Abstract

Mealworm (Tenebrio molitor L.) has great potential as a new sustainable protein and is currently introduced in commercially available burgers in a number of European countries. Recently, we showed there is risk of mealworm allergy in the majority of patients with shrimp allergy.1 Here we address the risk of mealworm sensitization and allergy in subjects without shrimp allergy. We evaluated primary sensitization in mice (C3H/HeOuJ) and allergy in 4 domestic and professional mealworm breeders with allergic symptoms on occupational exposure or ingestion.

Mealworm (Tenebrio molitor L.) has great potential as a new sustainable protein and is currently introduced in commercially available burgers in a number of European countries. Recently, we showed there is risk of mealworm allergy in the majority of patients with shrimp allergy.1 Here we address the risk of mealworm sensitization and allergy in subjects without shrimp allergy. We evaluated primary sensitization in mice (C3H/HeOuJ) and allergy in 4 domestic and professional mealworm breeders with allergic
symptoms on occupational exposure or ingestion. Mealworm or shrimp extract in PBS (20 mg of protein) with 10 mg of cholera toxin (CT; List Biological Laboratories, Campbell, Calif) was administered by means of gavage. Control animals received PBS and CT only. Sensitization profiles (specific IgE [sIgE], immunoblot, and the basophil activation test [BAT]), allergens involved (liquid chromatography–mass spectrometry) and the development of clinical allergy to shrimp (open food challenge) and mealworm
(double-blind, placebo-controlled food challenger [DBPCFC]) were investigated (for details, see the Methods section in this article’s Online Repository at www.jacionline.org). The study was approved by the local ethics committee (NL43731.041.13). Mealworm induced extract–specific IgG1 in 3 of 6 animals and
extract-specific IgE in 2 of 6 animals (Fig 1). Shrimp led to the
induction of extract-specific IgG1 and IgE in 5 of 6 animals, illustrating
the potency of both extracts to induce primary sensitization. We used a model previously described by Bowman and Selgrade,2 which is known to successfully differentiate between known allergenic proteins (eg, Ara h 1 and b-lactoglobulin) and
low/nonallergenic proteins (eg, gelatin and beef tropomyosin) in C3H/HeOuJ mice.
All 4 human subjects had mealworm allergy during professional or domestic mealworm breeding. The 2 professional breeders had symptoms of inhalant allergy when entering the mealworm rearing room. They sporadically consumed mealworms in small amounts without symptoms (2-5 years of exposure and sporadic ingestion of ;1 g of mealworm per
serving). One of the domestic breeders (subject 4) had rhinoconjunctivitis after 2 years of exposure, which progressed to dyspnea after starting domestic rearing. Both domestic breeders consumed higher amounts of mealworm than the professional farmers and
encountered progressive food-induced allergic symptoms when eating mealworm (7-9 years of exposure and 10-50 g of mealworm per serving on 5-10 occasions); anaphylaxis did not
occur. None reported shrimp or any other food allergy. Only subject 4 had mild rhinoconjunctivitis to house dust mite (HDM) and birch pollen (Fig 2 and see Table E1 in this article’s Online Repository at www.jacionline.org). All 4 were sensitized to mealworm in CAP tests, skin prick tests (SPTs; Fig 2), and BATs. Only subject 2 showed minor sensitization with no clinical allergy to shrimp. Although all subjects were sensitized to some common inhalant allergens (tree and grass pollen and animal dander), titers were very low (see Table E1). Immunoblotting showed that IgE from all subjects bound to similar proteins in mealworm, although with different intensities (Fig 1). When comparing the subjects with a reported respiratory allergy to mealworm (subjects 1
and 2) with those with food allergy (subjects 3 and 4), on the immunoblot of subject 3, additional 15-kDa and 10-kDa proteins were observed in the immunoblot of subject 4. All
subjects had a negative oral food challenge result for shrimp, and only subject 3 and 4 had positive DBPCFC results for mealworm. Because 3 of our subjects had higher sIgE levels to mealworm than to any other food or inhalant allergen, mealworm might be
the primary sensitizer. Although subject 4 showed slightly higher HDM sensitization compared with mealworm sensitization, this subject had IgE to Der p 1 and 2 (fecal components), but not Der p 10 (tropomyosin) and arginine kinase, which makes cross-reactivity to HDM less likely. Furthermore, immunoblots from patients with shrimp allergy1 showed different binding patterns compared with those from the 4 primary patients with mealworm allergy, and their basophil activation to mealworm was stronger than for shrimp (see Fig E1 in this article’s Online Repository at www.jacionline.org). Because levels of sIgE to shrimp and HDM were low, inhibition studies were not feasible. However, a role for shrimp as a sensitizer was unlikely because results of
food challenges with shrimp were negative. The 2 subjects with food allergy had higher sIgE levels to mealworm, consumed larger amounts of mealworm (up to c50 g per meal), and were exposed for a longer period of time (7-9 years) than the 2 subjects with respiratory allergy (c1 g of mealworm and 2-5 years of exposure). This might suggest that occupational exposure for longer periods or oral exposure with higher doses are required for development of mealworm allergy.
In line with our study, long occupational exposure resulted in respiratory allergy to mealworm, green bottle fly, and waxmoth.3,4 For both mice and human subjects, IgE binding was shown to known allergens, such as tropomyosin, arginine kinase, and the
myosin light and heavy chains (see Tables E2 and E3 in this article’s Online Repository at www.jacionline.org). Additionally,both mouse and human IgE recognized 3 new allergens with convincingly high scores and high sequence coverage (c90%). They were identified as highly the homologous (>88% sequence identity) mealworm larval cuticle proteins (LCPs) A1A, A2B, and A3A. IgE binding to LCP A1A was confirmed in 3 of the 4 subjects by using immunoblotting (see Fig E2 in this article’s Online Repository at www.jacionline.org). LCPs might be the dominant allergens in primary mealworm allergy, which is in contrast to cross-reactive mealworm allergy, where the panallergens tropomyosin, arginine kinase, and myosin heavy chain were dominant.1 LCPs have not been previously identified as allergens in insects or crustaceans. LCPs are proteins from the mealworm with a conserved domain in arthropod cuticles known as R&R consensus, and
they bind chitin.5 The chitin-binding complex links the soft internal tissue to the exoskeleton of the larvae. LCPs from mealworm are most likely both inhalant and food allergens since they were identified (in the Methods section in this article’s Online Repository at www.jacionline.org) in subjects with respiratory as well as
food allergy to mealworm. In conclusion, mealworm exposure can lead to primary
sensitization in both a mouse model and human subjects and can result in both food and inhalant allergy. LCPs might be major allergens in patients with mealworm allergy.

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