Usefulness of COI gene based molecular method for identification of field collected maggots, Puducherry, South India

Usefulness of COI gene based molecular method for identification of field collected maggots, Puducherry, South India | 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 Usefulness of COI gene based molecular method for identification of field collected maggots, Puducherry, South India Sudha Bhuvaneshwaran, Visa Shalini Padmanaban, Ranjana Devi Radja, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4416407/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 May, 2025 Read the published version in Frontiers in Insect Science → Version 1 posted You are reading this latest preprint version Abstract Flies and maggots are of medical importance, and it is often necessary to identify them at species level. Conventionally, this is carried out based on morphological features using taxonomic keys. However, identification of maggots based on morphology is difficult and required entomological expertise is often lacking in many clinical settings. Molecular methods can be an alternative to morphology based identification. We explored the utility of mitochondrial COI gene based molecular method, as a tool for identifying field collected maggots in Puducherry, India. Maggots were collected from different locations in Puducherry using rotten fish and kitchen waste as baits and a segment of the COI gene was amplified and phylogenetic analysis was performed. The identified maggots belonged to Sarcophaga peregrina , Hemipyrellia ligurriens and Chrysomya megacephala . We highlight the usefulness of molecular methods in precise identification of maggots in certain settings is highlighted. Maggots Mitochondrial cytochrome oxidase subunit 1 (COI) DNA barcoding Phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Flies are of great ecological significance as several species perform environmental services as pollinators and scavengers. They act as predators of agricultural pests in their larval forms, maggots. Several species of fruit flies act as pests of horticulture crops. The adult flies also transmit a variety of deadly diseases to animals and humans as mechanical and obligatory vectors. Maggots, the larva of some fly species are also of medical importance. Maggots of flesh flies and blue bottle flies found on dead bodies have been used for estimation of post-mortem interval (PMI) (Shang et al. 2019). Occasionally, maggots infect live humans and animals causing myiasis, which can be facultative or obligatory (Francesconi and Lupi 2012). On the contrary, maggots of certain flies are utilized for the maggot debridement therapy where sterile larvae are applied to clean up leg ulcers, pressure sores and infected surgical wounds (Sherman et al. 2001). Because of their importance to humans in several ways, accurate identification of flies and maggots is important and often required. Conventionally, this is carried out based on morphological features using taxonomic keys. Species identification is challenging when specimens are damaged while collection or transportation or without proper preservation and when the morphological features are lacking in such specimens. Also, unlike adult flies, species identification of maggots is difficult as the larvae of different flies, especially the first instar stage appears similar. Entomological expertise is required and qualified professionals are often lacking in many settings. We recently came across a case of extensive myiasis of the leg in a patient with filariasis related elephantiasis where we could not identify the larva because of the practical difficulties in collection of the maggots on his first visit to the Filariasis Management Clinic at our centre, and later he did not turn up for follow up (Kuttiatt et al. 2021). This is not an isolated scenario, and in many published reports on myiasis worldwide, larval species identification is missing, often due to lack of entomological expertise in clinical settings. Molecular techniques offer the best alternative in this scenario (Otranto and Stevens 2002;Meiklejohn et al. 2013). Molecular barcoding-based identification has been applied for animals including insects, however, studies on flies have been limited, globally and more so in the Indian settings (Smith and Baker 2008, Archana et al. 2016, Yussef-Vanegas and Agnarsson 2017;Sontigun et al. 2018). This prodded us to carry out this preliminary study in Puducherry to apply cytochrome C oxidase subunit I (COI) gene based molecular analysis as an alternative to morphological identification of maggots. Since we don’t come across patients with myiasis so frequently in our clinic, we resorted to a proxy method for convenience and ease, where we utilized maggots of necrophagous flies collected from the field instead of that from patients. This study was conducted in Puducherry, a union territory on the eastern coast in South India. We collected one set of maggots each, from four different locations in Puducherry namely Vanarapet, Indira Nagar, Mudaliarpet and Moolakulam, utilizing rotten fish and kitchen waste as baits. The maggots were reared to adult flies under laboratory condition by previously described methods (Frings 1941). Morphological evaluation was performed using the published taxonomic keys (Dodge 1953). Maggots and flies were processed individually for isolation of the mitochondrial DNA and a partial 710 bp segment of the COI gene was amplified using a set of universal primers as previously described (Tamura and Aotsuka 1988;Folmer et al. 1994). The amplicons were subjected to bidirectional cycle sequencing reaction using BigDye™ Terminator v3.1 cycle sequencing kit and capillary electrophoresis was performed on a 3130XL Genetic Analyser (Applied Biosystems, USA). The forward and reverse sequences were edited and trimmed using BioEdit version 7.7.1 software and consensus sequences generated. High quality sequences were available for 11 specimens and were subjected to BLAST analysis to identify matches from the database for identification of the species. Two sequences belonged to Chrysomya megacephala (VCRC IM3, IM5), three were identified as Hemipyrellia ligurriens (VCRC IFOG2A, IFOG3AF, IIF4), and six were Sarcophaga peregrina (IFOG4A, IRF3, IIRF4, F1IRM1F, IIRM2, IFOGM1). The COI nucleotide sequences generated from the maggots and the corresponding emerged adults were found to be matching completely. The maggots collected from Vanarapet belonged to Sarcophaga peregrina , and the ones from Indira Nagar were Hemipyrellia ligurriens , the ones from Mudaliarpet and Moolakulam were Chrysomya megacephala . Newly generated sequences have been submitted to GenBank (PP593642 to PP593652). For comparative analysis, COI gene sequences of each of these species available in GenBank from India and other countries were downloaded. There was a total of 31 COI sequences of C. megacephala , 16 of H. ligurriens and 19 sequences of S. peregrina available in GenBank. Among them, 27 sequences of C. megacephala , 15 of H. ligurriens and 18 of S. peregrina were selected for further analysis with newly generated sequences, as other sequences were of length shorter than 500 base pairs. The multiple alignment was carried out using the ClustalW Multiple alignment program. MEGA 11 was used for molecular evolutionary analysis using the Neighbor-Joining (NJ) approach (Saitou and Nei 1987;Tamura et al. 2021). Using the Kimura 2-parameter (K2P) method (Kimura 1980), pairwise nucleotide sequence divergences and mean evolutionary distances were calculated. Using 1000 bootstrap replicates, bootstrap support values (BSV) were calculated to quantify the statistical support for the clusters identified in the NJ tree. Initially, we carried out a phylogenetic analysis of the newly generated 11 sequences with one representative Indian sequence each of C. megacephala, H. ligurriens and S . peregrina selected from GenBank ( Fig.1 ). An Indian sequence of M. domestica is selected as an out-group. The identified species are shown in the phylogenetic tree ( Fig.1 ), each independently clustered with their matching species.Then we carried outphylogenetic analysis separately for the three species with sequences from India and other countries. Both C. megacephala sequencesexhibited a strong clustering pattern and strong associations with every sequence analysed ( Fig.2). In BLAST analysis two sequences of C. megacephala showed a high similarity of over 97% with all 24 sequences from GenBank. However, there was a noticeable divergence of over 3% with sequences from Punjab and Uttar Pradesh (DQ119584.1, FJ842472.1, and GQ466584.1). In case of H. ligurriens , the three newly generated sequences from Puducherry showed a very close match of over 99% with the 13 sequences selected from GenBank. However, their resemblance to sequences from Taiwan and USA were much less ranging from 92% to 94%, placing them in a different grouping. Close relatedness was noted between a sequence from Bangalore and VCRC-IIF4 ( Fig.3 ). Six S. peregrina sequences from this study showed 97% similarity with selected 15 GenBank sequences whereas more than 3% divergence observed with sequences from Punjab, Thailand and China (JX507334.1, MH686501.2, and KC966393.1). Six sequences from Puducherry and those from Maharashtra and Karnataka formed one cluster ( Fig.4 ). A previous survey on filth flies conducted in Puducherry has identified presence of several different species- Musca domestica, Stomoxys calcitrans, Musca sorbens, Calliphora sp, Fannia cannicularis, Ophyra sp, Sarcophaga sp and Hippelates sp in Puducherry (Velayudham et al. 2016). To our knowledge, no molecular study has been published on flies or maggots in Puducherry. A study undertaken to barcode five fly species prevalent in poultry farms in and around Bengaluru district in Karnataka state has identified 5 species which include Musca domestica, Chrysomya megacephala, Hydrotaea capensis, Hermetia illucens and Sarcophaga ruficornis ( Archana et al . 2016). Two studies have been published on molecular barcoding of forensically important flies in India, one from Maharashtra and the other from North India (Himachal Pradesh, Punjab, Haryana and Uttarakhand) (Abd-Algalil and Zambare 2017;Khullar 2016). Among the different flies identified in the current study, the presence of Sarcophaga peregrina, the flesh fly has been recorded throughout India. However, there are only a limited number of gene sequences of this species from India in the public data base. It has been used for PMI estimation and is also known to cause myiasis in both humans and animals (Chigusa et al. 2005;Shang et al. 2019). There is limited published data on Hemipyrrelia ligurriens from India and only a handful of gene sequences from the country are available in the public data base (Sinha and Nandi 2007). This fly is also of forensic significance and myiasis as H. ligurriens has also been reported in animals (Sinha 2012;Sontigun et al. 2018). Chrysomya megacephala, commonlyknown asthe oriental latrine fly is of forensic importance and is also known to cause accidental myiasis in open wounds (Ramaraj et al. 2014;Sangmala et al. 2021). There are several partial COI gene sequences of this fly species from different regions of India in the public database, but not from Puducherry. Molecular methods may find application especially for larval identification in myiasis and for post mortem interval estimation in forensic investigations. Apart from the convenience of specimen storage and analysis at a later point of time without the need of morphological features, molecular methods have special application in epidemiological investigations and evolutionary studies. Molecular epidemiological studies may be especially relevant in the context of the recent reports on expansion of the myiasis causing fly Chrysomya bezziana into newer geographical regions with increased cattle trade, and other reasons like global warming, environmental and climate changes as highlighted and predicted by a modelling study (Hosni et al. 2020). Molecular genetic analysis would be useful in ascertaining the genetic relatedness between strains from different geographical regions. However, molecular methods are not without limitations. This also requires technical expertise and specialized instruments and is costly, but is being rapidly made available in routine settings as well. In conclusion, we found COI based molecular method as a versatile tool for identification of maggots and this need to be promoted in clinical and forensic settings in India. Additionally, we generated molecular sequence data for two less studied fly species of medical importance, S. peregrina and H. ligurriens from South India. In future, there is a need for further detailed molecular studies on flies in the diverse epidemiological and geographic settings in India. Declarations Acknowledgment The authors thank Mr A Mathivanan, Technical Officer, ICMR-VCRC for his assistance in sequencing. Conflict of ineterst None declared References Abd-Algalil FM, Zambare SP (2017) Molecular identification of forensically important flesh flies (Diptera: Sarcophagidae) using COI Gene. J Entomol Zool Stud 5(2):263–267 Archana M, D’Souza PE, Ojha R, Jalali SK (2016) DNA barcoding of flies commonly prevalent in poultry farms of Bengaluru District. J Entomol Zool Stud 4(4):228–233 Chigusa Y, Kirinoki M, Matsuda H (2005) Nosocomial myiasis due to Sarcophaga peregrina in an intensive care unit (ICU) in Japan. 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Int J Curr Res 8(03):27256–27262 Yussef-Vanegas SZ, Agnarsson I (2017) DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region. PeerJ 5:e3516. https://doi.org/10.7717/peerj.3516 Cite Share Download PDF Status: Published Journal Publication published 13 May, 2025 Read the published version in Frontiers in Insect Science → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4416407","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":302892774,"identity":"821b3d3a-8697-401f-8959-65f5bd3a7049","order_by":0,"name":"Sudha Bhuvaneshwaran","email":"","orcid":"","institution":"ICMR-Vector Control Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Sudha","middleName":"","lastName":"Bhuvaneshwaran","suffix":""},{"id":302892775,"identity":"825699ab-2236-4883-ab29-18ac631870e7","order_by":1,"name":"Visa Shalini Padmanaban","email":"","orcid":"","institution":"ICMR-Vector Control Research 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of the three species—\u003cem\u003eC. megacephala\u003c/em\u003e, \u003cem\u003eH. ligurriens \u003c/em\u003eand\u003cem\u003e S. peregrina\u003c/em\u003e.\u003cem\u003e An M. domestica\u003c/em\u003e species selected as an out-group.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4416407/v1/164e41beadf029ece391dc57.png"},{"id":57063794,"identity":"ca2340e2-cf1c-4fba-8b32-19697c595677","added_by":"auto","created_at":"2024-05-24 06:45:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17912,"visible":true,"origin":"","legend":"\u003cp\u003eBootstrapped Neighbour-joining tree of 27 COI sequences from \u003cem\u003eC. megacephala\u003c/em\u003e belonging to India and other countries compared with 2 COI sequences of \u003cem\u003eC. megacephala \u003c/em\u003eof this study. Bootstrap values less than 50% are not shown. COI sequence cytochrome c oxidase subunit I of an \u003cem\u003eM. domestica \u003c/em\u003especies is selected as shown as an outgroup.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4416407/v1/16c83512e3c997f5508d61d2.png"},{"id":57063796,"identity":"3cd19249-9343-44bc-a228-9efcfaf33219","added_by":"auto","created_at":"2024-05-24 06:45:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9070,"visible":true,"origin":"","legend":"\u003cp\u003e15 COI sequences from \u003cem\u003eH. ligurriens\u003c/em\u003e that are from India and other countries compared with 3 COI sequences of this study are shown in a bootstrapped neighbour-joining tree. Values of the bootstrap less than 50% are not displayed. \u003cem\u003eM. domestica\u003c/em\u003e is chosen as an outgroup.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4416407/v1/b464fdacc4a51d5acec7a974.png"},{"id":57064556,"identity":"ca6f3863-d83e-4dc7-a99f-b0898e9e6dd6","added_by":"auto","created_at":"2024-05-24 06:53:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12836,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbour-Joining tree bootstrapped using 18 COI sequences from \u003cem\u003eS. peregrin\u003c/em\u003ea that are from India and other countries compared with 6 COI sequences of \u003cem\u003eS. peregrina\u003c/em\u003e of this study. Less than 50% of the bootstrap value is not shown. \u003cem\u003eM. domestica\u003c/em\u003e species is chosen as an outgroup.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4416407/v1/e2a607c89fc17ab2abe4b4cf.png"},{"id":83152395,"identity":"7029b455-9398-4a63-848c-7ffb192af0fb","added_by":"auto","created_at":"2025-05-20 14:05:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":489753,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4416407/v1/7a2ac4c2-4a49-428d-8ace-784a644d2b14.pdf"}],"financialInterests":"","formattedTitle":"Usefulness of COI gene based molecular method for identification of field collected maggots, Puducherry, South India","fulltext":[{"header":"Full Text","content":"\u003cp\u003eFlies are of great ecological significance as several species perform environmental services as pollinators and scavengers. They act as predators of agricultural pests in their larval forms, maggots. Several species of fruit flies act as pests of horticulture crops. The adult flies also transmit a variety of deadly diseases to animals and humans as mechanical and obligatory vectors. Maggots, the larva of some fly species are also of medical importance. Maggots of flesh flies and blue bottle flies found on dead bodies have been used for estimation of post-mortem interval (PMI) (Shang et al. 2019). Occasionally, maggots infect live humans and animals causing myiasis, which can be facultative or obligatory (Francesconi and Lupi 2012). On the contrary, maggots of certain flies are utilized for the maggot debridement therapy where sterile larvae are applied to clean up leg ulcers, pressure sores and infected surgical wounds (Sherman et al. 2001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause of their importance to humans in several ways, accurate identification of flies and maggots is important and often required. Conventionally, this is carried out based on morphological features using taxonomic keys. Species identification is challenging when specimens are damaged while collection or transportation or without proper preservation and when the morphological features are lacking in such specimens. Also, unlike adult flies, species identification of maggots is difficult as the larvae of different flies, especially the first instar stage appears similar. Entomological expertise is required and qualified professionals are often lacking in many settings. We recently came across a case of extensive myiasis of the leg in a patient with filariasis related elephantiasis where we could not identify the larva because of the practical difficulties in collection of the maggots on his first visit to the Filariasis Management Clinic at our centre, and later he did not turn up for follow up (Kuttiatt et al. 2021).\u0026nbsp;This is not an isolated scenario, and in many published reports on myiasis worldwide, larval species identification is missing, often due to lack of entomological expertise in clinical settings. Molecular techniques offer the best alternative in this scenario (Otranto and Stevens 2002;Meiklejohn et al. 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMolecular barcoding-based identification has been applied for animals including insects, however, studies on flies have been limited, globally and more so in the Indian settings (Smith and Baker 2008, Archana et al. 2016, Yussef-Vanegas and Agnarsson 2017;Sontigun et al. 2018). This prodded us to carry out this preliminary study in Puducherry to apply cytochrome C oxidase subunit I (COI) gene based molecular analysis as an alternative to morphological identification of maggots. Since we don\u0026rsquo;t come across patients with myiasis so frequently in our clinic, we resorted to a proxy method for convenience and ease, where we utilized maggots of necrophagous flies collected from the field instead of that from patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted in Puducherry, a union territory on the eastern coast in South India. We collected one set of maggots each, from four different locations in Puducherry namely Vanarapet, Indira Nagar, Mudaliarpet and Moolakulam, utilizing rotten fish and kitchen waste as baits. The maggots were reared to adult flies under laboratory condition by previously described methods (Frings 1941). Morphological evaluation was performed using the published taxonomic keys (Dodge 1953). Maggots and flies were processed individually for isolation of the mitochondrial DNA and a partial 710 bp segment of the COI gene was amplified using a set of universal primers as previously described (Tamura and Aotsuka 1988;Folmer et al. 1994). The amplicons were subjected to bidirectional cycle sequencing reaction using BigDye\u0026trade; Terminator v3.1 cycle sequencing kit and capillary electrophoresis was performed on a 3130XL Genetic Analyser (Applied Biosystems, USA). \u0026nbsp;The forward and reverse sequences were edited and trimmed using BioEdit version 7.7.1 software and consensus sequences generated. High quality sequences were available for 11 specimens and were subjected to BLAST analysis to identify matches from the database for identification of the species. Two sequences belonged to\u003cem\u003e\u0026nbsp;Chrysomya megacephala\u003c/em\u003e (VCRC IM3, IM5), three were identified as\u0026nbsp;\u003cem\u003eHemipyrellia ligurriens\u003c/em\u003e (VCRC IFOG2A, IFOG3AF, IIF4), and six were\u0026nbsp;\u003cem\u003eSarcophaga peregrina\u003c/em\u003e (IFOG4A, IRF3, IIRF4, F1IRM1F, IIRM2, IFOGM1). The COI nucleotide sequences generated from the maggots and the corresponding emerged adults were found to be matching completely. The maggots collected from Vanarapet belonged to \u003cem\u003eSarcophaga peregrina\u003c/em\u003e, and the ones from Indira Nagar were \u003cem\u003eHemipyrellia ligurriens\u003c/em\u003e, the ones from Mudaliarpet and Moolakulam were \u003cem\u003eChrysomya megacephala\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNewly generated sequences have been submitted to GenBank (PP593642 to PP593652). For comparative analysis, COI gene sequences of each of these species available in GenBank from India and other countries were downloaded. There was a total of 31 COI sequences of\u0026nbsp;\u003cem\u003eC. megacephala\u003c/em\u003e, 16 of\u0026nbsp;\u003cem\u003eH. ligurriens\u0026nbsp;\u003c/em\u003eand 19 sequences of\u0026nbsp;\u003cem\u003eS. peregrina\u003c/em\u003e available in GenBank. Among them, 27 sequences of\u0026nbsp;\u003cem\u003eC. megacephala\u003c/em\u003e, 15 of\u0026nbsp;\u003cem\u003eH. ligurriens\u003c/em\u003e and 18 of\u0026nbsp;\u003cem\u003eS. peregrina\u0026nbsp;\u003c/em\u003ewere selected for further analysis with newly generated sequences, as other sequences were of length shorter than 500 base pairs.\u0026nbsp;The multiple alignment was carried out using the ClustalW Multiple alignment program. MEGA 11 was used for molecular evolutionary analysis using the Neighbor-Joining (NJ) approach (Saitou and Nei 1987;Tamura et al. 2021). Using the Kimura 2-parameter (K2P) method (Kimura 1980), pairwise nucleotide sequence divergences and mean evolutionary distances were calculated. Using 1000 bootstrap replicates, bootstrap support values (BSV) were calculated to quantify the statistical support for the clusters identified in the NJ tree. Initially, we carried out a phylogenetic analysis of the newly generated 11 sequences with one representative Indian sequence each of \u003cem\u003eC. megacephala, H. ligurriens\u0026nbsp;\u003c/em\u003eand S\u003cem\u003e. peregrina\u0026nbsp;\u003c/em\u003eselected from GenBank (\u003cstrong\u003eFig.1\u003c/strong\u003e). An Indian sequence of \u003cem\u003eM. domestica\u003c/em\u003e is selected as an out-group. The identified species are shown in the phylogenetic tree (\u003cstrong\u003eFig.1\u003c/strong\u003e), each independently clustered with their matching species.Then we carried outphylogenetic analysis separately for the three species with sequences from India and other countries. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth \u003cem\u003eC. megacephala\u0026nbsp;\u003c/em\u003esequencesexhibited a strong clustering pattern and strong associations with every sequence analysed (\u003cstrong\u003eFig.2).\u003c/strong\u003e In BLAST analysis two sequences of \u003cem\u003eC. megacephala\u003c/em\u003e showed a high similarity of over 97% with all 24 sequences from GenBank. However, there was a noticeable divergence of over 3% with sequences from Punjab and Uttar Pradesh (DQ119584.1, FJ842472.1, and GQ466584.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn case of \u003cem\u003eH. ligurriens\u003c/em\u003e, the three newly generated sequences from Puducherry showed a very close match of over 99% with the 13 sequences selected from GenBank. However, their resemblance to sequences from Taiwan and USA were much less ranging from 92% to 94%, placing them in a different grouping. Close relatedness was noted between a sequence from Bangalore and VCRC-IIF4 (\u003cstrong\u003eFig.3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eSix \u003cem\u003eS. peregrina\u003c/em\u003e sequences from this study showed 97% similarity with selected 15 GenBank sequences whereas more than 3% divergence observed with sequences from Punjab, Thailand and China (JX507334.1, MH686501.2, and KC966393.1). Six sequences from Puducherry and those from Maharashtra and Karnataka formed one cluster (\u003cstrong\u003eFig.4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eA previous survey on filth flies conducted in Puducherry has identified presence of several different species- \u003cem\u003eMusca domestica, Stomoxys calcitrans, Musca sorbens, Calliphora sp, Fannia cannicularis, Ophyra sp, Sarcophaga sp and Hippelates sp\u0026nbsp;\u003c/em\u003ein Puducherry (Velayudham et al. 2016). To our knowledge, no molecular study has been published on flies or maggots in Puducherry. A study undertaken to barcode five fly species prevalent in poultry farms in and around Bengaluru district in Karnataka state has identified 5 species which include \u003cem\u003eMusca domestica, Chrysomya megacephala, Hydrotaea capensis, Hermetia illucens and Sarcophaga ruficornis (\u003c/em\u003eArchana et al\u003cem\u003e.\u0026nbsp;\u003c/em\u003e2016). Two studies have been published on molecular barcoding of forensically important flies in India, one from Maharashtra and the other from North India (Himachal Pradesh, Punjab, Haryana and Uttarakhand) (Abd-Algalil and Zambare 2017;Khullar 2016).\u003c/p\u003e\n\u003cp\u003eAmong the different flies identified in the current study, the presence of\u003cem\u003e\u0026nbsp;Sarcophaga peregrina,\u0026nbsp;\u003c/em\u003ethe flesh fly has been recorded throughout India. However, there are only a limited number of gene sequences of this species from India in the public data base. It has been used for PMI estimation and is also known to cause myiasis in both humans and animals (Chigusa et al. 2005;Shang et al. 2019). There is limited published data on \u003cem\u003eHemipyrrelia ligurriens\u003c/em\u003e from India and only a handful of gene sequences from the country are available in the public data base (Sinha and Nandi 2007). This fly is also of forensic significance and myiasis as \u003cem\u003eH. ligurriens\u0026nbsp;\u003c/em\u003ehas also been reported in animals (Sinha 2012;Sontigun et al. 2018). \u003cem\u003eChrysomya megacephala,\u0026nbsp;\u003c/em\u003ecommonlyknown asthe oriental latrine fly is of forensic importance and is also known to cause accidental myiasis in open wounds (Ramaraj et al. 2014;Sangmala et al. 2021). There are several partial COI gene sequences of this fly species from different regions of India in the public database, but not from Puducherry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMolecular methods may find application especially for larval identification in myiasis and for post mortem interval estimation in forensic investigations. Apart from the convenience of specimen storage and analysis at a later point of time without the need of morphological features, molecular methods have special application in epidemiological investigations and evolutionary studies. Molecular epidemiological studies may be especially relevant in the context of the recent reports on expansion of the myiasis causing fly \u003cem\u003eChrysomya bezziana into\u003c/em\u003e newer geographical regions with increased cattle trade, and other reasons like global warming, environmental and climate changes as highlighted and predicted by a modelling study (Hosni et al. 2020). Molecular genetic analysis would be useful in ascertaining the genetic relatedness between strains from different geographical regions. However, molecular methods are not without limitations. This also requires technical expertise and specialized instruments and is costly, but is being rapidly made available in routine settings as well.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, we found COI based molecular method as a versatile tool for identification of maggots and this need to be promoted in clinical and forensic settings in India. Additionally, we generated molecular sequence data for two less studied fly species of medical importance, \u003cem\u003eS. peregrina and H. ligurriens\u003c/em\u003e from South India. In future, there is a need for further detailed molecular studies on flies in the diverse epidemiological and geographic settings in India. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Mr A Mathivanan, Technical Officer, ICMR-VCRC for his assistance in sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of ineterst\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd-Algalil FM, Zambare SP (2017) Molecular identification of forensically important flesh flies (Diptera: Sarcophagidae) using COI Gene. 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Int J Curr Res 8(03):27256\u0026ndash;27262\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYussef-Vanegas SZ, Agnarsson I (2017) DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region. PeerJ 5:e3516. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.3516\u003c/span\u003e\u003cspan address=\"10.7717/peerj.3516\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Maggots, Mitochondrial cytochrome oxidase subunit 1 (COI), DNA barcoding, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-4416407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4416407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlies and maggots are of medical importance, and it is often necessary to identify them at species level. Conventionally, this is carried out based on morphological features using taxonomic keys. However, identification of maggots based on morphology is difficult and required entomological expertise is often lacking in many clinical settings. Molecular methods can be an alternative to morphology based identification. We explored the utility of mitochondrial COI gene based molecular method, as a tool for identifying field collected maggots in Puducherry, India. Maggots were collected from different locations in Puducherry using rotten fish and kitchen waste as baits and a segment of the COI gene was amplified and phylogenetic analysis was performed. The identified maggots belonged to \u003cem\u003eSarcophaga peregrina\u003c/em\u003e, \u003cem\u003eHemipyrellia ligurriens\u003c/em\u003eand \u003cem\u003eChrysomya megacephala\u003c/em\u003e. We highlight the usefulness of molecular methods in precise identification of maggots in certain settings is highlighted.\u003c/p\u003e","manuscriptTitle":"Usefulness of COI gene based molecular method for identification of field collected maggots, Puducherry, South India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-24 06:45:47","doi":"10.21203/rs.3.rs-4416407/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":"3a18156d-f457-434c-a9cf-919a67a0c80e","owner":[],"postedDate":"May 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-20T14:05:19+00:00","versionOfRecord":{"articleIdentity":"rs-4416407","link":"https://doi.org/10.3389/finsc.2025.1551807","journal":{"identity":"frontiers-in-insect-science","isVorOnly":true,"title":"Frontiers in Insect Science"},"publishedOn":"2025-05-14 00:00:00","publishedOnDateReadable":"May 14th, 2025"},"versionCreatedAt":"2024-05-24 06:45:47","video":"","vorDoi":"10.3389/finsc.2025.1551807","vorDoiUrl":"https://doi.org/10.3389/finsc.2025.1551807","workflowStages":[]},"version":"v1","identity":"rs-4416407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4416407","identity":"rs-4416407","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}