Electron Microscopy and Molecular Phylogenetic Characterization of the Allergenic Dust Mite Species Suidasia pontifica (Acari: Suidasiidae)

Electron Microscopy and Molecular Phylogenetic Characterization of the Allergenic Dust Mite Species Suidasia pontifica (Acari: Suidasiidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electron Microscopy and Molecular Phylogenetic Characterization of the Allergenic Dust Mite Species Suidasia pontifica (Acari: Suidasiidae) John Wayne Dela Cruz, Erika Pauline Malit, Chanie Patanindagat, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7056104/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jan, 2026 Read the published version in Experimental and Applied Acarology → Version 1 posted You are reading this latest preprint version Abstract The clinical significance of house dust mites (HDMs) as sources of allergens for medical diagnostics, allergen-specific immunotherapy, and allergology research is dependent on accurate morphological and molecular data analysis for species identification and characterization. Here, we report the species identification and allergenicity of a tropical HDM, Suidasia pontifica ( Sp ), via morphological-molecular characterization tandem and IgE ELISA, respectively. Electron microscopy of monocultures of HDM samples collected from Laguna, Philippines, revealed five different traits related to chaetotaxy and body anatomy, such as the presence of scapular setae, cuticle patterns, vertical setae, ω 1 setae, and anal suckers, suggesting that the sample identity is Sp . In addition, PCR amplification from the HDM monoculture genomic DNA and bidirectional sequencing of the 18S and COI gene markers were performed, which were subjected to sequence assembly, consensus acquisition, sequence alignment, and phylogenetic inference. The COI gene showed an exact match and phylogenetic attachment of the sample assembly to Sp , confirming the species-level identification, corroborated with morphological data. Furthermore, 18S gene character analysis was able to prove phylogenetic demarcation of the Sp 18S gene from the sister species S. nesbitti and other allergenic sarcoptiforman species. Our molecular phylogenetic analysis, which is strongly supported by electron microscopy data, indicates the identity of our monocultures as Sp . Interestingly, the Sp allergenicity profile of allergic patients and controls (n = 200) suggests 47% IgE-binding reactivity, confirming its allergenicity and clinical importance among atopic patients. This study emphasizes the resolving power of the morphological-molecular phylogenetic approach and IgE-reactivity to objectively verify Sp species identity and allergenicity for downstream immunological studies. Suidasia pontifica allergy IgE reactivity house dust mite DNA barcoding phylogenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Allergenic HDMs are a major cause of indoor asthma and allergic rhinitis worldwide, affecting as many as 50% of all global atopic sensitivities to approximately 90% of atopic patients in Asian populations (Calderón et al., 2015 ; Salo et al., 2014 ), with the prevalence varying across regions due to seasonal and environmental factors (Calderón et al., 2015 ; Salo et al., 2014 ; Tham et al., 2017 ). Traditionally, the term HDM is associated with species in the Pyroglyphidae family, but members of storage mites have also been medically recognized as having a comparative risk and degree of allergenicity due to their generalistic ability to proliferate indoors (Warner et al., 1999 ). Such addition of paraphyletic groups broadens the scope of allergenic HDMs to become taxonomically scattered under the mite Order Sarcoptiformes from Class Arachnida, particularly on the three main superfamilies Glycyphagoidea, Acaroidea, and Analgoidea (Colloff, 2009 ). One of the least studied storage mite species, Suidasia pontifica ( Sp ), is regarded as one of the major causes of food and storage product contamination worldwide and a significant factor in oral mite anaphylaxis (Barrera et al., 2015 ; Mangoba & Alvindia, 2020 ). Its reported prevalence in contaminated food and house dust of most Asian countries such as Korea, China, India, Hong Kong, Singapore, Thailand, Malaysia, and the Philippines (Chmielewski, 2009 ; Ebrahimi & Noei, 2022 ; Ernieenor et al., 2018 ) suggest the need to implicate a broader perspective in its identification, control, and allergenicity standards. An increasing number of studies regarding the characterization of HDM species, including Sp , have focused on the combination of morphological, molecular (DNA barcoding), and serological data to elucidate taxonomy (identification) and allergenicity. In tropical regions like the Philippines, warm and humid conditions create an ideal environment for HDM proliferation and cause sensitization in approximately 33–47% of atopic Filipino patients (Patanindagat et al., 2024 ). In line with this, Sp has also been shown to contain allergenic peptides (Puerta et al., 2005 ) and elicit strong IgE-mediated hypersensitivity responses that worsen allergic symptoms. However, recent studies on Sp among Filipino patients have not been conducted since 2014 (Yap et al., 2014 ). To date, this species has been found to have positive IgE binding among allergic patients (Mariana et al., 2000 ; Yap et al., 2014 ) with an inhibitory effect against Blomia tropicalis ( Bt ) and Dermatophagoides farinae ( Df ) extracts (Reginald et al., 2004 ), demonstrating the allergenic potential of its proteins compared with other potent allergen sources. Despite this, the Sp allergome is has rarely been investigated, with only one characterized allergen (Bajao & Ramos, 2020 ), rendering such a proteome unknown for its wide range of allergenic potential and clinical applications, implicating the need to update allergenicity profiles and enhance the spectrum for searching intraspecific allergens. The current study focuses on holistic morphological and molecular identification of autapomorphic and genetic (COI & 18S rRNA) characters of Sp isolated from Philippine acarofauna, along with subsequent updates on plasma IgE reactivity of whole mite extract and its cross-reactivity to other allergenic HDM species among the atopic Filipino population. METHODS Collection, Culture, and Purification of Mites Sp mites were obtained from house dust samples collected from San Pedro, Laguna, Philippines, via the vacuum extraction method. A 25-micron nylon filter mesh was placed between the vacuum cleaner hose and the nozzle adapter to serve as a dust trap, and entrapped dust was deposited into a sterile container with powdered TetraMin® culture medium and incubated for one month at 28 ± 4°C and 78 ± 7% relative humidity. Mites were placed on individual 1.5mL microcentrifuge tubes for oviposition and monoculture preparation for another month. Afterward, monocultures were expanded subsequently via transfer to increasing container volumes in the following order: first to a 15mL conical tube (with 5 g TetraMin®) for 2 weeks, followed by a Falcon® 70mL Vented Flask (with 30 g TetraMin®) for another 2 weeks. Mite bodies were separated in spent TetraMin® media via heat extraction using a modified Tullgren-Berlese setup and subsequently purified from the remaining food debris. Purified mites were stored in -20°C until use. 2. Scanning electron microscopy Adult mite bodies obtained from the monocultures were prepared for morphological analysis and observation via a scanning electron microscope (Hitachi TM3000 Tabletop Electron Microscope). Whole mite samples were affixed to conductive carbon adhesive tape mounted on coverslips and oriented in dorsal, ventral, and lateral positions. Microscopy-based species identification was then derived from the taxonomic keys provided by Hughes ( 1976 ). Optimal imaging resolution was achieved with an accelerating voltage of 15 kV and EDX mode. 3. Genomic DNA isolation Frozen Sp mite bodies (25 mg) were fully suspended in precooled PBS (1x) (Sigma-Aldrich, St. Louis, USA) and homogenized via a probe sonicator at 30 kHz for 10 seconds (one cycle) immersed in an ice bath. Genomic DNA was then extracted from the mite lysate using Solarbio® Animal Tissues/Cells Genomic DNA Extraction Kit (Beijing, China) according to the manufacturer's protocol. The quantity and quality of extracted DNA were determined using the SPECTROstar Nano LVis Plate (Ortenberg, Germany). 4. Allergen extract preparation Allergen extract was prepared by mechanically grinding the frozen Sp mites weighing 0.8 grams in a precooled mortar and pestle for 1 hour. Ground mite bodies were suspended in 10 mL of 1× PBS and 100 µL of phenylmethylsulfonyl fluoride protease inhibitor (1 mM). The resulting mite extract suspension was incubated at 4°C with gentle shaking for 16 hours. The suspension was centrifuged at 14,000 rpm for 20 minutes in a refrigerated centrifuge (DLAB D3024R, USA) to isolate the supernatant containing the aqueous Sp allergen extract. Finally, the Sp allergen extract was quantified via a Bradford assay with a concentration of 679.69 ug/mL. 5. PCR and Sequencing of 18s rRNA and Cox1 Genes Polymerase chain reaction (PCR) amplification of the 18S rRNA gene was conducted using forward (5’-CGCGAATGGCTCATTAAATC-3’) and reverse (5’-GATCGCCTTCGAACCTCTAA-3’) primers (Klimov & OConnor, 2008 ). Similarly, the cytochrome oxidase I ( COI ) mitochondrial gene was amplified using forward (5’-GTTTTGGGATATCTCTCATAC-3’) and reverse (5’-GAGCAACAACATAATAAGTAT-3’) universal primers (Ernieenor et al., 2018 ). The PCR amplicons were visualized on a 1% agarose gel and purified using Promega Wizard® SV Gel and PCR Clean-Up System, and triplicate samples from each gene were sent to Macrogen, South Korea, for bidirectional Sanger sequencing. 6. Phylogenetic analysis The 18S rRNA and COI gene sequencing reads were trimmed and assembled via the CodonCode Aligner according to patterns of peak quality. Triplicated assembly was then aligned to generate a consensus contig used for BLAST query and library preparation. A BLAST-derived compiled gene library fasta file was created by selecting allergenic species found in domestic acarofauna. The alignment was performed via the local alignment algorithm of MAFFT, and autotrimmed via TrimAI at default settings. Selection of a highly fit model was determined via PartitionFinder using Akaike information criterion (AICc). Bayesian analysis was performed with 5 million instances sampled per 100 generations. Tree topology analysis and annotations were performed via tvBOT (Xie et al., 2023 ). 7. IgE Enzyme-Linked Immunosorbent Assay Sp allergen extracts were prepared to a final concentration of 10 µg/mL in a 50 mM carbonate-bicarbonate coating buffer (pH 9.6). A 50 µL aliquot of the diluted Sp extract was added to each well of a 96-well plate (Corning Costar Inc., NY, USA) and incubated overnight on an orbital shaker at 200 rpm and 4°C. The plates were subsequently blocked with 100 µL of 1% BSA (diluted in 1× PBS) for 1 hour. Following blocking, 50 µL of 5× diluted human plasma in 1% BSA (Sigma-Aldrich, St. Louis, USA) was added to each well in duplicate and incubated for 2 hours. The reaction was then incubated with 100 µL of anti-human IgE-HRP conjugate (1:1000 dilution in PBS-T), for 1 hour. Colorimetric reaction was then initiated by adding 100 µL of 3,3',5,5'-Tetramethylbenzidine (TMB) and TMB substrate solution (Sigma-Aldrich, St. Louis, USA) to each well. All steps were conducted at room temperature, with three washes between steps using 1× PBS-T (0.05% Tween-20) in an ELISA washer (Lisawash 3000, IN). Absorbance at 605 nm was measured in an ELISA plate reader (SPECTROstar Nano, Germany). Cutoff values for allergenicity were based on the Mean + 1SD of ELISA results from non-atopic subjects. 8. IgE Inhibition Assay An IgE inhibition ELISA was conducted to evaluate the capacity of Sp extract to inhibit IgE reactivity against other allergen extracts. Seven plasma samples with positive IgE reactions to either Dermatophagoides pteronyssinus ( Dp ), Df, Bt , and Sp were used. All steps were carried out at room temperature unless specified otherwise. ELISA plates (Corning Costar Inc., NY, USA) were coated with Sp extract at a concentration of 10 µg/mL, diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6), at 50 µL per well, and incubated overnight at 4°C. Simultaneously, each plasma sample was preabsorbed with 5 µg/mL of Dp , Df , and Bt extracts and also incubated overnight at 4°C. The coated plates were then blocked with 1% BSA (Fisher BioReagents, MA, USA) diluted in 1× PBS for 1 hour. Preabsorbed plasma samples and their corresponding unabsorbed duplicates were added to the plates and incubated for 2 hours. Following this, 100 µL of horseradish peroxidase-conjugated anti-human IgE was added to each well and incubated for 1 hour. A colorimetric reaction was initiated by adding 100 µL of TMB (Sigma-Aldrich, St. Louis, USA) per well. Between steps, plates were washed three times with 1× PBS containing 0.05% Tween 20 (Loba Chemie Pvt Ltd, IN) using an ELISA washer (Lisawash 3000, IN). The absorbance at 605 nm was measured 30 minutes after TMB addition using an ELISA plate reader (SPECTROstar Nano, Germany). The percent inhibition was calculated via the following formula: % Inhibition = [(unabsorbed plasma OD - absorbed plasma OD) / unabsorbed plasma OD] * 100. RESULTS SEM reveals morphological features of Sp Several important morphological characteristics of the surmised Sp samples were identified by scanning electron microscopy. These findings are consistent with several other studies that taxonomically described the family Suidasiidae and outlined the specific features of Sp . Key distinctive features were the length difference of the scapular internal seta ( sci ) and scapular external seta ( sce ), with the sci being considerably shorter than the sce (Fig. 1 A). These hair-like structures have been well established as a hallmarks of Suidasia species (Hughes, 1961 ). Additionally, the external vertical setae, ( ve ), were located lateroposterior to the internal vertical setae, ( vi ), on the gnathosoma (Fig. 1 C) as previously described for this genus (Fain & Philips, 1978 ). These setal arrangements are essential for distinguishing Sp from other morphologically similar mites needed for classification. The cuticular morphology of suspected Sp , as shown in Fig. 1 B features reticulated patterns found on the dorsal surface of the mites covered with scale-like verrucae. This reticulated pattern is also present at the ventral surface of the mite but is less distinct than the dorsal cuticle (Ahamad et al., 2011 ). The smooth propodosomal shield at the anterior dorsal surface of the mite was also evident upon observing the mite’s head region. The omega I (ω¹) spine was found at the base of Tarsus I, close to the claw, upon closer inspection of the tarsal morphology (Fig. 1 D). This structure is one of the three conspicuous ventral spines of Sp , appearing as a slender curved rod (Hughes, 1961 ). Ventrally, a pair of oval-shaped anal suckers (Fig. 1 E), was observed in adult male mites. These structures are used for holding on to the female during copulation, which is crucial for successful reproduction. These anal suckers are surrounded by three pairs of anal setae. Unlike male Suidasia, females lack these anal structures but have bursa copulatrix to receive the sperm of males during copulation. COI gene sequence identifies the species Suidasia pontifica Identification relied on the COI gene consensus from triplicate sequencing data results. Amplification via the primers yielded an ~340bp amplicon, with sequence data suggesting a GC content of 39.4%. BLAST results yielded Sp (100% similarity) and S. medanensis (100% similarity) sequences. Sequence alignment and trimming generated an MSA alignment with 363 parsimony informative characters, with the best model GTR+I+G (General time reversible with invariant sites and gamma distribution). The generated phylogenetic tree (Figure 2) accommodates high topological bootstrap values ranging from 81.9% to 100%. Cladistic placement of suspected Sp shows monophyletic inclusion of both Sp and S. medanensis (BS=99.9%). 18S RNA sequence supports a separate clade of S. pontifica to S. nesbitii Amplification and sequencing of the triplicated partial 18S rRNA gene resulted in an 856bp sequence consensus with a GC content of 51.3%. Sequence alignment and trimming result in a global MSA length of 1478 parsimony informative sites, with the best model: GTR+G (General time reversible with gamma distribution) for Bayesian inference. A cladogram (Figure 3) generated from Bayesian phylogenetics suggests that the bootstrap value (BS) ranges from 43% to 100%. Interestingly, the sequenced sample assembly of Suidasia pontifica was able to form a monophyletic group with Suidasia nesbitti (BS=98.5%), yet strong support also suggests a separation of Sp to S. nesbitti (BS=99.8%) within the clade. Furthermore, 18S rRNA cladogram basal topology analysis suggests a correct monophyletic clade for class Arachnida (BS=79.8%). The superorder Acariformes (Trombidiformes+Sarcoptiformes) (BS=79.8%), distinct from arachnid orders Araneae (Spiders) and Scorpiones (Scorpions). Paraphyletic topology of Trombidiforman mites, indicated by arrows in Figure 3, may be explained by its relatively low bootstrap support (BS=61.4%). Independently, the monophyly of the 18S rRNA of Sarcoptiforman mites contain all common allergenic house dust/storage mites with emphasis on the common node ancestry of the traditional Pyroglyphidae HDM family (BS=96.6%), separate from other families containing species with allergenic importance (e.g. Echimyopodidae, Glycyphagidae, Acaridae). Sp exhibits IgE binding reactivity Approximately 47% of the 100 allergic patients tested had a positive reaction against Sp extract, with a relative cutoff value of 0.3063 based on the mean + 1 standard deviation (SD) of the relative Sp-specific IgE concentration of the 100 nonallergic patients (Figure 4). Sp-specific IgE levels of the allergic patients were significantly higher than those of nonallergic controls ( P < 0.001). Compared with the nonallergic controls, the allergic patients presented a higher relative Sp-specific IgE concentration, reaching 1.19, and the lowest, reaching 0.129; In comparison with the nonallergic controls, which reached a maximum and minimum Sp-specific IgE concentration of 0.426 and 0.094, respectively. Interestingly, among the allergic cases, 1 plasma reached relative Sp-specific IgE levels greater than 1, whereas none were recorded among the nonallergic patients. Sp cross-reacts with other HDM species Significant IgE cross-reactivity of the allergens from the Sp extract was observed against allergens from three other HDM species. Among the seven IgE reactive blood plasmas, the Sp extract had the greatest average inhibitory effect on Dp extract, with an average IgE inhibition of 22.34%, followed by Df, with 14.47%, and Bt with 11.28% average inhibition. While the degree of inhibitory effect varies among patients, the results consistently suggest a potential parallel sensitization mechanism in which Sp allergens may cross-react with other HDM species to induce allergic manifestations. Statistical analysis revealed no significant difference (p = 0.195) in the observed cross-reactivity of the Sp allergen extract with the allergens from Bt , Dp , and Df . DISCUSSION Sp was first described in 1905 by Oudemans and initially placed in the family Acaridae due to the presence of an external vertical (ve) setae; however, the discovery of a well-developed pretarsus bearing the claw led Fain (1978) to provisionally recircumscribe the species in the family Saproglyphidae. The description of additional intermediate characters from both families consequently placed the taxon into a separate family Suidasiidae, genus Suidasia (Fain & Philips, 1978; Pacia & Corpuz-Raros, 1998). Similarly, changes within the specific epithet caused convoluting identification in the literature, which remains contemporarily prevalent. Aphelana medanensis was previously described by Oudemans in 1923, who synonymized it with the genus Suidasia. However, upon examining the type specimen, no differences were found to differentiate S. medanensis from Sp , classifying S. medanensis as a junior synonym (not a separate species) to Sp . Although names are interchangeable, as given by the first and second publications of Hughes in Sp dichotomous keys (Hughes, 1961, 1976), proper identification and taxonomic nomenclature are not met and subsequently affect even the downstream nomenclature of allergens in the NCBI Protein Database owing to the presence of two different allergen names for one species (Sui p & Sui m). Our results show that morphological evidence of Sp supports nonplasticity in five utilized autapomorphies, which is well corroborated by molecular identification via COI gene characters and an exact BLAST match result with Sp submitted in GenBank. Given this, the valid nomenclature for the species should be prioritized to Sp , in accordance with Fain & Philips’s synonymization in 1978. However, HDM DNA barcoding remains crucially limited to molecular characters, which becomes hard to describe morphologically, therefore to reconcile the autapomorphic demarcation of Sp to its sister species S. nesbitti and other allergenic HDM families, this study further explored the use of a different primer that amplifies Sarcoptiforman 18S rRNA, and interestingly, the results show that Sp does indeed demarcate phylogenetically from the sister species S. nesbitti with strong support, which may indicate the reliability of molecular markers for HDM identification coupled with electron microscopy. Moreover, to prove its medical importance, Sp proteins were tested with specific IgE ELISA from Filipino atopic patients, which revealed that 47% of allergic patients exhibited relative reactivity as compared with the mean+1SD OD values of nonallergic patients, which implies their state of sensitization and the ability of Sp to produce potent allergens. To investigate this further, we performed cross-reactivity reactivity profiling to explore the possibility of homology in allergen protein structure and IgE epitopes for future detailed research and characterization. As anticipated, Sp protein extracts from the allergenic species Bt, Df, and Dp exhibited some degree of reactivity, with no significant differences across species combinations, possibly resulting from a parallel sensitization mechanism. Overall, the use of a multicombinatorial approach involving morphological, molecular, and proteomic/allergomic methods further opens new possibilities for a holistic identification of mite species and the subsequent examination of their medical importance, which may help expedite allergen source discovery. The sensitization profiles of Sp extract exhibited in this study were greater than those reported in previously conducted allergenicity studies, which describe approximately 39% sensitization in allergic individuals (Yap et al., 2014). This recent study further validated the positive reaction of allergic patients to Sp extracts, suggesting that Sp proteins play an important role in sensitization among allergic individuals. These findings highlight the need for more comprehensive studies to identify specific allergens within Sp extracts that contribute to this heightened sensitivity. Additionally, variability in sensitization rates among studies may be attributed to differences in sample populations, regional dietary habits, or environmental factors that influence allergen exposure (Calderón et al., 2015). The current results underscore the clinical relevance of Sp proteins and their potential as biomarkers for the diagnosis and management of allergic conditions. Moreover, these findings emphasize the importance of including Sp extracts in allergen panels for skin-prick tests to ensure accurate diagnosis (Bajao & Ramos, 2020; Yap et al., 2014). Future investigations should focus on characterizing individual allergenic proteins in Sp extracts and exploring their structural properties to better understand the mechanisms underlying sensitization. The accurate identification of HDMs at the species level via molecular and morphological data analysis is critical for evaluating the clinical significance of allergen sources in medical diagnostics, allergen-specific immunotherapy, and basic allergology research. In this study, we report the proper identification of Sp using scanning electron microscopy and phylogenetic analysis. The Sp identified in the study is a source of allergens with significant IgE binding reactivity among allergic patients. Declarations Author Contribution J.W.R. Dela Cruz and E.P.B. Malit carried out the collection and purification of Sp cultures, extracted Sp DNA, amplified 18S and COI genes, constructed and analyzed the phylogenetic trees, and collaborated with D.D.G. Boongaling to capture and analyze SEM images of Sp. C.Y. Patanindagat, C.F.L. Arevalo, and A.J.I. Manalo performed the Sp protein extraction, IgE ELISA, and IgE inhibition assay. J.D.A. Ramos conceptualized, supervised, and reviewed the manuscript before submission. All authors contributed to their corresponding interpretation of the results and to the writing of the manuscript. Acknowledgement The authors would like to express their gratitude to the UST Analytical Services Laboratory for their provision of Hitachi TM3000 Tabletop Electron Microscope throughout this research. Data Availability Sequence data that support the findings of this study are available in the National Center for Biotechnology Information GenBank ® database under the following accession codes: Suidasia pontifica SPL-1 isolate 18S (PV876202) and Suidasia pontifica SPL-1 isolate COI (PV876203). References Ahamad, M., Louis, S. R., Hamid, Z., & Ho, T. M. (2011). 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Mite fauna in the home and sensitivity to house‐dust and storage mites. Allergy , 54 (7), 681–690. https://doi.org/10.1034/j.1398-9995.1999.00850.x Yap, J. M., Ching, M., Cruz, R., & Ramos, J. D. (2014). Specific IgE against the house dust mite Suidasia pontifica as a risk factor for asthma and allergies in the tropics. Acta Manilana , 62 , 1–8. https://doi.org/10.53603/actamanil.62.2014.lghm5611 Xie, J., Chen, Y., Cai, G., Cai, R., Hu, Z., & Wang, H. (2023). Tree Visualization By One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic acids research, 51(W1), W587-W592. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7056104","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485367707,"identity":"52d34238-2f03-4e62-bd4a-232dbe2d7cea","order_by":0,"name":"John Wayne Dela Cruz","email":"data:image/png;base64,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","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":true,"prefix":"","firstName":"John","middleName":"Wayne Dela","lastName":"Cruz","suffix":""},{"id":485367708,"identity":"4adddb7e-e72f-49bf-95ea-8c470df687e4","order_by":1,"name":"Erika Pauline Malit","email":"","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"Pauline","lastName":"Malit","suffix":""},{"id":485367709,"identity":"4c985321-df62-4baa-bb20-3898d6076cf4","order_by":2,"name":"Chanie Patanindagat","email":"","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"Chanie","middleName":"","lastName":"Patanindagat","suffix":""},{"id":485367710,"identity":"5c6aa551-696f-4a26-b938-02afc9c26388","order_by":3,"name":"Cristian Floren Arevalo","email":"","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"Cristian","middleName":"Floren","lastName":"Arevalo","suffix":""},{"id":485367712,"identity":"9c963ee0-74f0-4b0a-b6ef-f11569115615","order_by":4,"name":"Arianne Joy Manalo","email":"","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"Arianne","middleName":"Joy","lastName":"Manalo","suffix":""},{"id":485367714,"identity":"f43ca839-0704-482a-976d-851ba28ce283","order_by":5,"name":"Deve Diane Boongaling","email":"","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"Deve","middleName":"Diane","lastName":"Boongaling","suffix":""},{"id":485367716,"identity":"068fdaa2-a519-48fe-94ba-e24e228a9a05","order_by":6,"name":"John Donnie Ramos","email":"","orcid":"","institution":"University of Santo Tomas","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"Donnie","lastName":"Ramos","suffix":""}],"badges":[],"createdAt":"2025-07-06 06:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7056104/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7056104/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10493-025-01106-7","type":"published","date":"2026-01-21T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87033005,"identity":"7115e457-0801-4611-89f7-57d661804158","added_by":"auto","created_at":"2025-07-18 13:03:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":277115,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy of whole mite samples. Scanning electron micrographs of \u003cem\u003eSp\u003c/em\u003e in the dorsal, lateral, and ventral positions, highlighting five key morphological features: (A) \u003cem\u003esci\u003c/em\u003e is notably shorter than \u003cem\u003esce\u003c/em\u003e; (B) distinct reticulated cuticle pattern; (C) \u003cem\u003eve\u003c/em\u003e setae located lateroposterior to \u003cem\u003evi\u003c/em\u003e setae; (D) ω¹ setae visible at the base of Tarsus I near the claw; (E) presence of anal suckers, characteristic of the species.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7056104/v1/dee49f4e6037e3e1a650ce8e.png"},{"id":87033008,"identity":"2085984e-ef29-4609-ab85-eaa0e6778c4d","added_by":"auto","created_at":"2025-07-18 13:03:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272196,"visible":true,"origin":"","legend":"\u003cp\u003eCOI gene Bayesian phylogenetic analysis. Tree topology of partial mitochondrial COI gene sequences from taxonomically representative samples obtained via BLAST, alongside the inferred sample \u003cem\u003eSuidasia pontifica\u003c/em\u003e (boxed). Node support is indicated by bootstrap values, with black circles denoting strong support (\u0026gt;90%) and gray circles indicating moderate support (70–90%). Node circles with support values \u0026lt;70% are omitted. The terminal taxa are color-coded by clade: red for insects, pink for storage mites, and blue for pyroglyphid HDMs. The de novo sequence (Contig_COI) groups within the Storage Mite clade, supporting its identity as Suidasia. Unresolved relationships within the Astigmatid clade are presented as polytomies. Outgroup arachnid taxa were excluded due to limited sequence availability and clarity improvement. The tree scale bar represents the number of nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7056104/v1/f1dbad8e2052c93899b1915a.png"},{"id":87033007,"identity":"76445aa6-986b-477f-8e0c-117674021f1b","added_by":"auto","created_at":"2025-07-18 13:03:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":322783,"visible":true,"origin":"","legend":"\u003cp\u003e18S rRNA Bayesian Phylogenetic analysis. Resulting tree topology of taxonomic representative samples from BLAST alongside suspected sample \u003cem\u003eSp\u003c/em\u003e (boxed). Bootstrap values are placed in node separations based on criterion of \u0026gt;90% (strongly supported) and 70-90% (well supported). Nodes with bootstrap values below 70% are not shown in the diagram. Grouping based on common names for clades is highlighted in each leaf, known taxons are listed on top of the respective branch. Short black arrows represent the paraphyletic Trombidiformes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7056104/v1/290159b561124e2dddb1299f.png"},{"id":87033010,"identity":"4849b10e-ac2a-4a11-be8c-c65040343755","added_by":"auto","created_at":"2025-07-18 13:03:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":151466,"visible":true,"origin":"","legend":"\u003cp\u003eSerological profiling of Sp-specific IgE levels among allergic and nonallergic individuals. Sp-specific IgE in serum samples from allergic (n = 100) and nonallergic (n = 100) participants, as measured by ELISA at OD605. Each dot in the scatter plots represents an individual, with sensitized individuals (Turquoise) and non-sensitized individuals (Yellow). The dotted horizontal line marks the ELISA cutoff value of 0.306 OD, used to discriminate sensitized from non-sensitized individuals. Boxplots indicate the median and interquartile ranges for each group. A donut chart summarizes the proportion of Sp-sensitized individuals within the allergic group, revealing that 47.0% (47 out of 100) of allergic patients tested positive for Sp-specific IgE, highlighting a substantial rate of Sp sensitization among allergic individuals.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7056104/v1/e30b9e2cdd3608ceafad9c94.png"},{"id":87034757,"identity":"9390443c-f2ec-4f05-89f1-b6f00ec39400","added_by":"auto","created_at":"2025-07-18 13:11:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32957,"visible":true,"origin":"","legend":"\u003cp\u003eCross-reactivity profile of Sp-specific IgE against common mite allergens. Percent inhibition of IgE binding to the Sp allergen extract in the presence of the heterologous mite allergen extracts, \u003cem\u003eBlomia tropicalis (Bt), Dermatophagoides pteronyssinus (Dp), and Dermatophagoides farinae (Df),\u003c/em\u003e as assessed by an inhibition ELISA of seven plasma samples from patients with the highest degree of reactivity. The y-axis represents the relative inhibition (%) of IgE binding calculated from the formula explained in the methods. Higher values indicate greater cross-reactivity between Sp and the respective mite extract. Each dot represents an individual patient's response, and boxplots depict the median, interquartile range, and full range of inhibition values across patients for each allergen tested. While the \u003cem\u003eDp\u003c/em\u003eextract showed the widest variation and greater median inhibition, a one-way ANOVA test revealed no statistically significant difference among the three groups (p = 0.2035), suggesting that there was no significant difference in cross-reactivity between \u003cem\u003eSp\u003c/em\u003e and these mite allergens across reactive individuals.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7056104/v1/78466ef80f8b0b12cb186b92.png"},{"id":101153075,"identity":"e7188715-c42b-4d4a-9df7-bc1b92b71b8a","added_by":"auto","created_at":"2026-01-26 16:14:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1612896,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7056104/v1/9843455f-75d5-433b-83db-35e8252f763c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electron Microscopy and Molecular Phylogenetic Characterization of the Allergenic Dust Mite Species Suidasia pontifica (Acari: Suidasiidae)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAllergenic HDMs are a major cause of indoor asthma and allergic rhinitis worldwide, affecting as many as 50% of all global atopic sensitivities to approximately 90% of atopic patients in Asian populations (Calderón et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Salo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with the prevalence varying across regions due to seasonal and environmental factors (Calderón et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Salo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tham et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Traditionally, the term HDM is associated with species in the Pyroglyphidae family, but members of storage mites have also been medically recognized as having a comparative risk and degree of allergenicity due to their generalistic ability to proliferate indoors (Warner et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Such addition of paraphyletic groups broadens the scope of allergenic HDMs to become taxonomically scattered under the mite Order Sarcoptiformes from Class Arachnida, particularly on the three main superfamilies Glycyphagoidea, Acaroidea, and Analgoidea (Colloff, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). One of the least studied storage mite species, \u003cem\u003eSuidasia pontifica\u003c/em\u003e (\u003cem\u003eSp\u003c/em\u003e), is regarded as one of the major causes of food and storage product contamination worldwide and a significant factor in oral mite anaphylaxis (Barrera et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mangoba \u0026amp; Alvindia, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Its reported prevalence in contaminated food and house dust of most Asian countries such as Korea, China, India, Hong Kong, Singapore, Thailand, Malaysia, and the Philippines (Chmielewski, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ebrahimi \u0026amp; Noei, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ernieenor et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) suggest the need to implicate a broader perspective in its identification, control, and allergenicity standards.\u003c/p\u003e\u003cp\u003eAn increasing number of studies regarding the characterization of HDM species, including \u003cem\u003eSp\u003c/em\u003e, have focused on the combination of morphological, molecular (DNA barcoding), and serological data to elucidate taxonomy (identification) and allergenicity. In tropical regions like the Philippines, warm and humid conditions create an ideal environment for HDM proliferation and cause sensitization in approximately 33–47% of atopic Filipino patients (Patanindagat et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In line with this, Sp has also been shown to contain allergenic peptides (Puerta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and elicit strong IgE-mediated hypersensitivity responses that worsen allergic symptoms. However, recent studies on Sp among Filipino patients have not been conducted since 2014 (Yap et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To date, this species has been found to have positive IgE binding among allergic patients (Mariana et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Yap et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with an inhibitory effect against \u003cem\u003eBlomia tropicalis\u003c/em\u003e (\u003cem\u003eBt\u003c/em\u003e) and \u003cem\u003eDermatophagoides farinae\u003c/em\u003e (\u003cem\u003eDf\u003c/em\u003e) extracts (Reginald et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), demonstrating the allergenic potential of its proteins compared with other potent allergen sources. Despite this, the \u003cem\u003eSp\u003c/em\u003e allergome is has rarely been investigated, with only one characterized allergen (Bajao \u0026amp; Ramos, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), rendering such a proteome unknown for its wide range of allergenic potential and clinical applications, implicating the need to update allergenicity profiles and enhance the spectrum for searching intraspecific allergens.\u003c/p\u003e\u003cp\u003eThe current study focuses on holistic morphological and molecular identification of autapomorphic and genetic (COI \u0026amp; 18S rRNA) characters of \u003cem\u003eSp\u003c/em\u003e isolated from Philippine acarofauna, along with subsequent updates on plasma IgE reactivity of whole mite extract and its cross-reactivity to other allergenic HDM species among the atopic Filipino population.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"METHODS","content":"\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eCollection, Culture, and Purification of Mites\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003cp\u003e\u003cem\u003eSp\u003c/em\u003e mites were obtained from house dust samples collected from San Pedro, Laguna, Philippines, via the vacuum extraction method. A 25-micron nylon filter mesh was placed between the vacuum cleaner hose and the nozzle adapter to serve as a dust trap, and entrapped dust was deposited into a sterile container with powdered TetraMin® culture medium and incubated for one month at 28 ± 4°C and 78 ± 7% relative humidity. Mites were placed on individual 1.5mL microcentrifuge tubes for oviposition and monoculture preparation for another month. Afterward, monocultures were expanded subsequently via transfer to increasing container volumes in the following order: first to a 15mL conical tube (with 5 g TetraMin®) for 2 weeks, followed by a Falcon® 70mL Vented Flask (with 30 g TetraMin®) for another 2 weeks. Mite bodies were separated in spent TetraMin® media via heat extraction using a modified Tullgren-Berlese setup and subsequently purified from the remaining food debris. Purified mites were stored in -20°C until use.\u003c/p\u003e\n\u003ch3\u003e2. Scanning electron microscopy\u003c/h3\u003e\n\u003cp\u003eAdult mite bodies obtained from the monocultures were prepared for morphological analysis and observation via a scanning electron microscope (Hitachi TM3000 Tabletop Electron Microscope). Whole mite samples were affixed to conductive carbon adhesive tape mounted on coverslips and oriented in dorsal, ventral, and lateral positions. Microscopy-based species identification was then derived from the taxonomic keys provided by Hughes (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Optimal imaging resolution was achieved with an accelerating voltage of 15 kV and EDX mode.\u003c/p\u003e\n\u003ch3\u003e3. Genomic DNA isolation\u003c/h3\u003e\n\u003cp\u003eFrozen Sp mite bodies (25 mg) were fully suspended in precooled PBS (1x) (Sigma-Aldrich, St. Louis, USA) and homogenized via a probe sonicator at 30 kHz for 10 seconds (one cycle) immersed in an ice bath. Genomic DNA was then extracted from the mite lysate using Solarbio\u0026reg; Animal Tissues/Cells Genomic DNA Extraction Kit (Beijing, China) according to the manufacturer's protocol. The quantity and quality of extracted DNA were determined using the SPECTROstar Nano LVis Plate (Ortenberg, Germany).\u003c/p\u003e\n\u003ch3\u003e4. Allergen extract preparation\u003c/h3\u003e\n\u003cp\u003eAllergen extract was prepared by mechanically grinding the frozen Sp mites weighing 0.8 grams in a precooled mortar and pestle for 1 hour. Ground mite bodies were suspended in 10 mL of 1\u0026times; PBS and 100 \u0026micro;L of phenylmethylsulfonyl fluoride protease inhibitor (1 mM). The resulting mite extract suspension was incubated at 4\u0026deg;C with gentle shaking for 16 hours. The suspension was centrifuged at 14,000 rpm for 20 minutes in a refrigerated centrifuge (DLAB D3024R, USA) to isolate the supernatant containing the aqueous Sp allergen extract. Finally, the Sp allergen extract was quantified via a Bradford assay with a concentration of 679.69 ug/mL.\u003c/p\u003e\n\u003ch3\u003e5. PCR and Sequencing of 18s rRNA and Cox1 Genes\u003c/h3\u003e\n\u003cp\u003ePolymerase chain reaction (PCR) amplification of the 18S rRNA gene was conducted using forward (5\u0026rsquo;-CGCGAATGGCTCATTAAATC-3\u0026rsquo;) and reverse (5\u0026rsquo;-GATCGCCTTCGAACCTCTAA-3\u0026rsquo;) primers (Klimov \u0026amp; OConnor, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similarly, the cytochrome oxidase I (\u003cem\u003eCOI\u003c/em\u003e) mitochondrial gene was amplified using forward (5\u0026rsquo;-GTTTTGGGATATCTCTCATAC-3\u0026rsquo;) and reverse (5\u0026rsquo;-GAGCAACAACATAATAAGTAT-3\u0026rsquo;) universal primers (Ernieenor et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The PCR amplicons were visualized on a 1% agarose gel and purified using Promega Wizard\u0026reg; SV Gel and PCR Clean-Up System, and triplicate samples from each gene were sent to Macrogen, South Korea, for bidirectional Sanger sequencing.\u003c/p\u003e\n\u003ch3\u003e6. Phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eThe 18S rRNA and \u003cem\u003eCOI\u003c/em\u003e gene sequencing reads were trimmed and assembled via the CodonCode Aligner according to patterns of peak quality. Triplicated assembly was then aligned to generate a consensus contig used for BLAST query and library preparation. A BLAST-derived compiled gene library fasta file was created by selecting allergenic species found in domestic acarofauna. The alignment was performed via the local alignment algorithm of MAFFT, and autotrimmed via TrimAI at default settings. Selection of a highly fit model was determined via PartitionFinder using Akaike information criterion (AICc). Bayesian analysis was performed with 5\u0026nbsp;million instances sampled per 100 generations. Tree topology analysis and annotations were performed via tvBOT (Xie et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e7. IgE Enzyme-Linked Immunosorbent Assay\u003c/h3\u003e\n\u003cp\u003eSp allergen extracts were prepared to a final concentration of 10 \u0026micro;g/mL in a 50 mM carbonate-bicarbonate coating buffer (pH 9.6). A 50 \u0026micro;L aliquot of the diluted Sp extract was added to each well of a 96-well plate (Corning Costar Inc., NY, USA) and incubated overnight on an orbital shaker at 200 rpm and 4\u0026deg;C. The plates were subsequently blocked with 100 \u0026micro;L of 1% BSA (diluted in 1\u0026times; PBS) for 1 hour. Following blocking, 50 \u0026micro;L of 5\u0026times; diluted human plasma in 1% BSA (Sigma-Aldrich, St. Louis, USA) was added to each well in duplicate and incubated for 2 hours. The reaction was then incubated with 100 \u0026micro;L of anti-human IgE-HRP conjugate (1:1000 dilution in PBS-T), for 1 hour. Colorimetric reaction was then initiated by adding 100 \u0026micro;L of 3,3',5,5'-Tetramethylbenzidine (TMB) and TMB substrate solution (Sigma-Aldrich, St. Louis, USA) to each well. All steps were conducted at room temperature, with three washes between steps using 1\u0026times; PBS-T (0.05% Tween-20) in an ELISA washer (Lisawash 3000, IN). Absorbance at 605 nm was measured in an ELISA plate reader (SPECTROstar Nano, Germany). Cutoff values for allergenicity were based on the Mean\u0026thinsp;+\u0026thinsp;1SD of ELISA results from non-atopic subjects.\u003c/p\u003e\n\u003ch3\u003e8. IgE Inhibition Assay\u003c/h3\u003e\n\u003cp\u003eAn IgE inhibition ELISA was conducted to evaluate the capacity of Sp extract to inhibit IgE reactivity against other allergen extracts. Seven plasma samples with positive IgE reactions to either \u003cem\u003eDermatophagoides pteronyssinus\u003c/em\u003e (\u003cem\u003eDp\u003c/em\u003e), \u003cem\u003eDf, Bt\u003c/em\u003e, and \u003cem\u003eSp\u003c/em\u003e were used. All steps were carried out at room temperature unless specified otherwise. ELISA plates (Corning Costar Inc., NY, USA) were coated with Sp extract at a concentration of 10 \u0026micro;g/mL, diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6), at 50 \u0026micro;L per well, and incubated overnight at 4\u0026deg;C. Simultaneously, each plasma sample was preabsorbed with 5 \u0026micro;g/mL of \u003cem\u003eDp\u003c/em\u003e, \u003cem\u003eDf\u003c/em\u003e, and Bt extracts and also incubated overnight at 4\u0026deg;C. The coated plates were then blocked with 1% BSA (Fisher BioReagents, MA, USA) diluted in 1\u0026times; PBS for 1 hour. Preabsorbed plasma samples and their corresponding unabsorbed duplicates were added to the plates and incubated for 2 hours. Following this, 100 \u0026micro;L of horseradish peroxidase-conjugated anti-human IgE was added to each well and incubated for 1 hour. A colorimetric reaction was initiated by adding 100 \u0026micro;L of TMB (Sigma-Aldrich, St. Louis, USA) per well. Between steps, plates were washed three times with 1\u0026times; PBS containing 0.05% Tween 20 (Loba Chemie Pvt Ltd, IN) using an ELISA washer (Lisawash 3000, IN). The absorbance at 605 nm was measured 30 minutes after TMB addition using an ELISA plate reader (SPECTROstar Nano, Germany). The percent inhibition was calculated via the following formula: \u003cem\u003e% Inhibition = [(unabsorbed plasma OD - absorbed plasma OD) / unabsorbed plasma OD] * 100.\u003c/em\u003e\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eSEM reveals morphological features of Sp\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeveral important morphological characteristics of the surmised \u003cem\u003eSp\u003c/em\u003e samples were identified by scanning electron microscopy. These findings are consistent with several other studies that taxonomically described the family Suidasiidae and outlined the specific features of \u003cem\u003eSp\u003c/em\u003e. Key distinctive features were the length difference of the scapular internal seta (\u003cem\u003esci\u003c/em\u003e) and scapular external seta (\u003cem\u003esce\u003c/em\u003e), with the \u003cem\u003esci\u003c/em\u003e being considerably shorter than the \u003cem\u003esce\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These hair-like structures have been well established as a hallmarks of Suidasia species (Hughes, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1961\u003c/span\u003e). Additionally, the external vertical setae, (\u003cem\u003eve\u003c/em\u003e), were located lateroposterior to the internal vertical setae, (\u003cem\u003evi\u003c/em\u003e), on the gnathosoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) as previously described for this genus (Fain \u0026amp; Philips, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). These setal arrangements are essential for distinguishing \u003cem\u003eSp\u003c/em\u003e from other morphologically similar mites needed for classification. The cuticular morphology of suspected \u003cem\u003eSp\u003c/em\u003e, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB features reticulated patterns found on the dorsal surface of the mites covered with scale-like verrucae. This reticulated pattern is also present at the ventral surface of the mite but is less distinct than the dorsal cuticle (Ahamad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The smooth propodosomal shield at the anterior dorsal surface of the mite was also evident upon observing the mite\u0026rsquo;s head region. The omega I (ω\u0026sup1;) spine was found at the base of Tarsus I, close to the claw, upon closer inspection of the tarsal morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This structure is one of the three conspicuous ventral spines of \u003cem\u003eSp\u003c/em\u003e, appearing as a slender curved rod (Hughes, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1961\u003c/span\u003e). Ventrally, a pair of oval-shaped anal suckers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), was observed in adult male mites. These structures are used for holding on to the female during copulation, which is crucial for successful reproduction. These anal suckers are surrounded by three pairs of anal setae. Unlike male Suidasia, females lack these anal structures but have bursa copulatrix to receive the sperm of males during copulation.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCOI\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene sequence identifies the species \u003cem\u003eSuidasia pontifica\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdentification relied on the COI gene consensus from triplicate sequencing data results. Amplification via the primers yielded an ~340bp amplicon, with sequence data suggesting a GC content of 39.4%. BLAST results yielded \u003cem\u003eSp\u003c/em\u003e (100% similarity) and \u003cem\u003eS. medanensis\u003c/em\u003e (100% similarity) sequences. Sequence alignment and trimming generated an MSA alignment with 363 parsimony informative characters, with the best model GTR+I+G (General time reversible with invariant sites and gamma distribution). The generated phylogenetic tree (Figure 2) accommodates high topological bootstrap values ranging from 81.9% to 100%. Cladistic placement of suspected \u003cem\u003eSp\u003c/em\u003e shows monophyletic inclusion of both\u003cem\u003e\u0026nbsp;Sp\u003c/em\u003e and \u003cem\u003eS. medanensis\u0026nbsp;\u003c/em\u003e(BS=99.9%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e18S RNA sequence supports a separate clade of S. pontifica to S. nesbitii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmplification and sequencing of the triplicated partial 18S rRNA gene resulted in an 856bp sequence consensus with a GC content of 51.3%. \u0026nbsp;Sequence alignment and trimming result in a global MSA length of 1478 parsimony informative sites, with the best model: GTR+G (General time reversible with gamma distribution) for Bayesian inference. A cladogram (Figure 3) generated from Bayesian phylogenetics suggests that the bootstrap value (BS) ranges from 43% to 100%. Interestingly, the sequenced sample assembly of \u003cem\u003eSuidasia pontifica\u0026nbsp;\u003c/em\u003ewas able to form a monophyletic group with \u003cem\u003eSuidasia nesbitti\u0026nbsp;\u003c/em\u003e(BS=98.5%), yet strong support also suggests a separation of \u003cem\u003eSp\u0026nbsp;\u003c/em\u003eto \u003cem\u003eS. nesbitti\u003c/em\u003e (BS=99.8%) within the clade. Furthermore, 18S rRNA cladogram basal topology analysis suggests a correct monophyletic clade for class Arachnida (BS=79.8%). The superorder Acariformes (Trombidiformes+Sarcoptiformes) (BS=79.8%), distinct from arachnid orders Araneae (Spiders) and Scorpiones (Scorpions). Paraphyletic topology of Trombidiforman mites, indicated by arrows in Figure 3, may be explained by its relatively low bootstrap support (BS=61.4%). Independently, the monophyly of the 18S rRNA of Sarcoptiforman mites contain all common allergenic house dust/storage mites with emphasis on the common node ancestry of the traditional Pyroglyphidae HDM family (BS=96.6%), separate from other families containing species with allergenic importance (e.g. Echimyopodidae, Glycyphagidae, Acaridae).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSp exhibits IgE binding reactivity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 47% of the 100 allergic patients tested had a positive reaction against \u003cem\u003eSp\u003c/em\u003e extract, with a relative cutoff value of 0.3063 based on the mean + 1 standard deviation (SD) of the relative Sp-specific IgE concentration of the 100 nonallergic patients (Figure 4). Sp-specific IgE levels of the allergic patients were significantly higher than those of nonallergic controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Compared with the nonallergic controls, the allergic patients presented a higher relative Sp-specific IgE concentration, reaching 1.19, and the lowest, reaching 0.129; In comparison with the nonallergic controls, which reached a maximum and minimum Sp-specific IgE concentration of 0.426 and 0.094, respectively. Interestingly, among the allergic cases, 1 plasma reached relative Sp-specific IgE levels greater than 1, whereas none were recorded among the nonallergic patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSp cross-reacts with other HDM species\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant IgE cross-reactivity of the allergens from the \u003cem\u003eSp\u003c/em\u003e extract was observed against allergens from three other HDM species. Among the seven IgE reactive blood plasmas, the \u003cem\u003eSp\u003c/em\u003e extract had the greatest average inhibitory effect on \u003cem\u003eDp\u003c/em\u003e extract, with an average IgE inhibition of 22.34%, followed by\u003cem\u003e\u0026nbsp;Df,\u0026nbsp;\u003c/em\u003ewith\u003cem\u003e\u0026nbsp;\u003c/em\u003e14.47%, and \u003cem\u003eBt\u003c/em\u003e with 11.28% average inhibition. While the degree of inhibitory effect varies among patients, the results consistently suggest a potential parallel sensitization mechanism in which \u003cem\u003eSp\u003c/em\u003e allergens may cross-react with other HDM species to induce allergic manifestations. Statistical analysis revealed no significant difference (p = 0.195) in the observed cross-reactivity of the Sp allergen extract with the allergens from \u003cem\u003eBt\u003c/em\u003e, \u003cem\u003eDp\u003c/em\u003e, and \u003cem\u003eDf\u003c/em\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cem\u003eSp\u003c/em\u003e was first described in 1905 by Oudemans and initially placed in the family Acaridae due to the presence of an external vertical (ve) setae; however, the discovery of a well-developed pretarsus bearing the claw led Fain (1978) to provisionally recircumscribe the species in the family Saproglyphidae. The description of additional intermediate characters from both families consequently placed the taxon into a separate family Suidasiidae, genus Suidasia (Fain \u0026amp; Philips, 1978; Pacia \u0026amp; Corpuz-Raros, 1998). Similarly, changes within the specific epithet caused convoluting identification in the literature, which remains contemporarily prevalent. \u003cem\u003eAphelana medanensis\u003c/em\u003e was previously described by Oudemans in 1923, who synonymized it with the genus Suidasia. However, upon examining the type specimen, no differences were found to differentiate \u003cem\u003eS. medanensis\u003c/em\u003e from \u003cem\u003eSp\u003c/em\u003e, classifying \u003cem\u003eS. medanensis\u003c/em\u003e as a junior synonym (not a separate species) to \u003cem\u003eSp\u003c/em\u003e. Although names are interchangeable, as given by the first and second publications of Hughes in \u003cem\u003eSp\u003c/em\u003e dichotomous keys (Hughes, 1961, 1976), proper identification and taxonomic nomenclature are not met and subsequently affect even the downstream nomenclature of allergens in the NCBI Protein Database owing to the presence of two different allergen names for one species (Sui p \u0026amp; Sui m).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur results show that morphological evidence of \u003cem\u003eSp\u0026nbsp;\u003c/em\u003esupports nonplasticity in five utilized autapomorphies, which is well corroborated by molecular identification via COI gene characters and an exact BLAST match result with \u003cem\u003eSp\u003c/em\u003e submitted in GenBank. Given this, the valid nomenclature for the species should be prioritized to \u003cem\u003eSp\u003c/em\u003e, in accordance with Fain \u0026amp; Philips’s synonymization in 1978. However, HDM DNA barcoding remains crucially limited to molecular characters, which becomes hard to describe morphologically, therefore to reconcile the autapomorphic demarcation of \u003cem\u003eSp\u0026nbsp;\u003c/em\u003eto its sister species \u003cem\u003eS. nesbitti\u0026nbsp;\u003c/em\u003eand other allergenic HDM families, this study further explored the use of a different primer that amplifies Sarcoptiforman 18S rRNA, and interestingly, the results show that \u003cem\u003eSp\u0026nbsp;\u003c/em\u003edoes indeed demarcate phylogenetically from the sister species \u003cem\u003eS. nesbitti\u0026nbsp;\u003c/em\u003ewith strong support, which may indicate the reliability of molecular markers for HDM identification coupled with electron microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, to prove its medical importance, \u003cem\u003eSp\u0026nbsp;\u003c/em\u003eproteins were tested with specific IgE ELISA from Filipino atopic patients, which revealed that 47% of allergic patients exhibited relative reactivity as compared with the mean+1SD OD values of nonallergic patients, which implies their state of sensitization and the ability of \u003cem\u003eSp\u0026nbsp;\u003c/em\u003eto produce potent allergens. To investigate this further, we performed cross-reactivity reactivity profiling to explore the possibility of homology in allergen protein structure and IgE epitopes for future detailed research and characterization. As anticipated, \u003cem\u003eSp\u003c/em\u003e protein extracts from the allergenic species \u003cem\u003eBt, Df,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eDp\u0026nbsp;\u003c/em\u003eexhibited some degree of reactivity, with no significant differences across species combinations, possibly resulting from a parallel sensitization mechanism. Overall, the use of a multicombinatorial approach involving morphological, molecular, and proteomic/allergomic methods further opens new possibilities for a holistic identification of mite species and the subsequent examination of their medical importance, which may help expedite allergen source discovery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sensitization profiles of \u003cem\u003eSp\u003c/em\u003e extract exhibited in this study were greater than those reported in previously conducted allergenicity studies, which describe approximately 39% sensitization in allergic individuals (Yap et al., 2014). This recent study further validated the positive reaction of allergic patients to \u003cem\u003eSp\u003c/em\u003e extracts, suggesting that \u003cem\u003eSp\u003c/em\u003e proteins play an important role in sensitization among allergic individuals. These findings highlight the need for more comprehensive studies to identify specific allergens within \u003cem\u003eSp\u003c/em\u003e extracts that contribute to this heightened sensitivity. Additionally, variability in sensitization rates among studies may be attributed to differences in sample populations, regional dietary habits, or environmental factors that influence allergen exposure (Calderón et al., 2015). The current results underscore the clinical relevance of \u003cem\u003eSp\u003c/em\u003e proteins and their potential as biomarkers for the diagnosis and management of allergic conditions. Moreover, these findings emphasize the importance of including \u003cem\u003eSp\u003c/em\u003e extracts in allergen panels for skin-prick tests to ensure accurate diagnosis (Bajao \u0026amp; Ramos, 2020; Yap et al., 2014). Future investigations should focus on characterizing individual allergenic proteins in \u003cem\u003eSp\u003c/em\u003e extracts and exploring their structural properties to better understand the mechanisms underlying sensitization.\u003c/p\u003e\n\u003cp\u003eThe accurate identification of HDMs at the species level via molecular and morphological data analysis is critical for evaluating the clinical significance of allergen sources in medical diagnostics, allergen-specific immunotherapy, and basic allergology research. In this study, we report the proper identification of \u003cem\u003eSp\u003c/em\u003e using scanning electron microscopy and phylogenetic analysis. \u0026nbsp;The \u003cem\u003eSp\u003c/em\u003e identified in the study is a source of allergens with significant IgE binding reactivity among allergic patients.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.W.R. Dela Cruz and E.P.B. Malit carried out the collection and purification of Sp cultures, extracted Sp DNA, amplified 18S and COI genes, constructed and analyzed the phylogenetic trees, and collaborated with D.D.G. Boongaling to capture and analyze SEM images of Sp. C.Y. Patanindagat, C.F.L. Arevalo, and A.J.I. Manalo performed the Sp protein extraction, IgE ELISA, and IgE inhibition assay. J.D.A. Ramos conceptualized, supervised, and reviewed the manuscript before submission. All authors contributed to their corresponding interpretation of the results and to the writing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their gratitude to the UST Analytical Services Laboratory for their provision of Hitachi TM3000 Tabletop Electron Microscope throughout this research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequence data that support the findings of this study are available in the National Center for Biotechnology Information GenBank \u0026reg; database under the following accession codes: Suidasia pontifica SPL-1 isolate 18S (PV876202) and Suidasia pontifica SPL-1 isolate COI (PV876203).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhamad, M., Louis, S. R., Hamid, Z., \u0026amp; Ho, T. M. (2011). Scanning electron micrographs of medically important dust mite, Suidasia pontifica (Acari: Astigmata: Saproglyphidae) in Malaysia. \u003cem\u003eTropical Biomedicine\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(2), 275\u0026ndash;282.\u003c/li\u003e\n\u003cli\u003eBajao, J. E. M., \u0026amp; Ramos, J. D. A. (2020). cDNA Cloning and Characterization of the House Dust Mite Allergen Sui p 2. \u003cem\u003eAmerican Journal of Biochemistry and Biotechnology\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(2), 222\u0026ndash;234. https://doi.org/10.3844/ajbbsp.2020.222.234\u003c/li\u003e\n\u003cli\u003eBarrera, O. M., Murgas, I. L., Berm\u0026uacute;dez, S., \u0026amp; Miranda, R. J. (2015). Anafilaxia oral por ingesti\u0026oacute;n de alimentos contaminados con \u0026aacute;caros en Ciudad de Panam\u0026aacute;, 2011-2014. \u003cem\u003eRevista Alergia M\u0026eacute;xico\u003c/em\u003e, \u003cem\u003e62\u003c/em\u003e(2), 112\u0026ndash;117. https://doi.org/10.29262/ram.v62i2.71\u003c/li\u003e\n\u003cli\u003eCalder\u0026oacute;n, M. A., Linneberg, A., Kleine-Tebbe, J., De Blay, F., Hernandez Fernandez De Rojas, D., Virchow, J. C., \u0026amp; Demoly, P. (2015). Respiratory allergy caused by house dust mites: What do we really know? \u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e, \u003cem\u003e136\u003c/em\u003e(1), 38\u0026ndash;48. https://doi.org/10.1016/j.jaci.2014.10.012\u003c/li\u003e\n\u003cli\u003eChmielewski, W. (2009). 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PCR identification and phylogenetic analysis of the medically important dust mite Suidasia medanensis (Acari: Suidasiidae) in Malaysia. \u003cem\u003eExperimental and Applied Acarology\u003c/em\u003e, \u003cem\u003e76\u003c/em\u003e(1), 99\u0026ndash;107. https://doi.org/10.1007/s10493-018-0285-4\u003c/li\u003e\n\u003cli\u003eFain, A., \u0026amp; Philips, J. R. (1978). Notes on the genus \u003cem\u003eSuidasia\u003c/em\u003e Oudemans, 1905 with descriptions of a new species from Australia (Acari, Astigmata, Saproglyphidae). \u003cem\u003eInternational Journal of Acarology\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(2), 115\u0026ndash;123. https://doi.org/10.1080/01647957808684031\u003c/li\u003e\n\u003cli\u003eHughes, A. M. (1961). \u003cem\u003eThe mites of stored food.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eHughes, A. M. (1976). \u003cem\u003eThe mites of stored food and houses.\u003c/em\u003e (Issue No. 9, Ed. 2).\u003c/li\u003e\n\u003cli\u003eKlimov, P. B., \u0026amp; OConnor, B. M. (2008). Origin and higher-level relationships of psoroptidian mites (Acari: Astigmata: Psoroptidia): Evidence from three nuclear genes. \u003cem\u003eMolecular Phylogenetics and Evolution\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(3), 1135\u0026ndash;1156. https://doi.org/10.1016/j.ympev.2007.12.025\u003c/li\u003e\n\u003cli\u003eMangoba, M. A. A., \u0026amp; Alvindia, D. D. G. (2020). Mititoxic properties of \u003cem\u003eCucurma longa\u003c/em\u003e against a major contributor of oral mite anaphylaxis, the grain mite \u003cem\u003eSuidasia pontifica\u003c/em\u003e. \u003cem\u003eInternational Journal of Acarology\u003c/em\u003e, \u003cem\u003e46\u003c/em\u003e(5), 313\u0026ndash;317. https://doi.org/10.1080/01647954.2020.1802509\u003c/li\u003e\n\u003cli\u003eMariana, A., Ho, T., Gendeh, B., Iskandar, H., \u0026amp; Zainuldin-Taib, M. (2000). 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Correlation of Blomia tropicalis-specific immunoglobulin epsilon profiles with family history of atopy in a Filipino population. \u003cem\u003eAsia Pacific Allergy\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(1), 12\u0026ndash;20. https://doi.org/10.5415/apallergy.0000000000000133\u003c/li\u003e\n\u003cli\u003ePuerta, L., Lagares, A., Mercado, D., Fern\u0026aacute;ndez‐Caldas, E., \u0026amp; Caraballo, L. (2005). Allergenic composition of the mite \u003cem\u003eSuidasia medanensis\u003c/em\u003e and cross‐reactivity with \u003cem\u003eBlomia tropicalis\u003c/em\u003e. \u003cem\u003eAllergy\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e(1), 41\u0026ndash;47. https://doi.org/10.1111/j.1398-9995.2004.00636.x\u003c/li\u003e\n\u003cli\u003eReginald, K., Gao, Y., Sew, Y., Shang, H., \u0026amp; Chew, F. (2004). Cross comparison of the IgE binding profiles to recombinant allergens from Suidasia medanensis, Blomia tropicalis and Dermatophagoides farinae using sera from Blomia-and Dermatophagoides-predominant environments. \u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e, \u003cem\u003e113\u003c/em\u003e(2), S228\u0026ndash;S229.\u003c/li\u003e\n\u003cli\u003eSalo, P. M., Arbes, S. J., Jaramillo, R., Calatroni, A., Weir, C. H., Sever, M. L., Hoppin, J. A., Rose, K. M., Liu, A. H., Gergen, P. J., Mitchell, H. E., \u0026amp; Zeldin, D. C. (2014). Prevalence of allergic sensitization in the United States: Results from the National Health and Nutrition Examination Survey (NHANES) 2005-2006. \u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e, \u003cem\u003e134\u003c/em\u003e(2), 350\u0026ndash;359. https://doi.org/10.1016/j.jaci.2013.12.1071\u003c/li\u003e\n\u003cli\u003eTham, E., Lee, A. J., \u0026amp; Bever, H. (2017). 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Tree Visualization By One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic acids research, 51(W1), W587-W592.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Suidasia pontifica, allergy, IgE reactivity, house dust mite, DNA barcoding, phylogenetics","lastPublishedDoi":"10.21203/rs.3.rs-7056104/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7056104/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe clinical significance of house dust mites (HDMs) as sources of allergens for medical diagnostics, allergen-specific immunotherapy, and allergology research is dependent on accurate morphological and molecular data analysis for species identification and characterization. Here, we report the species identification and allergenicity of a tropical HDM, \u003cem\u003eSuidasia pontifica\u003c/em\u003e (\u003cem\u003eSp\u003c/em\u003e), via morphological-molecular characterization tandem and IgE ELISA, respectively. Electron microscopy of monocultures of HDM samples collected from Laguna, Philippines, revealed five different traits related to chaetotaxy and body anatomy, such as the presence of scapular setae, cuticle patterns, vertical setae, ω\u003csup\u003e1\u003c/sup\u003e setae, and anal suckers, suggesting that the sample identity is \u003cem\u003eSp\u003c/em\u003e. In addition, PCR amplification from the HDM monoculture genomic DNA and bidirectional sequencing of the 18S and COI gene markers were performed, which were subjected to sequence assembly, consensus acquisition, sequence alignment, and phylogenetic inference. The COI gene showed an exact match and phylogenetic attachment of the sample assembly to \u003cem\u003eSp\u003c/em\u003e, confirming the species-level identification, corroborated with morphological data. Furthermore, 18S gene character analysis was able to prove phylogenetic demarcation of the \u003cem\u003eSp\u003c/em\u003e 18S gene from the sister species \u003cem\u003eS. nesbitti\u003c/em\u003e and other allergenic sarcoptiforman species. Our molecular phylogenetic analysis, which is strongly supported by electron microscopy data, indicates the identity of our monocultures as \u003cem\u003eSp\u003c/em\u003e. Interestingly, the \u003cem\u003eSp\u003c/em\u003e allergenicity profile of allergic patients and controls (n\u0026thinsp;=\u0026thinsp;200) suggests 47% IgE-binding reactivity, confirming its allergenicity and clinical importance among atopic patients. This study emphasizes the resolving power of the morphological-molecular phylogenetic approach and IgE-reactivity to objectively verify \u003cem\u003eSp\u003c/em\u003e species identity and allergenicity for downstream immunological studies.\u003c/p\u003e","manuscriptTitle":"Electron Microscopy and Molecular Phylogenetic Characterization of the Allergenic Dust Mite Species Suidasia pontifica (Acari: Suidasiidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 13:03:11","doi":"10.21203/rs.3.rs-7056104/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9374937-b5d4-4342-b195-e4b9d9724595","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:11:36+00:00","versionOfRecord":{"articleIdentity":"rs-7056104","link":"https://doi.org/10.1007/s10493-025-01106-7","journal":{"identity":"experimental-and-applied-acarology","isVorOnly":false,"title":"Experimental and Applied Acarology"},"publishedOn":"2026-01-21 15:57:50","publishedOnDateReadable":"January 21st, 2026"},"versionCreatedAt":"2025-07-18 13:03:11","video":"","vorDoi":"10.1007/s10493-025-01106-7","vorDoiUrl":"https://doi.org/10.1007/s10493-025-01106-7","workflowStages":[]},"version":"v1","identity":"rs-7056104","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7056104","identity":"rs-7056104","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}