Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.

A N Belozersky Institute of Physico Chemical Biology Lomonosov Moscow State University Moscow 119991 Russia

Faculty of Bioengineering and Bioinformatics Lomonosov Moscow State University Moscow 119991 Russia

Faculty of Chemistry Lomonosov Moscow State University Moscow 119991 Russia

Institute of Plant Molecular Biology Biology Centre of the Czech Academy of Sciences Branišovská 1160 31 370 05 České Budejovice Czech Republic

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Ikeda M., Yaginuma T., Kobayashi M., Yamashita O. cDNA cloning, sequencing and temporal expression of the protease responsible for vitellin degradation in the silkworm, Bombyx mori. Comp. Biochem. Physiol. B. 1991;99:405–411. doi: 10.1016/0305-0491(91)90062-i. PubMed DOI

Choo Y.M., Lee K.S., Yoon H.J., Lee S.B., Kim J.H., Sohn H.D., Jin B.R. A serine protease from the midgut of the bumblebee, Bombus ignites (Hymenoptera: Apidae): cDNA cloning, gene structure, expression and enzyme activity. Eur. J. Entomol. 2007;104:1–7. doi: 10.14411/eje.2007.001. DOI

Waterhouse R.M., Kriventseva E.V., Meister S., Xi Z., Alvarez K.S., Bartholomay L.C., Barillas-Mury C., Bian G., Blandin S., Christensen B.M., et al. Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science. 2007;316:1738–1743. doi: 10.1126/science.1139862. PubMed DOI PMC

Kan H., Kim C.H., Kwon H.M., Park J.W., Roh K.B., Lee H., Park B.J., Zhang R., Zhang J., Söderhäll K., et al. Molecular control of phenoloxidase-induced melanin synthesis in an insect. J. Biol. Chem. 2008;283:25316–25323. doi: 10.1074/jbc.M804364200. PubMed DOI

Jiang H., Vilcinskas A., Kanost M.R. Immunity in lepidopteran insects. Adv. Exp. Med. Biol. 2010;708:181–204. doi: 10.1007/978-1-4419-8059-5_10. PubMed DOI PMC

Veillard F., Troxler L., Reichhart J.M. Drosophila melanogaster clip-domain serine proteases: Structure, function and regulation. Biochimie. 2016;22:255–269. doi: 10.1016/j.biochi.2015.10.007. PubMed DOI

Clark K.D. Insect Hemolymph Immune Complexes. Subcell Biochem. 2020;94:123–161. doi: 10.1007/978-3-030-41769-7_5. PubMed DOI

Contreras E.G., Glavic Á., Brand A.H., Sierralta J.A. The serine protease homolog, scarface, is sensitive to nutrient availability and modulates the development of the Drosophila blood-brain barrier. J. Neurosci. 2021;41:6430–6448. doi: 10.1523/JNEUROSCI.0452-20.2021. PubMed DOI PMC

Perona J.J., Craik C.S. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995;4:337–360. doi: 10.1002/pro.5560040301. PubMed DOI PMC

Bao Y.Y., Qin X., Yu B., Chen L.B., Wang Z.C., Zhang C.X. Genomic insights into the serine protease gene family and expression profile analysis in the planthopper, Nilaparvata lugens. BMC Genom. 2014;15:507. doi: 10.1186/1471-2164-15-507. PubMed DOI PMC

Ross J., Jiang H., Kanost M., Wanga Y. Serine proteases and their homologs in the Drosophila melanogaster genome: An initial analysis of sequence conservation and phylogenetic relationships. Gene. 2003;304:117–131. doi: 10.1016/s0378-1119(02)01187-3. PubMed DOI

Cao X., Jiang H. Building a platform for predicting functions of serine protease-related proteins in Drosophila melanogaster and other insects. Insect Biochem. Mol. Biol. 2018;103:53–69. doi: 10.1016/j.ibmb.2018.10.006. PubMed DOI PMC

Christophides G.K., Zdobnov E., Barillas-Mury C., Birney E., Blandin S., Blass C., Brey P.T., Collins F.H., Danielli A., Dimopoulos G., et al. Immunity-related genes and gene families in Anopheles gambiae. Science. 2002;298:159–165. doi: 10.1126/science.1077136. PubMed DOI

Cao X., Gulati M., Jiang H. Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect Biochem. Mol. Biol. 2017;88:48–62. doi: 10.1016/j.ibmb.2017.07.008. PubMed DOI PMC

Brackney D.E., Isoe J., Black W.C., Zamora J., Foy B.D., Miesfeld R.L., Olson K.E. Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito, Aedes aegypti. J. Insect Physiol. 2010;56:736–744. doi: 10.1016/j.jinsphys.2010.01.003. PubMed DOI PMC

Soares T.S., Watanabe R.M.O., Lemos F.J.A., Tanaka A.S. Molecular characterization of genes encoding trypsinlike enzymes from Aedes aegypti larvae and identification of digestive enzymes. Gene. 2011;489:70–75. doi: 10.1016/j.gene.2011.08.018. PubMed DOI

Cao X., He Y., Hu Y., Zhang X., Wang Y., Zou Z., Chen Y., Blissard G.W., Kanost M.R., Jiang H. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta. Insect Biochem. Mol. Biol. 2015;62:51–63. doi: 10.1016/j.ibmb.2014.10.006. PubMed DOI PMC

Miao Z., Cao X., Jiang H. Digestion-related proteins in the tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 2020;126:103457. doi: 10.1016/j.ibmb.2020.103457. PubMed DOI PMC

Zhao P., Wang G.H., Dong Z.M., Duan J., Xu P.Z., Cheng T.C., Xiang Z.H., Xia Q.Y. Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori. BMC Genom. 2010;11:405. doi: 10.1186/1471-2164-11-405. PubMed DOI PMC

Liu H., Heng J., Wang L., Tang X., Guo P., Li Y., Xia Q., Zhao P. Identification, characterization, and expression analysis of clip-domain serine protease genes in the silkworm, Bombyx mori. Dev. Comp. Immunol. 2020;105:103584. doi: 10.1016/j.dci.2019.103584. PubMed DOI

Lin H., Xia X., Yu L., Vasseur L., Gurr G.M., Yao F., Yang G., You M. Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.) BMC Genom. 2015;16:1054. doi: 10.1186/s12864-015-2243-4. PubMed DOI PMC

Yang L., Xing B.Q., Wang L.K., Yuan L.L., Manzoor M., Li F., Wu S. Identification of serine protease, serine protease homolog and prophenoloxidase genes in Spodoptera frugiperda (Lepidoptera: Noctuidae) J. Asia-Pac. Entomol. 2021;24:1144–1152. doi: 10.1016/j.aspen.2021.10.010. DOI

Zou Z., Lopez D.L., Kanost M.R., Evans J.D., Jiang H. Comparative analysis of serine protease-related genes in the honey bee genome: Possible involvement in embryonic development and innate immunity. Insect Mol. Biol. 2006;15:603–614. doi: 10.1111/j.1365-2583.2006.00684.x. PubMed DOI PMC

Yang L., Lin Z., Fang Q., Wang J., Yan Z., Zou Z., Song Q., Ye G. The genomic and transcriptomic analyses of serine proteases and their homologs in an endoparasitoid, Pteromalus puparum. Dev. Comp. Immunol. 2017;77:56–68. doi: 10.1016/j.dci.2017.07.014. PubMed DOI

Oppert B., Muszewska A., Steczkiewicz K., Šatović-Vukšić E., Plohl M., Fabrick J.A., Vinokurov K.S., Koloniuk I., Johnston J.S., Smith T.P.L., et al. The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success. Genes. 2022;13:446. doi: 10.3390/genes13030446. PubMed DOI PMC

Tribolium Sequencing Consortium The genome of the model beetle and pest Tribolium castaneum. Nature. 2008;452:949–955. doi: 10.1038/nature06784. PubMed DOI

Prabhakar S., Chen M.-S., Elpidina E.N., Vinokurov K.S., Smith C.M., Marshall J., Oppert B. Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L. Insect Mol. Biol. 2007;16:455–468. doi: 10.1111/j.1365-2583.2007.00740.x. PubMed DOI

Tsybina T.A., Dunaevsky Y.E., Belozersky M.A., Zhuzhikov D.P., Oppert B., Elpidina E.N. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: Purification and characterization of a trypsin-like proteinase. Biochemistry. 2005;70:300–305. doi: 10.1007/s10541-005-0115-2. PubMed DOI

Elpidina E.N., Tsybina T.A., Dunaevsky Y.E., Belozersky M.A., Zhuzhikov D.P., Oppert B. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie. 2005;87:771–779. doi: 10.1016/j.biochi.2005.02.013. PubMed DOI

Oppert B., Dowd S.E., Bouffard P., Li L., Conesa A., Lorenzen M.D., Toutges M., Marshall J., Huestis D.L., Fabrick J., et al. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin. PLoS ONE. 2012;7:e34624. doi: 10.1371/journal.pone.0034624. PubMed DOI PMC

Zhiganov N.I., Tereshchenkova V.F., Oppert B., Filippova I.Y., Belyaeva N.V., Dunaevsky Y.E., Belozersky M.A., Elpidina E.N. The dataset of predicted trypsin serine peptidases and their inactive homologs in Tenebrio molitor transcriptomes. Data Brief. 2021;38:107301. doi: 10.1016/j.dib.2021.107301. PubMed DOI PMC

Gorbunov A.A., Akentyev F.I., Gubaidullin I.I., Zhiganov N.I., Tereshchenkova V.F., Elpidina E.N., Kozlov D.G. Biosynthesis and Secretion of Serine Peptidase SerP38 from Tenebrio molitor in the Yeast Komagataella kurtzmanii. Appl. Biochem. Microbiol. 2021;57:917–924. doi: 10.1134/S0003683821090039. DOI

Tereshchenkova V.F., Zhiganov N.I., Akentyev P.I., Gubaidullin I.I., Kozlov D.G., Belyaeva N.V., Filippova I.Y., Elpidina E.N. Preparation and properties of the recombinant Tenebrio molitor SerPH122—Proteolytically active homolog of serine peptidase. Appl. Biochem. Microbiol. 2021;57:579–585. doi: 10.1134/S0003683821050161. DOI

Wu C.Y., Xiao K.R., Wang L.Z., Wang J., Song Q.S., Stanley D., Wei S.J., Zhu J.Y. Identification and expression profiling of serine protease-related genes in Tenebrio molitor. Arch. Insect Biochem. Physiol. 2022;111:e21963. doi: 10.1002/arch.21963. PubMed DOI

Errico S., Spagnoletta A., Verardi A., Moliterni S., Dimatteo S., Sangiorgio P. Tenebrio molitor as a source of interesting natural compounds, their recovery processes, biological effects, and safety aspects. Compr. Rev. Food Sci. Food Saf. 2022;21:148–197. doi: 10.1111/1541-4337.12863. PubMed DOI

Moncada-Pazos A., Cal S., Lopez-Otín C. Polyserases. In: Rawlings N.D., Salvesen G., editors. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press; London, UK: 2013. pp. 2990–2994.

Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967;27:157–162. doi: 10.1016/s0006-291x(67)80055-x. PubMed DOI

Botos I., Meyer E., Nguyen M., Swanson S.M., Koomen J.M., Russell D.H., Meyer E.F. The structure of an insect chymotrypsin. J. Mol. Biol. 2000;298:895–901. doi: 10.1006/jmbi.2000.3699. PubMed DOI

Rawlings N.D., Barrett A.J. Introduction: Serine peptidases and their clans. In: Rawlings N.D., Salvesen G., editors. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press; London, UK: 2013. pp. 2491–2523.

Baird T.T., Jr., Craik C.S. Trypsin. In: Rawlings N.D., Salvesen G., editors. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press; London, UK: 2013. pp. 2594–2600.

Kanost M.R., Jiang H. Clip-domain serine proteases as immune factors in insect hemolymph. Curr. Opin. Insect Sci. 2015;11:47–55. doi: 10.1016/j.cois.2015.09.003. PubMed DOI PMC

Lopes A.R., Sato P.M., Terra W.R. Insect chymotrypsins: Chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants. Arch. Insect Biochem. Physiol. 2009;70:188–203. doi: 10.1002/arch.20289. PubMed DOI

Whitworth S.T., Blum M.S., Travis J. Proteolytic enzymes from larvae of the fire ant, Solenopsis invicta. Isolation and characterization of four serine endopeptidases. J. Biol. Chem. 1998;273:14430–14434. doi: 10.1074/jbc.273.23.14430. PubMed DOI

Tereshchenkova V.F., Zhiganov N.I., Gubaeva A.S., Akentyev F.I., Dunaevsky Y.E., Kozlov D.G., Belozersky M.A., Elpidina E.N. Characteristics of recombinant chymotrypsin-like peptidase from the midgut of Tenebrio molitor larvae. Appl. Biochem. Microbiol. 2024;60:420–430. doi: 10.1134/S0003683824603652. DOI

Tsu C.A., Perona J.J., Schellenberger V., Turck C.W., Craik C.S. The substrate specificity of Uca pugilator collagenolytic serine protease 1 correlates with the bovine type I collagen cleavage sites. J. Biol. Chem. 1994;269:19565–19572. doi: 10.1016/S0021-9258(17)32206-8. PubMed DOI

Tsu C.A., Craik C.S. Substrate recognition by recombinant serine collagenase 1 from Uca pugilator. J. Biol. Chem. 1996;271:11563–11570. doi: 10.1074/jbc.271.19.11563. PubMed DOI

Bode W., Meyer E., Jr., Powers J.C. Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors. Biochemistry. 1989;28:1951–1963. doi: 10.1021/bi00431a001. PubMed DOI

Oliveira E.B., Salgado M.C.O. Pancreatic elastases. In: Rawlings N.D., Salvesen G., editors. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press; London, UK: 2013. pp. 2639–2645. DOI

DeLotto R. Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila. EMBO Rep. 2001;2:721–726. doi: 10.1093/embo-reports/kve153. PubMed DOI PMC

Reynolds S.L., Fischer K. Pseudoproteases: Mechanisms and function. Biochem. J. 2015;468:17–24. doi: 10.1042/BJ20141506. PubMed DOI

Cal S., Moncada-Pazos A., Lopez-Otin C. Expanding the complexity of the human degradome: Polyserases and their tandem serine protease domains. Front. Biosci. 2007;12:4661–4669. doi: 10.2741/2415. PubMed DOI

Chen L.M., Skinner M.L., Kauffman S.W., Chao J., Chao L., Thaler C.D., Chai K.X. Prostasin is a glycosylphosphatidylinositol-anchored active serine protease. J. Biol. Chem. 2001;276:21434–21442. doi: 10.1074/jbc.M011423200. PubMed DOI

Scarman A.L., Hooper J.D., Boucaut K.J., Sit M., Webb G.C., Normyle J.F., Antalis T.M. Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis. Eur. J. Biochem. 2001;268:1250–1258. doi: 10.1046/j.1432-1327.2001.01986.x. PubMed DOI

Rickert K.W., Kelley P., Byrne N.J., Diehl R.E., Hall D.L., Montalvo A.M., Reid J.C., Shipman J.M., Thomas B.W., Munshi S.K., et al. Structure of human prostasin, a target for the regulation of hypertension. J. Biol. Chem. 2008;283:34864–34872. doi: 10.1074/jbc.M805262200. PubMed DOI PMC

Lee K.Y., Zhang R., Kim M.S., Park J.W., Park H.Y., Kawabata S., Lee B.L. A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae. Eur. J. Biochem. 2002;269:4375–4383. doi: 10.1046/j.1432-1033.2002.03155.x. PubMed DOI

Piao S., Song Y.-L., Kim J.H., Park S.Y., Park J.W., Lee B.L., Oh B.-H., Ha N.-C. Crystal structure of a clip-domain serine protease and functional roles of the clip domains. EMBO J. 2005;24:4404–4414. doi: 10.1038/sj.emboj.7600891. PubMed DOI PMC

Huang R., Lu Z., Dai H., Velde D.V., Prakash O., Jiang H. The solution structure of clip domains from Manduca sexta prophenoloxidase activating proteinase-2. Biochemistry. 2007;46:11431–11439. doi: 10.1021/bi7010724. PubMed DOI

Kim C.H., Kim S.J., Kan H., Kwon H.M., Roh K.B., Jiang R., Yang Y., Park J.W., Lee H.H., Ha N.C., et al. A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect. J. Biol. Chem. 2008;283:7599–7607. doi: 10.1074/jbc.M710216200. PubMed DOI

He Y., Wang Y., Yang F., Jiang H. Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism, mediates immune responses. Insect Biochem. Mol. Biol. 2017;84:23–31. doi: 10.1016/j.ibmb.2017.03.008. PubMed DOI PMC

Bork P., Beckmann G. The CUB domain: A widespread module in developmentally regulated proteins. J. Mol. Biol. 1993;231:539–545. doi: 10.1006/jmbi.1993.1305. PubMed DOI

Blanc G., Font B., Eichenberger D., Moreau C., Ricard-Blum S., Hulmes D.J., Moali C. Insights into how CUB domains can exert specific functions while sharing a common fold: Conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity. J. Biol. Chem. 2007;282:16924–16933. doi: 10.1074/jbc.M701610200. PubMed DOI

Park J.W., Kim C.H., Kim J.H., Je B.R., Roh K.B., Kim S.J., Lee H.H., Ryu J.H., Lim J.H., Oh B.H., et al. Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects. Proc. Natl. Acad. Sci. USA. 2007;104:6602–6607. doi: 10.1073/pnas.0610924104. PubMed DOI PMC

Cho Y.S., Stevens L.M., Sieverman K.J., Nguyen J., Stein D. A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity. Curr. Biol. 2012;22:1013–1018. doi: 10.1016/j.cub.2012.03.065. PubMed DOI PMC

Benjamini Y., Hochberg Y. Controlling the false Discovery rate: A practical and powerful approach to multiple testing. J. R. Statist. Soc. B. 1995;57:289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x. DOI

Keller M., Sneh B., Strizhov N., Prudovsky E., Regev A., Koncz C., Schell J., Zilberstein A. Digestion of δ-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC. Insect Biochem. Mol. Biol. 1996;26:365–373. doi: 10.1016/0965-1748(95)00102-6. PubMed DOI

Zalunin I.A., Elpidina E.N., Oppert B. The role of proteolysis in the biological activity of Bt insecticidal crystal proteins. In: Soberón M., Gao Y., Bravo A., editors. Bt Resistance—Characterization and Strategies for GM Crops Producing Bacillus thuringiensis Toxins. CAB International Publishers; Wallingford, UK: 2015. pp. 107–118.

Oppert B., Elpidina E.N., Toutges M., Mazumdar-Leighton S. Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors. Comp. Biochem. Physiol. D Genom. Proteom. 2010;5:280–287. doi: 10.1016/j.cbd.2010.08.001. PubMed DOI

Martynov A.G., Elpidina E.N., Perkin L., Oppert B. Functional analysis of C1 family cysteine peptidases in the larval gut of Tenebrio molitor and Tribolium castaneum. BMC Genom. 2015;16:75. doi: 10.1186/s12864-015-1306-x. PubMed DOI PMC

Broehan G., Arakane Y., Beeman R.W., Kramer K.J., Muthukrishnan S., Merzendorfer H. Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference. Insect Biochem. Mol. Biol. 2010;40:274–283. doi: 10.1016/j.ibmb.2009.10.009. PubMed DOI

LeMosy E.K., Tan Y.Q., Hashimoto C. Activation of a protease cascade involved in patterning the Drosophila embryo. Proc. Natl. Acad. Sci. USA. 2001;98:5055–5060. doi: 10.1073/pnas.081026598. PubMed DOI PMC

Muta T., Hashimoto R., Miyata T., Nishimura H., Toh Y., Iwanaga S. Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization. J. Biol. Chem. 1990;265:22426–22433. doi: 10.1016/S0021-9258(18)45722-5. PubMed DOI

Munier A.I., Medzhitov R., Janeway C.A., Doucet D., Capovilla M., Lagueux M. Graal: A Drosophila gene coding for several mosaic serine proteases. Insect Biochem. Mol. Biol. 2004;34:1025–1035. doi: 10.1016/j.ibmb.2003.09.009. PubMed DOI

Ewels P., Magnusson M., Lundin S., Käller M. MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–3048. doi: 10.1093/bioinformatics/btw354. PubMed DOI PMC

Bolger A.M., Lohse M., Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Pertea M., Kim D., Pertea G.M., Leek J.T., Salzberg S.L. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 2016;11:1650–1667. doi: 10.1038/nprot.2016.095. PubMed DOI PMC

Trapnell C., Williams B., Pertea G., Mortazavi A., Kwan G., van Baren M.J., Salzberg S.L., Wold B.J., Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 2010;28:511–515. doi: 10.1038/nbt.1621. PubMed DOI PMC

Pertea M., Pertea G.M., Antonescu C.M., Chang T.C., Mendell J.T., Salzberg S.L. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015;33:290–295. doi: 10.1038/nbt.3122. PubMed DOI PMC

Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. PubMed DOI

Hall T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. 1999;41:95–98.

Kaur S., Stinson S.A., di Cenzo G.C. Whole genome assemblies of Zophobas morio and Tenebrio molitor. G3. 2023;13:jkad079. doi: 10.1093/g3journal/jkad079. PubMed DOI PMC

Eriksson T., Andere A.A., Kelstrup H., Emery V.J., Picard C.J. The yellow mealworm (Tenebrio molitor) genome: A resource for the emerging insects as food and feed industry. J. Insects Food Feed. 2020;6:445–455. doi: 10.3920/JIFF2019.0057. DOI

McWilliam H., Li W., Uludag M., Squizzato S., Park Y.M., Buso N., Cowley A.P., Lopez R. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res. 2013;41:W597–W600. doi: 10.1093/nar/gkt376. PubMed DOI PMC

Almagro Armenteros J.J., Tsirigos K.D., Sønderby C.K., Petersen T.N., Winther O., Brunak S., von Heijne G., Nielsen H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019;37:420–423. doi: 10.1038/s41587-019-0036-z. PubMed DOI

Krogh A., Larsson B., von Heijne G., Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001;305:567–580. doi: 10.1006/jmbi.2000.4315. PubMed DOI

Käll L., Krogh A., Sonnhammer E.L. Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server. Nucleic Acids Res. 2007;35:W429–W432. doi: 10.1093/nar/gkm256. PubMed DOI PMC

Lomize A.L., Hage J.M., Pogozheva I.D. Membranome 2.0: Database for proteome-wide profiling of bitopic proteins and their dimers. Bioinformatics. 2018;34:1061–1062. doi: 10.1093/bioinformatics/btx720. PubMed DOI

Paysan-Lafosse T., Blum M., Chuguransky S., Grego T., Pinto B.L., Salazar G.A., Bileschi M.L., Bork P., Bridge A., Colwell L., et al. InterPro in 2022. Nucleic Acids Res. 2023;51:418–427. doi: 10.1093/nar/gkac993. PubMed DOI PMC

Wang J., Chitsaz F., Derbyshire M.K., Gonzales N.R., Gwadz M., Lu S., Marchler G.H., Song J.S., Thanki N., Yamashita R.A., et al. The conserved domain database in 2023. Nucleic Acids Res. 2023;51:D384–D388. doi: 10.1093/nar/gkac1096. PubMed DOI PMC

Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R.D., Bairoch A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31:3784–3788. doi: 10.1093/nar/gkg563. PubMed DOI PMC

Katoh K., Rozewicki J., Yamada K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2019;20:1160–1166. doi: 10.1093/bib/bbx108. PubMed DOI PMC

Trifinopoulos J., Nguyen L.T., von Haeseler A., Minh B.Q. W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016;44:W232–W235. doi: 10.1093/nar/gkw256. PubMed DOI PMC

Mortazavi A., Williams B.A., McCue K., Schaeffer L., Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods. 2008;5:621–628. doi: 10.1038/nmeth.1226. PubMed DOI

Chen C., Chen H., Zhang Y., Thomas H.R., Frank M.H., He Y., Xia R. TBtools: An Integrative Tool kit Developed for Interactive Analyses of Big Biological Data. Mol. Plant. 2020;13:1194–1202. doi: 10.1016/j.molp.2020.06.009. PubMed DOI

Kruskal W.H., Wallis W.A. Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 1952;47:583–621. doi: 10.1080/01621459.1952.10483441. DOI

Statistics Kingdom. [(accessed on 1 April 2024)]. Available online: https://www.statskingdom.com/index.html.