Allergology International (1997) 46: 43-49 Original Article Allergenic importance of 22 species of Japanese chironomid midges Koichiro Kawai,1 Gyokei Murakami,2 Shiro Kasuya,3 Hidetoyo Teranishi4 and Atsushi Muraguchi5 'Laboratory of Ecology , Faculty of Applied Biological Science, Hiroshima University, Hiroshima, 2Department of Pediatrics , Toyama Red Cross Hospital, Toyama, 3Department of Parasitology, School of Medicine, Gifu University, Gifu, and Departments of 4Public Health and 5lmmunology, School of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan ABSTRACT INTRODUCTION Twenty-two Japanese chironomid species were exam- A dipteran insect family Chironomidae has been proved ined for their allergenicities using ELISA with the sera of worldwide to be a potent human allergen.1-4 However, of 32 asthmatic patients. The species Paratrichocladius the several thousands of species recorded worldwide, rufiventris and Cricotopus sylvestris showed high posi- only 38 species belonging to 19 genera have been tive rates of specific IgE, high average IgE reactivities examined for allergenicities using prick tests and radio- and high frequencies of strong IgE reactivity and the allergosorbent test (RAST).5-11Cranston et al. examined highest IgE reactivity of the 22 species, suggesting a the allergenicities of eight Nilotic chironomid species, high rate of contact with humans and the possession of including Cladotanytarsus lewisi, a midge that emerge highly allergenic components by these two species. In from the River Nile Basin in an enormous mass.7 They contrast, Tanypus punctipennis and Rheotanytarsus used skin prick tests in Sudanese and Egyptian asthmatic kyotoensis showed low allergenicities, suggesting a subjects, and demonstrated that about 80% of asthmatics low level of human contact and/or a lack of allergenic showed positive reactions to C. lewisi and that more than components. Furthermore, species that emerge from 40% of C. lewisi-positive subjects showed positive reac- eutrophic waters in a large mass, such as Macropel- tions to the species of the genera Dicrotendipes, Pro- opia paranebulosa, Paratrichocladius rufiventris and cladius and Conchapelopia, while less than 20% of Chironomus yoshimatsui, showed strong allergenicities C. lewisi-positive subjects showed positive reactions to in all the tests. This suggests that eutrophic water be genera Paracladopelma, Nanocladius, and Cryptochiro- regarded as an important reservoir to allergenic nomus.7 Baur et al. also examined the allergenicities of chironomids. 25 species of different taxa using RASTand the sera of German and American asthmatics, thought to be sensi- Key words: allergen, chironomid, cross-allergenicity, tized by Chironomus thummi thummi and C. plumosus, ELISA. respectively, and demonstrated that evolutionarily closely related species, such as species of the genera Chirono- mus, Glyptotendipes and Polypedilum, showed moderate to strong binding to the patients' IgE.6 Corespondence: Dr Koichiro Kawai, Laboratory of Ecology, In the present study, 22 species of different genera, dis- Faculty of Applied Biological Science, Hiroshima University, tributed widely in various types of waters in Japan, includ- 1-4-4 Kagamiyama, Higashihiroshima-shi, Hiroshima 739, Japan. ing 11 genera that had not been previously examined for Received 1 August 1996. Accepted for publication 19 allergenicities, were examined for their reactivity with IgE December 1996. in asthmatic patients' sera using ELISA. 44 K KAWAI ET AL. METHODS attacks of wheezing, dyspnea, cough and expectoration of mucoid sputum and showed positive IgE reactions to at Chironomid midges least one species of chironomid in BAST, RAST enzyme Twenty-twospecies, each belonging to a different genus, immunoassay or ELISA tests. of three chironomid subfamilies were used (Table 1). Among these, midges of CS, CaB, ChY, GT, PeS, PoN, Midge extracts PtG and TaO species were cultured in the laboratory. ToA midges were obtained by rearing the larvae purchased Midge extracts were prepared by the methods described from a commercial source in Osaka, Japan. Midges of previously.27 Briefly, triturated midges were defatted with other species were obtained by rearing the larvae ether and extraction was performed with phosphate- hatched from egg masses collected in the field. The evo- buffered soline (PBS), pH 7.5, for 48h at 4℃, with gentle lutionary relationships between the 21 genera, using the stirring. After centrifugation, the supernatant was dialysed systems of Fittkau24and Saether,25,26are shown in Fig. 1. against PBS using Spectrapor 3 tubing (Spectrum Medi- cal Industries, USA; MW exclusion 3500 Da). The mater- Patients ial retained in the tubing was centrifuged, and the supernatant was used as the crude extract. Sera of 32 asthmatics (range, 6-69 years old, mean, 25.1; 25 males, 7 females) were obtained from the ELISA Department of Internal Medicine of Tohno Hospital, from the Departments of First Internal Medicine and Pediatrics [LISA was performed using 96-well microtiter plates. of Toyama Medical and Pharmaceutical University Hos- After coating each well with midge crude extract at pital and from the Department of Pediatrics of Toyama 10μgP/mL in a coating buffer containing 0.1mol/L Red Cross Hospital. All the patients had recurrent acute NaHCO3 and 0.02%NaN3' pH 9.6, overnight at 4℃, Table 1. The 22 chironomid species studied and their major habitats ALLERGENICITY OF CHIRONOMID MIDGES 45 Table 2. IgE reactivity of a pooled cord serum Fig. 1 Evolutionary relationships between 21 genera of Chironomid midges. The genus Tokunagayusurika was excluded due to no information on its evolution. Quite high IgE reactivities were shown to HB, CaB, ChY, DP, GT, MiP, PdA, PeS and PtK. Low values were observed for the other 13 species. the wells were washed and filled with patients' sera at 1/10 dilution with diluent buffer containing 0.05mol/L Tris, 0.001mol/L MgCl2, 0.15mol/L NaCI, 0.05% Positive rates of specific IgE in patients' sera Tween20, 0.02% NaN3 and 1% bovine serum albumin, (Fig. 2) and incubated for 2h at room temperature. After wash- When the [LISAvalue of a patient's serum to a species ing, the wells were filled with HRP-conjugated goat anti- was higher than twice that of the pooled cord serum to humon ∈-choin IgG antibody (DAKO, Glostrup, that same species, itwas regarded as positiveIgE reactiv- Denmark) at 1/2000 dilution pnd incubated for 2h at ity,and positiverates were compared among the species. room temperature. After washing, a substrate solution High positive rates were observed to almost all the contoining 2.5g of o-phenylenediamine and 500μL of species belonging to the subfamilies Tanypodinaeand hydrogen peroxide per liter of 0.033mol/L citrate and Orthocladiinae. In contrast, that positive rate differed 0.066mol/L Na2HPO4 was pipetted in, and the reaction from species to species for Chironominae.Of the species was stopped with 2 N sulfuric acid. Absorbance at of Chironominae,the highest positiverate was shown to 492nm subtracted by that at 405nm, both measured by PoN. Positiverates higher than 50% were also shown to an ELISA reader (Bichromatic-348, Labsystems ChY,PaD, SM, PtG and TaO. Only low positiverates were Multiskan, Helsinki, Finland), were represented as the observed for DP,PdT and PeS. [LISAvalues. IgE reactivities of the patients' sera (Fig. 3) RESULTS For each species, the ELISAvalue in a patient, obtained IgE reactivity of a pooled cord serum by subtracting the ELISAvalue for that species in the A pooled cord serum from eight infants was examined for pooled cord serum, was regarded as the specific IgE IgE reactivity with extracts of the 22 species (Table 2). reactivity,and specificreactivities were compared among 46 K KAWAI ET AL. the species. The highest average reactivity was shown frequencies. Specific[LISA values higher than 1.0 were to PR. High average reactivities were also observed for never observed for the other 13 species. AM, MP, CS and ChY. Average reactivity differed from species to species for subfamilies Tanypodinae and Frequencies of the highest ELISAvalue in each Orthocladiinae and tribe Chironomini. In contrast, weak patient for the 22 species (Fig. 4) reactivities were observed for all the species of tribe Thefrequency of the highestELISA value was examined in Tanytarsini. each patient for each of the 22 species, and PRshowed Extremelystrong IgE reactivity (i.e. specific ELISAvalues by far the highestfrequency of 9. CS, ToA,ChY and PoN higher than 1.0) was shown to PRat the highest frequency also showed high frequencies of 4-5. AM, HB, PsA,CT of 5. The species AM, MP and MiP also showed strong and MiP also showed high values, although at low fre- reactivities at the high frequency of 3; CS, CaB, ChY, DP quencies. Theother 12.speciesnever showed high values and SM also showed strong reactivities, although at low in any patient. Fig. 2 Positive rates of specific IgE reactive to crude extracts of Fig. 4 Frequencies of the highest specific ELISA value in each 22 species for 32 patients as assayed by ELISA.When the ELISA patient for 22 species. value of a patient's serum to a species was higher than twice that for a pooled cord serum it was regarded as positive. (See Table 1 for key to abbreviations.) Fig. 3 IgE reactivity of patients' sera to crude extracts of the 22 species. The ELISAvalue of a patient's serum to a species subtracted by that for a pooled cord serum was regarded as the specific ELISAvalue. A horizontal bar represents the mean specific value of 32 patients. (See Table 1 for key to abbreviations.) ALLERGENICITY OF CHIRONOMID MIDGES 47 DISCUSSION various highly eutrophicated waters14-16,28located in the Japanese metropolitan areas. MP, ChY and PoN also In the study reported here, a total of 22 species of chi- showed high allergenicities and these species also ronomid midges, each belonging to a different genus, emerge from eutrophicated waters,13,16,18,21Therefore, were examined by ELISAfor allergenicities in humans eutrophicated water should be regarded as a reservoir for from different aspects; that is, the positive rate of specific air-borne allergenic chironomid midges.