table 2.

Identified Proteins from Salivary Gland Profiles of the Aedes aegypti Colonies

			Molecular weight (kDa)
Band number excised	Protein name	Accession no. ^a	Theoretical	Observed	Coverage (%)	Number of MS/MS peptide sequences identified	Significance (Mascot score)
1	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878633	222.35	225	20	35	2028.06
	SGS1 (Ae. aegypti)^b	gi\|66828491	346.51		5	15	733.86
	Conserved hypothetical protein (Ae. aegypti)	gi\|108873896	369.18		4	13	643.09
	na+/k+ atpase alpha subunit (Ae. aegypti)	gi\|108871576	111.86		4	3	239.84
	Actin (Ae. aegypti)^d	gi\|108879763	42.15		12	4	172.45
2	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878633	222.35	211	14	22	1256.12
	Conserved hypothetical protein (Ae. aegypti)	gi\|108873896	369.18		5	16	705.79
3	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878633	222.35	201	36	63	3664.63
	Vitellogenin-B (Ae. aegypti)^b	gi\|37528873	250.39		4	8	389.27
	Glutamate synthase (Ae. aegypti)	gi\|108868750	231.67		2	4	145.47
4	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878633	222.35	168	9	18	1106.45
	Mitochondrial ATP synthase alpha subunit (Ae. aegypti)	gi\|94468442	59.52		7	3	151.76
	Actin (Ae. aegypti)^d	gi\|108879764	42.05		7	3	138.12
5	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878633	222.35	141	10	21	1005.36
	Pyruvate carboxylase (Ae. aegypti)	gi\|550486	132.85		6	7	312.53
	na+/k+ atpase alpha subunit (Ae. aegypti)	gi\|108871577	111.91		5	4	311.51
	Conserved hypothetical protein (Ae. aegypti)	gi\|108879668	71.58		10	6	289.44
	Stretchin-mlck (Ae. aegypti)	gi\|108876801	80.19		6	4	262.23
	Pupal-specific flight muscle actin (Ae. aegypti)	gi\|41393662	41.84		18	6	242.55
	Fructose-bisphosphate aldolase (Ae. aegypti)	gi\|108878478	39.97		13	4	226.9
6	na+/k+ atpase alpha subunit (Ae. aegypti)	gi\|108871577	111.91	124	19	15	1105.81
	2-Oxoglutarate dehydrogenase (Ae. aegypti)	gi\|108877402	115.04		19	17	842.19
	Paramyosin, long form (Ae. aegypti)	gi\|108872770	103.39		13	11	592.78
	Calcium-transporting atpase sarcoplasmic/endoplasmic reticulum type (calcium pump) (Ae. aegypti)	gi\|108877602	110.55		11	10	540.78
	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878633	222.35		2	4	258.77
7	Glycogen phosphorylase (Ae. aegypti)	gi\|108884025	97.35	111	20	17	784.28
	Alpha-actinin (Ae. aegypti)	gi\|108876788	104.20		12	10	612.73
	Elongation factor 2 (Ae. aegypti)	gi\|12667408	95.31		9	8	335.54
	Spermatogenesis associated factor (Ae. aegypti)	gi\|108873203	89.43		9	7	275.19
	Dynamin (Ae. aegypti)	gi\|108876817	94.24		6	4	243.88
	Actin (Ae. aegypti)^d	gi\|108879763	42.15		12	4	220.54
	Paramyosin, long form (Ae. aegypti)	gi\|108872770	103.39		4	4	199.61
8	Aconitase, mitochondrial (Ae. aegypti)	gi\|108870681	86.41	102	56	31	1736.69
	Glutamate semialdehyde dehydrogenase (Ae. aegypti)	gi\|108877298	87.53		20	14	766.36
	Heat shock protein (Ae. aegypti)	gi\|108868694	81.82		10	7	354.58
	Heat shock cognate 70 (Ae. aegypti)^d	gi\|94468818	72.35		10	6	284.42
	Conserved hypothetical protein (Ae. aegypti)	gi\|108873896	369.18		1	3	216.65
	Conserved hypothetical protein (Ae. aegypti)	gi\|108873839	81.32		10	7	218.96
	Myosin heavy chain, nonmuscle or smooth muscle (Ae. aegypti)	gi\|108878534	222.99		2	2	159.16
9	Conserved hypothetical protein (Ae. aegypti)	gi\|108873896	369.18	93	6	18	1169.56
	NADH-ubiquinone oxidoreductase (Ae. aegypti)	gi\|108871047	80.11		18	10	611.62
	3-Hydroxyacyl-coa dehyrogenase (Ae. aegypti)	gi\|108873690	82.87		9	6	324.3
	Heat shock cognate 70 (Ae. aegypti)^d	gi\|94468966	71.38		12	7	357.15
	Moesin/ezrin/radixin (Ae. aegypti)	gi\|108876119	69.13		9	6	272.3
	Glycerol-3-phosphate dehydrogenase (Ae. aegypti)	gi\|108880571	81.65		7	5	265.87
	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881790	29.54		15	4	207.76
10	Transferrin precursor (Ae. aegypti)^b	gi\|2645497	71.70	81	32	18	942.53
	Apyrase, putative (Ae. aegypti)^b–d	gi\|108877845	63.17		23	11	547.61
	Proline oxidase (Ae. aegypti)	gi\|108870085	44.60		21	8	408.27
	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881789	32.75		18	5	292.78
	Vitellogenin-C (Ae. aegypti)^b	gi\|37528871	243.28		3	6	273.14
	Succinate dehydrogenase (Ae. aegypti)	gi\|108873156	72.80		8	5	230.48
11	Chaperonin-60kD, ch60 (Ae. aegypti)	gi\|108872102	61.15	72	14	7	307.99
	Transketolase (Ae. aegypti)	gi\|108879967	68.46		11	6	285.15
	Bifunctional purine biosynthesis protein (Ae. aegypti)	gi\|108870755	64.64		7	4	185.01
	Proline oxidase (Ae. aegypti)	gi\|108870085	44.60		8	3	173.47
	Apyrase, putative (Ae. aegypti)^b–d	gi\|108877845	63.17		5	2	110.22
	Lamin (Ae. aegypti)	gi\|108881054	64.01		4	3	100.61
	Failed axon connections protein, putative (Ae. aegypti)	gi\|108881007	34.51		12	3	98.32
	Cytochrome P450 (Ae. aegypti)	gi\|108877345	62.65		5	2	95.91
	Actin (Ae. aegypti)^d	gi\|108879764	42.05		5	2	86.32
	adp,atp carrier protein (Ae. aegypti)	gi\|108872852	28.85		9	2	73.15
12	Pyrroline-5-carboxylate dehydrogenase (Ae. aegypti)	gi\|108878912	63.63	66	27	14	666.26
	Chaperonin-60kD, ch60 (Ae. aegypti)	gi\|108872102	61.15		19	9	526.56
	Malic enzyme (Ae. aegypti)	gi\|108883625	72.55		10	6	320.16
	Adenosine deaminase (Ae. aegypti)^b,d	gi\|108878609	59.94		17	7	331.37
	Pyruvate kinase (Ae. aegypti)	gi\|108868621	58.04		18	7	305.23
	α-tubulin (Ae. aegypti)	gi\|94468850	50.56		11	4	198.02
	Actin (Ae. aegypti)^d	gi\|108879764	42.05		9	3	191.16
13	Mitochondrial ATP synthase alpha subunit (Ae. aegypti)	gi\|94468442	59.52	57	62	26	1626.05
	ATP synthase beta subunit (Ae. aegypti)	gi\|108881686	53.87		55	19	1251.26
	β-4 tubulin (Ae. aegypti)	gi\|111035022	50.59		39	12	670.51
14	ATP synthase beta subunit (Ae. aegypti)	gi\|108881105	53.94	51	35	13	844.37
	Mitochondrial ATP synthase alpha subunit (Ae. aegypti)	gi\|94468442	59.52		25	13	777.66
	Enolase (Ae. aegypti)	gi\|108882996	46.87		28	9	546.64
	Phosphoglycerate kinase (Ae. aegypti)	gi\|18091771	44.05		25	10	503.85
	Pupal-specific flight muscle actin (Ae. aegypti)	gi\|41393662	41.84		31	9	502.26
	Mitochondrial processing peptidase beta subunit (Ae. aegypti)	gi\|108878872	52.84		18	8	487.53
	Serine protease inhibitor (serpin-4), putative (Ae. aegypti)^b	gi\|108881841	50.03		23	9	484.3
	Translation elongation factor EF-1 alpha/Tu (Ae. aegypti)	gi\|94468780	50.78		25	10	458.31
	Succinyl-coa synthetase beta chain (Ae. aegypti)	gi\|108871926	48.64		16	7	420.09
	Putative serpin (Ae. aegypti)^b,d	gi\|18568304	47.31		17	6	320.72
	Aspartate ammonia lyase (Ae. aegypti)	gi\|108875839	54.78		14	5	308.42
	Actin (Ae. aegypti)^d	gi\|108879764	42.05		17	5	260.23
	Imaginal disc growth factor (Ae. aegypti)	gi\|108882601	48.28		10	4	239.51
15	Ubiquinol-cytochrome c reductase complex core protein (Ae. aegypti)	gi\|108879066	45.88	45	33	11	780.32
	Pupal-specific flight muscle actin (Ae. aegypti)	gi\|41393662	41.84		36	10	686.74
	Citrate synthase (Ae. aegypti)	gi\|108881547	51.85		29	12	577.39
	Fructose-bisphosphate aldolase (Ae. aegypti)	gi\|108878478	39.97		21	6	400.63
	Creatine kinase (Ae. aegypti)	gi\|94468822	40.19		29	8	351.2
16	Fructose-bisphosphate aldolase (Ae. aegypti)	gi\|108878479	39.55	41	48	14	954.41
	Creatine kinase (Ae. aegypti)	gi\|94468822	40.19		51	14	832.83
	Isocitrate dehydrogenase (Ae. aegypti)	gi\|108884328	44.04		32	10	535.44
	Pyruvate dehydrogenase (Ae. aegypti)	gi\|108869893	42.80		10	4	211.72
	d-3-phosphoglycerate dehydrogenase (Ae. aegypti)	gi\|108878977	35.72		15	4	184.92
	Sodium/potassium-dependent atpase beta-2 subunit (Ae. aegypti)	gi\|108872996	36.37		22	5	174.87
	Putative purine hydrolase (Ae. aegypti)	gi\|18568280	38.16		7	3	123.13
	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881790	29.54		8	2	84.51
17	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881788	31.03	40	31	9	396.92
	ATP synthase beta subunit (Ae. aegypti)	gi\|108881105	53.94		6	3	181.52
18	Glycerol-3-phosphate dehydrogenase (Ae. aegypti)	gi\|108883108	39.44	39	34	9	614.75
	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881786	32.47		29	9	477.96
	D7 protein, putative (Ae. aegypti)^b–d	gi\|108877769	37.44		14	5	210.98
19	ATP synthase beta subunit (Ae. aegypti)	gi\|108881105	53.94	38	27	11	687.94
	D7 protein, putative (Ae. aegypti)^b–d	gi\|108877769	37.44		28	10	435.98
	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881783	32.61		24	7	415.68
	Glycerol-3-phosphate dehydrogenase (Ae. aegypti)	gi\|108883107	39.83		19	5	261.53
20	D7 protein, putative (Ae. aegypti)^b–d	gi\|108877768	39.17	36	6	2	88.63
	Malate dehydrogenase (Ae. aegypti)	gi\|108875864	45.04		19	7	427.58
	Electron transport oxidoreductase (Ae. aegypti)	gi\|108869776	34.44		12	3	207.17
	F0F1-type ATP synthase b subunit (Ae. aegypti)^d	gi\|94468834	53.93		6	3	177.09
	Putative 34 kDa secreted protein (Ae. aegypti)^b,d	gi\|18568296	36.38		10	3	172.42
	Succinyl-CoA synthetase alpha subunit (Ae. aegypti)	gi\|94468890	34.79		9	3	163.5
	Prohibitin (Ae. aegypti)	gi\|108871326	33.12		12	4	141.06
	Tropomyosin invertebrate (Ae. aegypti)	gi\|108881790	29.54		12	3	116.27
21	Malate dehydrogenase (Ae. aegypti)	gi\|108875864	45.04	34	37	13	768.73
	14-3-3 protein sigma, gamma, zeta, beta/alpha (Ae. aegypti)	gi\|108872598	29.60		21	5	271.81
	Vacuolar ATP synthase subunit E (Ae. aegypti)	gi\|94469084	25.72		17	4	191.63
	Succinyl-CoA synthetase alpha subunit (Ae. aegypti)	gi\|94468890	34.80		11	3	183.81
	Mitochondrial porin (Ae. aegypti)	gi\|94468842	30.79		16	4	173.1
22	14-3-3 protein sigma, gamma, zeta, beta/alpha (Ae. aegypti)	gi\|108872598	29.60	31	41	9	550.16
	ATP synthase gamma subunit (Ae. aegypti)	gi\|108875130	33.01		31	8	499.79
	Multifunctional 14-3-3 family chaperone (Ae. aegypti)	gi\|94468884	28.32		33	7	443.26
	Mitochondrial porin (Ae. aegypti)	gi\|94468842	30.79		29	7	363.65
	Putative 30 kDa allergen-like protein (Ae. aegypti)^b–d	gi\|18568322	23.79		23	5	239.35
	adp,atp carrier protein (Ae. aegypti)	gi\|108872852	28.85		19	5	172.44
	30 kDa salivary gland allergen variant 3 (Ae. aegypti)^b,d	gi\|94468552	27.91		11	3	167.84
	ADP/ATP translocase (Ae. aegypti)	gi\|94468376	33.21		14	4	166.24
	Vacuolar ATP synthase subunit E (Ae. aegypti)	gi\|94469084	25.72		20	4	151.98
	Malate dehydrogenase (Ae. aegypti)	gi\|108875864	45.04		7	3	149.5
23	Phosphoglycerate mutase (Ae. aegypti)	gi\|108878150	28.59	28	40	9	562.34
	ADP/ATP translocase (Ae. aegypti)	gi\|94468376	33.21		31	10	441.23
	Multifunctional 14-3-3 family chaperone (Ae. aegypti)	gi\|94468884	28.32		34	8	433.29
	adp, atp carrier protein (Ae. aegypti)	gi\|108872852	28.85		29	8	369.73
	30 kDa salivary gland allergen variant 2 (Ae. aegypti)^b,c	gi\|94468546	29.42		17	5	276.02
	Electron transfer flavoprotein beta-subunit (Ae. aegypti)	gi\|108879274	22.90		22	5	204.1
	Putative 30 kDa allergen-like protein (Ae. aegypti)^b–d	gi\|18568322	23.79		15	3	173.73
	Conserved hypothetical protein (Ae. aegypti)	gi\|108873666	23.63		12	3	163.2
	NADH-ubiquinone oxidoreductase 24 kda subunit (Ae. aegypti)	gi\|108878789	23.08		18	3	116.94
24	Triosephosphate isomerase (Ae. aegypti)	gi\|108882001	26.70	26	32	6	340.44
	Peroxiredoxin 6, prx-6 (Ae. aegypti)	gi\|108882310	25.10		29	5	210.96
	Secreted ferritin G subunit precursor, putative (Ae. aegypti)	gi\|108876699	17.68		27	3	186.04
	Creatine kinase (Ae. aegypti)	gi\|94468822	40.19		6	2	108.69
	Mitochondrial ATP synthase alpha subunit (Ae. aegypti)	gi\|94468442	59.52		4	2	105.66
	Conserved hypothetical protein (Ae. aegypti)	gi\|108883588	21.79		14	2	104.93
	adp, atp carrier protein (Ae. aegypti)	gi\|108872852	28.85		9	2	102.97
	Troponin i (Ae. aegypti)	gi\|108872887	24.66		9	2	95.02
	Ribosomal protein S5 (Ae. aegypti)	gi\|94468378	24.79		6	1	85.13
25	Ferritin subunit 1, putative (Ae. aegypti)	gi\|108876700	23.74	23	31	6	313.14
	Adenylate kinase isoenzyme (Ae. aegypti)	gi\|108874575	22.57		22	4	257.7
	Glutathione s-transferase (Ae. aegypti)	gi\|108871931	23.26		23	4	196.64
	40S ribosomal protein S8 (Ae. aegypti)	gi\|94468438	23.40		11	2	122.15
26	Hypothetical protein AaeL_AAEL004249 (Ae. aegypti)	gi\|157105256	17.24	21	26	3	146.76
	Myosin light chain 1, putative (Ae. aegypti)	gi\|157167811	18.15		15	2	108.88
	Calcium-binding protein, putative (Ae. aegypti)	gi\|157119961	22.12		10	3	94.79
27	Myosin light chain 1, putative (Ae. aegypti)	gi\|157167807	18.30	17	29	4	228.82
	Superoxide dismutase (Ae. aegypti)	gi\|157127037	15.62		18	2	129.98
	Histone H2B (Ae. aegypti)	gi\|157137739	13.86		25	3	124.59
	Cytochrome c oxidase subunit iv (Ae. aegypti)	gi\|157108935	46.92		8	3	119.64
	Histone H4 (Ae. aegypti)	gi\|157138406	11.40		21	2	106.07
28	Histone H4 (Ae. aegypti)	gi\|157138406	11.40	14	40	4	195.63
	Myosin light chain 1, putative (Ae. aegypti)	gi\|157167807	18.30		22	3	163.55
	Mitochondrial NADH-ubuiquinone oxidoreductase 13 kDa-B subunit (Ae. aegypti)	gi\|94469096	13.87		13	2	116.65
29	Cytochrome c oxidase, subunit VB, putative (Ae. aegypti)	gi\|157119566	14.01	11	20	2	83.15
30	n.i.				7

The proteins were identified by mass spectrometry after in-gel trypsin digestion. The band name corresponds to the same numbers as indicated in Fig. 1.

The identities of the bands, their ^aNCBi accession numbers, the theoretical and observed molecular weight values, as well as the number of peptide sequences, the corresponding percentage sequence coverage and the Mascot score are listed for MS/MS analysis (Protein scores greater than 67 were considered to be significant (p < 0.05)).

Proteins identified previously in Ae. aegypti saliva or salivary glands by ^bAlmeras et al. (2008), ^cValenzuela et al. (2002), and ^dRibeiro et al. (2007).

n.i., no identification; MS, mass spectrometry.

The proteins identified were classified according to their known or predicted cellular localization and biological function (Fig. 2). Although more than 50% of the proteins identified were assigned in the cytoplasmic and mitochondrial compartments, 15 proteins were classified as secreted salivary proteins representing the third group in terms of the number of proteins identified (12.5%). Of these secreted proteins, 11 (9.2%) were involved in mosquito blood-feeding. Some of these secreted proteins have been described as modulating host immune response (members of the D7 family (gi|108877769 and gi|108877768) (Calvo et al. 2006a), adenosine deaminase (gi|108878609) (Hasko et al. 2000), apyrase (gi|108877845) (Sun et al. 2006), purine hydrolase (gi|18568280), and 30 kDa allergen (gi|94468546, gi|94468552, and gi|18568322) (Ribeiro et al. 2007). Members of the serpin family (gi|108881841 and gi|18568304) have been implicated in the regulation of blood coagulation (Gettins 2002). The secreted ferritin G subunit (gi|108876699) has an antioxidant function and could be used to store iron (Dunkov et al. 2002). For the other secreted proteins (e.g., vitellogenin-C [gi|37528871], vitellogenin-B [gi|37528873], SGS1 [gi|66828491], and 34 kDa secreted protein [gi|18568296]), their functions in mosquito saliva are unknown (Ribeiro et al. 2007).

FIG. 2.

Pie chart representing the proteins identified by mass spectrometry in the salivary glands of Ae. aegypti mosquitoes (n = 120). The proteins have been classified according to their cellular localization (A) and biological function (B). The percentage of proteins associated with each part of the pie chart is indicated in parentheses.

Numerous proteins identified previously in saliva were not found in the salivary glands (Almeras et al. 2008). This could be attributed to the presence of housekeeping proteins in the salivary glands that could mask the detection of the secreted proteins present in smaller quantities. Effectively, numerous proteins involved in cytoskeleton maintenance, transporter function, or nuclear regulation have been characterized by MS analysis (Table 2). However, most of the secreted proteins identified here corresponded to proteins that were detected as predominant bands in the saliva (Almeras et al. 2008). Taken together, these data suggest that only proteins expressed at high levels in Ae. aegypti saliva are also detected in their salivary glands. One secreted protein (e.g., ferritin G subunit, gi|108876699), not detected in saliva, was identified in the salivary glands.

It is interesting to note that certain secreted proteins identified in both the saliva and salivary glands of the same Ae. aegypti colonies were detected at several molecular weights (Table 2) (Almeras et al. 2008). This observation was more frequently found in the saliva than in the salivary glands. It was the case for D7 protein (detected in three bands in SGH vs. seven bands in the saliva) and the SGS1 protein (one vs. eight bands). These differences could be attributed to maturation phenomena, including posttranslational modifications, which could occur during the transit of the secreted proteins from salivary gland cells to saliva, as evoked previously (Almeras et al. 2008).

Few studies have investigated Ae. aegypti salivary gland composition at the protein level. As a result, comparisons of our results with those of others is limited. Valenzuela et al. (2002) identified 10 aminoterminal sequences obtained by Edman degradation of 1D SDS-PAGE separated salivary gland proteins. Ribeiro et al. (2007), using 2D gel electrophoresis, succeeded in identifying 24 salivary gland proteins. Several of these proteins, such as apyrase, members of the D7 protein family, and certain salivary allergens, were also identified in the present study (Table 2). This indicates that only the more abundant proteins were found in these different studies. Interestingly, of the 120 salivary gland proteins identified, 106 were described at the protein level for the first time (Table 2). This proteomic approach confirms the translation of several transcripts predicted until now only by means of the cDNA base strategy (Valenzuela et al. 2002, Ribeiro et al. 2007).

The salivary gland protein repertoire from other mosquito species and genera using proteomic approaches has been performed for Ae. albopictus (Arca et al. 2007), An. gambiae (Kalume et al. 2005, Choumet et al. 2007), An. stephensi (Valenzuela et al. 2003), and Culex pipiens quinquefasciatus (Ribeiro et al. 2004). Regardless of the method (i.e., Edman degradation or MS), the number of secreted proteins identified in the salivary glands in the present and previous studies did not exceed 20, while more than 40 secreted proteins were identified by MS in the saliva of Ae. aegypti (Almeras et al. 2008). This suggests that secreted salivary proteins should be screened for preferentially in saliva rather than in the salivary glands.

Concluding remarks

Genome sequencing has made it conceivable to develop proteomic analysis of several mosquito species (Holt et al. 2002, Lawson et al. 2007, Nene et al. 2007). In this study, to investigate the salivary gland protein repertoire of adult female Ae. aegypti mosquitoes, a proteomic analysis was performed. One hundred and twenty proteins were identified in these salivary glands, representing the largest description of the Ae. aegypti salivary gland protein catalog. Finally, this paper plays a part in identifying 15 secreted proteins, some of which have already been reported to be involved in blood-feeding. Some of these secreted proteins could be used as antigenic markers for the serological estimation of human exposure to Ae. aegypti mosquitoes.

Footnotes

Acknowledgments

This study was financially supported by the Délégation Générale pour l'Armement (DGA, SALIVAPULS project, Grant 07CO406). This article has been reviewed and corrected by K. Snaith (Médicis Traduction), member of the French Society of Translators.

Disclosure Statement

No competing financial interests exist.

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