RESEARCH ARTICLE Open Access Genome wide identification and gene editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus Guichun Liu1,2†, Wei Liu2,3†, Ruoping Zhao2†, Jinwu He[.]
Liu et al BMC Genomics (2021) 22:120 https://doi.org/10.1186/s12864-021-07400-z RESEARCH ARTICLE Open Access Genome-wide identification and geneediting of pigment transporter genes in the swallowtail butterfly Papilio xuthus Guichun Liu1,2†, Wei Liu2,3†, Ruoping Zhao2†, Jinwu He1,2†, Zhiwei Dong2, Lei Chen1, Wenting Wan1,2, Zhou Chang2, Wen Wang1,2,4* and Xueyan Li2* Abstract Background: Insect body coloration often functions as camouflage to survive from predators or mate selection Transportation of pigment precursors or related metabolites from cytoplasm to subcellular pigment granules is one of the key steps in insect pigmentation and usually executed via such transporter proteins as the ATP-binding cassette (ABC) transmembrane transporters and small G-proteins (e.g Rab protein) However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies Results: Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus This is the first case of genome-wide gene copy number identification of ABC transporters in swallowtail butterflies and Rab family in lepidopteran insects Aiming to investigate the contribution of the five genes which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) of fruit fly or silkworm during the development of swallowtail butterflies, we performed CRISPR/Cas9 gene-editing of these genes using P xuthus as a model and sequenced the transcriptomes of their morphological mutants Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color The transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 can lead to the accumulation of abnormal transcripts and the decrease or dosage compensation of normal transcripts at gene expression level Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid GO and KEGG enrichment analysis showed that DEGs in ABCG transporter mutants enriched to the oxidoreductase activity, heme binding, iron ion binding process possibly related to the color display, and DEGs in lightoid mutants are enriched in glycoprotein binding and protein kinases (Continued on next page) * Correspondence: lixy@mail.kiz.ac.cn; wwang@mail.kiz.ac.cn † Guichun Liu, Wei Liu, Ruoping Zhao and Jinwu He contributed equally to this work School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710072, Shanxi, China State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China Full list of author information is available at the end of the 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made available in this article, unless otherwise stated in a credit line to the data Liu et al BMC Genomics (2021) 22:120 Page of 18 (Continued from previous page) Conclusions: Our data indicated these transporter proteins play an important role in body color of P xuthus Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies Keywords: ATP-binding cassette (ABC) transporters, Rab transporters, Papilio xuthus, CRISPR/Cas9, Transcriptome Background Butterflies display a diversity of body color among and within species in their different development stages, especially larvae and adults, serving diverse and crucial functions in sexual selection, predator avoidance, and thermoregulation [1] Like other insects, the metabolites from three main pigmentation pathways (i.e., tyrosinederived melanin, tryptophan-derived ommochromes and guanine-derived pteridines) and other related metabolites (i.e., uric acid etc.) mainly contribute to color pattern in butterflies [2, 3] Tyrosine-derived melanin metabolites are well known to play central roles in body color of all kinds of insects [4] Tryptophan-derived ommochromes and guanine-derived pteridine have been verified to contribute to eye color in many insects independently (e.g., flour beetle Tribolium casstaneum) [5–9], or jointly (e.g., fruit fly Drosophila melanogaster, cotton ballworm Helicoverpa armigera, water strider Limnogonus franciscanus) [10–14]; they also play important roles in coloration of larval epidermis and wing etc [15, 16] In addition, the fourth pigment, i.e., papiliochrome, is unique to swallowtail butterflies (Papilionidae) and biosynthesized from one tyrosine-derived metabolite (N-β-alanyldopamine) and one tryptophan-derived metabolite (kynurenine) [17, 18] In insects, pigments are biosynthesized in epidermal cells through a development process that includes pigment patterning and synthesis [18] During the process, one of the key steps is the transportation of pigment precursors or related metabolites, which are usually executed via such transporter such as ATP-binding cassette (ABC) proteins, Rab proteins etc [12, 19] ABC family is one of the largest transporter families and present in all living organisms [20, 21] They can be classified into seven subfamilies in human [22, 23] or eight subfamilies (A-H) in arthropods [24] The majority of these ABC proteins function as primary-active transporters For ABC transporters, ATP binding and hydrolyzing in the nucleotide-binding domains (NBDs) is a necessary process to transport a wide spectrum of substrates (e.g., amino acids, sugars, heavy metal ions and conjugates, peptides, lipids, polysaccharides, xenobiotic and chemotherapeutic drugs) via the integral transmembrane domains (TMDs) across lipid membranes [24, 25] Notably, ABCG subfamily includes such well-studied ABC members as white, scarlet and brown in D melanogaster, which are involved in the uptake of pigment precursors in ommochromes and pteridines pathways in the development of cells of Malpighian tubules and compound eyes [26–29] The functional experiments from such a few non-dipteran insects as Lepidoptera (including a few moths and one nymphid African butterfly Bicyclus anynana), Coleoptera, Hemiptera, Orthoptera also confirmed the important roles of these ABCG members (especially white and scarlet) in pigmentation [8– 10, 14, 30–35] It is very interesting that no morphologically mutated phenotypes were observed in H armigera of Lepidoptera after the brown gene was disrupted [10] Nevertheless, another ABCG gene, ok, a paralog of brown, was identified in Lepidoptera (B mori, H armigera) and verified to play an important role in the development of larval epidermis or/and adult eyes [3, 10] Another kind of notable transporter proteins are Rab proteins, which are small (21–25 kDa) monomeric GTPase/GTP-binding proteins and found in organisms ranging from yeast to humans with different gene copies [19] They are known to be involved in intracellular vesicle transport [36] Among 33 Rab genes identified in the genome of D melanogaster, Rab32/RP1, encoded by gene lightoid, plays an important role in eye color via participating in biogenesis or degradation of pigment granules [37–39] However, nothing is known for function of lightoid in other insects except for fruit fly and silkworm The experiments from Drosophila and other insects demonstrate the important roles of such transporter proteins as ABCG members and Rab proteins in pigmentations [12, 18, 19] However, it is still not sure whether these findings hold for swallowtail butterflies (Papilionidae), the most historically significant group of butterflies (Papilionoidea) because of their phylogenetic basal position to all other butterflies and their morphological diversity Moreover, it is not known how these transporter genes affect the expression profiling of other related genes In addition, we aim to test if these transporters contribute to the biosynthesis of papiliochrome in swallowtail butterflies by transporting the precursor (kynurenine) of tryptophan-derived metabolites, as postulated in our previous work [2] The swallowtail butterfly P xuthus is an intriguing species commonly used in butterfly research because of both their enigmatically morphological changes in ontogeny and their wellstudied biology as well as ease of breeding [2, 40–42] (2021) 22:120 Page of 18 alignment with those of D melanogaster and B mori (Fig 1; Additional file 1: Table S1 and Table S2) Like that of most other insects, the most expanded subfamily in P xuthus genome is ABCG (30% of total ABC, 17 members), and the next is ABCC (~ 21% of total), while the most expanded subfamily in other arthropods (e.g Arachnida, Branchiopoda, Copepoda) and even in human is ABCC (Additional file 1: Table S1) These data suggest ABCG may play a more important role in the evolution of diverse insects All ABC transporter genes of P xuthus vary in length from 1841 bp (Px_01485_ CG10226) to 33,147 bp (Px_12497_CG7627) and each of them possesses at least one nucleotide binding domain (NBD) (Additional file 1: Table S2) There are 21 full transporters (each full transporter including two NBDs and two transmembrane domains (TMDs)) in ABCA, ABCB and ABCC subfamilies, 28 half transporters (each half transporter including one NBD and one TMD) in ABCA, ABCB, ABCC, ABCD, ABCG, ABCH subfamilies, and seven atypical transporters (each only including ~ Here, we systematically identified potential ABC transporters and Rab protein family in the genome of P xuthus Then, we investigate the contributions of five of them, which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) in fruit fly, in the development of P xuthus via CRISPR/Cas9 technology which is widely used in insect [43] Combining comparative transcriptomics of mutants and wild-types, we provide new insights into the function of ABC transporters and small G-proteins in the morphological development of swallowtail butterflies Results Identification and phylogenetic analysis of ABC and Rab transporters in P xuthus 100 100 98 99 84 100 72 31 64 31 02 CG 52 64 90_ A00 59 G tet t CGx_0 BM 9_A Ate 094 P GI 96 27_ 005 B G2 60 GA 89 C x_0 M 316 P GIB 689 _CG 10 100 B G31 961 482 C _05 G Px 482 91_C 05203 00 10 81 CG _059 GA0 4822 0 Px IBM 0_CG 091 BG _1649 _CG32 Px 16489 23 100 10 Px_ 32091 32091 CG 17808_CG 35 100 Px_ MGA0120 IB 00 G 100 B 78 100 58 98 58 37 43 27 01 56 A 31 G M CG 52 IB 156 39_ 008 CB BG G3 160 GA 82 C x_ B M AB G1 P I 24 _C 414 10 BGG18 7609 A00 50 C x_0 MG Mdr r50 10 100 P GIB 23_ Md B 85 72_ dr5 CG _132 71_M 07494 94 00 100 Px _132 GA0 Px IBM BG 22 0 CG110181 dr49 100 10 CG 3879_M dr49 CG 01396_M 100 52 Px_ A0094 G M BGIB 58_Mdr49 Px_144 GA011228 BGIBM dr49 Px_01486_M Px_09896_Mdr49 BGIBMGA000725 73 32 Px B _1 Px GIB 245 Px _14 MG 4_C BG _143 383 A0 G4 IB 73 _CG 07 562 M _C 78 GA G 62 CG 011 627 CG 105 220 82 BG BG IBMG CG1 1189 05 10 IBM A 18 10 Px_ GA00103397 BGI 059 103 BM 02_M 31 95 00 Px_ GA01 RP 99 0590 033 3_M R 100 Px_1 6284 CG6214P _C 00 BGIB 100 MGA G7806 010636 100 90 100 BGIBMGACG7806 006882 75 100 Px_07339_S ur 100 CG5772_Sur Px_01485_CG10226 96 BGIBMGA000724 94 10 47 69 10 88 100 63 100 100 61 CG1703 BGIBMGA007869 Px_10653_CG17 03 CG9330 100 BG 100 Px IBMGA006964 _07205 ABCF _CG93 CG92 100 30 BGIB 81 47 Px_0 MGA00 100 CG 8532_ 2004 98 BG 5651_ CG9281 100 Px IBMG Pixie 100 CG _0429 A010 10 BG 231 7_pix 129 10 C IB ABC 10 Px G12 MGA E BG _10 703 0046 99 16 C IB 21 10 BGG42 MG 8_CG Px IB 25 A0 12 C _ M 12 703 A BC 68 Px G7 053 GA _1 95 66_ 00 D 02 Hm 547 81 t -1 _A BC B7 100 100 C 18 56 57 BC 100 47 96 A 44 Px B _06 BG GIB 94 P I M 9_ Px x_0 BM GA CG _ 69 G BG 1125 53_ A00 041 718 BG IBM 8_C CG 418 88 40 IB GA G3 605 MG 41 A 09 20 90 CG 0095 503 88 02 C 10 100 CG G14920 Px_ 4 Px_ 06952_ CG18672 76 10 03 C Px_1 164_CG G17181 49 32 12 46 CG99 58_CG9 186 BGIB 90_Snust990 56 o MGA Px 01076 rr 100 ABCH _01414_CG33 970 BGIBMGA 65 99 010726 CG33970 Px_01415_CG1114 92 100 100 BGIBMGA010825 100 CG11147 CG31793 100100 100 CG9270 100 CG14709 CG45622 59 100 65 79 31 G C 27 76 G C 9 100 8 CG 627 68 37 _CG 0849 2497 100 Px_1 MGA0 07769 627 IB A G G B 23 99 96 85 BM CG7 59 BGI 2656_ 0033709 A Px_ IBMG CG1 7793 0 10 BG 2436_ A00 0779 62 10 G Px_ GIBM GA CG4 7735 B IBM 35_ 00 78 98 BG 124 GA CG 773 89 _ M Px GIB 439_ A0 G57 784 B _12 MG C 007 Px GIB GA B M IB BG 100 A CG3327_E23 100 BGIBMGA000220 31121 100 Px_10204_CG 31121 CG 100 11069 05_CG 100 Px_102 GA0004729 M 87 100 1106 BGIB G C 1 0072 59 MGA 07308 32 BGIB MGA0 1718 44 35 G 99 BGIB1770_C 00721 1 9 Px_ BMGAA0072718 I BG BMG _CG16052 74 I BG 11769 CG 171 CG 3121 16 Px_ 10 CG 42 908 CG G8 1731 C 05 CGCG6 278 _ 71 A0 21 G _1 M Px GIB B A BC CG2759_White Px_03417_w BGIBMGA002922 white CG4314_S 100 Px_03415 carlet 100 B _st 79 GIB scarlet Px_1 MGA002924 71 100 BGIB7844_st 83 CG1 MGA00 ok 100 Px_ 7632_b 2581 BG 17845 rown 94 CG IBM _w 100 24 Px_ 9664 GA002 712 163 brow B 83 n Px GIB 06_ 10 C _16 MGA CG9 10 CGG966 305_ 010 664 10 CG 557 BG 17 966 10 P I 64 C x_ BM P G 16 G BG x_0 585 304_ A01 AB CG 055 IB 596 CG 17 M 0_ 64 G C A G 00 58 52 53 26 We comprehensively identified copy number of ABC gene family in the genome of the swallowtail butterfly P xuthus The genome has a total of 56 ABC transporters, which, like those of other insects, is classified into eight subfamilies (A-H) based on the multiple sequence 96 Liu et al BMC Genomics Papilio xuthus Bombyx mori Drosophila melanogaster Fig Phylogenetic tree of ATP-binding cassette (ABC) transporters of Papilio xuthus (Px), Bombyx mori (BGIBM) and Drosophila melanogaster (CG) The maximum likelihood tree was calculated on the basis of multiple alignments of the ABC transporter protein sequences All ABCs were clustered into eight subfamilies (ABCA-H) The green pentagrams represent the genes belongs to the P xuthus, the blue circles indicate the genes among B mori, and the orange boxs show the genes in the genome of D melanogaster Four Px genes highlighted in grey in ABCG subfamily were selected to investigate their function in the development of P xuthus via CRISPR/Cas9 gene-editing technology Liu et al BMC Genomics (2021) 22:120 Page of 18 melanogaster (33) [38] and nematode Caenorhabditis elegans (29), but near to that in human (70) [44] This is the first two cases of genome-wide identification of copy number of Rab gene in lepidopteran insects Phylogenetic analysis indicates that both genomes showed an expansion of specific-lineage close to clades of Rab32 (lightoid) and Rab23 (Fig 2) Both clades of Rab32 and Rab23 include single-copy orthologs within three investigated species Among them, Px_17846_ltd, together with its ortholog of silkworm (BGIBMGA002711), is single-copy orthologous to lightoid of fruit fly (i.e Rab32), which was found to be essential in eye development, autophagy and lipid storage via vesicle trafficking regulation in Drosophila [37, 39] and in silkworm’s response to bacterial challenge [45] Rab23 is involved in the regulation of the number and planar polarization of the adult cuticular hairs in Drosophila [46] and lipid metabolism [39] 100 100 28 38 22 eb 57 Rh 34 9_ 00 2l 03 GA Rap 257 _1 M 6_ 06 Px GIB 381 GA0 B x_1 M _R P GIB 020 _Ric 980 B _02 636 003 10 Px 07 GA 7169 Px_ IBM GA00 a 9 BG IBM 1_Ral 18 BG 1345 A0099 10 Px_ IBMG 00872 43 57 BG BMGA as64B I R 77 BG 08827_ 9923 10 Px_ MGA00 74 BGIB 77_Ras64B 97 Px_13 100 100 99 95 54 75 49 28 28 Px_17373_Rab27 BGIBMGA012463 Rab27 Px_12840 _Rab3 Rab3 100 B 48 P GIBMGA00 x_12 0295 RabX 841_Rab 100 BG 100 Px IBMG A _ Rab 17436 002449 _ 79 Rab Px X4 BG _ 91 I 10 98 B Rab8 BMG _Rab1 A0 0 GI 046 P B 66 10 R x_ M B ab 1572 GA0 Px GIB 3_R 048 ab _1 M 66 GA 44 _R 07 ab 021 40 cluster_C 73 ste r_ B 100 P BG x_0 IB 722 M 9_ Ra GA R b1 BG 00 ab2 Px IBM 6 _0 26 GA Ra 956 58 96 _R 0019 b26 BG ab - 03 IB R 99 Px_ MGA Rab P3 037 98 63_ 09361 Rab BGI 18 BM Ra 30 Px_1 GA006 b30 5216 _Rab 21 10 00 14 Rab1 100 Rab BGIBM GA0019 37 71 28 Px_02634 _Rab 100 98 BGIBMGA006 641 100 Px_01846_Rab4 76 100 04 29 01 35 GA ab b3 M 5_R Ra GIB 196 ab 15 B x_1 _R P ab1 078 005 R _10 GA 10 99 M x P B I BG Fb 90 00 Rab9 bX Ra 9Db Rab 9Fa Rab E 100 Rab D 100100 b9 69 47 Ra b11 ab11 Ra 039_R Px_02 GA002209 100 GIBM 77 B Rab39 280 BGIBMGA011 Px_04756_Rab39 Rab4 77 clu 30 100 BGIBMGA007505 Px_09252_Rho1 966 100 BGIBMGA001 _Rab23 10 Px_09272 2644 100 GA01 BGIBM Rab23 81 00 11 0027 d MGA lt 48 BGIB _17846_ 32 Px Rab 51 100 006 ran oid GA _ IBM 7680 ab8 100 light G B 4_R 60 10 57 33 72 Px_ 112 014 1F 92 Px_ GA rf5 A IBM 24_ 036 2F 10 BG 141 A0 rf10 36 _ MG A 21 Px _ B I 93 22 BG 066 GA CG 107 _ BM 9_ 00 Px I A BG 177 MG _ Px GIB B D r_ te us cl P BG x_1 Px IBM 651 _0 _ 17 GA Ar BG Px_ 20_ 000 f84 F BG IBM 131 Arf8 786 Px IBM GA0 1_G 4F _12 GA 10 ie 00 953 00 94 _C 036 53 G CG 4789 00 10 Px_ 07 CG15 89 84 Px_ 230_R 399 a 67 BGIB 13227_ b26 99 MGA RfC3 0054 14 Rab 100 BGIBM 15 GA00 X5 7712 Px_123 99 56_Rab 100 Rab7 BGIBMGA007 672 99 90 Px_10558_Rab9 17 100 Rab9 RabX6 X6 100 Px_15083_Rab 000682 90100 BGIBMGA ab21 66 _R 84 Px_081 Rab21 96 6_chrw6 31 Px_0 A00326 MG RabX1 100100 12 BGIB 45 35 A00 b5 MG 96_Ra b5 B I BG _043 Ra 23 100 Px 84 001 ab5 10 GA 6_R ab6 M B R I _ 10 BG x_08 169 035 b6 a P _1 A0 R 203 Px MG 02 R P B I GA Rab BG M _ IB 34 BG 020 _ x P Px_12525_RhoL Px_05147_Rho BTB BGIBMGA 006475 Px_0 30 62 100 BG 4000_Mtl IBM Px_0 GA012295 63 97 100 Px_16 59_Cdc42 55 BGI 711_Ra 16 Px_ BMGA c1 30 99 BG 08971_ 007110 Px IBMG Rgk2 65 Px _062 A00 32 10 BG _121 22_CG 0952 516 Px IBM 38_ 67 P _12 G CG1 10 10 Px x_17 861 A000 3375 _ _ B C BG GIB 765 7_C G85 IB MG 7_C G86 00 M A G1 41 GA 00 33 00 657 75 47 03 NBD but not TMD) ABCE and ABCF subfamilies contain atypical ABC transporters characterized by a pair of linked NBDs with no TMDs In addition, three ABC genes (ABCA: Px_03164_CG32186; ABCB: Px_01485_CG10226; ABCG: Px_10205_CG11069) also show ABC domains with only one NBD (Additional file 1: Table S2) Seventeen members of ABCG span in five scaffolds with to genes in each, and 16 of them are typical half transporters, except one with a single NBD (Px_10205_CG11069) (Additional file 1: Table S2) Phylogenetic analysis indicates that the four pigmentation related genes (scarlet, white, brown and ok), which are all single-copy in P xuthus, form a cluster among three species (P xuthus, B mori, and D melanogaster) (Fig 1) We identified 58 and 51 Rab members in the genomes of P xuthus and B mori, respectively (Additional file 1: Table S3), which are nearly twice as much as that in D 65 cluster_B clu ste r_ A Papilio xuthus Bombyx mori Drosophila melanogaster Fig Phylogenetic tree of Rab family of Papilio xuthus (Px), Bombyx mori (BGIBM) and Drosophila melanogaster (CG) The green pentagrams represent the genes belongs to the P xuthus, the blue circles indicate the genes among B mori, and the orange boxs show the gene in the genome of D melanogaster Lightoid, highlighted in red in cluster D, was selected to investigate its function in the development of P xuthus via CRISPR/Cas9 gene-editing technology Liu et al BMC Genomics (2021) 22:120 Page of 18 Somatic mutations of four ABCG transporters and one Rab protein in P xuthus The experiments from Drosophila and other insects demonstrate the important roles of five genes (scarlet, white, brown, ok and lightoid) in pigmentations [12, 18, 19] However, it is still not sure whether these findings hold for Papilionidae butterflies To investigate the potential functions of these transporter proteins in swallowtails butterflies, we performed CRISPR/Cas9 gene-editing for these five single-copy genes (white, scarlet, brown, ok and lightoid) using P xuthus as a model (Tables and 2; Figs 3, 4, 5, 6; Additional file 1: Tables S4–5; Additional file 2: Fig S1; Additional file 3: Fig S2) Mutations in the white gene We injected the mixed sgRNAs of three target sites (2nd exon: T_8165, T_8232; 3rd exon: T_8700) of white gene and Cas9 protein into eggs (Table 1; Additional file 1: Table S4) Compared with wild-types, the edited individuals showed some morphologically changes in both larvae and adults of G0 generation (directly developing from injected eggs) (Fig 3) In details, the mosaic mutants of the fourth-instar larvae showed a disappearance of V-shaped white markings in their dorsal sides (Fig 3a), which originally made them mimic to birds dropping to avoid predators The fifth-instar larvae showed a translucent cuticle instead of green camouflage coloration in wild-types (Fig 3b) We also observed that the testis of the fifth-instar larval mutants showed part or complete disappearance of white external sheath and red follicular epithelium (Fig 3c) No changes in shape and color were observed in the pupa and adult wing (Additional file 2: Fig S1B) Some of adults developed from larval mutants showed abnormal eyes with white and black mosaic color stripes instead of black eyes in wildtypes (Fig 3d) Mutations in the scarlet gene We injected the mixed sgRNA of two target sites (2nd exon: T_661, T_684) of scarlet gene and Cas9 protein into eggs (Table 1; Additional file 1: Table S4) No morphological changes were observed in the injected G0 larvae, but 36.36% (four individuals: three females and one male) emerged adults of G0 showed abnormal eyes with mosaic stripes of white and black/red-brown (Table and Fig 4b, c), but their wing pattern show no changes (Additional file 2: Fig S1C) Because of the discordance of emergence time for male and female mutants, we made a cross of a wild-type female (Fwt) adult with G0 male adult mutant (MG0) of mosaic white and redbrown eye color and get six G1 adults, for which no morphological change was observed We further made a cross between G1 female and male adults (FG1, MG1) to obtain four G2 adults (one female and three males), all of which showed the complete white eyes (Fig 4d) Mutations in the brown and ok genes We injected the mixed sgRNA of two target sites (3rd exon: T_16066; 5th exon: T_15076) of brown gene and Cas9 protein into eggs (Table 1; Additional file 1: Table S4) We observed that 22.86% of the fifth-instar larvae of G0 showed a translucent cuticle (Table and Fig 5b), similar to that of white mutants However, unlike those of white mutants, mutated fifth-instar larvae of brown have normal testis, and all mosaic G0 adults have normal black eyes and wing (Additional file 2: Fig S1D) We also injected the mix sgRNA of two target sites (3rd exon: T_4354, T4454) of ok gene and Cas9 protein into eggs (Table 1; Additional file 1: Table S4) Similar to that of its close paralog brown mutants, G0 fifth-instar larvae of ok also showed a translucent cuticle (Fig 5c), but normal testis and normal wing pattern (Additional file 2: Fig S1E), and the mosaic G0 adults also have normal black eyes Mutations in the lightoid gene We injected the mixed sgRNAs of four target sites (2nd exon: T_2307, T_2271; 3rd exon: T_3154, T3097) and Cas9 protein into eggs (Table 1; Additional file 1: Table S4) Like that of white disruption, we observed the Table Summary of injected sgRNA and Cas9 mRNA and mutants in CRISPR/Cas9-gene editing experiment The bracket is the number of larvae and adult which showed phenotypic changes Gene Gene ID Target sites Final concentration Injected Hatching larva L5 Mutation Pupa Adult Mutation of injected sgRNA eggs (hatching rate) (Mutants) rate in L5 (Mutants) rate in a (ng/μl) (%) adult (%) white Px_03417_w T_8165, T_8232, T_8700 990 245 76 (31.02%) 69 (21) 30.43 52 39 (5) 12.83 scarlet Px_03415_st T_661, T_684 814 260 48 (18.46%) 28 (0) 24 11 (4) 36.36 brown Px_17845_w T_15076, T_16066 925 485 119 (24.54%) 70 (16) 22.86 65 61 (0) ok Px_17844_st T_4354, T_4454 925 250 40 (16%) 12 (3) 25 10 (0) lightoid Px_17846_ltd T_2271, T_2307, T_3097, 800 T_3154 260 98 (37.69%) 27 (15) 55.55 27 25 (0) 31 18 (58.06%) 12 NA 10 10 NA Control NA a NA Cas9 protein concentration is 1000 (ng/μl) NA Liu et al BMC Genomics (2021) 22:120 Page of 18 Table CRISPR/Cas9 induced phenotype changes of five genes in Papilio xuthus Tissue Wild-type white mutant scarlet mutant brown mutant ok mutant lightoid mutant The epidermal tissues of brownish black the fourth instar larvae (L4) integuments with white V-markers white V-markers change to transparent NA NA NA white V-markers change to transparent The epidermal tissues of the fifth instar larvae (L5) green transparent mosaic NA transparent mosaic transparent mosaic transparent mosaic Testes of L5 red white, white and red mosaic NA NA NA white and red mosaic Eyes of adults black white and black mosaic white and black, pink and white, white NA NA NA Wings of adults black and yellow NA NA NA NA NA disappearance of V-shape white markings in the fourthinstar larvae of G0 (Fig 6a) and a translucent cuticle in their fifth-instar larvae (Table 1, Fig 6b), but the adult wing pattern is unaffected (Additional file 2: Fig S1F) Anatomy of these mutated fifth-instar larval testis also showed partially disappearance of white external sheath and red follicular epithelium (Table 1, Fig 6c), just like that of white mutants But unlike white mutants, no morphological changes were observed in G0 adults of lightoid developed from the fifth-instar larval mutants Genotyping of mutants Genomic DNA was isolated from mutant adults/larvae, and PCR amplicons including the region of target sites were cloned and sequenced The sequenced data validated that these five genes were disrupted in their corresponding mutants (Additional file 1: Table S5; Additional file 3: Fig S2) All six G0 mutants of white (three 5th-instar larvae and three adults) showed the disruption (10–100% mutated rate) in all or part of target sites with numerous deletions (1–84 bp), inserts (1–30 bp) or substitutions in the targeted regions (Additional file 3: Fig S2A) Four G2 adult mutants of scarlet showed a deletion of 8–11 bp in the target site T_684 in all clones (Additional file 3: Fig S2B), suggesting that these G2 adults may be homozygous mutants of scarlet locus All G0 larval mutants of brown were disrupted (mutated rate: 80–100%) in two target sites (T_15076 and T_16066) with numerous deletions (1–52 bp), inserts (2–21 bp) or substitutions (Additional file 3: Fig S2C) All three larval mutants of ok were disrupted in target sites T_4354 and T_4454 with numerous deletions (2–25 bp), inserts (3–8 bp) or substitutions (Additional file 3: Fig S2D) All three larval mutants of lightoid gene showed numerous deletions (1–24 bp), inserts (3–25 bp) or substitutions in all or part of target sites (T_3154, T_3097, T2307 and T_2271) (Additional file 3: Fig S2E) Transcriptome profiling of the mutants To further investigate transcriptomic profiles involved with these pigment-related transporters, we dissected the epidermal tissues of the fifth-instar larval mutants induced by the disruption of white, brown, ok and lightoid genes and head tissues of adult mutants induced by the disruption of scarlet gene for transcriptomic sequencing In total, 172 Gbp transcriptomic data and average 51 M reads per library were generated for 22 individuals (Additional file 1: Table S6), which are verified to be mutated at genomic DNA level The average mapping depth of RNA reads in exon regions varied from 125× to 204× with the reads alignment ratio varying at 83.56– 90.80% for both mutants and wild-types (Additional file 1: Table S7), suggesting that the transcriptomic data is adequate for transcriptomic analysis and identification of differentially expressed genes (DEGs) between mutants and wild-types Variations in transcripts in mutants of five disrupted pigment transporting genes The analysis of the transcriptomic sequencing depth indicate that most mutated individuals showed a deletion of several bases or reduced mapping depths in target regions than those of wild-types (Additional file 4: Fig S3) Further analysis of nuclear variant calling (including SNPs and INDELs) for all the samples confirmed INDE Ls in the transcripts of most target regions, and also identified some SNP mutation in the regions of some targets (Additional file 1: Table S8) Specifically, a homozygous 8-bp deletion was identified at the region of target site T_684 in the transcripts of four investigated scarlet mutants of G2 (Additional file 1: Table S8; Additional file 4: Fig S3B), as shown in PCR genotyping (Additional file 3: Fig S2B) For G0 mutants of other four genes (white, brown, ok, and lightoid), a deletion of several bases or reduced mapping depths in target regions can be detected (Additional file 4: Fig S3A, C, D, E) To further explore how the mutations introduced by Liu et al BMC Genomics (2021) 22:120 Page of 18 Fig CRISPR/Cas9 disruption of white gene resulted in mosaic depigmented phenotypes in larval epidermis, testes and adult eyes of P xuthus a The fourth instar larva (L4) b The fifth instar larva (L5) c Testes of the fifth instar larva d Adult eyes Left panel: wild types; right panel: white mutants The area with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red circle in the panels of (a) and (d) and in red square (b) Testes with obviously morphological mutation in mutants and their corresponding part in wildtype were highlighted in red arrow (c) Scale bars: mm The photo credit is provided by Zhiwei Dong CRISPR/Cas9 gene-editing affect the expression of the genes, the expression level (Fragments per Kilobase Million, FPKM) of the exon involved with target sites were acquired by manually distinguishing the mutated reads and normal reads in the mutant samples (Fig 7) Our data indicated that except T_16066 and T_15076 of brown, the exons of all other target sites showed a lower expression in mutated individuals than in wild-type individuals Among them, the exons of most target sites (excl T_8165 and T_8232 of white) showed a significantly (t-test, Pvalue < 0.05) decreased expression of normal transcript in mutated individuals than in wild-type individuals (Fig 7a, b, d, e), suggesting that the normal transcripts were less transcribed after CRISPR/Cas9-induced mutations, thus leading to the down-expression of the five genes For T_ 16066 and T_15076 of brown, they showed a slightly higher expression of normal transcripts in mutant samples than in wild-type samples (Fig 7c), which may be caused by the dosage compensation [47] In summary, these transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 at genomic level can produce abnormal expression with accumulation of abnormal transcripts and decrease or dosage compensation of normal transcripts at transcriptomic level ... known how these transporter genes affect the expression profiling of other related genes In addition, we aim to test if these transporters contribute to the biosynthesis of papiliochrome in swallowtail. .. copy number of ABC gene family in the genome of the swallowtail butterfly P xuthus The genome has a total of 56 ABC transporters, which, like those of other insects, is classified into eight subfamilies... subfamilies (ABCA-H) The green pentagrams represent the genes belongs to the P xuthus, the blue circles indicate the genes among B mori, and the orange boxs show the genes in the genome of D melanogaster