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The present study aimed to evaluate the essential oil (EO) of Piper hemmendorffii and its major constituent, limonene, for their activity against R. microplus larvae. The EO of P. hemmendorffii and its major compound, limonene, were evaluated against pyrethroid-resistant R. microplus larvae using larval immersion bioassays, biochemical analyses of detoxification enzymes and acetylcholinesterase, and scanning electron microscopy (SEM). The EO showed higher larvicidal potency (LC50 = 10.46 mg\/mL; 95% CI: 9.86\u201311.09) compared to limonene (LC50 = 31.62 mg\/mL; 95% CI: 30.86\u201332.40). Piper hemmendorffii EO affected the following classes of enzymes investigated: \u03b1-Esterase (\u03b1-EST) activity increased at LC25 and LC50, reaching its highest level at LC50, whereas \u03b2-esterase (\u03b2-EST) activity increased only at LC25. Cytochrome P450 (CYP450)-associated heme content activity showed a significant increase exclusively at LC50, suggesting a response of the surviving larvae to EO exposure, although this increase does not by itself demonstrate the activation of detoxification pathways. In contrast, limonene did not significantly affect the activity of the three evaluated enzymes. The inhibition of AChE by the EO suggests a possible effect on the cholinergic system, which may be associated with neurotoxic activity, whereas limonene had no significant effect. The absence of detectable ultrastructural changes by SEM suggests that treatment with either EO or limonene did not induce evident external morphological damage to the larval cuticle under the evaluated conditions. EO toxicity seems to be associated with changes in detoxification enzyme and AChE activities, suggesting the involvement of these biochemical pathways in larval response.<\/jats:p>","DOI":"10.3390\/pathogens15080862","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T09:30:53Z","timestamp":1787131853000},"page":"862","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Effects of Piper hemmendorffii (Piperales: Piperaceae) Essential Oil and Limonene on Modulation of Phase I Detoxifying Enzymes and Acetylcholinesterase Activity of Rhipicephalus microplus (Acari: Ixodidae)"],"prefix":"10.3390","volume":"15","author":[{"given":"Adalberto Alves","family":"Pereira Filho","sequence":"first","affiliation":[{"name":"Laborat\u00f3rio de Artr\u00f3podes Hemat\u00f3fagos, Departamento de Parasitologia\/ICB, Universidade Federal de Minas Gerais, Av. Ant\u00f4nio Carlos 6627, Pampulha, Belo Horizonte 31270-901, MG, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6505-5999","authenticated-orcid":false,"given":"Vladimir Fazito do","family":"Vale","sequence":"additional","affiliation":[{"name":"Grupo de Pesquisa em Triatom\u00edneos, Instituto Ren\u00e9 Rachou, Fiocruz, Belo Horizonte 30190-009, MG, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5253-5451","authenticated-orcid":false,"given":"Caio Marcio de Oliveira","family":"Monteiro","sequence":"additional","affiliation":[{"name":"Instituto de Patologia Tropical e Sa\u00fade P\u00fablica, Universidade Federal de Goi\u00e1s, Rua 235, s\/n, Setor Leste Universit\u00e1rio, Goi\u00e2nia 74605-050, GO, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0340-6666","authenticated-orcid":false,"given":"Mayara Mac\u00eado","family":"Barrozo","sequence":"additional","affiliation":[{"name":"Instituto de Patologia Tropical e Sa\u00fade P\u00fablica, Universidade Federal de Goi\u00e1s, Rua 235, s\/n, Setor Leste Universit\u00e1rio, Goi\u00e2nia 74605-050, GO, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel Sobreira","family":"Rodrigues","sequence":"additional","affiliation":[{"name":"Empresa de Pesquisa Agropecu\u00e1ria de Minas Gerais, EPAMIG Campo Experimental Santa Rita, Prudente de Morais 34701-970, MG, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lydia Fumiko","family":"Yamaguchi","sequence":"additional","affiliation":[{"name":"Instituto de Pesquisas Tecnol\u00f3gicas do Estado de S\u00e3o Paulo, NUTABES, Avenida Professor Almeida Prado, 532, Butant\u00e3, S\u00e3o Paulo 05508-901, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3315-2129","authenticated-orcid":false,"given":"Massuo Jorge","family":"Kato","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Qu\u00edmica de Produtos Naturais, Departamento de Qu\u00edmica Fundamental, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Avenida Professor Lineu Prestes, 748, Butant\u00e3, S\u00e3o Paulo 05508-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1272-4386","authenticated-orcid":false,"given":"Ricardo Nascimento","family":"Araujo","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Artr\u00f3podes Hemat\u00f3fagos, Departamento de Parasitologia\/ICB, Universidade Federal de Minas Gerais, Av. Ant\u00f4nio Carlos 6627, Pampulha, Belo Horizonte 31270-901, MG, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2421","DOI":"10.1007\/s00436-020-06758-5","article-title":"Biological Parameters for Rhipicephalus microplus in the Field and Laboratory and Estimation of Its Annual Number of Generations in a Tropical Region","volume":"119","author":"Cruz","year":"2020","journal-title":"Parasitol. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1186\/s13071-024-06220-w","article-title":"Biology of the Non-Parasitic Phase of the Cattle Tick Rhipicephalus (Boophilus) microplus in an Area of Amazon Influence","volume":"17","author":"Sales","year":"2024","journal-title":"Parasit. 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