Sand flies infect more than 1 million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled, and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: cytochrome P450s (CYPs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs), carboxyl/cholinesterases (CCEs), and ATP-binding cassette (ABC) transporters. Using this comparative approach, we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of (i) CYP4G genes involved in cuticular hydrocarbon biosynthesis, (ii) ABCB genes involved in xenobiotic toxicity, and (iii) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and voltage gated sodium channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Department of Biology University of Crete Heraklion 71409 Greece

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Eck Institute for Global Health Department of Biological Sciences University of Notre Dame Notre Dame IN USA

Institute of Computer Science Foundation for Research and Technology Hellas Heraklion Greece

Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas Heraklion 70013 Greece

Laboratory of Agrozoology Department of Plants and Crops Faculty of Bioscience Engineering Ghent University Ghent 9000 Belgium

Pesticide Science Laboratory Department of Crop Science Agricultural University of Athens Athens 11855 Greece

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Ahn  SJ, Marygold  SJ. The UDP-glycosyltransferase family in Drosophila melanogaster: nomenclature update, gene expression and phylogenetic analysis. Front Physiol.  2021:12:648481. 10.3389/fphys.2021.648481. PubMed DOI PMC

Akhoundi  M, Kuhls  K, Cannet  A, Votýpka  J, Marty  P, Delaunay  P, Sereno  D. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis.  2016:10(3):e0004349. 10.1371/journal.pntd.0004349. PubMed DOI PMC

Amezian  D, Nauen  R, Van Leeuwen  T. The role of ATP-binding cassette transporters in arthropod pesticide toxicity and resistance. Curr Opin Insect Sci.  2024:63:101200. 10.1016/j.cois.2024.101200. PubMed DOI

Balaska  S, Calzolari  M, Grisendi  A, Scremin  M, Dottori  M, Mavridis  K, Bellini  R, Vontas  J. Monitoring of insecticide resistance mutations and pathogen circulation in sand flies from Emilia-Romagna, a leishmaniasis endemic region of Northern Italy. Viruses. 2023:15(1):148. 10.3390/v15010148. PubMed DOI PMC

Balaska  S, Fotakis  EA, Chaskopoulou  A, Vontas  J. Chemical control and insecticide resistance status of sand fly vectors worldwide. PLoS Negl Trop Dis.  2021:15(8):e0009586. 10.1371/journal.pntd.0009586. PubMed DOI PMC

Buchfink  B, Reuter  K, Drost  H-G. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods.  2021:18(4):366–368. 10.1038/s41592-021-01101-x. PubMed DOI PMC

Bushmanova  E, Antipov  D, Lapidus  A, Prjibelski  AD. rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data. GigaScience. 2019:8(9):giz100. 10.1093/gigascience/giz100. PubMed DOI PMC

Cantalapiedra  CP, Hernández-Plaza  A, Letunic  I, Bork  P, Huerta-Cepas  J. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol.  2021:38(12):5825–5829. 10.1093/molbev/msab293. PubMed DOI PMC

Capella-Gutiérrez  S, Silla-Martínez  JM, Gabaldón  T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009:25(15):1972–1973. 10.1093/bioinformatics/btp348. PubMed DOI PMC

Charamis  J. PhyloSuite: suite of tools that provide a unified framework of analyzing, manipulating and visualizing phylogenetic data. 2024. [accessed 204 Feb 05]. https://github.com/JasonCharamis/PhyloSuite/tree/main/OliveTRee.

Clarkson  CS, Miles  A, Harding  NJ, O'Reilly  AO, Weetman  D, Kwiatkowski  D, Donnelly  MJ; Anopheles gambiae 1000 Genomes Consortium . The genetic architecture of target-site resistance to pyrethroid insecticides in the African malaria vectors Anopheles gambiae and Anopheles coluzzii. Mol Ecol.  2021:30(21):5303–5317. 10.1111/mec.15845. PubMed DOI PMC

Coudert  E, Gehant  S, de Castro  E, Pozzato  M, Baratin  D, Neto  T, Sigrist  CJA, Redaschi  N, Bridge  A; UniProt Consortium . Annotation of biologically relevant ligands in UniProtKB using ChEBI. Bioinformatics. 2023:39(1):btac793. 10.1093/bioinformatics/btac793. PubMed DOI PMC

Coutinho-Abreu  IV, Balbino  VQ, Valenzuela  JG, Sonoda  IV, Ramalho-Ortigão  JM. Structural characterization of acetylcholinesterase 1 from the sand fly Lutzomyia longipalpis (Diptera: Psychodidae). J Med Entomol.  2007:44(4):639–650. 10.1603/0022-2585(2007)44[639:SCOAFT]2.0.CO;2. PubMed DOI

Cruaud  A, Lehrter  V, Genson  G, Rasplus  J-Y, Depaquit  J. Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers. PLoS Negl Trop Dis.  2021:15(7):e0009479. 10.1371/journal.pntd.0009479. PubMed DOI PMC

Cruse  C, Moural  TW, Zhu  F. Dynamic roles of insect carboxyl/cholinesterases in chemical adaptation. Insects. 2023:14(2):194. 10.3390/insects14020194. PubMed DOI PMC

Darriba  D, Posada  D, Kozlov  AM, Stamatakis  A, Morel  B, Flouri  T. ModelTest-NG: a new and scalable tool for the selection of DNA and protein evolutionary models. Mol Biol Evol.  2020:37(1):291–294. 10.1093/molbev/msz189. PubMed DOI PMC

Denecke  S, Bảo Lương  HN, Koidou  V, Kalogeridi  M, Socratous  R, Howe  S, Vogelsang  K, Nauen  R, Batterham  P, Geibel  S, et al.  Characterization of a novel pesticide transporter and P-glycoprotein orthologues in Drosophila melanogaster. Proc R Soc Lond B Biol Sci.  2022:289(1975):20220625. 10.1098/rspb.2022.0625. PubMed DOI PMC

Dermauw  W, Van Leeuwen  T. The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance. Insect Biochem Mol Biol. 2014:45:89–110. 10.1016/j.ibmb.2013.11.001. PubMed DOI

Dermauw  W, Van Leeuwen  T, Feyereisen  R. Diversity and evolution of the P450 family in arthropods. Insect Biochem Mol Biol.  2020:127:103490. 10.1016/j.ibmb.2020.103490. PubMed DOI

Dong  K, Du  Y, Rinkevich  F, Nomura  Y, Xu  P, Wang  L, Silver  K, Zhorov  BS. Molecular biology of insect sodium channels and pyrethroid resistance. Insect Biochem Mol Biol.  2014:50:1–17. 10.1016/j.ibmb.2014.03.012. PubMed DOI PMC

Douris  V, Denecke  S, Van Leeuwen  T, Bass  C, Nauen  R, Vontas  J. Using CRISPR/Cas9 genome modification to understand the genetic basis of insecticide resistance: Drosophila and beyond. Pestic Biochem Physiol.  2020:167:104595. 10.1016/j.pestbp.2020.104595. PubMed DOI

Dvořák  V, Shaw  J, Volf  P. Parasite biology: the vectors. In: Bruschi  F, Gradoni  L, editors. The leishmaniases: old neglected tropical diseases. New York (NY): Springer; 2018. p. 31–77.

Eddy  SR. Accelerated profile HMM searches. PLoS Comput Biol.  2011:7(10):e1002195. 10.1371/journal.pcbi.1002195. PubMed DOI PMC

Emms  DM, Kelly  S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol.  2019:20(1):238. 10.1186/s13059-019-1832-y. PubMed DOI PMC

Feyereisen  R. Arthropod CYPomes illustrate the tempo and mode in P450 evolution. Biochim Biophys Acta.  2011:1814(1):19–28. 10.1016/j.bbapap.2010.06.012. PubMed DOI

Feyereisen  R. Origin and evolution of the CYP4G subfamily in insects, cytochrome P450 enzymes involved in cuticular hydrocarbon synthesis. Mol Phylogenet Evol.  2020:143:106695. 10.1016/j.ympev.2019.106695. PubMed DOI

Feyereisen  R, Dermauw  W, Van Leeuwen  T. Genotype to phenotype, the molecular and physiological dimensions of resistance in arthropods. Pestic Biochem Physiol.  2015:121:61–77. 10.1016/j.pestbp.2015.01.004. PubMed DOI

Freedman  AH, Clamp  M, Sackton  TB. Error, noise and bias in de novo transcriptome assemblies. Mol Ecol Resour.  2021:21(1):18–29. 10.1111/1755-0998.13156. PubMed DOI

Fu  L, Niu  B, Zhu  Z, Wu  S, Li  W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012:28(23):3150–3152. 10.1093/bioinformatics/bts565. PubMed DOI PMC

Gabaldón  T, Koonin  EV. Functional and evolutionary implications of gene orthology. Nat Rev Genet.  2013:14(5):360–366. 10.1038/nrg3456. PubMed DOI PMC

Gilbert  D. Gene-omes built from mRNA seq not genome DNA. 7th Annual Arthropod Genomics Symposium, Notre Dame.  F1000Res. 2013:1695(5). 10.7490/f1000research.1112594.1. DOI

Grabherr  MG, Haas  BJ, Yassour  M, Levin  JZ, Thompson  DA, Amit  I, Adiconis  X, Fan  L, Raychowdhury  R, Zeng  Q, et al.  Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol.  2011:29(7):644–652. 10.1038/nbt.1883. PubMed DOI PMC

Grace-Lema  DM, Yared  S, Quitadamo  A, Janies  DA, Wheeler  WC, Balkew  M, Hailu  A, Warburg  A, Clouse  RM. A phylogeny of sand flies (Diptera: Psychodidae: Phlebotominae), using recent Ethiopian collections and a broad selection of publicly available DNA sequence data. Syst Entomol.  2015:40(4):733–744. 10.1111/syen.12135. DOI

Grau-Bové  X, Lucas  E, Pipini  D, Rippon  E, van't Hof  AE, Constant  E, Dadzie  S, Egyir-Yawson  A, Essandoh  J, Chabi  J, et al.  Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus. PLoS Genet.  2021:17(1):e1009253. 10.1371/journal.pgen.1009253. PubMed DOI PMC

Guang  A, Howison  M, Zapata  F, Lawrence  C, Dunn  CW. Revising transcriptome assemblies with phylogenetic information. PLoS One. 2021:16(1):e0244202. 10.1371/journal.pone.0244202. PubMed DOI PMC

Guittard  E, Blais  C, Maria  A, Parvy  JP, Pasricha  S, Lumb  C, Lafont  R, Daborn  PJ, Dauphin-Villemant  C. CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis. Dev Biol. 2011:349(1):35–45. 10.1016/j.ydbio.2010.09.023. PubMed DOI

Haag  J, Höhler  D, Bettisworth  B, Stamatakis  A. From easy to hopeless—predicting the difficulty of phylogenetic analyses. Mol Biol Evol.  2022:39(12):msac254. 10.1093/molbev/msac254. PubMed DOI PMC

Haas  BJ. TransDecoder. 2022. [accessed 2022 Sept 15]. https://github.com/TransDecoder/TransDecoder.

Hackl  T, Ankenbrand  M, Van Adrichem  B. Hackl T, Ankenbrand M, van Adrichem, B. gggenomes: a grammar of graphics for comparative genomics. R Package Version 0.9.9.9000. 2023 [accessed 2023 Oct 10]. https://github.com/thackl/gggenomes.

Hassan  F, Singh  KP, Shivam  P, Ali  V, Dinesh  DS. Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance. Sci Rep.  2019:9(1):19636. 10.1038/s41598-019-56209-0. PubMed DOI PMC

Hassan  F, Singh  KP, Shivam  P, Ali  V, Dinesh  DS. Amplification and characterization of DDT metabolizing delta class GST in sand fly, Phlebotomus argentipes (Diptera: Psychodidae) from Bihar, India. J Med Entomol.  2021:58(6):2349–2357. 10.1093/jme/tjab124. PubMed DOI

Hölzer  M, Marz  M. De novo transcriptome assembly: a comprehensive cross-species comparison of short-read RNA-Seq assemblers. GigaScience. 2019:8(5):giz039. 10.1093/gigascience/giz039. PubMed DOI PMC

Huang  M, Kingan  S, Shoue  D, Nguyen  O, Froenicke  L, Galvin  B, Lambert  C, Khan  R, Maheshwari  C, Weisz  D, et al.  Improved high quality sand fly assemblies enabled by ultra low input long read sequencing. Sci Data. 2024:11(1):918. 10.1038/s41597-024-03628-y. PubMed DOI PMC

Huerta-Cepas  J, Serra  F, Bork  P. ETE 3: reconstruction, analysis, and visualization of phylogenomic data. Mol Biol Evol.  2016:33(6):1635–1638. 10.1093/molbev/msw046. PubMed DOI PMC

Katoh  K, Standley  DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol.  2013:30(4):772–780. 10.1093/molbev/mst010. PubMed DOI PMC

Kefi  M, Balabanidou  V, Douris  V, Lycett  G, Feyereisen  R, Vontas  J. Two functionally distinct CYP4G genes of Anopheles gambiae contribute to cuticular hydrocarbon biosynthesis. Insect Biochem Mol Biol.  2019:110:52–59. 10.1016/j.ibmb.2019.04.018. PubMed DOI

Kefi  M, Balabanidou  V, Sarafoglou  C, Charamis  J, Lycett  G, Ranson  H, Gouridis  G, Vontas  J. ABCH2 transporter mediates deltamethrin uptake and toxicity in the malaria vector Anopheles coluzzii. PLoS Pathog.  2023:19(8):e1011226. 10.1371/journal.ppat.1011226. PubMed DOI PMC

Koirala  BKS, Moural  T, Zhu  F. Functional and structural diversity of insect glutathione S-transferases in Xenobiotic adaptation. Int J Biol Sci.  2022:18(15):5713–5723. 10.7150/ijbs.77141. PubMed DOI PMC

Kozlov  AM, Darriba  D, Flouri  T, Morel  B, Stamatakis  A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019:35(21):4453–4455. 10.1093/bioinformatics/btz305. PubMed DOI PMC

Labbé  F, Abdeladhim  M, Abrudan  J, Araki  AS, Araujo  RN, Arensburger  P, Benoit  JB, Brazil  RP, Bruno  RV, Bueno da Silva Rivas  G, et al.  Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World. PLoS Negl Trop Dis.  2023:17(4):e0010862. 10.1371/journal.pntd.0010862. PubMed DOI PMC

Liu  W, Sun  X, Sun  W, Zhou  A, Li  R, Wang  B, Li  X, Yan  C. Genome-wide analyses of ATP-Binding Cassette (ABC) transporter gene family and its expression profile related to deltamethrin tolerance in non-biting midge Propsilocerus akamusi. Aquatic Toxicology. 2021:239:105940. 10.1016/j.aquatox.2021.105940. PubMed DOI

Lumjuan  N, Stevenson  BJ, Prapanthadara  LA, Somboon  P, Brophy  PM, Loftus  BJ, Severson  DW, Ranson  H. The Aedes aegypti glutathione transferase family. Insect Biochem Mol Biol.  2007:37(10):1026–1035. 10.1016/j.ibmb.2007.05.018. PubMed DOI

Maia  C, Conceição  C, Pereira  A, Rocha  R, Ortuño  M, Muñoz  C, Jumakanova  Z, Pérez-Cutillas  P, Özbel  Y, Töz  S, et al.  The estimated distribution of autochthonous leishmaniasis by Leishmania infantum in Europe in 2005–2020. PLoS Negl Trop Dis.  2023:17(7):e0011497. 10.1371/journal.pntd.0011497. PubMed DOI PMC

Manni  M, Berkeley  MR, Seppey  M, Simão  FA, Zdobnov  EM. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol.  2021:38(10):4647–4654. 10.1093/molbev/msab199. PubMed DOI PMC

Maroli  M, Feliciangeli  MD, Bichaud  L, Charrel  RN, Gradoni  L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol.  2013:27(2):123–147. 10.1111/j.1365-2915.2012.01034.x. PubMed DOI

Medlock  JM, Hansford  KM, Van Bortel  W, Zeller  H, Alten  B. A summary of the evidence for the change in European distribution of phlebotomine sand flies (Diptera: Psychodidae) of public health importance. J Vector Ecol.  2014:39(1):72–77. 10.1111/j.1948-7134.2014.12072.x. PubMed DOI

Musdal  Y, Ismail  A, Sjödin  B, Mannervik  B. Potent GST ketosteroid isomerase activity relevant to ecdysteroidogenesis in the malaria vector Anopheles gambiae. Biomolecules. 2023:13(6):976. 10.3390/biom13060976. PubMed DOI PMC

Nauen  R, Bass  C, Feyereisen  R, Vontas  J. The role of cytochrome P450s in insect toxicology and resistance. Annu Rev Entomol.  2022:67(1):105–124. 10.1146/annurev-ento-070621-061328. PubMed DOI

Nauen  R, Zimmer  CT, Vontas  J. Heterologous expression of insect P450 enzymes that metabolize xenobiotics. Curr Opin Insect Sci. 2021:43:78–84. 10.1016/j.cois.2020.10.011. PubMed DOI

Neafsey  DE, Waterhouse  RM, Abai  MR, Aganezov  SS, Alekseyev  MA, Allen  JE, Amon  J, Arcà  B, Arensburger  P, Artemov  G, et al.  Mosquito genomics. Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes. Science. 2015:347(6217):1258522. 10.1126/science.1258522. PubMed DOI PMC

Nouzova  M, Edwards  MJ, Michalkova  V, Ramirez  CE, Ruiz  M, Areiza  M, DeGennaro  M, Fernandez-Lima  F, Feyereisen  R, Jindra  M, et al.  Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness. Proc Natl Acad Sci U S A. 2021:118(45):e2109381118. 10.1073/pnas.2109381118. PubMed DOI PMC

Oakeshott  JG, Johnson  RM, Berenbaum  MR, Ranson  H, Cristino  AS, Claudianos  C. Metabolic enzymes associated with xenobiotic and chemosensory responses in Nasonia vitripennis. Insect Mol Biol.  2010:19(s1):147–163. 10.1111/j.1365-2583.2009.00961.x. PubMed DOI

Öztürk-Çolak  A, Marygold  SJ, Antonazzo  G, Attrill  H, Goutte-Gattat  D, Jenkins  VK, Matthews  BB, Millburn  G, Dos Santos  G, Tabone  CJ, et al.  FlyBase: updates to the Drosophila genes and genomes database. Genetics. 2024:227(1):iyad211. 10.1093/genetics/iyad211. PubMed DOI PMC

Paradis  E, Schliep  K. ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019:35(3):526–528. 10.1093/bioinformatics/bty633. PubMed DOI

Paronyan  L, Babayan  L, Vardanyan  H, Manucharyan  A, Papapostolou  KM, Balaska  S, Vontas  J, Mavridis  K. Molecular monitoring of insecticide resistance in major disease vectors in Armenia. Parasit Vectors. 2024:17(1):54. 10.1186/s13071-024-06139-2. PubMed DOI PMC

Patro  R, Duggal  G, Love  MI, Irizarry  RA, Kingsford  C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods.  2017:14(4):417–419. 10.1038/nmeth.4197. PubMed DOI PMC

Pavlidi  N, Vontas  J, Van Leeuwen  T. The role of glutathione S-transferases (GSTs) in insecticide resistance in crop pests and disease vectors. Curr Opin Insect Sci.  2018:27:97–102. 10.1016/j.cois.2018.04.007. PubMed DOI

Pignatelli  P, Ingham  VA, Balabanidou  V, Vontas  J, Lycett  G, Ranson  H. The Anopheles gambiae ATP-binding cassette transporter family: phylogenetic analysis and tissue localization provide clues on function and role in insecticide resistance. Insect Mol Biol.  2018:27(1):110–122. 10.1111/imb.12351. PubMed DOI

Ranson  H, Claudianos  C, Ortelli  F, Abgrall  C, Hemingway  J, Sharakhova  MV, Unger  MF, Collins  FH, Feyereisen  R. Evolution of supergene families associated with insecticide resistance. Science. 2002:298(5591):179–181. 10.1126/science.1076781. PubMed DOI

Reid  E, Deb  RM, Ali  A, Singh  RP, Mishra  PK, Shepherd  J, Singh  AM, Bharti  A, Singh  C, Sharma  S, et al.  Molecular surveillance of insecticide resistance in Phlebotomus argentipes targeted by indoor residual spraying for visceral leishmaniasis elimination in India. PLoS Negl Trop Dis.  2023:17(11):e0011734. 10.1371/journal.pntd.0011734. PubMed DOI PMC

Revell  LJ. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol.  2012:3(2):217–223. 10.1111/j.2041-210X.2011.00169.x. DOI

Sadlova  J, Homola  M, Myskova  J, Jancarova  M, Volf  P. Refractoriness of Sergentomyia schwetzi to Leishmania spp. is mediated by the peritrophic matrix. PLoS Negl Trop Dis. 2018:12(4):e0006382. 10.1371/journal.pntd.0006382. PubMed DOI PMC

Shi  Y, Liu  Q, Lu  W, Yuan  J, Yang  Y, Oakeshott  J, Wu  Y. Divergent amplifications of CYP9A cytochrome P450 genes provide two noctuid pests with differential protection against xenobiotics. Proc Natl Acad Sci. 2023:120(37):e2308685120. 10.1073/pnas.2308685120. PubMed DOI PMC

Smith-Unna  R, Boursnell  C, Patro  R, Hibberd  JM, Kelly  S. TransRate: reference-free quality assessment of de novo transcriptome assemblies. Genome Res.  2016:26(8):1134–1144. 10.1101/gr.196469.115. PubMed DOI PMC

Temeyer  KB, Tong  F, Totrov  MM, Tuckow  AP, Chen  QH, Carlier  PR, Pérez de León  AA, Bloomquist  JR. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant. Parasit Vectors. 2014:7(1):577. 10.1186/s13071-014-0577-4. PubMed DOI PMC

Thomas  GWC, Dohmen  E, Hughes  DST, Murali  SC, Poelchau  M, Glastad  K, Anstead  CA, Ayoub  NA, Batterham  P, Bellair  M. Gene content evolution in the arthropods. Genome Biol. 2020:21(1):15. 10.1186/s13059-019-1925-7. PubMed DOI PMC

Volf  P, Volfova  V. Establishment and maintenance of sand fly colonies. J Vector Ecol.  2011:36(Suppl 1):S1–S9. 10.1111/j.1948-7134.2011.00106.x. PubMed DOI

Vontas  J, Katsavou  E, Mavridis  K. Cytochrome P450-based metabolic insecticide resistance in Anopheles and Aedes mosquito vectors: muddying the waters. Pestic Biochem Physiol. 2020:170:104666. 10.1016/j.pestbp.2020.104666. PubMed DOI

Voshall  A, Behera  S, Li  X, Yu  XH, Kapil  K, Deogun  JS, Shanklin  J, Cahoon  EB, Moriyama  EN. A consensus-based ensemble approach to improve transcriptome assembly. BMC Bioinformatics. 2021:22(1):513. 10.1186/s12859-021-04434-8. PubMed DOI PMC

Wickham  H. ggplot2: elegant graphics for data analysis. New York: Springer-Verlag; 2016. https://ggplot2.tidyverse.org.

Wilson  AL, Courtenay  O, Kelly-Hope  LA, Scott  TW, Takken  W, Torr  SJ, Lindsay  SW. The importance of vector control for the control and elimination of vector-borne diseases. PLoS Negl Trop Dis.  2020:14(1):e0007831. 10.1371/journal.pntd.0007831. PubMed DOI PMC

Wu  L, Jia  Q, Zhang  X, Zhang  X, Liu  S, Park  Y, Feyereisen  R, Zhu  KY, Ma  E, Zhang  J, et al.  CYP303A1 has a conserved function in adult eclosion in Locusta migratoria and Drosophila melanogaster. Insect Biochem Mol Biol. 2019:113:103210. 10.1016/j.ibmb.2019.103210. PubMed DOI

Yu  G. Using ggtree to visualize data on tree-like structures. Curr Protoc Bioinformatics. 2020:69(1):e96. 10.1002/cpbi.96. PubMed DOI

Zhou  Y, Fu  WB, Si  FL, Yan  ZT, Zhang  YJ, He  QY, Chen  B. UDP-glycosyltransferase genes and their association and mutations associated with pyrethroid resistance in Anopheles sinensis (Diptera: Culicidae). Malar J. 2019:18(1):62. 10.1186/s12936-019-2705-2. PubMed DOI PMC

Zhou  L, Yu  G. ggmsa: a visual exploration tool for multiple sequence alignment and associated data. Brief Bioinform.  2022:23(4):bbac222. 10.1093/bib/bbac222. PubMed DOI