Immunoblot analysis of salivary allergens in 10 mosquito species with worldwide distribution and the human IgE responses to these allergens

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1 Immunoblot analysis of salivary allergens in 10 mosquito species with worldwide distribution and the human IgE responses to these allergens Zhikang Peng, MD, a,b Hongbing Li, RT, a and F. Estelle R. Simons, MD a,b Winnipeg, Manitoba, Canada Background: Most people develop skin reactions to mosquito bites, however, little is known about mosquito salivary allergens and the IgE responses to them. Objectives: We sought to identify these allergens and the specific IgE responses they elicit. Methods: Saliva or salivary gland extracts were prepared from 10 mosquito species, including seven species with worldwide distribution: Aedes () aegypti, vexans, albopictus, togoi, triseriatus, Culex (Cx.) quinquefasciatus, Cx. pipiens, Cx. tarsalis, Anopheles (An.) sinensis, and Culiseta (Cs.) inornata. Proteins from these preparations were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, which were immunoblotted by sequential incubations with human serum, monoclonal anti-human IgE, and enzyme-conjugated goat anti-mouse IgG. Salivary allergens were analyzed by using a pooled serum from subjects allergic to mosquitos. Individual IgE responses to each allergen were evaluated in 12 subjects allergic to mosquitos living in Canada, the United States, and China, as well as in five subjects not allergic to mosquito bites. To study species-shared allergens, the membranes were immunoblotted with two rabbit antibodies specific to recombinant mosquito salivary proteins. Results: Three to sixteen salivary allergens with molecular masses ranging from 16 to 95 kd were found in each species. Both species-shared and species-specific allergens were identified by molecular masses, binding to the two rabbit antibodies, and individual IgE responses to species indigenous to and absent from the regions where the subjects lived. Salivary allergens, especially from aegypti, vexans, and albopictus, elicited higher IgE responses in subjects allergic to mosquitos than in nonallergic subjects. Conclusions: Species-shared and species-specific allergens that cause IgE responses in subjects allergic to mosquitos are immunologically identified. Species-shared allergens are the most important for potential use in diagnosis and immunotherapy. (J Allergy Clin Immunol 1998;101: ) Key words: Salivary allergen, antigen, mosquito allergy, immunoblot, Western blot, aegypti, albopictus, vexans, togoi, Cx. pipiens, Cx. quinquefasciatus, Cx. tarsalis, An. sinensis, triseriatus, Cs. inornata From a the Section of Allergy and Clinical Immunology, and b the Department of Immunology, Faculty of Medicine, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg. Supported by the Children s Hospital Foundation, Winnipeg, Manitoba, Canada. Received for publication Aug. 11, 1997; revised Oct. 16, 1997; accepted for publication Nov. 19, Reprint requests: Zhikang Peng, MS746A-820 Sherbrook St., Winnipeg, Manitoba, Canada R3A 1R9. Copyright 1998 by Mosby, Inc /98 $ /1/87709 Abbreviations used BSA: Bovine serum albumin PBS: 0.02 mol/l phosphate-buffered saline (ph 7.2) PBST: 0.02 mol/l phosphate-buffered saline (ph 7.2) supplemented with 0.5% (vol/ vol) Tween SDS-PAGE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis There are more than 3000 mosquito species worldwide. As human pests, some species, such as Aedes () aegypti and vexans, are more important than others, such as Culex (Cx.) quinquefasciatus (Fig. 1). Their bites cause immediate and delayed local cutaneous and, rarely, systemic reactions. 1-5 The immediate cutaneous reaction consists of a pruritic wheal and surrounding flare that appear within several minutes, peak at 20 minutes, and subside. The delayed reaction consists of an indurated papule that appears a few hours after the bite, reaches a peak at 24 to 36 hours, then diminishes over several days. 6 Reactions to mosquito bites are immunologic in nature, and are caused by specific sensitization to mosquito saliva. Initial exposure to species to which the subjects have not been previously exposed causes no reaction. 7, 8 Mosquito bite induced immediate wheal and flare reactions correlate with both mosquito salivary gland specific IgE and IgG levels, whereas bite-induced delayed indurations correlate with lymphocyte proliferation responses to mosquito allergens. 6, 9 Serum mosquito specific IgE has been identified in humans by using passive cutaneous anaphylaxis transfer tests (Prausnitz-Küstner tests), 10, 11 immunoassays such as ELISA 9, 12 and RAST, 2 histamine release from basophils, 11 and immunoblot techniques There are more than 20 polypeptides in mosquito saliva 18 and 26 to 37 polypeptides in mosquito whole body extracts. 14 Immunoblot techniques have revealed that there are up to 19 allergens in mosquito salivary gland extracts. 15 However, there are only a few reports dealing with mosquito salivary allergens, and each study 15, 16 was limited to a small number of mosquito species. Although reactions to mosquito bites occur frequently, mosquito salivary allergens have not been studied extensively, and the prevalence of IgE responses to 498

2 J ALLERGY CLIN IMMUNOL VOLUME 101, NUMBER 4, PART 1 Peng, Li, and Simons 499 TABLE I. Distribution of the 10 mosquito species Mosquito species Distribution aegypti* A worldwide cosmotropical distribution 31 between 30 NL and 30 SL (R. Brust ) vexans* Worldwide north temperature zone between 60 and 20 NL (Brust); North America, Eurasia and Africa 32 albopictus* Australia and throughout the Orient 33 ; southern and eastern United States 34 togoi Throughout eastern Asia 33 triseriatus The eastern half of southern Canada and the United States 32 Cx. pipiens* Throughout the Holarctic Region, Africa, and South America 33 Cx. quinquefasciatus* A cosmotropical distribution 33 Cx. tarsalis Throughout much of the United States and western Canada 32 An. sinensis China, Indochina, Sumatra, Thailand, India, Japan, Manchuria, and Siberia 33 Cs. inornata From the Yukon to northern Mexico 32 *The most important mosquito species with worldwide distribution. Other important species. Personal communication, each allergen in subjects allergic to mosquitos has not yet been ascertained. We hypothesized that (1) mosquito salivary proteins cause IgE responses, (2) saliva from some species is more important than that from others, and (3) there are species-shared and species-specific allergens. In this study, using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we analyzed the IgE-binding allergens in saliva or salivary gland extracts of 10 mosquito species, seven of which are important pests in North America, worldwide, or both (Table I, Fig. 1). We also studied individual IgE responses to these allergens in subjects allergic to mosquitos living in Canada, the United States, and China. METHODS Mosquitos, mosquito saliva, and salivary gland extracts Female vexans, triseriatus, Cx. tarsalis, and Culiseta (Cs.) inornata mosquitos were collected in local fields and identified by scientists in the Department of Entomology at the University of Manitoba. The aegypti colony was obtained from the same Department and maintained in our laboratory. The Cx. quinquefasciatus colony was imported from Dr. Robert J. Novak s laboratory at the University of Illinois (Champaign, Ill.) and maintained in our laboratory. Adult aegypti and Cx. quinquefasciatus mosquitos (4 to 12 days old) were used for saliva collection or salivary gland dissection. Mosquito saliva or salivary gland extracts were prepared for the study. Saliva was collected from female adult aegypti, vexans, triseriatus, Cx. tarsalis, and Cs. inornata mosquitos as follows. A mosquito was aspirated from the cage, placed in a test tube with water, and shaken gently. The wet mosquito was then confined to the wall of a plastic box by placing vaseline on FIG. 1. Maps showing worldwide distribution of aegypti, 31 vexans, 32 and Cx. quinquefasciatus. 33 its legs and wings, and its proboscis was inserted into a capillary tube filled with 20 l of water. Salivation was induced by applying a small amount of 0.5% malathion in acetone (vol/vol) to the thorax. 19 One hour later, the contents of the capillary tubes were collected, pooled, and lyophilized. The saliva was reconstituted by dissolving the lyophilized proteins in 0.02 mol/l phosphate-buffered saline (ph 7.2) (PBS) before use. Cx. quinquefasciatus did not salivate when we used this method, therefore we prepared a salivary gland extract from this species as previously described. 12 The salivary gland extracts of albopictus, togoi, Cx. pipiens, and Anopheles (An.) sinensis were provided by Dr. Sulan Liu (Shanghai Medical University) using the same method. 12 The protein concentrations of these saliva and salivary gland extracts ranged between 0.07 and 0.40 mg/ml as measured by a Bio-Rad Protein Assay kit (Bio-Rad Labs., Richmond, Calif.). Subjects This project was approved by the University of Manitoba Faculty Committee on the Use of Human Subjects in Research, and the participants gave written, informed consent before

3 500 Peng, Li, and Simons J ALLERGY CLIN IMMUNOL APRIL 1998 FIG. 2. Salivary allergens of 10 mosquito species. Proteins in saliva or salivary gland extracts of 10 species were separated by SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were sequentially incubated with pooled serum from subjects allergic to mosquitos, monoclonal anti-human IgE, and enzyme-conjugated goat anti-mouse IgG as described in Methods section. Aed a 1 (68 kd) and Aed a 2 (37 kd) of aegypti are indicated by arrows a and b, respectively. entering the study. vexans skin bite tests were performed, and serum vexans salivary gland-specific IgE was measured in 41 subjects living in Winnipeg. The skin reactions to mosquito bites ranged from none to strongly positive. 6 Among them, six subjects allergic to mosquitos with strongly positive skin reactions (wheal diameter, 10 to 18 mm) and five nonallergic subjects with absent or minimal reactions (wheal diameter, 0 to 3 mm and no flare) in the bite tests were selected for the study. Four sera provided from the United States by Dr. Kim-Lien Nguyen (Wyomissing, Pa.) and two sera provided from China by Dr. Ruenmai Tian (Shanghai Medical University) were obtained from subjects with a history of severe skin reactions to mosquito bites. The Health Sciences Centre Clinical Chemistry Laboratory provided a residual serum sample from a 5-month-old infant who had never been bitten by mosquitos. SDS-PAGE and immunoblot analyses of salivary allergens and human individual IgE responses The proteins in the mosquito extracts were separated by SDS-PAGE in a discontinuous system according to the method previously described 17 by using a BioRad Mini-PROTEAN II apparatus. Portions of each mosquito extract (1 to 2 g/well), boiled 4 minutes in a reducing sample buffer, were loaded and separated by 12% gel SDS-PAGE. These proteins were then electrotransferred onto nitrocellulose membranes. Free binding sites on the membranes were blocked by incubation of the membranes with 3% bovine serum albumin (BSA) dissolved in 0.2 mol/l PBS supplemented with 0.5% (vol/vol) Tween (PBST) for 2 hours at room temperature. The immunoblot was completed by incubation of the membranes with human sera (1:10 dilution) at 4 C overnight. After washing three times with PBST, the membranes were incubated with monoclonal antihuman IgE (1:15,000; ascites, clone No. 7.12, obtained from Dr. Andrew Saxon, University of California) for 1.5 hours at room temperature. The membranes were washed again and then incubated with horseradish peroxidase-conjugated goat antimouse IgG (1:5000 dilution; Calbiochem Corporation, San Diego, Calif.) for 1.5 hours. After washing, the membranes were incubated with ECL detecting reagents (Amersham Life Science, Buckinghamshire, England) and then exposed to X- Omat film (Kodak, Rochester, N.Y.). Prestained SDS-PAGE standards (Bio-Rad) were used to determine the relative molecular weights of the electrophoresed components. In the allergen analysis, we used a pooled serum obtained from subjects living in Canada and the southern United States who exhibited severe skin reactions to mosquito bites; this serum contained antibodies to all the major North American mosquito species. Incubations of the membranes containing each mosquito species extract with infant serum, umbilical cord serum, or 1% BSA in PBST served as negative controls. In the study of IgE responses in individual sera, the membranes containing mosquito salivary allergens were incubated with individual serum samples. Analysis of species-shared allergens (Aed a1and Aeda2) A 68 kd salivary protein of aegypti, which was identified as apyrase, 20 was cloned by Dr. Anthony James and his colleagues. 21 In our laboratory the recombinant protein was expressed by using a baculovirus/insect cell system, purified, and demonstrated to bind to IgE and to elicit skin immediate and delayed reactions in subjects allergic to mosquitos, 22, 23 indicating that it is an allergen. According to current allergen nomenclature, 24 we have named the 68 kd apyrase Aed a A 37 kd salivary protein of aegypti, which had previously been named D7, was also cloned by Dr. Anthony James. 25 This protein was expressed by using a baculovirus system and purified in our laboratory. The recombinant 37 kd protein

4 J ALLERGY CLIN IMMUNOL VOLUME 101, NUMBER 4, PART 1 Peng, Li, and Simons 501 TABLE II. Molecular masses of salivary allergens in 10 mosquito species and their IgE responses in subjects allergic to mosquitos and nonallergic subjects Marker (kd) aegypti vexans albopictus togoi triseriatus Cx. quinquefasciatus Cx. pipiens Cx. tarsalis An. sinensis Cs. inornata (1/12,0/5) (4/12,0/5) (1/11,0/5) 70 (2/12,0/5) (11/12,5/5)* 68 (11/12,4/5) 68 (6/12,0/5) (6/12,1/5) 61.5 (11/12,4/5) 61.5 (4/12,0/5) (3/12,0/5) 57.5 (4/12,0/5) (11/12,3/5) 55.5 (1/11,0/5) 55.5 (9/12,1/5) (6/12,0/5) (2/12,0/5) (11/12,5/5) 50 (12/12,4/5) 50 (8/11,1/5) (3/11,0/5) (7/11,5/5) (12/12,5/5) 39 (9/12,2/5) (1/12,0/5) (12/12,5/5) 37 (12/12,5/5) 37 (11/12,4/5) 37 (12/12,5/5) (10/11,5/5) (12/12,4/5) 35.5 (7/11,2/5) (12/12,4/5) (7/12,0/5) 33 (3/12,0/5) 33 (5/12,0/5) 33 (10/11,5/5) 33 (11/11,4/5) (5/12,0/5) 30.5 (11/12,4/5)30.5 (9/12,0/5) 30.5 (5/12,0/5) (1/12,0/5) (1/12,0/5) (1/12,0/5) (6/12,0/5) (4/12,1/5) 20.5 (11/12,0/5) (3/12,0/5) (12/12,4/5)19.5 (7/12,0/5) (11/11,4/ 5) (5/12,2/5) 18.5 (2/12,0/5) 18.5 (10/12,0/5) (9/11,4/5) (4/12,0/5) 17.5 (4/12,0/5) (8/11,5/5) 16 (6/12,0/5) Total no SGE, Salivary gland extract. *Number of subjects allergic to mosquitos with IgE response to the antigen/number of subjects allergic to mosquitos, number of nonallergic subjects with IgE response to the antigen/number of nonallergic subjects. bound to human IgE and elicited fewer and smaller skin reactions than raed a 1. It was named Aed a The two recombinant proteins, raed a 1 and raed a 2, and their rabbit antibodies (gifts from Dr. Anthony James, University of California, Irvine) were used to determine if there are shared allergens in other mosquito species. The same SDS-PAGE and immunoblot methods as described above, with some modifications, were used. Proteins (1 to 2 g/well) in each of the 4 mosquito extracts were studied ( aegypti, vexans, albopictus, and Cx. quinquefasciatus), and each of the two recombinant proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then incubated with rabbit anti-raed a 1 (1:5000) or rabbit anti-raed a 2 (1:5000). Incubation of the membranes containing each of the four mosquito extracts or each recombinant protein with 1% BSA in PBST served as negative controls. This incubation was followed by incubation with horseradish peroxidase conjugated goat antirabbit IgG (1:5000 dilution; Jackson ImmunoResearch, West Grove, Pa.). The remaining steps were the same as described above. RESULTS Salivary allergens of 10 mosquito species As illustrated in Fig. 2 and Table II, immunoblot analysis revealed that there were 3 to 16 allergens in each of the 10 mosquito salivary extracts. There were no bands, or a few very faint bands, found in the control strips (Fig. 3, strips 18 to 20). The molecular masses of these allergens ranged from 16 to 95 kd. In Fig. 2, the 68 kdaeda1andthe37kdaeda2of aegypti, which have now been cloned and sequenced, are indicated by arrows a and b (strip 1), respectively. The number of

5 502 Peng, Li, and Simons J ALLERGY CLIN IMMUNOL APRIL 1998 FIG. 3. IgE responses to salivary allergens of six mosquito species in subjects allergic to mosquitos and nonallergic subjects. Proteins of saliva or salivary gland extracts separated by SDS-PAGE were transferred to nitrocellulose membranes. Membranes were sequentially incubated with sera from subjects allergic to mosquitos (strips 1 to 12) or sera from nonallergic subjects (strips 13 to 17), monoclonal anti-human IgE, and enzyme-conjugated goat anti-mouse IgG as described in Methods section. Infant serum (strip 18), cord serum (strip 19), and PBST (strip 20) were used as negative controls. Six mosquito species used were aegypti (A), vexans (B), albopictus (C), togoi (D), Cx. quinquefasciatus (E), and Cx. pipiens (F). In vexans a 68 kd species-shared allergen, Aed a 1, is indicated by arrow a, and a 23 kd vexans specific allergen is indicated by arrow b. allergens in each species and the molecular mass of each allergen is summarized in Table II. By molecular size, both species-unique and species-shared allergens were present. Species-shared Aed a 1 and Aed a 2 allergens As shown in Fig. 4, the existence of species-shared allergens was confirmed by immunoblot analysis with rabbit anti-raed a 1 and raed a 2 antibodies, respectively. A 68 kd allergen, which was recognized by rabbit anti-raed a 1, was found not only in the aegypti extract (Fig. 4, A, strip 1) but also in the extracts of vexans and albopictus (Fig. 4, A, strips 2 and 3), however, not in the extract of Cx. quinquefasciatus (Fig. 4, A, strip 4). No allergen bands were found in the PBS control strips (data not shown). A 37 to 39 kd protein recognized by rabbit anti-raed a 2 was identified in all 3 Aedes species studied (Fig. 4, B, strips 1 to 3) and in Cx. quinquefasciatus as well (Fig. 4, B, strip 4). Recently, the two recombinant allergens have been found to be important in mosquito allergy. 23 Among 31 subjects with a positive mosquito bite test response, 29% (9 of 31) had a positive skin test response to raed a1(p 0.05) and 10% (3 of 31) had a positive skin test response to raed a 2, whereas none (0 of 15) of the subjects with a negative bite test response had a positive skin test response to either raed a1orraed a Species-shared and species-specific allergens identified by individual sera Each mosquito species studied has a different geographic distribution as shown in Table I. By using the sera from individual subjects who live in Canada, the United States, and China, species-shared and speciesspecific allergens were identified. The sera from Canada were obtained from subjects who lived in Winnipeg, Manitoba, where only vexans is found as a major pest, representing up to 80% of the indigenous mosquito population (unpublished data from the Insect Control Branch, Parks and Recreation Department, The City of Winnipeg). The sera from Manitobans allergic to mosquitos (Fig. 3, strips 1 to 6) reacted not only with vexans but also with the allergens of five other species that are not found in Manitoba (Fig. 3). Similarly, the sera from Shanghai, China (Fig. 3, strips 11 and 12) reacted with the vexans allergens, a species that is not an indigenous pest in China. These data strongly suggest the existence of species-shared allergens. Species-specific allergens also existed, as evidenced by a23kd vexans allergen, which reacted only with the sera from Canada where vexans is abundant (Fig. 3, B, indicated by arrow b). IgE responses in subjects allergic to mosquitos As illustrated in Fig. 3, the 12 subjects allergic to mosquitos had stronger IgE responses than the nonal-

6 J ALLERGY CLIN IMMUNOL VOLUME 101, NUMBER 4, PART 1 Peng, Li, and Simons 503 lergic subjects as shown by the more intense and broader bands and the number of bands. Although the patterns of IgE responses were similar in most of the subjects, some individuals responded to more allergens than others. For example, subject 2 reacted to more albopictus allergens than the remaining subjects (Fig. 3, C, strip 2). Similarly, subjects 2, 6, and 9 reacted to more togoi allergens than the others (Fig. 3, D). The positivity rates of IgE response to each allergen in the allergic and nonallergic groups are summarized in Table II. In parentheses, the first fraction shows the number of subjects allergic to mosquitos with visible IgE responses to the allergen over the total number of subjects allergic to mosquitos, and the second fraction shows the number of nonallergic subjects with IgE responses to the allergen over the total number of nonallergic subjects. Allergens were more likely to elicit IgE responses in the subjects allergic to mosquitos than in the nonallergic subjects. Cord blood should contain few IgE antibodies, because IgE does not cross the placenta. The strong IgE reactivity to a mosquito aegypti antigen in the cord serum (Fig. 3, A, strip 19) may have occurred because of a mixture of maternal and fetal blood during delivery, which, although a rare event, is more common than in utero sensitization. 26 DISCUSSION Mosquito salivary secretions are directly responsible for skin reactions to mosquito bites. 27 Lack of commercially available, specific, high-quality mosquito saliva allergens has been a major rate-limiting step in the study of mosquito allergy to date. Collection of mosquito saliva or dissection of salivary glands is time-consuming and labor-intensive. By using these materials, it has been found that mosquito salivary gland specific IgE is significantly increased in subjects allergic to mosquitos. 9, 12 Mosquito saliva contains more than 20 polypeptides, 18 most of which are allergens, as shown in this study (Fig. 2). Even if mosquito saliva or salivary gland extracts were readily available, it would be impossible to study the IgE responses to each and every allergen in saliva by using immunoassays such as ELISAs or RASTs. It is also not practical to purify each allergen from saliva. The SDS-PAGE and immunoblot techniques provide a unique opportunity for analyzing multiple allergens in the saliva. These techniques can also be used to study the IgE response to each salivary allergen by incubating the saliva-containing membranes with individual sera. The immunoblot techniques we established were specific for the allergens, as shown by the dose-dependent inhibition tests in our previous report. 17 In the inhibition tests, before immunoblotting, sera from subjects allergic to mosquitos was preincubated with different dilutions of mosquito antigens. Mosquito allergen treated sera were then incubated with nitrocellulose membranes containing mosquito salivary allergens separated by SDS- PAGE. The results showed that preincubation of the FIG. 4. Analysis of shared Aed a 1 and Aed a 2 allergens in different mosquito species. Proteins in four mosquito saliva or salivary gland extracts were separated by SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were incubated with rabbit anti-raed a 1 (left) or rabbit anti-raed a 2 (right) followed by incubation with enzyme-conjugated goat anti-rabbit IgG as described in Methods section. sera with mosquito allergens inhibited the binding of serum IgE to the allergens in the nitrocellulose membranes. In this study although positivity rates were qualitatively evaluated by the naked eye and may not be precise, a screening picture of IgE responses to each allergen was obtained. Most allergens elicited IgE responses only in subjects allergic to mosquitos. For example, the 75, 57.5, 54, 28, 23, and 17.5 kd allergens of vexans appeared only in subjects allergic to mosquitos (Table II, Fig. 3, B). This information about the prevalence of each allergen in these subjects will become important as more mosquito salivary proteins are cloned. Because SDS-PAGE and immunoblot techniques are qualitative assays, there were high percentages of IgE response to some allergens in nonallergic subjects. For example, the percentage of IgE response to the 68 kd allergen of vexans was 92% (11 of 12) in allergic subjects and 80% (4 of 5) in nonallergic subjects. Although there was no significant difference in reactivity between the two groups, this band was much more intense and broader in the allergic group than in the nonallergic group (Fig 3, B, indicated by arrow a). The salivary allergens in the three major human-biting Aedes species ( aegypti, vexans, and albopictus), which are also present in North America, elicited the strongest IgE responses. The allergen bands were broader and more intense, and there was a higher number of allergen bands than in other species (Fig. 2,

7 504 Peng, Li, and Simons J ALLERGY CLIN IMMUNOL APRIL 1998 strips 1 to 3). Because togoi is distributed only in eastern Asia, it follows that some allergens in togoi may not be identified by the pooled serum from subjects allergic to mosquitos obtained from North America. triseriatus, Cx. tarsalis, and Cs. inornata are minor species (Fig. 2, strips 5, 8, and 10). Although Cx. quinquefasciatus and Cx. pipiens (Fig. 2, strips 6 and 7) are distributed globally, they are not found in Canada and are less important as human-biting species. An. sinensis (Fig. 2, strip 9) is not present in North America and is also less important as a human-biting species, although it is a very important disease vector. Hence, these species elicited fewer IgE responses than the major Aedes species (Figs. 2 and 4). 28, 29 Cross-reactivity with respect to bite reactions and IgE responses 7 from different mosquito species has been reported previously. Sensitization of animals by mosquito bites or injection of allergenic extracts from one species can confer reactivity against another species. 28, 29 aegypti and Cx. quinquefasciatus specific IgE antibodies were detected in subjects allergic to mosquitos from Manitoba, where these two mosquito species are absent and vexans is the major pest. 7 The immunologic basis for the cross-reactive skin and IgE responses is the existence of speciesshared allergens. Such allergens, based on molecular mass measurements, have been previously identified among various mosquito species However, proteins with the same molecular mass may not be immunologically identical. We previously produced a monoclonal antibody that bound to a 37 kd saliva protein of aegypti in SDS-PAGE and immunoblotting, but not to the recombinant 37 kd protein. 30 In the present study allergens shared among species were immunologically identified by the reactions of the mosquito preparations to the specific antibodies against two recombinant aegypti saliva proteins, raed a 1 and raed a 2 (Fig. 4). Furthermore, when using sera from subjects allergic to mosquitos living in various areas where mosquito species are distributed differently, both species-shared and species-specific allergens were immunologically identified. As illustrated in Fig. 3, the IgE in Manitobans allergic to mosquitos reacted with not only the indigenous species, vexans, but also the five other species not present locally, suggesting that these allergens are immunologically identical with the allergens of vexans. Study of these species-shared allergens is more important than study of species-specific allergens for potential diagnosis and immunotherapy of subjects sensitive to multiple mosquito species. We thank Dr. Reinhart Brust for valuable advice and for preparing maps of vexans and Cx. quinquefasciatus distribution; Dr. Anthony James for providing rabbit antibodies to two recombinant mosquito salivary proteins; Dr. Sulan Liu and Andrew Mackay, MSc, for providing mosquito saliva or salivary gland extracts; and Drs. Kim-Lien Nguyen and Ruenmai Tian for providing sera from subjects allergic to mosquitos. REFERENCES 1. Frazier CA. Biting insects. Arch Dermatol 1973;107: Suzuki S, Negishi K, Tomizawa S, Shibaski M, Kuroume T, Matsumura T. A case of mosquito allergy. Acta Allergol 1976;31: Gluck JC, Pacin MP. Asthma from mosquito bites: a case report. Ann Allergy 1986;56: Lelong M. Severe local reactions to mosquito bites. Apropos of 20 cases. 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