{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T18:51:27Z","timestamp":1767034287595,"version":"3.48.0"},"reference-count":46,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T00:00:00Z","timestamp":1766966400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"DOI":"10.13039\/100007917","name":"Agricultural Research Service","doi-asserted-by":"publisher","award":["2072-21000-061-000-D"],"award-info":[{"award-number":["2072-21000-061-000-D"]}],"id":[{"id":"10.13039\/100007917","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007917","name":"Agricultural Research Service","doi-asserted-by":"publisher","award":["0440860"],"award-info":[{"award-number":["0440860"]}],"id":[{"id":"10.13039\/100007917","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"\n The two-spotted spider mite (\n Tetranychus urticae<\/jats:italic>\n Koch) is a globally significant agricultural pest with high reproductive capacity, rapid development, and frequent evolution of miticide resistance. Breeding and selection of resistant host cultivars represent a promising complement to chemical control, but widespread adoption is limited primarily due to the labor-intensive nature of conventional\n in vitro<\/jats:italic>\n phenotyping methods. Here, we present a high-throughput, semi-automated image analysis pipeline integrating the Blackbird CNC Microscopy Imaging Robot with computer vision models for mite life stage identification. We developed a publicly available dataset of over 1,500 annotated images (nearly 32,000 labeled instances) spanning five biologically relevant classes across 10 host species and >25 cultivars. Three YOLO11-based object detection models (three-, four-, and five-class configurations) were trained and evaluated using real and synthetic data. The three-class model achieved the highest overall performance on the hold out test set (precision = 0.875, recall = 0.871, mAP50 = 0.883), with detection accuracy robust to host background and moderate object densities. Application to miticidal assays demonstrated reliable fecundity estimation but reduced accuracy for mortality assessment due to misclassification of dead mites. In hop cultivar assays, the pipeline detected significant differences in fecundity, aligning with manual counts (\n R<\/jats:italic>\n 2<\/jats:sup>\n \n \n \n \n \n \u2265<\/mml:mo>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n 0.98). Performance declined on hosts absent from training data and at densities exceeding\n \n \n \n \n \n ~<\/mml:mi>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n 80 objects per image, underscoring the need for host-specific fine-tuning and density-aware assay experimental design. By enabling rapid, standardized, and reproducible quantification of mite life stages, this system offers a scalable alternative to manual scoring, particularly for resistance breeding programs targeting antibiosis traits. Our approach addresses major throughput bottlenecks in\n T. urticae<\/jats:italic>\n phenotyping and establishes a framework for integrating automated imaging into broader pest management and plant breeding pipelines. Dataset, code, and trained models are publicly available to facilitate adoption and extension.\n <\/jats:p>","DOI":"10.1371\/journal.pone.0333253","type":"journal-article","created":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T18:39:47Z","timestamp":1767033587000},"page":"e0333253","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Automated detection and quantification of two-spotted spider mite life stages using computer vision for high-throughput in vitro assays"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7009-2594","authenticated-orcid":true,"given":"Michele S.","family":"Wiseman","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joanna L.","family":"Woods","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carly R.","family":"Hartgrave","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Briana J.","family":"Richardson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9529-9831","authenticated-orcid":true,"given":"David H.","family":"Gent","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,12,29]]},"reference":[{"key":"pone.0333253.ref001","doi-asserted-by":"crossref","first-page":"1206","DOI":"10.3389\/fpls.2018.01206","article-title":"The digestive system of the two-spotted spider mite, Tetranychus urticae Koch, in the context of the mite-plant interaction","volume":"9","author":"N Bensoussan","year":"2018","journal-title":"Front Plant Sci."},{"key":"pone.0333253.ref002","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/B978-0-12-802441-6.00005-X","volume-title":"Sustainable management of arthropod pests of tomato","author":"GE Brust","year":"2018"},{"issue":"7374","key":"pone.0333253.ref003","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1038\/nature10640","article-title":"The genome of Tetranychus urticae reveals herbivorous pest adaptations","volume":"479","author":"M Grbi\u0107","year":"2011","journal-title":"Nature."},{"issue":"2","key":"pone.0333253.ref004","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1093\/aesa\/69.2.362","article-title":"Biology, life table, and intrinsic rate of increase of Tetranychus urticae1, 3","volume":"69","author":"CT Shih","year":"1976","journal-title":"Ann Entomol Soc Am."},{"issue":"5","key":"pone.0333253.ref005","first-page":"35","article-title":"A review on reproductive strategies in two spotted spider mite, Tetranychus urticae Koch 1836 (Acari: Tetranychidae","volume":"2","author":"K Tehri","year":"2014","journal-title":"). 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