Arthropod Pax Gene Evolution: A Role for Vanessa Cardui Twin of Eyeless in Eye Development

Ximena Gutiérrez Ramos; Leslie Pick

doi:10.1093/molbev/msaf220

. 2025 Sep 12;42(10):msaf220. doi: 10.1093/molbev/msaf220

Arthropod Pax Gene Evolution: A Role for Vanessa Cardui Twin of Eyeless in Eye Development

Ximena Gutiérrez Ramos ¹, Leslie Pick ^2,^✉,^b

Editor: Patricia Wittkopp

PMCID: PMC12486386 PMID: 40966593

Abstract

Pax genes encode transcription factors involved in diverse processes. First identified in Drosophila, they have been found throughout the animal kingdom, suggesting highly conserved functions. Pax proteins are defined by a DNA-binding PRD domain along with variable presence of a homeodomain and octapeptide motif. Although some Pax genes have been studied in selected arthropod groups, less is known about phylogenetic relationships among arthropod Pax genes. Here, we analyzed their distribution and classification and established the painted lady butterfly, Vanessa cardui, to study Pax gene expression and function. Our phylogenetic analysis grouped arthropod Pax genes into 6 clades: Pax3/7, Pax1/9, Poxn, Pax6-like/eyg, Pax2/5/8, and Pax4/6. This large-scale analysis confirmed that the Pax3/7 gene paired was lost in Lepidoptera, which retain other Pax3/7 family members as well as all other Pax groups. Expression of Vcar-Pax genes during Vanessa embryonic development was largely similar to previous reports for Drosophila. To test functional conservation, we focused on the eye development master regulator, twin of eyeless (toy). Vcar-toy was expressed in the head lobes and embryonic RNA interference resulted in loss of larval eyes. In addition, Vcar-ey, a candidate downstream target of toy, was downregulated after Vcar-toy knockdown. Ectopic expression of Vcar-toy in Drosophila resulted in ectopic, Drosophila-like adult eyes, supporting the notion that gene regulatory networks regulating larval and adult eyes are conserved and also shared between Vanessa and Drosophila. Overall, these results suggest that Pax genes are highly conserved in arthropods and provide a butterfly model to study eye regulatory networks in Lepidoptera.

Keywords: Pax genes, butterfly, Pax 6, gene family, gene evolution

Introduction

Pax proteins are important transcription factors with a wide variety of roles in development, homeostasis, and disease (reviewed in [Blake and Ziman 2014; Thompson et al. 2021]). The founding member of this family, paired (prd) was identified in Drosophila melanogaster (Drosophila), leading to the discovery of a family of related proteins that have diverse functions in Drosophila development (Bopp et al. 1986; Frigerio et al. 1986; Baumgartner et al. 1987). Pax genes have since been found throughout the animal kingdom, including Porifera, Cnidaria, Annelida, Arthropoda and Chordata (Balczarek et al. 1997; Hoshiyama et al. 1998; Breitling and Gerber 2000). These genes encode a conserved paired domain (PRD), an ∼128 amino acid DNA-binding domain at the N-amino termini of PRD family proteins. The PRD domain includes 2 subdomains, the PAI and the RED subdomains, separated by a linker region, each of which folds into a DNA-binding helix-turn-helix (Chalepakis et al. 1991; Treisman et al. 1991; Czerny et al. 1993; Epstein et al. 1994a; Czerny and Busslinger 1995; Xu et al. 1995, 1999; Jun and Desplan 1996; Luscombe et al. 2000). In addition, some Pax proteins have complete or partial homeodomains, the Paired-class homeodomain (HOX domain). These HOX domains share a serine at position 50 of the homeodomain, distinguishing them from other homeodomains (Bopp et al. 1986; Frigerio et al. 1986). The PRD and HOX domains can each bind DNA independently, although they may also interact cooperatively, enabling binding to different types of target sequences that contain binding sites for one, the other, or both domains (Treisman et al. 1991; Jun and Desplan 1996) (reviewed in [Mayran et al. 2015]). These domains interact with different protein partners, further diversifying their regulatory potential (Czerny et al. 1993; Lechner and Dressler 1996; Underhill and Gros 1997; Hollenbach et al. 1999; Eberhard et al. 2000). In addition to the PRD and HOX domains, some Pax proteins have an 8-residue motif, the Octapeptide/Engrailed Homology-1 (OP/EH-1) motif (Burri et al. 1989; Smith and Jaynes 1996; Tolkunova et al. 1998), first identified in Drosophila Pax proteins, that is located downstream of the PRD domain and between the PRD and HOX domains in proteins that contain all 3 (Noll 1993; Copley 2005; Underhill 2012). The OP appears to mediate transcriptional repression by the recruitment of Groucho/Transducin-Like Enhancer of split family (Lechner and Dressler 1996; Eberhard et al. 2000). Pax7 proteins also have an Orthopedia, Aristaless and Rax motif at the C- terminus of the HOX domain that has a role in transcription inhibition (reviewed in [Underhill 2012]).

The Pax gene family is an ancient gene family, thought to have originated from the insertion of a transposable element encoding a PRD domain-like sequence into a gene encoding a PRD-type homeodomain and an OP in an early metazoan (Breitling and Gerber 2000; Vorobyov and Horst 2006; Wang et al. 2010) (reviewed in [Underhill 2012; Bürglin and Affolter 2016; Thompson et al. 2021]). Since that time, Pax genes have diversified across the animal kingdom, such that different combinations of the PRD, HOX, and OP domains are present in different subfamilies of Pax genes. The exact cohort of Pax genes varies within phyla due to gene duplication and loss. In addition, alternate splicing results in unique isoforms for many Pax genes, often including different combinations of domains for the same gene (Epstein et al. 1994b).

Pax genes have been classified based on the structure of the domains they encode, sequence similarity, genomic organization, and likely evolutionary origin (Gruss and Walther 1992; Noll 1993; Wang et al. 2010). For our analysis, we began with the classification of Pax genes used by Blake & Ziman (Blake and Ziman 2014): Group I Pax genes encode a PRD domain and an OP but no HOX domain, represented in vertebrates by Pax1 and Pax9, and in insects by Drosophila melanogaster (Dmel) Pox meso (Poxm). Group II Pax proteins encode a PRD domain, an OP and a partial HOX domain, represented by vertebrate Pax2, Pax5 and Pax8, and Dmel shaven (sv). Group III Pax genes encode a PRD domain, OP and full HOX domain, represented by vertebrate Pax3 and 7, and Dmel gooseberry (gsb) and gooseberry neuro (gsbn), as well as other genes related by sequence, such as Dmel prd, which lacks the OP. Group IV Pax genes encode PRD and HOX domains but lack the OP, represented by vertebrate Pax4 and 6 and Dmel twin of eyeless (toy) and eyeless (ey) (reviewed in [Noll 1993; Blake and Ziman 2014; Thompson et al. 2021]). Lastly, there are 3 Drosophila Pax genes that are not present in the vertebrates: Pox neuro (Poxn), Pax6-like or eyegone (eyg), and twin of eyegone (toe). These form Group V (Poxn, [Balczarek et al. 1997]) and VI (eyg and toe) (Jun et al. 1998; Aldaz et al. 2003) in our analysis. These genes were likely lost in vertebrate lineages (Friedrich and Caravas 2011).

Arthropod Pax genes have been best studied in Drosophila, where they were first characterized in the 1980s based on their roles in embryogenesis. Dmel-prd, a classic pair-rule gene, was the first PRD domain-encoding gene identified, followed by the identification and study of other family members, such as Dmel-gsb, which is expressed in segmental stripes and functions as a segment polarity gene in Drosophila (Nüsslein-Volhard and Wieschaus 1980; Kilchherr et al. 1986). Other Pax family members also play roles in Drosophila embryonic development such as gsbn, involved in the development of the central nervous system; Poxm, specification of the somatic mesoderm; and Poxn, with roles in central and peripheral nervous systems (Nüsslein-Volhard and Wieschaus 1980; Bopp et al. 1986; Frigerio et al. 1986; Kilchherr et al. 1986; Bopp et al. 1989; Duan et al. 2007). prd itself has been characterized in other arthropods, where expression and/or functional studies point to a highly conserved role in pair-rule patterning (Osborne and Dearden 2005; Choe et al. 2006; Keller et al. 2010; Xiang et al. 2015). Our lab previously showed that an exception to this is found in the mosquito lineage, in which no prd ortholog could be identified (Cheatle Jarvela et al. 2020). Further, in the mosquito Anopheles stephensi, gsb has taken on pair-rule expression and functionally replaced the pair-rule role of prd, while retaining its role as a segment polarity gene (Cheatle Jarvela et al. 2020). In the hemipteran Oncopeltus fasciatus, like other Drosophila pair-rule gene orthologs, prd is expressed segmentally, as is gsb (Reding et al. 2019). The individual roles of prd and gsb remain unclear in more basally branching insects and noninsect arthropods, as most studies of these genes in these species were done using an antibody that recognizes the Pax3/7 HOX domain and thus were unable to distinguish family members. The Pax3/7 genes thus identified have been referred to as pairberry1 (pby1) and pairberry2 (pby2) and are expressed in segmental stripes in a basally-branching insect, several crustaceans, a centipede, and several chelicerates (Davis et al. 2001, 2005; Dearden et al. 2002). However, within Lepidoptera, a group more closely related to Drosophila, no prd ortholog was identified in the genome assemblies of several species: Danaus plexippus and Manduca sexta (Cheatle Jarvela et al. 2020), the butterfly Bicyclus anynana (Matsuoka et al. 2023), and the silk moth Bombyx mori (Yang et al. 2024).

Due to its widely conserved role in eye development (reviewed in [Gehring and Ikeo 1999]), the Pax gene that has received the most attention is Pax6. First identified in mammals (Hill et al. 1991; Walther and Gruss 1991; Ton et al. 1992), Pax6 is the vertebrate ortholog of Dmel-ey (Quiring et al. 1994) and has been identified in the coral, jellyfish, nematode, zebrafish, frog, chicken, rat, and human genomes (Dressler et al. 1988; Burri et al. 1989; Catmull et al. 1998; Miller et al. 2000; Suga et al. 2010). Mutations in Pax6 result in eye abnormalities in vertebrates: in mouse, the gene was named Small eye (Sey), based on the reduction of eye size in heterozygous mutant animals and the absence of eyes in homozygotes (Hill et al. 1991). In humans, Pax6 mutations are responsible for aniridia, a condition affecting the iris, optic nerve and lens (reviewed in [Landsend et al. 2021]). Ectopic expression of Dmel-ey or of vertebrate Pax6 orthologs resulted in the formation of ectopic, Drosophila-like eyes, reflecting striking functional conservation of these genes across taxa (Halder et al. 1995). Thus, Pax6 is considered a master regulator of eye development (reviewed in [Gehring and Ikeo 1999]). Dmel-ey expression is activated in the eye primordia by another Pax6 family gene, toy, that, along with ey regulates a set of downstream target genes necessary for formation of the eye (reviewed in [Kumar 2009] and see below).

Here, we have examined the distribution of Pax genes in arthropods. Using annotated genomes, we identified Pax sequences from 47 arthropod species. We found that prd was lost in Lepidoptera, which have only 2 Pax3/7 genes, gsb and gsbn. We found that Pax6-like genes group with Dmel-eyg in a separate clade from Pax4/6 genes ey and toy. We next used Vanessa cardui, the painted lady butterfly, for expression and functional analysis. Poxn was expressed in the peripheral nervous system, while both gsb and gsbn were expressed segmentally. The Pax6 family genes ey, toy and Pax6-like were expressed in the eye primordia, as expected. Using embryonic RNA interference (eRNAi), we showed that Vcar-toy is important in the development of larval eyes and that mis-expression in Drosophila generated ectopic eyes.

Results

Evolutionary Distribution of Pax Genes Across the Arthropods

We identified Pax genes that are present across the arthropod lineage by searching for PRD domain sequences, the only domain present in all Pax genes. We searched for PRD domain sequences from 28 arthropods and 1 outgroup species. The species were chosen from arthropod orders that have at least 1 annotated genome available through NCBI. The selected species fell into the major arthropod groups: 2 chelicerates, 2 crustaceans and 24 hexapods (including 1 species of Collembola and 23 insect species), with the chordate Branchiostoma floridae as the outgroup. For the PRD domain search we used the pattern R-P-C-x(11)-C-V-S (Prosite: PS00034), the consensus amino acid sequence associated with the DNA-binding activity of the PRD domain that is conserved across different animals (Epstein et al. 1994a; Czerny and Busslinger 1995). This identified 239 PRD domain-encoding genes, including multiple sequences from the 29 selected species (Table S1).

To determine the relationship of these genes to each other, we performed a maximum likelihood phylogenetic inference analysis based on their PRD domains. The Pax genes distributed in 2 major nodes: a single clade in Node I and 6 nested clades in Node II (Fig. 1). Clade A within Node I includes all sequences corresponding to previously annotated Pax3/7 genes (Fig. 1 orange). In Node I are basally branching Pax sequences for the outgroup species B. floridae annotated as Pax7 (Fig. 1, square), and in clade A, sequences for the crustacean Eurytemora carrolleeae are on basal branches (Fig. 1, star).

Node II is the common ancestor for 6 clades where the rest of the Pax genes are distributed. All genes in clade B were previously annotated as Pax1/9 (Fig. 1 dark green). The outgroup species and noninsect arthropod sequences are not localized at the base of the clade, possibly because the sequences from these species lack complete PRD domains (see Figure S1). Branching from node III are 5 clades. Clade C and clade D are sister groups defined by node IV. Clade C includes all the arthropod genes previously annotated as Poxn (Fig. 1 light green). Sequences annotated as Pax6-like clustered in clade D (Fig. 1 blue-gray). However, the most basal branches of clade D include sequences annotated as Poxn (light green highlight) from Chelicerata, the common house spider Parasteatoda tepidariorum and the black-legged tick Ixodes scapularis, suggesting a common origin of Poxn and Pax6-like genes, although we cannot rule out previous mis-annotation of Chelicerate Poxn genes. Our analysis indicates that Pax6-like is present not just in insects, as previously proposed (Aldaz et al. 2003), but rather in all arthropods.

Node V includes the next 3 clades (E, F, G). Sequences annotated as Pax2/5/8 or sv all fell into clade E (Fig. 1 pink). Node VI includes clade F and clade G both of which contain sequences previously annotated as Pax4/6 (Fig. 1 blue). The PRD domains in clade F and G have 89.1% amino acid similarity, but the proteins differ in motif structure (see below).

Our phylogenetic analysis placed Pax3/7 genes at the base of the tree, suggesting that they diverged first from the ancestral Pax gene, followed by Pax1/9. Poxn and Pax6-like clades have a common origin in the arthropods but form distinct Pax groups. Similarly, Pax2/5/8 and Pax 4/6 are in sister clades branching from Node V, representing a common ancestor. Previous analyses of Pax evolution support our observations (Matus et al. 2007; Suga et al. 2010; Wang et al. 2010). Our results also support the proposal that there are 6 Pax gene groups (Friedrich and Caravas 2011; Bürglin and Affolter 2016), present in arthropods: Pax3/7, Pax1/9, Poxn, Pax6-like, Pax2/5/8, and Pax4/6. In addition, we found that all previously annotated Pax gene groups can be found in all arthropod orders used in this analysis.

Toy, Ey and Pax6-like/Eyg are Conserved Across the Arthropods

We next focused on the distribution of Pax4/6 genes across arthropods, all of which were in Node VI in sister clades F and G. This gene family is known to have a conserved role in eye development in vertebrates and invertebrates (Gehring and Ikeo 1999). Drosophila Pax6 orthologs toy and ey (Quiring et al. 1994; Czerny et al. 1999), both encode a PRD domain, an OP motif, and a HOX domain (Hill et al. 1991; Walther and Gruss 1991). Drosophila has a second set of Pax6 genes that are also involved in eye development, eyg and toe, but these are proposed to have arisen from a duplication event in insects (Aldaz et al. 2003; Jang et al. 2003; Bao and Friedrich 2009).

Analysis of the sequences in Node VI suggested that the genes in sister clade F correspond to toy and in G correspond to ey (Fig. 1, Table S2). To test this, we further analyzed the complete sequences previously annotated as Pax4/6 from the 29 previously selected species. We manually re-annotated them to identify PRD and HOX domains, and the absence/presence and type of OP motifs. We identified the Pax4/6 PRD domain of 86 sequences from 29 species. The Toy OP motif was defined by signature amino acids (E/D)-x-VY(D/E)KLR(M/I)(L/F) (Fig. 2a) and the Ey OP motif identified based on the consensus sequence is x(4)-(D/E)(K/R)(L/I)R(L/V/I)L (Fig. 2b), with the most common differences at positions 1 to 4. Figure 2c shows a subset of the data from Fig. 1, to highlight the genes found in Node III. The Toy OP motif was found in 27 genes from 25 of the 29 species. Three species have 2 genes with a Toy OP. For 4 species, no Toy OP was identified: the mosquitoes Aedes aegypti and A. stephensi, the copepod E. carolleeae, and the outgroup species B. floridae (Fig. 2c). One of these, B. floridae, has one gene encoding an Ey OP motif. Twenty-two species have a sequence with the Ey OP. Our analysis did not identify an Ey OP in chelicerate or crustacean sequences, more basally branching arthropods; rather, these have an OP sequence more similar to Toy (Fig. 2c, red dots). Note that for P. tepidariorum, 2 Pax6 genes have been identified, thought to have resulted from a duplication event (Schwager et al. 2017). In a phylogenetic analysis, these were found in sister groups (Samadi et al. 2015).

Next, we mapped the sequences with the new annotations to the phylogenetic tree (Fig. 2c). Sequences that we annotated as toy based on the presence of the Toy OP were found in clade F, except for some chelicerate and crustacean (P. tepidariorum, Daphnia pulex, Thrips palmi, and I. scapularis) sequences with Toy OPs that were found in the basal branches of clade G. Genes that we annotated as ey based on the presence of the EY OP were all found in clade G. Based on these OP-sequences, we suggest renaming these sequence in clade G to Toy-like, rather than Ey (Fig. 2c red dots).

Once we re-annotated sequences based on the OP sequence (Toy and Ey), all arthropod Pax6-like genes were found in clade D (Fig. 2c). Pax6-like genes encode a PRD domain, similar in sequence to Pax6 but do not have an OP motif. This clade D includes Drosophila Eyg and Toe. For these reasons, we refer to this clade as Pax6-like/Eyg. In sum, the Pax4/6 genes toy and ey are highly conserved in arthropods. Both ey and toy orthologs encode PRD domains, OP motifs and Hox domains, but the sequence of the OP motifs differentiates them. Finally, Pax6-like/eyg genes are likely present in all arthropods, not just insects.

gsb is the Only Pax3/7 Gene Conserved Across All Arthropods

Initial analysis of the Pax3/7 genes in Node A (Fig. 1; Figure S1) suggested that gsb is present in all arthropods examined; gsbn is present in Pancrustacea, as we observed it in crustacea and insects; but prd is only present in hexapods. This suggested that gsb is the most conserved Pax3/7 family member in the arthropods. To test this, we selected Pax3/7 sequences from an additional 19 species (Table S3). These included sequences reported as prd/prd-like in genome annotations and sequences identified experimentally, the latter including O. fasciatus (Reding et al. 2019), Glomeris marginata (Janssen et al. 2011), Parhyale hawaiensis (Davis et al. 2005) and Euperipatoides rowelli (Franke et al. 2015). For these 19 new species as well as the original 28 species (47 arthropods total), and 2 outgroup species, including 1 new species, the velvet worm (Euperipatoides rowelli), we manually identified the Pax3/7 PRD domain, HOX domain, and OP sequences. This identified 188 Pax3/7 sequences, with PRD domains ranging from 78 to 151 amino acids in length. Halyomorpha halys, the brown marmorated stink bug, has the longest PRD domain, and the shortest domain is an isoform from M. sexta, the tobacco hornworm. Each individual domain was then subjected to a maximum likelihood phylogenetic analysis (Figure S2 PRD domain, S4 OP motif and S5 HOX domain). In the PRD domain phylogenetic analysis, sequences from the outgroup species (B. floridae and E. rowelli; Figure S2a squares) are in the most basal branches of the tree, as expected, and next are Gsb sequences from Chelicerata and Myriapoda (Figure S2a triangle and ring).

We identified the consensus amino acids sequence for each type of OP motif (Figure S3). For Gsb, the OP consensus is x-H(T/S/N)I-x(4)-(G/A/S); for Gsbn, the OP motif is DY(T/S)I(D/N/H)GILG (Figure S3a). The Prd/Prd-like proteins were classified by the presence of PRD and HOX domains, and absence of an OP motif. Multiple sequences alignment of sequences between the PRD and HOX domains from Prd/Prd-like proteins, revealed regions of 22 to 64 amino acids in length. In that region we identified 14 amino acids that are shared among 32 genes, localized upstream of the HOX domain (Figure S3b, light yellow box). To test whether there are evolutionary remnants of an OP motif present in Prd/Prd-like proteins we used MEME (Multiple Em for Motif Elicitation) (Bailey et al. 2015). This identified a region upstream of the HOX domain that is conserved (9 amino acids upstream of the HOX domain) (Figure S3b, asterisks) but its placement is offset from the OP in Gsb/Gsbn proteins.

When we compared the sequence annotation based on NCBI and our annotation using the OP motif, we observed several differences. NCBI annotations include 102 Gsb/Gsb-like (∼54% of total), 48 Gsbn/Gsbn-like (∼26%), 13 Prd (<7%), 23 Pax3/7 (∼12%), and 2 Pairberry proteins (Figure S2a colored bar NCBI; Table S3). Our annotation based on the OP motif identified 82 sequences as Gsb (43%), 43 as Gsbn (∼23%), 50 as Prd/Prd-like (26%), and 13 as Pax3/7 (<7%) (Figure S2a colored bar OP; Table S3). This re-annotation based on the OP sequences thus reclassified 37 sequences annotated in NCBI as Gsb or Gsbn to Prd/Prd-like. Further, the 2 Pairberry proteins are now classified as Gsb based on their OP sequences. Importantly, our analysis based on the OP led to a reclassification of genes previously reported Gsbn in crustacean, odonatan and ephemeropteran proteins, to Gsb or Prd/Prd-like. Based on this, Gsbn is only present in neopteran insects, suggesting that this gene arose at the base of this clade.

Next, we performed a phylogenetic analysis using the OP motif (Figure S4) and then independently the HOX domain (Figure S5). These revealed similar distributions as the PRD domain phylogeny (Figures S4 and S5). Together, the fact that onychophoran, chelicerate, myriapod, crustacean and collembolan species have gsb-like genes, while chelicerate, myriapod and lepidopteran species lack prd, suggests that gsb is the most broadly conserved Pax3/7 gene and likely the ancestral family member. gsbn is present only in neopteran insects and is thus likely the most recent Pax3/7 gene in arthropods. The finding that crustacean species such as E. carolleeae and D. pulex have prd/prd-like sequences, further suggests that prd/prd-like arose in the common ancestor of Crustaceans and Insecta, and gsbn arose in the common ancestor of neopteran insects (Figure S2b).

In sum, prd/prd-like sequences are found across Pancrustacea, gsb is distributed across all arthropods, and gsbn is found only in neopteran insects (Figure S2b). However, in the case of the 2 lepidopteran species in this analysis, we did identify gsb and gsbn genes, but no sequences similar to prd/prd-like, consistent with previous reports (Cheatle Jarvela et al. 2020). The order Lepidoptera is phylogenetically located between the Diptera, Coleoptera and Hymenoptera, orders that have reported prd genes, suggesting a loss of prd/prd-like gene in Lepidoptera.

prd is the Only Pax Gene that is not Conserved in Lepidoptera

As mentioned above, prd was not found in any lepidopteran genome. To further assess this, we made use of the fact that in recent years, a large number of lepidopteran genome sequences have been reported in public databases (Challis et al. 2016; Triant et al. 2018; Wright et al. 2024). This allowed us to expand the representation of this order beyond the 2 that were previously used for genomic analysis. We selected 20 lepidopteran species that have annotated genomes, representing the subfamilies Bombycoidea, Gelechioidea, Noctuoidea, Papilionoidea, Pyraloidea, Tortricoidea, and Yponomeutoidea (Table S4, Figure S6a). For the 20 genomes, we identified the PRD domain consensus pattern using HMMER (E value <1e−5 and identity percentage >70%). Next, for each sequence with a PRD domain we identified the HOX domain using HMMER and performed manual annotation of their OP motifs based on sequences reported previously (Keller et al. 2010). Next, we reviewed our previous results from the Siphonaptera Ctenocephalides felis, the cat flea, where we could not identify a prd/prd-like gene (Table S3). Because we did not find prd/prd-like in orders closely related to Lepidoptera, we selected the Drosophila Pax genes as the outgroup sequences for our phylogenetic analysis.

Phylogenetic analysis using the PRD domain grouped these lepidopteran Pax proteins into 2 major nodes (Fig. 3a): Node I forms a single clade (clade A) containing lepidopteran Gsbn (Pax3/7) proteins that share a Gsbn-like OP motif, NHSIDGILG, with outgroups of Dmel- Prd, Dmel- Gsb and Dmel- Gsbn. Node II includes 6 nested clades: Gsb (Pax3/7), Poxm (Pax1/9), Pax6-like (Pax6-like/Eyg), Poxn (Poxn), Sv (Pax2/5/8) and Ey and Toy (Pax4/6). The OP motif for these proteins is (if present): Gsb, NHSIDGILG; Poxm, no OP; Pax6-like/Eyg, no OP; Poxn, YSIEELLK; Sv, YSINGILG, and Toy, DSVYEKLRMF; Ey, PVYERLRLL (Fig. 3b). Gsb-like sequences are the most basally branching (clade B). Clade C includes protein sequences for Poxm (Pax1/9 family) and Pax-like, differentiated by a partial PRD domain, lacking the PAI and/or RED subdomains. Poxm proteins are between 200 and 236 amino acids long: the Poxm PRD domain itself is 123 amino acids, such that the PRD domain is almost the complete protein, and the OP sequence is absent. All Poxn protein sequences, sharing a Poxn OP, clustered in clade D. Clade E includes Pax2/5/8 proteins that share a Sv OP.

Clade E and F genes encode closely related PRD domains. Clade F protein sequences have a PRD and HOX domain but no OP, similar to the group Pax6-like/Eyg proteins. Lepidopteran Pax6-like/Eyg sequences group with Drosophila Eyg/Toe sequences in the basal branch of clade F. Pax4/6 protein sequences were found in Clades G and H: Clade G protein sequences share an Ey OP while clade H sequences share a Toy OP (Fig. 3b, Table S4). The lepidopteran Ey OP consensus has only 5 conserved amino acids xYxxLRLL, compared with 7 amino acids (xxVYxKLRLL) in other insects (Fig. 3b).

Overall, the lepidopteran Pax genes that we identified are Poxn, Poxm, sv, gsb, gsbn, toy, ey, and Pax6-like/eyg. Most of the Pax genes encode OP motifs that are similar to those in the corresponding proteins from other hexapods. Lastly, no prd or prd-like gene was found in any of the selected lepidopteran species.

Vanessa Pax Gene Structure

Our next goal was to develop a lepidopteran model system to study the function of Pax genes in this large insect order. We selected Vanessa cardui (Vanessa), the painted lady butterfly, a species relatively easy to rear in the laboratory. The species has high fecundity—a colony of 40 females is able to lay up to 400 eggs in 3 h—a sequenced genome, and transcriptomes for some developmental stages (larval and pupal), and open chromatin data sets are available. Protocols for embryo injection and in situ hybridization have been worked out in our lab and others (Martin et al. 2020; Reding 2024). We identified 10 Vanessa genes that encode a PRD domain and classified them into groups based on the presence or absence of the OP motif and HOX domain (Fig. 4). Vanessa cardui (Vcar) Pax genes are: Vcar-Poxn (Poxn); Vcar-Poxm (Pax1/9); Vcar-sv (Pax2/5/8); Vcar-gsb and Vcar-gsbn (Pax3/7); Vcar-ey and Vcar-toy (Pax4/6); Vcar-757 (Pax6-like/eyg), and Pax-like Vcar-3947 and Vcar-13302. All experimentally isolated and sequenced Vanessa Pax genes exactly match predicted gene models (Fig. 4).

Fig. 4. — *Vanessa Pax* gene structure. Schematic representation of *V. cardui Pax* genes arranged by groups: a) *Poxn*; b) *Pax1/9*; c) *Pax2/5/8*; d) *Pax3/7*; e) *Pax4/6*; f) *Pax6-like/eyg* and g) *Pax-like*. Black line indicates transcript of indicated length (relative rather than exact scale). Protein domains are indicated as boxes, PRD domain (purple), HOX domain (green), OP motif (yellow). Asterisks indicate predicted isoforms.

Vcar-Poxn encodes a PRD domain and YSIEELK OP motif that is highly conserved across insects (Fig. 3b). The Vcar-Poxn complete cDNA is 1,751 bp with a coding DNA sequence (CDS) of 1,107 bp, encoding a complete PRD domain located in the amino terminal, the OP at the carboxyl-terminal of the protein and no HOX domain (Fig. 4a). The Vcar-Poxm complete cDNA is 1,057 bp (CDS 702 bp) encoding a complete PRD domain that is located in the middle of the protein. The PRD domain was the only domain found (Fig. 4b) and the OP motif is absent. We were only able to isolate Vcar-Poxm from pupal stage cDNA, compared with the other Pax genes that we isolated from embryonic stages. The Vcar-sv complete cDNA is 3,199 bp encoding a complete PRD domain, a highly conserved OP motif (YSINGILG) and no HOX domain. Vcar-sv is predicted to have 3 isoforms (Fig. 4c): sv-X1 CDS is 1,317 bp, sv-X2 is 1,302 bp and sv-X3 is 1,299 bp, all with complete PRD domains and OPs, differing from each other in absence of 5 amino acids in the amino-terminus of the protein in Sv-X2, and 6 amino acids in the carboxyl-terminal in Sv-X3.

Two Vanessa Pax3/7 genes were isolated, corresponding to gsb and gsbn. The Vcar-gsb complete cDNA is 1,733 bp encoding a complete PRD domain, a conserved OP motif (NHSIDGILG, Fig. 3b) and a HOX domain. Two isoforms were verified experimentally (Fig. 4d): a complete isoform gsb-X1 (CDS 1,416 bp) and gsb-X2 (CDS 1,260 bp) that is missing a region encoding 52 amino acids comprising the last 33 amino acids of the PRD domain through to the penultimate amino acid of the OP. The Vcar-gsbn complete cDNA is 2,071 bp encoding a complete PRD domain, OP motif (DYSIDGILG) and HOX domain. The OP differs from other Gsbn OPs in the third amino acid, which is Threonine (T) rather than the Serine (S) found in other insect Gsbn, a similar amino acid (Fig. 3b). Vcar-gsbn has 2 predicted isoforms (Fig. 4d), a complete isoform gsbn-X1 (CDS 1,386 bp) and gsbn-X2 (CDS 1,266 bp) that is missing the region encoding the first 40 amino acids, including the first 28 amino acids of the PRD domain. Both genes are located on chromosome 17. Vcar-gsbn (NC_061139.1: 6800037-6815975) is transcribed in the opposite direction of Vcar-gsb (NC_0611391: 6848517-6864169), with a distance between them of almost 32.5 kilobases. As mentioned above, no Vcar-prd has been annotated in any lepidopteran genome, including Vanessa. To further assess this, we used degenerate primers previously used for Coleoptera (Xiang et al. 2015) to amplify all Pax3/7 genes expressed in embryos at 16 to 24 and 40 to 48 h after egg laying (AEL). All sequences that we amplified correspond to Vcar-gsb, Vcar-gsbn, and other more distantly related Pax genes, supporting the absence of prd from this clade (Table S5).

Two Vanessa Pax4/6 genes were identified: toy and ey. The Vcar-toy complete cDNA is 3,082 bp (CDS 1,299 bp) encoding complete PRD and HOX domains, and an OP motif (DSVYEKLRMF) with conservative changes in the first and fifth amino acids compared with other insects (E to D and D to E, compared with Drosophila) (Fig. 3b). Vcar-toy has 1 isoform (Fig. 4e), encoding a 432 amino acid protein. Vcar-ey (1,134 bp) encodes a 377 amino acid protein with complete PRD and HOX domains, and an OP motif (PVYERLRLL) that differs from the other Ey insect octapeptides but conserves the LRLL terminal pattern (Fig. 3b). Vcar-Pax6-like (757) (Vcar-757), has a complete cDNA of 2,599 bp encoding a partial PRD domain and a complete HOX domain; no OP motif was identified. Three Vcar-757 isoforms are predicted (Fig. 4f).

The last Vanessa Pax genes we identified are Vcar-3947 and Vcar-13302, both encoding a partial PRD-like domain. These cannot be assigned to a Pax group because they lack a HOX domain and an OP motif (Fig. 4g). Vcar-3947 (867 bp) encodes a 288 amino acid protein, with an 81 amino acid PRD domain. Vcar-13302 has a predicted model of 2,244 bp that encodes a 748 amino acid protein. In sum, we identified 10 Vanessa Pax genes. We isolated cDNA encoding 8 of these Pax genes from 16 to 24 h and 40 to 48 h AEL and verified some of the predicted gene models. Poxm was isolated from pupal cDNA and Vcar-13302 could not be isolated from cDNA.

Next, we determined the similarity of the Vanessa Pax PRD domains. Within group Pax3/7, Gsb and Gsbn are 92.62% identical. For group Pax4/6, the identity between Toy and Ey is 91.13%. When compared with the Pax6-like/Eyg protein, 757, the identity is 59.18% with Ey and 56.12% with Toy. Thus, family member PRD domains are highly similar, making it is important to use the OP motif to differentiate between Pax proteins within each group. We next examined the PRD subdomains (PAI and RED) in each Vanessa Pax protein, using as a reference Dmel- Prd PAI and RED subdomains and the sequences reported previously (Friedrich and Caravas 2011) (Figure S6b). For 8 of the 10 Vanessa Pax proteins, complete PAI (49 amino acids) and RED (57 amino acids) subdomains were identified, suggesting conservation of DNA-binding potential. In the second isoform of Vcar- Gsb (X2), the last 43 amino acids of the RED subdomain are absent, such that the last 2 alpha helices are missing. In the second predicted isoform of Vcar- Gsbn (X2), the first ∼half (24 amino acids) of the PAI subdomain, corresponding to the first alpha helix, is absent. Changes in the subdomains in Gsb and Gsbn could have an impact on the interaction with protein partners and/or their target genes. Vcar- 3947 and 13302 have partial PRD domains and we could not identify PAI or RED subdomains.

Vanessa Embryonic Pax Gene Expression Patterns

The expression patterns of Vanessa Pax genes were assessed by in situ hybridization at 3 successive stages of germband development (mid- to late- germband elongated stages) corresponding to 16 to 24 h AEL (Fig. 5a). In mid-germband elongated embryos (left-most panels), a defined head region is evident. The gnathal segments are beginning to form and the segment addition zone (SAZ) can be seen at the posterior tip of the embryo. In the next stage (middle panels), gnathal and thoracic segments are defined, and abdominal segments begin to form. At late-germband elongated stages (right-most panels), there are clearly defined head lobes as well as labrum (la) and stomodeum (st); the gnathal, thoracic, abdominal segments are clearly defined, and the telson is evident. In most cases, the invagination of the stomodeum is observed. Vcar-Poxn expression was not detected at mid-germband elongation but was observed in dots in every segment at late-germband elongation, likely in the peripheral nervous system, and in the head lobes (arrowheads, Fig. 5b). These expression domains appear similar to Drosophila Poxn, which is expressed in the central and peripheral nervous systems and the precursors of poly-innervated external sensory organs (Bopp et al. 1989; Dambly-Chaudière et al. 1992). We were unable to detect Vcar-Poxm expression in any of the stages examined embryos (Fig. 5c). Vcar-sv expression was more variable than other genes studied. At the mid-germband elongated stage, expression was observed in the head and midline. As the germband continued to elongate, expression was retained in the head lobes in the antennal primordia; midline expression was retained and stronger expression was observed at the posterior tip. In late-germband elongated embryos stronger expression was observed in the stomodeum and telson (Fig. 5d). Since Vcar-sv expression was more variable than other genes examined, we generated a sense Vcar-sv probe as control. The Vcar-sv expression pattern reported here was not observed with the sense probe (see Figure S7).

Fig. 5. — *Vanessa Pax* gene embryonic expression patterns. Colorimetric in situ hybridization using digoxigenin-labelled probes was carried out on *V. cardui* germbands. a) Top panel: schematic drawing of embryos at mid- to late- stages of germband elongation. Mid-germband elongation embryos have defined head lobes (purple), antenna primordia (a) (gray), gnathal segments (red) and SAZ (light-green). As germband elongation initiates, the gnathal segments form, beginning with the mandibular segment (m). Next, 3 thorax segments (light-orange) and then the 10 abdominal segments (yellow) form. The midline (light blue), primordia of the central nervous system, becomes evident. Finally, in late- germband elongation, the head lobes gain more definition with the labrum (la) (brown) and stomodeum (st) (pink) apparent. The telson forms at the posterior tip of the embryo (green). b) *Vcar-Poxn.* Expression observed as dots in each segment (arrowheads) in elongating germbands (pattern expressed in embryos/total embryos: 318/355). c) *Vcar-Poxm*. No expression was detected at this stage of development (198/198 embryos). d) *Vcar-sv.* Expression was dynamic. Expression was first observed in the head and along the midline, later condensing in the labrum, stomodeum and telson with weak expression in the thoracic segments and along the midline (pattern expressed in 137/162 embryos). e) *Vcar-gsb.* Expression was detected in segmental stripes and in the intercalary segment (arrowhead), with strong expression in the mandibular segment (m) (251/251 embryos). f) *Vcar-gsbn.* Expression was first detected in segmental stripes (asterisk) in mid-germband elongated embryos. In late-germbands expression continued in the mid-region, with strong expression in the mandibular (m) and intercalary segments (arrowhead) (pattern expressed in 139/143 embryos). g) *Vcar-toy.* Expression detected mainly in the head lobes (h) throughout. Weak dot-like expression observed in every segment closer to the midline in late-germbands (arrowhead) (pattern expressed in 360/374 embryos). h) *Vcar-ey.* Expression was observed in each head lobe (arrowheads) after germband elongation progressed (pattern expressed in 173/216 embryos). i) *Vcar-757.* Expression detected in the mandibular segment (arrowheads [pattern expressed in 269/285 embryos]). j) *Vcar-3947*. Expression detected in the midline, with stronger expression in the head and telson (t) in late-germbands (pattern expressed in 189/204 embryos). Images oriented anterior, left; posterior, right. Scale bar: 100 μm.

Vcar-gsb was expressed at mid-germband elongation and continued through late-germband elongation in clear stripes in every segment that decreased in intensity somewhat over time. These stripes were centrally located within each segment and, except for the mandibular segment (m), the stripes did not reach the edges of the embryo. Expression in the intercalary segment was observed (arrowheads) and faint expression was seen in the labrum in late-germbands (Fig. 5e). Vcar-gsbn was expressed in faint stripes in mid-germband elongation stage embryos (asterisks). As the germbands elongated, segmental expression became more pronounced, with strong stripes in the central portion of each segment at late-germband elongation. In addition, strong expression in the mandibular segment and faint stripe expression in the intercalary segment were observed at these late stages (arrowhead, Fig. 5f). The stripes are similar to those of Vcar-gsb but are more centrally restricted. Vcar-gsb and Vcar-gsbn expression in stripes in every segment in the extended germ band is similar to Drosophila gsb and gsbn (Bopp et al. 1986). Expression of gsb in the butterfly head region has also been observed in B. anynana, in the mandibular segments, as well as in the prolegs of embryos 48 h AEL (Matsuoka et al. 2023).

Vcar-toy expression was detected early in mid-germband elongated embryos as a semicircle in each head lobe. This expression continued through late-germband elongation. In addition, 2 dots in every hemisegment (arrowhead), likely in the ventral nerve cord, were observed during late-germband elongation (Fig. 5g). This head expression is consistent with expression in the developing embryonic brain, seen for toy in Drosophila and Tribolium (Czerny et al. 1999; Yang et al. 2009). Vcar-ey expression was detected later and in a more restricted pattern than Vcar-toy as a dot in the posterior portion of each head lobe (arrowheads) (Fig. 5h). The expression pattern is similar to that of Dmel-ey, which is expressed in the optic primordia (Quiring et al. 1994). Note that Vcar-toy is expressed in a broader region, presumably the larval eye primordium and other portions of the brain, while Vcar-ey expression is more restricted. Vcar-757 expression was detected as a very faint dot in the mid-germband stage embryos in the gnathal region (arrowhead); as the germband elongated expression was detected in the mandibular segment along with faint expression in the labrum region (Fig. 5i). Lastly, Vcar-3947 was expressed in mid- to late- stage germbands along in the midline and in the head lobes; in late-germband embryos, the expression in the head lobes localized to the stomodeum region as well as in the telson (t) (Fig. 5j).

Vanessa Toy Functions in Eye Development

Pax6 genes regulate eye development in Drosophila in a well-studied process in which Dmel-toy activates the expression of Dmel-ey, which in turn regulates sine oculis (so), dachshund (dac) and eyes absent (eya) (reviewed in [Kumar 2009]). Vcar-toy expression was found in the head lobes, which is consistent with but does not demonstrate a role in eye development (Fig. 5g). While toy does not appear necessary for larval eye formation in Drosophila (Halder et al. 1998; Suzuki and Saigo 2000), it is required for the development of stemmata, larval eyes, in Tribolium (Yang et al. 2009; Luan et al. 2014), likely reflecting an ancestral role of Pax6 family genes (Yang et al. 2009). To determine the function of Vcar-toy, embryonic RNA interference (eRNAi) was carried out. Two nonoverlapping Vcar-toy double stranded RNAs (dsRNA) were generated, 1 targeting the region downstream of the HOX-domain-encoding region (dsRNA1) and the other matching the region between the PRD and HOX domains (dsRNA2) (Fig. 6a). Embryos were injected with Vcar-toy-dsRNA1, Vcar-toy-dsRNA2, or ds-tgfp as a negative control. Overall, the appearance and gross morphology of Vcar-toy-dsRNA1 and Vcar-toy-dsRNA2 larvae were similar to wild type. There was no change in the number or size of segments, legs or prolegs, and overall size and shape were similar to wild type (Figs. 6bi and S8a). We next examined first instar larval heads to determine whether Vcar-toy plays a role in development of the stemmata. The Vanessa larval visual system includes 6 stemmata distributed in a circular array on each head capsule (see Fig. 6bii, wild type) (Briscoe and White 2005). After control ds-tgfp eRNAi, the expected 6 stemmata were observed in almost 80% of the injected embryos (n = 432/557) (Fig. 6biii, Table S6). Knockdown of Vcar-toy resulted in a decreased number of stemmata. For Vcar-toy-dsRNA1, 22.67% (n = 129/569) of larvae had between 1 and 4 stemmata; for Vcar-toy-dsRNA2, 56.47% (n = 362/638) larvae had <5 stemmata (Fig. 6iv–vi). For both Vcar-toy dsRNAs, we observed developmental delay in some embryos (dsRNA1, 27.76% [n = 158/569]; dsRNA2, 4.70% [n = 30/638]) compared with ds-tgfp 2.51% (n = 14/557); or failure to develop (dsRNA1, 22.32% [n = 127/569]; dsRNA2, 12.38% [n = 79/638]), compared with ds-tgfp, 19.93% (n = 111/557). There were no defects observed in the overall head shape (Fig. 6b). When we allowed the injected embryos to reach hatching, ∼80% of ds-tgfp injected embryos hatched and continued to develop, but for both sets of Vcar-toy dsRNA-injected embryos, the larvae did not hatch, although movement could be observed inside the chorion. When we manually removed these larvae from the chorion and transferred them to a synthetic diet, they did not eat and died. These results demonstrate that Vcar-toy is important for development of the larval stemmata, as well as for the hatching and survival of the larva.

Fig. 6. — *Vcar-toy* plays a role in eye development in butterflies. a) Schematic representation of *Vcar-toy* transcript (black line), with protein domains indicated in boxes. The regions targeted by dsRNAs indicated below the line (gray boxes). Each dsRNA was injected at 3 μM into embryos collected over 3 h. (b) Photos of fixed larvae before hatching. n refers to number of stemmata observed in the example shown. Wild type larva (i) and schematic representation of the larval head (ii). Wild type heads have 6 stemmata. ds-*tgfp* (iii) injected embryos display 6 stemmata. (iv to vi) Representative phenotypes observed after *Vcar-toy* RNAi: iv. no stemmata observed; v, 2 stemmata developed; vi, 3 stemmata present, all smaller than wild type. c, d) Colorimetric in situ hybridization using digoxigenin-labelled probes for c, *Vcar-ey*, or d, *Vcar-eya* was carried out on ci, di) 21-24 h AEL wild type embryos or embryos injected with cii, dii) ds-*tgfp* or ciii, diii) *Vcar-toy* dsRNA2 at 3 μM. h, head; th, thoracic segments; st, stomodeum. All samples were hybridized at the same time. Expression of candidate target gene *Vcar-ey* decreased after *Vcar-toy* knockdown, and no change was observed for *Vcar-eya*. e) Ectopic expression of *Vcar-toy* in *Drosophila* generated ectopic eyes. *Vcar-toy* was expressed in *Drosophila* via the GAL4-UAS system with a *dpp-GAL4* driver. Ectopic *Drosophila*-like eyes were induced in the legs (i and ii). These are identified by the red eye color and the presence of ommatidia. ei and eii, detail of legs that present eye-like structures. Scale bar is 200 μm.

Vcar-toy has a Conserved Role in the Eye Gene Regulatory Network

The gene regulatory network that controls larval and adult eye development in insects are shared, likely reflecting common ancestry (Liu and Friedrich 2004; Friedrich 2006a, 2006b; Yang et al. 2009). Toy lies at the top of this network, which has been best studied in Drosophila (Gehring and Ikeo 1999). To ask whether Vanessa use a similar gene regulatory network for larval eye development, we examined the expression of candidate target genes after Vcar-toy knockdown. The expression of Dmel-ey, a master control gene in eye morphogenesis (Gehring 1996), is dependent upon Dmel-toy (Czerny et al. 1999). This interaction appears to be conserved for larval eye development in Vanessa; after knockdown of Vcar-toy, expression of Vcar-ey was undetectable (Fig. 6ciii). As expected, expression of Vcar-ey was similar to wild type in ds-tgfp control embryos (Figs. 5h and 6ci and cii, arrowheads).

To determine the extent of conservation of the eye regulatory network, we isolated additional candidate Toy target genes eyes absent (Vcar-eya) and dachshund (Vcar-dac) (reviewed in [Kumar 2001]) and performed in situ hybridization on Vcar-toy-dsRNA2 or control ds-tgfp knockdown embryos 20 to 24 h post injection. Vcar-eya was expressed in the most posterior part of the head lobes, in the labrum primordium, in the stomodeum, and in dots across the mandibular and thoracic segments (Figure S8b). Vcar-dac wild type expression was detected in the most posterior part of the head lobes, in each leg primordia, and as 2 parallel stripes in the telson (Figure S8c). For both Vcar-eya and Vcar-dac the expression pattern in Vcar-toy-dsRNA2 and ds-tgfp was indistinguishable from wild type (Figs. 6dii, iii and S8dii, iii). In sum, Vcar-toy appears to be required for patterned expression of ey, similar to Drosophila (Czerny et al. 1999). However, we did not observe a role for Vcar-toy in regulation of candidate pathway members eya or dac. Similarly, in Tribolium, Toy and Dac appear to act in together, rather than sequentially, to promote eye development (Luan et al. 2014).

Ectopic expression of Dmel-toy can induce formation of compound eye-like structures in Drosophila (Czerny et al. 1999). To ask if this function is shared with Vac-toy, we expressed Vcar-toy in Drosophila using the dpp-GAL4 driver (Czerny et al. 1999; Weasner et al. 2009; Hou et al. 2016). We found that Vcar-toy induced compound eye-like structures on the legs of the fly (Fig. 6e, arrowheads, detail in 6ei, ii), suggesting functional conservation of Toy in eye development.

Discussion

Our results demonstrated that all Pax gene groups are conserved in arthropods: Pax3/7, Pax1/9, Poxn, Pax6-like/eyg, Pax2/5/8, and Pax4/6 (Figs. 1–3). We observed differences in the cohort of arthropod Pax3/7 genes across arthropods: gsb is found in all arthropods, prd/prd-like is present in Pancrustaceans, while gsbn is found only in neopteran insects (Fig. 7). As prd/prd-like was not found in our analysis of 47 arthropod genomes, we manually annotated Pax genes from an additional 20 lepidopteran species. This identified representatives of all Pax gene groups in all lepidopteran species and confirmed the absence of prd in this large Order (Fig. 3). To assess lepidopteran Pax gene function, we carried out experiments with Vanessa cardui, the painted lady butterfly. We isolated 10 Vcar-Pax genes, 5 of which showed embryonic expression patterns similar to their orthologs in Drosophila (Fig. 5). We found that Vcar-toy, a Pax6-family gene, is expressed in the head lobes with 2 dots in every hemisegment (Fig. 5g). Embryonic RNA interference showed that Vcar-toy is important for the development of larval eyes, as knockdown led to reduction of the number of stemmata (Fig. 6b). Further, Vcar-ey expression decreased after knockdown of Vcar-toy, suggesting conservation of this aspect of the regulatory hierarchy seen in Drosophila. Consistent with this, ectopic expression of Vcar-toy in Drosophila generated ectopic eyes (Fig. 6e).

Fig. 7. — Arthropod *Pax* genes. Summary of the phylogenetic relationships of the *Pax* genes in arthropods. Top: phylogenetic relationships of the *Pax* gene groups: group III Pax3/7 with *gsb*, *prd* and *gsbn* (yellow box); group I Pax1/9 with *Poxm* (dark green box); group Poxn with *Poxn* (light green box); group Pax6/Eyg with *Pax6-like* (blue-gray); group II Pax2/5/8 with sv (pink box); and group IV Pax4/6 with ey and *toy* (blue box). Left side: arthropod orders and phylogenetic relationships between them. Blue shaded blocks indicated clade classification of arthropods: darker blue, Pancrustacea; blue, Hexapoda; light blue, Insects. Whitin the matrix, the presence of the indicated *Pax* gene is denoted by a dark gray box, the absence with a white box, and unclear presence/absence with a light gray box. The *Pax* genes present in all arthropods are *gsb*, *Poxm*, *Poxn*, *Pax6-like*, sv, with some unclear information ey and *toy* (as in Myriapoda and Chelicerata). Most changes are observed in group III Pax3/7 *prd* and *gsbn*, where *prd* is present in Pancrustacea and *gsbn* is present in neopteran insects, although *prd* has been lost in some lineages (Lepidoptera, white box; mosquitoes; hashed box).

Six Pax Gene Groups are Present in Arthropods

The origin of the Pax genes has been of interest since the discovery of the conservation of the PRD domain in mice and flies (Walther and Gruss 1991). In our phylogenetic analysis of arthropod Pax proteins, we identified 6 clades (Fig. 1): Pax3/7, Pax1/9, Poxn, Pax6-like/Eyg, Pax2/5/8, and Pax4/6. Arthropod Pax3/7 (Gsb) and Pax1/9 (Poxm) were the most basally branching in the arthropods, consistent with previous observations on Pax evolution (Hoshiyama et al. 1998; Breitling and Gerber 2000; Miller et al. 2000; Matus et al. 2007; Wang et al. 2010). Arthropod groups Poxn and Pax6-like, and Pax2/5/8 and Pax4/6, have a close relationship with each other (Fig. 1, Node III). Thus, we consider Poxn and Pax6-like as sister groups, which share a common ancestor (Fig. 1, Node IV). Similarly, Pax2/5/8 and Pax4/6 are sister groups, based on the shared common ancestor (Fig. 1, Node V). This is consistent with the earlier suggestion that Pax genes can be grouped as 2 supergroups in vertebrates, 1 including Pax 2/5/8/4/6 and a more basal supergroup including Pax 1/9/3/7 (Balczarek et al. 1997; Miller et al. 2000). The Pax4/6 group is highly conserved in animals, with both vertebrates and insects consistently having 2 genes involved in eye development (Breitling and Gerber 2000; Wang et al. 2010). The presence of toy-like sequences in noninsect arthropods D. pulex, E. carcharodonta and P. tepidariorum was previously suggested (Rivera et al. 2010). We observed that 3 noninsect arthropod species (P. tepidariorum, D. pulex, and T. palmi) have 2 Pax6 sequences similar to toy, but no ey sequence, based on our manual annotation using the OP (Fig. 2c), possibly reflecting variation in the sequence and function of the OP. Note that in a previous phylogenetic analysis based on full length sequences, a clear separation of P. tepidariorum Toy-like and Ey-like was observed (Samadi et al. 2015; Janeschik et al. 2022).

We also observed close phylogenetic relationships between Poxn and Pax6-like/Eyg (Fig. 2c). Studies in cnidarians parsed Pax genes into 4 groups: PaxA; PaxB, Pax2/5/8; PaxC, Poxn-like; PaxD, Pax3/7, and Pax1/9 (Catmull et al. 1998; Wang et al. 2010). In this analysis, PaxA and C were sister groups, consistent with our analysis. To date, the function of eyg has only been studied in Drosophila and Tribolium (Jun et al. 1998; ZarinKamar et al. 2011). The broad presence of Pax6-like/eyg that we observed throughout the arthropods, as well as its presence in sea urchin, sea anemone and acorn worm (Matus et al. 2007; Friedrich and Caravas 2011), suggests an ancestral and highly conserved role in eye development throughout the animal kingdom.

Arthropod Pax3/7: gsb Conservation, Emergence of prd in Hexapods and gsbn in Insects

Sequence analysis showed a close relationship between ancestral transposases and PaxD/3/7/gsb/prd genes (Breitling and Gerber 2000). Although best studied in vertebrates and insects, PaxD/3/7 group genes were identified in the cnidarian Acropora millepora and in the basal chordate, the ascidian Halocynthia roretzi. In this species, PaxD/3/7 genes are expressed during neural tube closure in the neurula stage (Wada et al. 1997; Miller et al. 2000). In noninsect arthropods, malacostracan and branchiopod crustaceans, lithobiomorph centipede and 2 chelicerates, Pax3/7 genes are expressed segmentally and in the CNS (Davis et al. 2005). Together, this suggests that the ancestral role of Pax3/7 genes was to determine CNS cell-fate, a role retained in extant H. roretzi, noninsect arthropods, Drosophila, and vertebrates. Later, Pax3/7 genes sub-functionalized to take on roles in segmentation, as is the case for Drosophila prd and gsb.

Our data show that all arthropods have gsb sequences, which share an OP motif consensus sequence (Figure S3). prd and prd-like genes, which lack an OP motif, are present in Pancrustacea, with the exceptions previously mentioned (e.g. mosquitoes, Lepidoptera), but are not present in close outgroups to the Pancrustacea, suggesting prd/prd-like arose in the last common ancestor of the Pancrustacea (Figs. 7 and S2).

It was suggested that the Pax3/7 family present in Drosophila arose from a duplication that generated prd and gsb, followed by a second duplication to produce gsb and gsbn (Balczarek et al. 1997). Our analysis suggests that gsb is the most basal Pax3/7 gene, because the Pax3/7 genes in the nonarthropod outgroups have Gsb-like OPs (KHSIDGILA and SHSIDGILG) compared with insect consensus xHSIxGILG OP motif (Figure S3). Pancrustaceans would then have gained a prd/prd-like gene by a duplication event of the ancestral gsb. In the selected species that have a chromosome-level genome assembly, prd/prd-like sequences are located on a different chromosome than gsb, suggesting a chromosomal translocation event. The second duplication event would have been a tandem gene duplication in insects, as gsb and gsbn are linked in the insect species where this has been examined (Bopp et al. 1986; Choe et al. 2006; Cheatle Jarvela et al. 2020).

The prd Gene is Absent in the Lepidoptera

We found that 20 lepidopteran species have gsb and gsbn genes but prd/prd-like is absent from all these species. The absence of prd in Lepidoptera is surprising because it is an order that is in between Diptera and Coleoptera, each of which has a highly conserved set of 3 Pax3/7 genes, prd, gsb and gsbn. Further, regulatory interactions between prd and gsb have been studied in Diptera (Drosophila), Hymenoptera (Apis), and Coleoptera (Tribolium), and are considered to be conserved between these species, with Prd required to activate gsb expression (Bopp et al. 1986; Osborne and Dearden 2005; Choe et al. 2006). The prd gene was also lost in the Culicidae (mosquitoes) (Cheatle Jarvela et al. 2020), suggesting that loss of prd seen in lepidopteran lineages occurred independently since loss of prd has not been reported in any other family of dipterans.

The emergence of genes by duplication events can lead to sub-functionalization and neofunctionalization (Force et al. 1999), possibly the situation for gsb, gsbn, and prd in insects. Future work will parse out how the functions of gsb, gsbn, and prd diversified in different insect groups, including how gsb functions in Lepidoptera in segment formation.

Pax6 Genes are Required for Larval Eye Development

Stemmata are the visual organs of the larvae of holometabolous insects. They are rhabdomere-based, and similar in structure to adult compound eyes, although the number and morphology of stemmata vary in different insect orders and species (reviewed in Gilbert 1994; Buschbeck and Friedrich 2008; Buschbeck 2014). These stemmata are functional eyes with a large literature documenting their spectral, polarization, motion, and photoperiodic sensitivity, as well the expression of different opsins in stemmata of different insects (e.g., see [Ichikawa and Tateda 1982; Hiraga 2005; Hirata and Shiga 2023]; reviewed in [Gilbert 1994; Friedrich 2006b; Buschbeck 2014]). While hemimetabolous insects retain nymphal eyes through to adulthood, the adult compound eyes of holometabolous insects develop from imaginal discs. However, there is compelling evidence that these different eye forms evolved from a common ancestral compound eye (Buschbeck and Friedrich 2008). In support of this notion is the finding that the gene regulatory network that directs formation of larval and adult eyes is shared (Liu and Friedrich 2004; Friedrich 2006a, 2006b, 2008).

In Drosophila and other cyclorrhaphous flies, the larval eye, referred to as the Bolwig's Organ, has been reduced and is thus evolutionarily derived (Liu and Friedrich 2004; Friedrich 2006a, 2006b; Yang et al. 2009). Interestingly, toy does not appear to be necessary for Bolwig's Organ development in Drosophila (Halder et al. 1998; Suzuki and Saigo 2000), although it is required for larval eye development in Tribolium (Yang et al. 2009; Luan et al. 2014). Our finding that toy is required for stemmata development in Vanessa (Fig. 6) is in keeping with an ancestral role for toy in larval eye development that was lost in lineages leading to Drosophila, perhaps in conjunction with the reduction of the larval eye that led to the Bolwig's Organ.

Roles of Lepidopteran Pax Genes

In addition to the Pax6 genes, other lepidopteran Pax genes have been studied in a small number of model species. In the silkworm Bombyx (Bmor), Bmor-gsb is necessary for silk gland development (Dhawan and Gopinathan 2003; Chai et al. 2008; Yang et al. 2024). Bmor-Pax6 was found to be expressed during apoptosis at different developmental stages (Zhang et al. 2010). In the butterfly Bicyclus (Bany), ey is important for eye development in pupal stages but is not required for eye maintenance in adults (Das Gupta et al. 2015). Bany-gsb is expressed in the proleg tips and mandible edge (Matsuoka et al. 2023) and Bany-sv is important in the development of scales and sockets in wings (Prakash et al. 2024). In Heliconius melpomene, the postman butterfly, a role for sv was suggested in eye development (McCulloch et al. 2022). sv was also shown to be important in scale formation in the alfalfa butterfly, Colias eurytheme (Loh et al. 2025). In our study, we expanded this survey by isolating and analyzing the expression patterns of 9 Pax genes in Vanessa embryonic development (Figs. 4 and 5 and Figures S7 and S8). Most of the Vcar-Pax were expressed in patterns similar to Drosophila, suggesting conserved roles in embryo development. An exception was Vcar-Poxm, that could only be isolated from pupae and no expression was detected in early embryos. The role in Vanessa may be more similar to Bombyx, as Bm-Poxm is expressed from 48 to 192 h AEL, in the midgut, fat body and testis (Yang et al. 2024). Vcar-gsb stripes in early embryos appeared similar to those seen in Drosophila but additionally, strong expression in the mandibular segment (Fig. 5e) observed is similar to that seen in Bicyclus and other arthropods (Davis et al. 2005; Matsuoka et al. 2023). Vanessa toy and ey are expressed in embryos (Figs. 5 and 6), but we do not know if they are expressed in other developmental stages, as observed in Bicyclus (Das Gupta et al. 2015).

In addition to providing information about Pax genes, our work in Vanessa established protocols for RNAi and in situ hybridization for early embryonic stages. This expands the genetic toolkit for this species, which is easy to rear and for which mutagenesis (Martin et al. 2020) and genome and transcriptomic data sets are available. Thus, Vanessa will serve as a good model system for future molecular genetic analyses of Pax and other regulatory genes in Lepidoptera.

Materials and Methods

Data Selection, PRD Domain Identification and Phylogenetic Analysis

Arthropod species selection was based on a representation of each order that had a genome annotation reported in NCBI (search April 2024). All selected genomes have a chromosome level assembly and annotation on the Ref-seq in NCBI. Each genome was searched for annotated genes that had a PRD domain using the interface of the NCBI portal, then the PRD domain sequence was used for the phylogenetic analysis (Table S1). PRD domain identification was made by XGR reviewing each amino acid sequence manually for some arthropod species using the PRD domain sequence reported in Prosite (PS51057) as a reference. Arthropod Pax4/6 HOX domain and OP motif (Keller et al. 2010) annotations were performed by XGR reviewing each amino acid sequence manually (Table S2). Twenty lepidopteran genomes that have genome annotations were selected to perform identification and annotation of PRD domain encoding-genes. Lepidopteran genome selection was based on a broad representation of phylogenetic distribution (Kawahara et al. 2019) (Table S4). Annotation was based on the identification of the PRD domain (PS51057) and HOX domain (PD50071) using HMMER v.3.3.2 (Eddy 2011), and manual identification of the OP motif (Table S4).

PRD domain phylogenetic reconstructions were done for each of the selected groups' (all arthropods or Lepidoptera) PRD domain sequences by alignment using MAFFT v.7.511 (Katoh and Standley 2013) with FFT-NS-2 models and arguments: –retree 2 –phylipout –reorder. The maximum likelihood trees were built with IQ-Tree v.2.2.6 (Minh et al. 2020), using the model Q.insect + I + R4, with 1,000 SH-aLRT (Guindon et al. 2010) and 1,000 ultrafast bootstraps (Hoang et al. 2018). Visualizations were carried out using ggtree v.4.4 (Yu et al. 2017). These parameters were also used for lepidopteran HOX domain and OP motif phylogenetic reconstructions.

Vanessa Rearing

Our Vanessa colony is maintained in an incubator (Percival) at 25 °C with a 12 h light/dark cycle at 50% humidity. Adults are fed with a diluted 50% sports drink (Gatorade:tap water). For embryo collection, the adults are exposed to a hollyhock plant, embryos are removed from the leaves and grown in a petri dish until the desired time at 25 °C. Larvae are maintained on an artificial diet (Multiple species diet, Southland Products) (Martin et al. 2020).

Vanessa Gene Isolation

The V. cardui genome annotation (PRJEB42869, ilVanCard2.1 [GCF_905220365.1]) was used to compare the PRD domain sequences that we identified by HMMER analysis (see Data selection, PRD domain identification and phylogenetic analysis) using reciprocal BLASTP. For gene isolation, primers (Table S7) were designed using the transcript sequences reported on NCBI. cDNA from 0 to 24 h AEL or 24 to 48 h AEL embryos, pupae or adult females was used as template for PCR amplification. Embryo, pupae and adult female total RNA was extracted using Trizol (Invitrogen) following manufacturer instructions. For embryos we used ∼100 μL of embryos. Pupa were cut with a sterile razor and the first third of the pupae (anterior to posterior—head) was used for RNA isolation. For adults, we anesthetized sexually mature adults in a cage (about 7 days after eclosion) by freezing them for 5 min or until they stopped flying. To identify females, we placed them under a dissection microscope and identified their sexual organs in the most posterior part of their body by a gently squeeze. We separated females, then removed the wings and cut a small piece of their abdomen with a sterile razor. The small female abdomen piece was used for RNA isolation. Tissue of the selected developmental stage was transferred to an Eppendorf tube and 500 μL of Trizol was added. Samples were then ground with a sterile pestle. Total RNA was DNase treated with TURBO DNA-free (Invitrogen). cDNA synthesis was done with NEB reagents using their random primer mix. All products were inserted into a pGEM vector (Promega) and Sanger sequenced. For probe synthesis, Vanessa Pax genes were amplified by PCR using the pGEM constructs as template (GenBank PV893039-PV893051). PCR products were gel extracted and cleaned using the Monarch DNA Gel Extraction kit (NEB). Probe synthesis was done with T7 RNA Polymerase (NEB) and digoxigenin RNA labeling mix (Roche), following standard manufacturer protocols.

Embryo Fixation and Gene Expression Analysis

Embryos used for the gene expression analysis were collected for 8 h and allowed to develop to the desired developmental time in petri dishes under the same conditions as the adults. Embryo fixation and in situ hybridization protocols were modified from those used by Reding et al. for O. fasciatus (Reding et al. 2019). Approximately 300 μL of embryos were put in an Eppendorf tube with 500 μL of water, which was then placed in boiling water for 3 min, then moved to ice for 10 min. We have observed that a wash with 1.5% bleach for 45 s softens the chorion making it easier to remove with forceps. Next, embryos were placed on a Pyrex spot plate in tap water and the chorions were manually removed under a dissection microscope using forceps (DUMONT #5, Fine Science Tools). After chorion removal, embryos were fixed by rocking in 4% paraformaldehyde in phosphate buffer saline with 0.1% Tween-20, pH 7.1 (PBST) for 20 min, washed with PBST and then dehydrated gradually into 100% methanol. Embryos were stored at −20 °C until use.

For in situ hybridization, embryos were serially rehydrated into PBST. Germbands were then dissected from the yolk with forceps in PBST, transferred to Eppendorf tubes and fixed in 4% paraformaldehyde in PBST for 1.5 h at room temperature by rocking. Next, the germbands were washed with PBST, then PBST and hybridization buffer (50% formamide, 5× saline-sodium citrate, 0.05 mg/mL yeast tRNA, 100 μg/mL heparin, 0.1% Tween-20) 1:1 v/v. Embryos were prehybridized for 3 h at 60 °C in hybridization buffer, then incubated overnight in hybridization buffer with 1 ng of digoxigenin labeled antisense RNA probe at 60 °C. The next day, embryos were washed twice for 30 min in hybridization buffer, 2 times with 2× SSC (300 mM NaCl, 30 mM sodium citrate, 0.1% Tween-20) for 30 min and 1 time with 0.2% SSC at room temperature for 30 min. The embryos were next washed 4 times for 10 min in PBST. Embryos were incubated with 10% bovine serum albumin in PBST for 1 h and then incubated 1 h with 1:2000 Anti-digoxigenin-AP Fab fragments (Roche) in PBST. After the incubation, embryos were rinsed 3 times with PBST and washed overnight in PBST at 4 °C. The following day embryos were washed with staining buffer (100 mM Tris 9.5 pH, 100 mM NaCl, 50 mM MgCl₂ and 0.1% Tween-20) before incubation with NBT/BCIP (Roche) in staining buffer. Incubation time with NBT/BCIP varied for different probes, as determined by visual inspection under a dissection microscope, but usually lasted 6 h at room temperature. To stop staining, samples were rinsed with PBST 3 times and then were dehydrated gradually into 100% methanol, rinsed twice with 100% methanol, once with 100% ethanol, twice with 100% methanol, and rehydrated gradually into PBST. We have observed that the alcohol rinses help to remove background. Embryos were photographed with differential interference contrast on a Zeiss Axio Imager M1 microscope; several photos taken for each embryo were stitched together using ImageJ v.1.54f (Preibisch et al. 2009; Schneider et al. 2012) and Inkscape v.1.3.2.

Embryonic RNA Interference Experiments

For synthesis of double stranded RNA, templates were PCR-amplified with Q5 high fidelity DNA polymerase (NEB) using the pGEM-Vcar plasmids as template. Both primers used to amplify dsRNA templates had T7 RNA polymerase promoter sequences at their 5′ ends. Primer sequences are in Table S7. PCR products were used as templates for RNA transcription reactions using the MEGAscript T7 Transcription kit (Invitrogen) at 37 °C overnight. Products were treated with TURBO DNase (Invitrogen) at 37 °C for 15 min and RNA was denatured by incubation at 95 °C for 3 min. RNA strands were allowed to anneal over the course of 1 h by slow cooling to 45 °C. dsRNA was precipitated with ethanol and lithium chloride and resuspended in water. A dsRNA template matching turbo gfp (tgfp) was amplified from plasmid 86453 (Addgene) as a negative control. Embryos used for eRNAi were collected for 3 h, removed from plant leaves and aligned on a 3% agar bed, as previously reported (Reding et al. 2023). For all eRNAi injections, 1.5 μM dsRNA of each dsRNA was injected first. The dose was later increased to 3 μM depending on the observed phenotypes. dsRNA was adjusted to the desired concentration in injection buffer (5 mM KCl, 0.1 mM phosphate buffer pH 6.8) with green food coloring (McCormick) diluted 1:50. Embryos were injected using a glass needle pulled from borosilicate glass capillary tubes (World Precision Instruments). After injection, the embryos were left in the agar bed overnight at 25 °C in a petri dish. The next day, injected embryos were moved with a fine paint brush from the agar bed to a petri dish to avoid mold and keep them growing for 3 days. For phenotype screening, dsRNA-injected embryos were boiled and cooled, as described for in situ hybridization. Embryos were dissected from the chorion and phenotypes scored. Late embryos were photographed in PBST using a Zeiss Discovery.V12 microscope. For gene expression analysis, embryos were boiled, fixed and subjected to in situ hybridization 24 h post injection, as described above.

Drosophila Ectopic Expression Analysis

The full length Vcar-toy cDNA that was previously isolated (see above, Vanessa gene isolation and GenBank PV893045) was inserted into the XbaI-EcoRI sites of pUAS-attB using the HiFi cloning system (NEB). Sequence of the insert was verified by Sanger sequencing. The plasmid was injected into Drosophila embryos (Rainbow Transgenic Flies) for insertion at Chr 2, 57F5, 2R:21645971. Three independent transformant lines were established. Adults emerging from crosses of injected (G0) male or females to w¹¹¹⁸ flies were screened for red eyes, which were then crossed to the balancer w; If/CyO. Emerging G2 red-eyed (w+), curly winged (CyO) flies were selected and self-crossed to generate homozygous lines, identified by absence of CyO. Homozygous transgenic males were crossed to virgin females carrying the dpp-GAL4 driver (BDSC1553). Flies were reared under standard conditions at 25 °C. For analysis, pupae were transferred to a clean vial to avoid adults getting stuck in the food. Flies were photographed using an Olympus SZX16 microscope. All fly lines were obtained from the Bloomington Stock Center.

Supplementary Material

msaf220_Supplementary_Data

msaf220_supplementary_data.zip^{(5MB, zip)}

Acknowledgments

Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. We thank Mihn Lê and Katie Reding for comments on the manuscript. We thank Mario Arteaga-Vazquez and Rebecca Mosher for their feedback on the phylogenetic analysis. We thank Arnaud Martin for the suggestions on Vanessa embryo fixations.

Contributor Information

Ximena Gutiérrez Ramos, Department of Entomology, University of Maryland, College Park, MD 20742, USA.

Leslie Pick, Department of Entomology, University of Maryland, College Park, MD 20742, USA.

Supplementary material

Supplementary material is available at Molecular Biology and Evolution online.

Author Contributions

Conceptualization: X.G.R. and L.P.; Methodology: X.G.R.; Formal analysis: X.G.R.; Validation: X.G.R.; Resources and supervision: L.P. Writing—original draft: X.G.R.; writing—review and editing: X.G.R. and L.P. Funding acquisition: L.P.

Funding

This work was supported by the National Institutes of Health grant R01GM113230 to L.P.

Data Availability

The data underlying this article is available in its online Supplementary material and GenBank PV893039-PV893051.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

msaf220_Supplementary_Data

msaf220_supplementary_data.zip^{(5MB, zip)}

Data Availability Statement

The data underlying this article is available in its online Supplementary material and GenBank PV893039-PV893051.

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PERMALINK

Arthropod Pax Gene Evolution: A Role for Vanessa Cardui Twin of Eyeless in Eye Development

Ximena Gutiérrez Ramos

Leslie Pick

Roles

Abstract

Introduction

Results

Evolutionary Distribution of Pax Genes Across the Arthropods

Fig. 1.

Toy, Ey and Pax6-like/Eyg are Conserved Across the Arthropods

Fig. 2.

gsb is the Only Pax3/7 Gene Conserved Across All Arthropods

prd is the Only Pax Gene that is not Conserved in Lepidoptera

Fig. 3.

Vanessa Pax Gene Structure

Fig. 4.

Vanessa Embryonic Pax Gene Expression Patterns

Fig. 5.

Vanessa Toy Functions in Eye Development

Fig. 6.

Vcar-toy has a Conserved Role in the Eye Gene Regulatory Network

Discussion

Fig. 7.

Six Pax Gene Groups are Present in Arthropods

Arthropod Pax3/7: gsb Conservation, Emergence of prd in Hexapods and gsbn in Insects

The prd Gene is Absent in the Lepidoptera

Pax6 Genes are Required for Larval Eye Development

Roles of Lepidopteran Pax Genes

Materials and Methods

Data Selection, PRD Domain Identification and Phylogenetic Analysis

Vanessa Rearing

Vanessa Gene Isolation

Embryo Fixation and Gene Expression Analysis

Embryonic RNA Interference Experiments

Drosophila Ectopic Expression Analysis

Supplementary Material

Acknowledgments

Contributor Information

Supplementary material

Author Contributions

Funding

Data Availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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