Toward a description of the sialome of the adult female mosquito Aedes aegypti

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Abstract

To describe the set of mRNA and protein expressed in the salivary glands (sialome) of Aedes aegypti mosquitoes, we randomly sequenced a full-length cDNA library of this insect and performed Edman degradation of PVDF-transferred protein bands from salivary homogenates. We found 238 cDNA clusters which contained those coding for 10 of the 11 proteins found by aminoterminal degradation. All six previously described salivary proteins were found in this library. Full-length sequences of 32 novel cDNA sequences are reported, one of which is the product of a transposable element. Among the 31 novel protein sequences are 4 additional members of the D7 protein family; 4 novel members of the antigen 5 family (a protein family not reported in Aedes); a novel serpin; a novel member of the 30-kDa allergen of Ae. Aegypti; a secreted calreticulin; 2 proteins similar to mammalian angiopoietins; adenosine deaminase; purine hydrolase; lysozyme; a C-type lectin; 3 serine proteases, including one with high similarity to Bombyx prophenoloxidase activating enzyme; 2 proteins related to invertebrate immunity; and several sequences that have no significant matches to known proteins. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed.

Introduction

Saliva of blood-sucking arthropods contains substances that counteract vertebrate host hemostasis. Accordingly, at least one anticlotting, one antiplatelet, and one vasodilator substance usually are found in the salivary glands of such animals. These substances presumably increase arthropod fitness by increasing the speed at which blood is found and imbibed and by decreasing the possibility of being killed by the host during feeding (Ribeiro, 1987, Ribeiro, 1995). These salivary components may also affect parasite transmission by arthropod vectors and thus may serve as vaccine targets against these diseases (Valenzuela et al., 2001). Mosquito salivary glands also contain enzymes associated with sugar feeding (Marinotti et al., 1990, Grossman and James, 1993) as well as lysozyme, which may help to control bacterial growth in the sugar meal while stored in the mosquito crop (Rossignol and Lueders, 1986). Salivary glands of mosquitoes have usually less than 3 μg of protein (Ribeiro et al., 2001). Identification and characterization of such compounds usually is accomplished by tedious accumulations of over 1000 pairs of dissected salivary glands as starting material for fractionation and/or bioassay experiments.

The protein composition of the salivary glands of blood-sucking diptera is not very complex. Usually, less than 20 main protein bands are seen by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Racioppi and Spielman, 1987, Mellink and Van Zeben, 1976, Poehling, 1979. Recent advances in proteomics research indicate that, in general, there is no significant correlation between the amount of cellular mRNA coding for a particular protein and the amount of the protein (Futcher et al., 1999, Gygi et al., 1999, Gygi et al., 2000). On the other hand, a significant correlation exists between amounts of RNA and the coded protein only when the 12 most abundant yeast proteins are considered (Gygi et al., 1999). If this correlation holds true for mosquito salivary glands, random sequencing of a non-normalized, non-subtracted organ library should yield information of the most abundant proteins found in the insect salivary proteome.

We report the results following sequencing of 456 clones from a full-length, PCR-constructed, cDNA library from adult female Aedes aegypti. The resulting database allowed identification of the cDNA clone corresponding to 10 of 11 salivary gland proteins isolated by SDS-PAGE that yielded aminoterminal sequencing by Edman’s degradation. Furthermore, 227 novel partial sequences from Ae. aegypti are described, including full-length sequence information for 32 of these. Interestingly, we cannot ascribe functions to the majority of these new sequences, indicating that these salivary proteins may have functions not related directly to hemostasis or sugar feeding, or, alternatively, that they may represent novel molecule classes acting on hemostasis, sugar digestion, or antibacterial activity. Finally, this paper contributes to defining the sialome (the set of RNA messages + proteins expressed in the salivary glands) from females of the mosquito, Ae. aegypti.

Section snippets

Reagents

All water used was of 18 MΩ quality and was produced using a MilliQ apparatus (Millipore, Bedford, MA, USA). Organic compounds were obtained from Sigma Chemical Corporation (St. Louis, MO, USA) or as stated otherwise.

Mosquitoes

The Liverpool/black eye strain of Ae. aegypti was reared under the expert supervision of Mr. André Laughinghouse. Insectary rooms were kept at 26±0.5°C, with a relative humidity of 70% and a 16 h:8 h light:dark photoperiod. Adult female mosquitoes used in the experiments were aged

Results

We sequenced 456 random clones from a PCR-based cDNA library from the salivary glands of adult female Aedes aegypti. After grouping this database in clusters, as described in the methods section, 238 individual clusters were found. The distribution of the clusters as a function of their decreasing number of sequences is shown in Fig. 1. Table 1A, Table 1B, Table 1C indicates the similarities of the putative translated products of such clusters when compared with the nonredundant (NR) protein

Discussion

Presently, there are 6 different sequences deposited in GenBank and known to be expressed in the salivary glands of Ae. aegypti. These are for amylase (Grossman and James, 1993), alpha-1,4 glucosidase (James et al., 1989), apyrase (Champagne et al., 1995), D7 protein (James et al., 1991), 30-kDa allergen (Simons and Peng, 2001), and the vasodilator sialokinin (Beerntsen et al., 1999, Champagne and Ribeiro, 1994). To extend the knowledge on the compositional diversity and complexity of the

Acknowledgements

We are grateful to Drs Robert Gwadz, Thomas Kindt and Louis Miller for encouragement and support, and to Nancy Shulman for editorial assistance. We are also thankful for helpful comments by two anonymous reviewers.

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