We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.

Department of Parasitology Faculty of Science Charles University Viničná 7 Prague 128 00 Czech Republic

Zobrazit více v PubMed

Jancarova, M., Polanska, N., Volf, P. & Dvorak, V. The role of sand flies as vectors of viruses other than phleboviruses. J. Gen. Virol.104, 001837 (2023). PubMed

Maroli, M., Feliciangeli, M. D., Bichaud, L., Charrel, R. N. & Gradoni, L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med. Vet. Entomol.27, 123–147 (2013). PubMed

Cecílio, P., Cordeiro-da-Silva, A. & Oliveira, F. Sand flies: basic information on the vectors of leishmaniasis and their interactions with Leishmania parasites. Commun. Biol.5, 305 (2022). PubMed PMC

Sloan, M. A. et al. The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal. Parasite. Vector. 14, 1–14 (2021). PubMed PMC

Coutinho-Abreu, I. V. et al. Leishmania infection induces a limited differential gene expression in the sand fly midgut. BMC Genom.21, 608 (2020). PubMed PMC

Labbé, F. et al. Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World. PLoS Negl. Trop. Dis.17, e0010862 (2023). PubMed PMC

Sayers, E. W. et al. Database resources of the national center for biotechnology information. Nucleic Acids Res.50, D20–D26 (2022). PubMed PMC

Amos, B. et al. VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center. Nucleic Acids Res.50, D898–D911 (2022). PubMed PMC

Artika, I. M., Dewi, Y. P., Nainggolan, I. M., Siregar, J. E. & Antonjaya, U. Real-time polymerase chain reaction: current techniques, applications, and role in COVID-19 diagnosis. Genes (Basel). 13, 2387 (2022). PubMed PMC

Taylor, S. C. et al. The ultimate qPCR experiment: producing publication quality, reproducible data the first time. Trends Biotechnol.37, 761–774 (2019). PubMed

Lü, J., Yang, C., Zhang, Y. & Pan, H. Selection of reference genes for the normalization of RT-qPCR data in gene expression studies in insects: a systematic review. Front. Physiol.9, 1560 (2018). PubMed PMC

Shakeel, M., Rodriguez, A., Tahir, U., Bin & Jin, F. Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects. Biotechnol. Lett.40, 227–236 (2018). PubMed

Rubenstein, A. P. The functional importance of multiple actin isoforms. BioEssays. 12, 309–315 (1990). PubMed

Joshi, H. C. & Cleveland, D. W. Diversity among tubulin subunits: toward what functional end? Cell. Motil. Cytoskeleton. 16, 159–163 (1990). PubMed

Tristan, C., Shahani, N., Sedlak, T. W. & Sawa, A. The diverse functions of GAPDH: views from different subcellular compartments. Cell. Signal.23, 317–323 (2011). PubMed PMC

Ühlein, M., Weglöhner, W., Urlaub, H. & Wotmann-Liebold, B. Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies. Biochem. J.331, 423–430 (1998). PubMed PMC

Dudov, K. P. & Perry, R. P. The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell. 37, 457–468 (1984). PubMed

Xu, B., Liu, L. & Song, G. Functions and regulation of translation elongation factors. Front. Mol. Biosci.8, 816398 (2022). PubMed PMC

Fitipaldi Veloso Guimaraes, V. C. et al. Lutzomyia migonei is a permissive vector competent for Leishmania infantum. Parasite. Vector. 9, 159 (2016). PubMed PMC

Dvorak, V., Shaw, J. & Volf, P. Parasite biology: the vectors. in The Leishmaniases: Old Neglected Tropical Diseases (eds Bruschi, F. & Gradoni, L.) 31–77. 10.1007/978-3-319-72386-0_3 (Springer, 2018).

Maia, C. & Depaquit, J. Can Sergentomyia (Diptera, Psychodidae) play a role in the transmission of mammal-infecting Leishmania? Parasite.23, 55 (2016). PubMed PMC

Jochim, R. C. et al. The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies. BMC Genom.9, 15 (2008). PubMed PMC

Xie, F., Wang, J. & Zhang, B. RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes. Funct. Integr. Genomics. 23, 125 (2023). PubMed

Silver, N., Best, S., Jiang, J. & Thein, S. L. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol. Biol.7, 33 (2006). PubMed PMC

Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnol. Lett.26, 509–515 (2004). PubMed

Andersen, C. L., Jensen, J. L. & Orntoft, T. F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res.64, 5245–5250 (2004). PubMed

Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol.3, research0034.1 (2002). PubMed PMC

Ramalho-Ortigao, J. M. et al. Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis. Mem. Inst. Oswaldo Cruz. 96, 105–111 (2001). PubMed

Boulanger, N. et al. Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major. Infect. Immun.72, 7140–7146 (2004). PubMed PMC

Anderson, J. M. et al. Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genom.7, 52 (2006). PubMed PMC

Ximenes, M. D. F. F. D. M., Maciel, J. C. & Jerônimo, S. M. B. Characteristics of the biological cycle of Lutzomyia evandroi Costa Lima & Antunes, 1936 (Diptera: Psychodidae) under experimental conditions. Mem. Inst. Oswaldo Cruz. 96, 883–886 (2001). PubMed

Balla, A. et al. The threat of pests and pathogens and the potential for biological control in forest ecosystems. Forests. 12, 1579 (2021).

Truman, J. W. The evolution of insect metamorphosis. Curr. Biol.29, R1252–R1268 (2019). PubMed

Ikegawa, Y., Ito, K., Himuro, C. & Honma, A. Sterile males and females can synergistically suppress wild pests targeted by sterile insect technique. J. Theor. Biol.530, 110878 (2021). PubMed

Napoleão, T. H. et al. Insect midgut structures and molecules as targets of plant-derived protease inhibitors and lectins. Pest Manag. Sci.75, 1212–1222 (2019). PubMed

Telleria, E. L., Martins-Da-Silva, A., Tempone, A. J. & Traub-Cseko, Y. M. Leishmania, microbiota and sand fly immunity. Parasitology. 145, 1336–1353 (2018). PubMed PMC

Vogel, E., Santos, D., Mingels, L., Verdonckt, T. W. & Broeck, J. Vanden. RNA interference in insects: protecting beneficials and controlling pests. Front. Physiol.9, 1912 (2019). PubMed PMC

Mueller, S. et al. RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila. Proc. Natl. Acad. Sci. U S A. 107, 19390–19395 (2010). PubMed PMC

Dostálová, A. & Volf, P. Leishmania development in sand flies: parasite-vector interactions overview. Parasite. Vector.51(5), 276 (2012). PubMed PMC

Magnarelli, L. A., Modi, G. B. & Tesh, R. B. Follicular development and parity in phlebotomine sand flies (Diptera: Psychodidae). J. Med. Entomol.21, 681–689 (1984). PubMed

Gao, Z., Deng, W. & Zhu, F. Reference gene selection for quantitative gene expression analysis in black soldier fly (Hermetia illucens). PLoS One. 14, e0221420 (2019). PubMed PMC

Wang, Z. et al. Identification and evaluation of reference genes for normalization of Gene expression in Developmental stages, sexes, and tissues of Diaphania caesalis (Lepidoptera, Pyralidae). J. Insect Sci.20, 6 (2020). PubMed PMC

Sellamuthu, G. et al. Reference gene selection for normalizing gene expression in Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) under different experimental conditions. Front. Physiol.12, 752768 (2021). PubMed PMC

Ponton, F., Chapuis, M. P., Pernice, M., Sword, G. A. & Simpson, S. J. Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J. Insect Physiol.57, 840–850 (2011). PubMed

Shi, C. et al. Evaluation of housekeeping genes for quantitative real-time PCR analysis of Bradysia odoriphaga (Diptera: Sciaridae). Int. J. Mol. Sci.17, 1034 (2016). PubMed PMC

Ma, K. S. et al. Identification and validation of reference genes for the normalization of gene expression data in qRT-PCR analysis in Aphis gossypii (Hemiptera: Aphididae). J. Insect Sci.16, 17 (2016). PubMed PMC

Wan, P. J. et al. Reference genes for quantitative real-time PCR analysis in symbiont entomomyces delphacidicola of Nilaparvata lugens (Stål). Sci. Rep.7, 42206 (2017). PubMed PMC

Sun, M., Lu, M. X., Tang, X. T. & Du, Y. Z. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae). PLoS One. 10, e0115979 (2015). PubMed PMC

Zhao, X. et al. Evaluation of optimal reference genes for qRT-PCR analysis in Hyphantria cunea (Drury). Insects. 13, 97 (2022). PubMed PMC

De Groef, S. et al. Reference genes to study the sex-biased expression of genes regulating Drosophila metabolism. Sci. Rep.14, 9518 (2024). PubMed PMC

Adeyinka, O. S. et al. Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus. Sci. Rep.9, 13629 (2019). PubMed PMC

Kyre, B. R., Rodrigues, T. B. & Rieske, L. K. RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis. Sci. Rep.9, 5640 (2019). PubMed PMC

Pinheiro, D. H. & Siegfried, B. D. Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae). Sci. Rep.10, 5070 (2020). PubMed PMC

Zhang, S. et al. Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae). Gene. 555, 393–402 (2015). PubMed

Zhao, Z. et al. Evaluation of reference genes for normalization of RT-qPCR gene expression data for Trichoplusia ni cells during Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus (AnpeNPV) infection. J. Insect Sci.19, 4 (2019). PubMed PMC

Osborne, C. et al. Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses. Sci. Rep.13, 16729 (2023). PubMed PMC

Habibi, J., Goodman, C. L. & Stuart, M. K. Distribution of elongation factor-1α in larval tissues of the fall armyworm, Spodoptera frugiperda. J. Insect Sci.6, 33 (2006). PubMed PMC

Dzaki, N., Ramli, K. N., Azlan, A., Ishak, I. H. & Azzam, G. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti. Sci. Rep.7, 43618 (2017). PubMed PMC

Dzaki, N. & Azzam, G. Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development. PLoS One. 13, e0194664 (2018). PubMed PMC

Sagri, E. et al. Housekeeping in Tephritid insects: the best gene choice for expression analyses in the medfly and the olive fly. Sci. Rep.7, 45634 (2017). PubMed PMC

Mounier, N. & Sparrw, J. C. Muscle actin genes in insects. Comp. Biochem. Physiol. Part. B Comp. Biochem.105, 231–238 (1993). PubMed

Picton, H., Briggs, D. & Gosden, R. The molecular basis of oocyte growth and development. Mol. Cell. Endocrinol.145, 27–37 (1998). PubMed

Sellamuthu, G., Bílý, J., Joga, M. R., Synek, J. & Roy, A. Identifying optimal reference genes for gene expression studies in eurasian spruce bark beetle, Ips typographus (Coleoptera: Curculionidae: Scolytinae). Sci. Rep.12, 4671 (2022). PubMed PMC

Majerowicz, D. et al. Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae). Insect Mol. Biol.20, 713–722 (2011). PubMed

Paim, R. M. et al. Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR. BMC Res. Notes. 5, 128 (2012). PubMed PMC

Liew, J. W. K., Fong, M. Y. & Lau, Y. L. Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes. PeerJ. 5, e3577 (2017). PubMed PMC

Wolmuth-Gordon, H. S., Nakabayashi, K. & Brown, M. J. F. Newly emerged bumblebees are highly susceptible to gut parasite infection. Insect. Soc.71, 85–96 (2024).

Tramuta, C. et al. A Set of Multiplex polymerase chain reactions for genomic detection of nine edible insect species in foods. J. Insect Sci.18, 3 (2018). PubMed PMC

Harish, A. & Caetano-Anollés, G. Ribosomal history reveals origins of modern protein synthesis. PLoS One. 7, e32776 (2012). PubMed PMC

Meireles-Filho, A. C. A., Amoretty, P. R., Souza, N. A., Kyriacou, C. P. & Peixoto, A. A. Rhythmic expression of the cycle gene in a hematophagous insect vector. BMC Mol. Biol.7, 38 (2006). PubMed PMC

da Silva Goncalves, D. et al. Wolbachia introduction into Lutzomyia longipalpis (Diptera: Psychodidae) cell lines and its effects on immune-related gene expression and interaction with Leishmania infantum. Parasite. Vector. 12, 33 (2019). PubMed PMC

Kykalová, B., Tichá, L., Volf, P. & Telleria, E. L. Phlebotomus papatasi antimicrobial peptides in Larvae and females and a gut-specific defensin upregulated by Leishmania major infection. Microorganisms. 9, 2307 (2021). PubMed PMC

Blum, M. et al. The InterPro protein families and domains database: 20 years on. Nucleic Acids Res.49, D344–D354 (2021). PubMed PMC

Lu, S. et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res.48, D265–D268 (2020). PubMed PMC

Edgar, R. C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res.32, 1792–1797 (2004). PubMed PMC

Felsenstein, J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol.17, 368–376 (1981). PubMed

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol.35, 1547–1549 (2018). PubMed PMC

Volf, P. & Volfova, V. Establishment and maintenance of sand fly colonies. J. Vector Ecol.36, S1–S9 (2011). PubMed

Juers, D. H., Matthews, B. W. & Huber, R. E. LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci.21, 1792–1807 (2012). PubMed PMC

Vomáčková Kykalová, B., Sassù, F., Volf, P. & Telleria, E. L. RNAi-mediated gene silencing of Phlebotomus papatasi defensins favors Leishmania major infection. Front. Physiol.14, 1182141 (2023). PubMed PMC

Molina-Cruz, A. et al. Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium. J. Biol. Chem.283, 3217–3223 (2008). PubMed

Sant’Anna, M. R. V., Alexander, B., Bates, P. A. & Dillon, R. J. Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections. Insect Biochem. Mol. Biol.38, 652–660 (2008). PubMed PMC