CRISPR/Cas9-Mediated Mutagenesis of Abdominal-A and Ultrabithorax in the Asian Corn Borer, Ostrinia furnacalis

Simple Summary Homeotic genes encode transcription factors that coordinated the anatomical structure formation during the early embryonic development of organisms. In this study, we functionally characterized two homeotic genes, Abdominal-A (Abd-A) and Ultrabithorax (Ubx), in the Asian corn borer, Ostrinia furnacalis (a maize pest that has devastated the Asia-Pacific region) by using a CRISPR/Cas9 genome editing system. Our results show that the mutagenesis of OfAbd-A and OfUbx led to severe morphological defects in O. furnacalis, which included fused segments and segmental twist during the larval stage, and hollowed and incision-like segments during the pupal stage in OfAbd-A mutants, as well as defects in the wing-pad development in pupal and adult OfUbx mutants. Overall, knocking out Abd-A and Ubx in O. furnacalis resulted in the embryonic lethality to, and pleiotropic impact on, other homeotic genes. This study not only confirms the conserved body planning functions in OfAbd-A and OfUbx, but it also strengthens the control implications of these homeotic genes for lepidopteran pests. Abstract (1) Background: Abdominal-A (Abd-A) and Ultrabithorax (Ubx) are homeotic genes that determine the identity and morphology of the thorax and abdomen in insects. The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), is a devastating maize pest throughout Asia, the Western Pacific, and Australia. Building on previous knowledge, we hypothesized that the knockout of Abd-A and Ubx would disrupt the abdominal body planning in O. furnacalis. (2) Methods: CRISPR/Cas9-targeted mutagenesis was employed to decipher the functions of these homeotic genes. (3) Results: Knockout insects demonstrated classical homeotic transformations. Specifically, the mutagenesis of OfAbd-A resulted in: (1) Fused segments and segmental twist during the larval stage; (2) Embryonic lethality; and (3) The pleiotropic upregulation of other homeotic genes, including Lab, Pd, Dfd, Antp, and Abd-B. The mutagenesis of OfUbx led to: (1) Severe defects in the wing pads, which limited the ability of the adults to fly and mate; (2) Female sterility; and (3) The pleiotropic upregulation of other homeotic genes, including Dfd, Abd-B, and Wnt1. (4) Conclusions: These combined results not only support our hypothesis, but they also strengthen the potential of using homeotic genes as molecular targets for the genetic control of this global insect pest.


Sample Collection
To investigate the spatial expressions of OfAbd-A and OfUbx genes, total RNA was isolated from eggs, from larvae during the first day of each instar, from prepupae (PP), from pupae (P), and from adults (A), by using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and it was treated with RNase-free DNase I (Ambion, Austin, TX, USA), according to the manufacturer's protocols. For tissue-specific analysis, fifth-instar larvae were dissected three days after ecdysis to obtain tissue from head, midgut, foregut, fat body, epidermis, testes, and ovaries, which were kept in liquid nitrogen for sample collection and total RNA extraction. To address the question of how Hox proteins are regulated by the mutagenesis of OfAbd-A and OfUbx, we collected pupal O. furnacalis for the total RNA extraction to analyze the relative transcript levels of the genes in the Hox family in OfAbd-A and OfUbx mutants.

Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Analysis
cDNAs were synthesized using 1 µg total RNA from each developmental stage as templates by using an Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany) in a 20 µL reaction mixture. The primers used for the RT-qPCR are listed in Table S1. RT-qPCR was performed on an Eppendorf Real-time PCR System using the following conditions: a 2 min denaturing cycle at 95 • C, 35 cycles of 1 min at 95 • C, 30 s at 55 • C, and 30 s at 72 • C, followed by a final extension at 72 • C for 10 min. Total RNA was isolated from fifth-instar larvae using Trizol Reagent (Invitrogen, Carlsbad, USA) and was treated with RNase-free DNase I (Ambion, Austin, TX, USA), according to the manufacturer's protocol. cDNA was synthesized with Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany), using manufacturer's instructions. Putative OfAbd-A and OfUbx genes were identified using the NCBI Blast system. OfAbd-A cDNA fragments were amplified by PCR with the following pair of primers: forward, 5 -ATGGCAGCGGCTGCCCAGTT-3 ; and reverse, 5 -TTACGTGGGGACTTTGTTCA-3 . OfUbx cDNA fragments were amplified by PCR with the following pair of primers: forward, 5 -ATGAACTCCTACTTTGAGCAGGGT-3 ; and reverse, 5 -TTACGTGGGGACTTTGTTCA -3 . PCR was carried out using KOD -Plus-polymerase (TOYOBO, Osaka, Japan) under the following conditions: 98 • C for 2 min, followed by 30 cycles at 98 • C for 30 s, 55 • C for 30 s, 68 • C for 1 min, and an elongation phase at 68 • C for 10 min. Amplified products were sequenced after cloning into a PJET1.2-T vector (Fermentas, Burlington, ON, Canada). The primers are listed in Table S1.

Synthesis of Cas9 mRNA and sgRNAs
We selected one 23 bp sgRNA to target the OfAbd-A genome locus on the third exon of the common region in the four spliced variants, and two 23 bp sgRNAs targeting the OfUbx genome locus in exon 1 of the common region for the two spliced variants. The sgRNA was subcloned into a 500 bp linearized CloneJet PJET1.2-T vector (Thermo Fisher, Waltham, MA, USA), upstream of the protospacer adjacent motif (PAM) sequence to allow sgRNA expression under the control of the T7 promoter. The sgRNA was synthesized in vitro with a MEGAScript T7 kit (Ambion, Austin, TX, USA), according to the manufacturer's instructions. Cas9 mRNA was synthesized in vitro using the mMESSAGE T7 Kit (Ambion, Austin, TX, USA) and a PTD1-T7-Cas9 vector as the template, according to the manufacturer's instructions.

Embryo Microinjection
A laboratory strain (Shanghai) of O. furnacalis larvae was reared with an artificial diet [46]. Insects were kept at 25 • C with 80% relative humidity and a 16:8 light:dark photoperiod [47]. Adults of O. furnacalis were maintained in transparent plastic bags and were fed using cotton balls soaked in sugar water (10% honey in distilled water) to allow them to lay eggs. Eggs were collected from the plastic bags, which were cut into pieces for easier collection, and were arranged in a row on sterilized cover slips, as described previously [48]. Eggs were injected on the lateral side with 10 nL of a mixture containing 300 ng/µL Cas9 mRNA and 150 ng/µL sgRNA, within 1 h of oviposition. After injection, eggs were incubated in a humidified chamber at 25 • C for 4 days until hatching.

Genomic DNA Extraction to Identify Successful Mutants
The genomic DNA of O. furnacalis larvae was extracted by using phenol:chloroform and an isopropanol precipitation extraction. Specifically, newly hatched larvae were collected and incubated with proteinase K, followed by DNA purification and RNaseA treatment. PCR was carried out to identify OfAbd-A and OfUbx mutant alleles by using primers spanning the target sites in OfAbd-A and OfUbx (Table S1). The PCR conditions were as follows: 98 • C for 2 min, followed by 35 cycles of 94 • C for 10 s, 55 • C for 30 s, and 72 • C for 1 min, followed by a final extension period of 72 • C for 10 min. The PCR products were cloned into pJET1.2-T vectors (Fermentas, Burlington, ON, Canada) and were sent for sequencing. The mutants were photographed with a digital stereoscope (Nikon AZ100).

Analysis of Hatch Rate after Mutagenesis
We analyzed the hatching rate of eggs injected with different concentrations of OfAbd-A sgRNA, and the hatching rate of eggs produced by adult OfUbx mutants. When OfUbx mutants reach adult stage, single-paired adults of different combinations, including mutant male with mutant female, mutant female with WT male, mutant male with WT female, and GFP male with GFP female, as control, were collected to examine hatching rate of G1. Each combination was set in five replicates. All adults were placed in a transparent plastic bag supplemented with cotton balls soaked in 10% honey water to allow them to lay eggs. After five days, we collected eggs from each bag and allowed them to hatch in order to analyze hatching rate of each combination.

Statistical Analysis
RT-qPCR results were collected and presented in figures by using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA). Data were analyzed using an unpaired Student's t test (SPSS Statistics 25.0 software, IBM, Armonk, NY, USA). Probability values of less than 0.05 were considered significant. Data are presented as means with SEMs.

Phylogenetic Analysis of OfAbd-A and OfUbx
Hox genes in both Drosophila and other invertebrates perform the same overall function of body organization along the anterior-posterior axis [49]. In Drosophila, bithorax complex (BX-C) includes Ubx, Abd-A, and Abd-B genes, which implies that Ubx and Abd-A have a close relationship [50]. We analyzed the genomic sequence of the Abd-A and Ubx genes in O. furnacalis and performed multiple alignments to carry out a sequence analysis. The genomic sequence of OfAbd-A is 41,431 base pairs (bps) in length, and it has three exons with four alternative splicing variants. OfAbd-A mRNA contains four ORFs that are 1050, 1050, 1035, and 1023 bp in length, which encode proteins of lengths of 349, 349, 344, and 340 amino acids (aa), respectively. Comparably, length of Abd-A in Drosophila melanogaster is 990 bp, which encodes a 330aa protein, which exhibits a highly conserved domain with OfAbd-A. Among other moth species, open reading frame (ORF) of Abd-A in Bombyx mori (NP_001166808.1) is 1056 bp, which encodes a protein of 351aa, while, in Plutella xylostella (XP_011569267), 1062 bp Abd-A encodes a protein of 352aa. Both genes are slightly larger than the O. furnacalis Abd-A gene. The genomic sequence of OfUbx is 123,000 bp in length and it has two exons, with two ORFs of 774 and 762 bp, which encode 257 and 254aa proteins, respectively. Ubx encodes a 389aa protein in D. melanogaster (NP_536752.1) and a 254aa protein in B. mori (NP_001107632.1). On the basis of our phylogenetic analysis, OfAbd-A is clustered with homologs in other Lepidopteran species, such as Plutella xylostella, Bombyx mori, and Papilio Xuthus ( Figure 1A), while OfUbx is grouped with homologs in Dendrolimus punctatus and Biston betularia ( Figure 1B). Our sequence alignment analysis shows that OfAbd-A and OfUbx both contain a Homeodomain and the respective Abdominal and Ultrabithorax domains, which demonstrates a structural conservation with their homologues in other insects (Figure 2A

Temporal-Spatial Distribution of OfAbd-A and OfUbx
To investigate the transcript changes of OfAbd-A and OfUbx during different developmental stages in Ostrinia furnacalis, we collected eggs, larval instars (first-fifth), from hatching to wandering stages, pupae, and adults of both females and males for the total RNA extraction and RT-qPCR analysis. The results of the RT-qPCR show that the OfAbd-A gene is highly expressed in the egg, wandering, pupal, and female adult stages ( Figure  3A), whereas the OfUbx gene is highly expressed in the pupal and adult stages of both females and males ( Figure 3B).

Temporal-Spatial Distribution of OfAbd-A and OfUbx
To investigate the transcript changes of OfAbd-A and OfUbx during different developmental stages in Ostrinia furnacalis, we collected eggs, larval instars (first-fifth), from hatching to wandering stages, pupae, and adults of both females and males for the total RNA extraction and RT-qPCR analysis. The results of the RT-qPCR show that the OfAbd-A gene is highly expressed in the egg, wandering, pupal, and female adult stages ( Figure  3A), whereas the OfUbx gene is highly expressed in the pupal and adult stages of both females and males ( Figure 3B).

Temporal-Spatial Distribution of OfAbd-A and OfUbx
To investigate the transcript changes of OfAbd-A and OfUbx during different developmental stages in Ostrinia furnacalis, we collected eggs, larval instars (first-fifth), from hatching to wandering stages, pupae, and adults of both females and males for the total RNA extraction and RT-qPCR analysis. The results of the RT-qPCR show that the OfAbd-A gene is highly expressed in the egg, wandering, pupal, and female adult stages ( Figure 3A), whereas the OfUbx gene is highly expressed in the pupal and adult stages of both females and males ( Figure 3B). To address the question of whether the transcript levels of OfAbd-A and OfUbx are tissue specific, we investigated the expression levels of these two genes from different tissue regions in O. furnacalis larvae by using RT-qPCR. Tissue from head, foregut (FG), midgut (MG), fat body (FB), epidermis (EPI), testes in male larvae (TE), and ovaries in female larvae (OV) were dissected using three-day-old fifth-instar larvae (L5D3). Results from RT-qPCR analysis show that OfAbd-A is highly expressed in the epidermis and ovaries ( Figure 3C), while OfUbx is highly expressed in fat body, epidermis, and ovaries (Figure 3D).

CRISPR/Cas9-Mediated Mutagenesis in O. furnacalis
We used CRISPR/Cas9 genome editing system to knock out Abd-A and Ubx genes in O. furnacalis [32]. There are four isoforms of OfAbd-A, and two isoforms of OfUbx ( Figures  4A and 5A). To identify the mutated alleles, we extracted and sequenced the genomic DNA from larvae with mutant phenotypes for OfAbd-A, and from pupae with mutant phenotypes for OfUbx. The genome sequencing showed the successful deletion of sequences within a single target site in the OfAbd-A gene ( Figure 4C), and within two target sites in the OfUbx gene ( Figure 5C). To address the question of whether the transcript levels of OfAbd-A and OfUbx are tissue specific, we investigated the expression levels of these two genes from different tissue regions in O. furnacalis larvae by using RT-qPCR. Tissue from head, foregut (FG), midgut (MG), fat body (FB), epidermis (EPI), testes in male larvae (TE), and ovaries in female larvae (OV) were dissected using three-day-old fifth-instar larvae (L5D3). Results from RT-qPCR analysis show that OfAbd-A is highly expressed in the epidermis and ovaries ( Figure 3C), while OfUbx is highly expressed in fat body, epidermis, and ovaries ( Figure 3D).

CRISPR/Cas9-Mediated Mutagenesis in O. furnacalis
We used CRISPR/Cas9 genome editing system to knock out Abd-A and Ubx genes in O. furnacalis [32]. There are four isoforms of OfAbd-A, and two isoforms of OfUbx ( Figures 4A and 5A). To identify the mutated alleles, we extracted and sequenced the genomic DNA from larvae with mutant phenotypes for OfAbd-A, and from pupae with mutant phenotypes for OfUbx. The genome sequencing showed the successful deletion of sequences within a single target site in the OfAbd-A gene ( Figure 4C), and within two target sites in the OfUbx gene ( Figure 5C).

Morphological Impacts
The OfAbd-A knockout resulted in twisted abdominal segments in the larvae via abnormal combination of adjacent segments from A2 to A7 ( Figure 6A). In the pupal st

Morphological Impacts
The OfAbd-A knockout resulted in twisted abdominal segments in the larvae via the abnormal combination of adjacent segments from A2 to A7 ( Figure 6A). In the pupal stage,

Morphological Impacts
The OfAbd-A knockout resulted in twisted abdominal segments in the larvae via the abnormal combination of adjacent segments from A2 to A7 ( Figure 6A). In the pupal stage, mutagenesis of OfAbd-A led to hollowed and incision-like segments at abdominal segments, A2 to A7 ( Figure 6B). OfUbx gene mutagenesis caused abnormal folding of wing in pupal stage, which hinders the complete covering of the thoracic region ( Figure 7A). Mutagenesis of OfUbx also led to the disruption of wing development in pupae. Moreover, severe mutagenesis prevented the adults from spreading their wings to fly, which thus limited their ability to mate and reproduce ( Figure 7B).
Insects 2022, 12, x FOR PEER REVIEW 9 of 16 mutagenesis of OfAbd-A led to hollowed and incision-like segments at abdominal segments, A2 to A7 ( Figure 6B). OfUbx gene mutagenesis caused abnormal folding of wing in pupal stage, which hinders the complete covering of the thoracic region ( Figure 7A). Mutagenesis of OfUbx also led to the disruption of wing development in pupae. Moreover, severe mutagenesis prevented the adults from spreading their wings to fly, which thus limited their ability to mate and reproduce ( Figure 7B).   mutagenesis of OfAbd-A led to hollowed and incision-like segments at abdominal segments, A2 to A7 ( Figure 6B). OfUbx gene mutagenesis caused abnormal folding of wing in pupal stage, which hinders the complete covering of the thoracic region ( Figure 7A). Mutagenesis of OfUbx also led to the disruption of wing development in pupae. Moreover, severe mutagenesis prevented the adults from spreading their wings to fly, which thus limited their ability to mate and reproduce ( Figure 7B).

Physiological Impacts
To investigate the physiological changes induced by OfAbd-A mutation in O. furnacalis, we analyzed the hatching rate of larvae post OfAbd-A injection by using GFP as a control ( Table 1). The hatching rate of the GFP control larvae was approximately 62%, while that of the embryos injected with OfAbd-A Cas9/sgRNA was approximately 30%, which suggests that the decrease in hatching rate was specifically caused by OfAbd-A mutagenesis. To further clarify the relationship between the hatching rate and OfAbd-A mutagenesis, O. furnacalis eggs were injected with different doses of Cas9 and Abd-A sgRNA. Results show that, when eggs were injected with Cas9/sgRNA at a concentration of 300 ng/µL, the hatching rate of the larvae decreased to 21% with a mutation rate of approximately 56% (Table 1). When the injection concentration of Cas9/sgRNA was decreased to 150 ng/µL, the hatching rate of the larvae reached 33%, and the mutation rate decreased to approximately 38%. Pupation rate of the treatment group was also lower than that of the GFP control group (Table 1). Our results demonstrate that mutagenesis of the OfAbd-A gene leads to embryonic lethality and affects larval development (Figure 8).

Physiological Impacts
To investigate the physiological changes induced by OfAbd-A mutation in O. furnacalis, we analyzed the hatching rate of larvae post OfAbd-A injection by using GFP as a control ( Table 1). The hatching rate of the GFP control larvae was approximately 62%, while that of the embryos injected with OfAbd-A Cas9/sgRNA was approximately 30%, which suggests that the decrease in hatching rate was specifically caused by OfAbd-A mutagenesis. To further clarify the relationship between the hatching rate and OfAbd-A mutagenesis, O. furnacalis eggs were injected with different doses of Cas9 and Abd-A sgRNA. Results show that, when eggs were injected with Cas9/sgRNA at a concentration of 300 ng/μL, the hatching rate of the larvae decreased to 21% with a mutation rate of approximately 56% (Table 1). When the injection concentration of Cas9/sgRNA was decreased to 150 ng/μL, the hatching rate of the larvae reached 33%, and the mutation rate decreased to approximately 38%. Pupation rate of the treatment group was also lower than that of the GFP control group (Table 1). Our results demonstrate that mutagenesis of the OfAbd-A gene leads to embryonic lethality and affects larval development (Figure 8).  Knocking out of OfAbd-A resulted in embryonic lethality, while OfUbx mutation led to adult sterility. From left to right, bars depict the hatching rates of eggs injected with different concentrations of OfAbd-A sgRNA, and the hatching rates of eggs produced by adult OfUbx mutants mated with each other. The asterisks (***) indicate significant differences (p < 0.01 or p < 0.001), compared to results from wild-type adults with a two-tailed t-test.
Similarly, higher concentrations of OfUbx sgRNA and Cas9 mRNA induced a higher mutation rate. Only 50% of the pupal OfUbx mutants were able to emerge to the adult stage (Table 1), compared to the emergence rate of GFP Cas9/sgRNA-treated pupae, which was 71%. We also observed that the adult male OfUbx mutants were unable to mate with either wild-type or mutant females, and that female OfUbx mutants could not mate with males from either group. On the basis of our observations, OfUbx mutation can induce sterility in both sexes of adult O. furnacalis (Figure 8).

Pleiotropic Impacts
To address the question of how Hox proteins are regulated by the mutagenesis of OfAbd-A and OfUbx, we analyzed the relative expression levels of the other Hox genes within the OfAbd-A and OfUbx mutants in the pupal stage. OfAbd-A mutagenesis gave rise to the pleiotropic upregulation of Labial (Lab), Proboscipedia (Pb), Deformed (Dfd), Antennapedia (Antp), and Abd-B ( Figure S1A), while OfUbx mutagenesis resulted in the upregulation of Dfd, Abd-B, and Wingless Integrated family member 1 (Wnt1) ( Figure S1B). The relative transcript level of Dfd increased dramatically following OfUbx and OfAbd-A mutagenesis. Our results show no significant transcript variation in Scr in response to OfUbx and OfAbd-A mutagenesis, while Antp was highly upregulated after OfAbd-A was knocked out ( Figure S1).

Discussion
In insects, homeotic genes, such as Abd-A and Ubx, specify the distinct identities of body segments [51,52]. We examined the transcription levels of OfAbd-A and OfUbx across O. furnacalis developmental stages to generate a temporal profile of expression for these two genes. OfAbd-A is highly expressed in eggs, wandering-stage larvae, pupae, and female adults ( Figure 3A). In other insect species, Abd-A has been reported to initiate its function of segmental identity determination during embryogenesis [53]. OfUbx is highly expressed in pupal and adult stages ( Figure 3B). This result is in accordance with characterized function of Ubx in rice planthoppers, where Ubx is expressed in both forewing and hindwing, and it acts as the key regulator for the switch between long and short wing forms in adult stages of both sexes [22]. In addition, Ubx was reported to regulate the development of A1 and T3 legs during adult stage in Oncopeltus fasciatus [52].
Spatial expression pattern of Hox family genes is relatively well understood in Drosophila [19,54] and other insects [55,56], especially in the thoracic and abdominal segments. However, studies on tissue-specific characterization in whole larvae are comparably lacking. We investigated the expression of OfAbd-A and OfUbx in seven larval tissue types in O. furnacalis ( Figure 3C,D). Our results show that the relative expression of OfAbd-A is dramatically higher in epidermis, and is comparably high in ovary tissue, compared to other body regions ( Figure 3C), which is in accordance with previous reports that show its expression in the epidermal and neural cells in Drosophila [19,26]. The OfUbx gene is highly expressed within epidermis, fat body, and ovary tissues ( Figure 3D), which is consistent with previous studies that show that Ubx is involved in gut and muscle development in Drosophila and that it has distinct identities in segmental and structural identification [26,57]. The extensive expression of Ubx was examined in Drosophila in a contiguous region from T3 to A6/A7 in abdomen and gonads, alongside the expression in the fat body [29]. The expression of Ubx was also reported to extend to cover anterior and posterior body regions, and to suppress limb and/or wing development [58][59][60].
CRISPR/Cas9 is a genome editing tool that has recently been exploited as an alternative for pest management [61][62][63]. In this study, we hypothesized that OfAbd-A and OfUbx genes modulate the morphological identity and development of thorax and abdomen in O. furnacalis. The first gene editing system in O. furnacalis was established by You et al. (2018) to characterize the functional role of Ago1 in pigmentation [48], which was then followed by precise gene manipulation targeting yellow [46]. In addition to Ago1 and yellow, research regarding the functional role of Bt toxin receptors, such as ABCC2 and cadherin in O. furnacalis, has been conducted by using gene editing systems [47,64]. By using the CRISPR/Cas9 genome editing system, we successfully induced deletion mutations of 59 and 250 bp into OfAbd-A, respectively (Figure 4), and deletion mutations of 9 and 82 bp into OfUbx, respectively ( Figure 5), in O. furnacalis. After knocking out OfAbd-A, the abnormal development of the abdominal segments was observed in larvae ( Figure 6), while the abnormal folding of the wings in pupae and adults was observed after OfUbx mutagenesis ( Figure 7A,B). In the latter case, adults with severely malformed wings were unable to fly, which restricted their ability to mate and reproduce.
Abd-A regulates wing disc cuticle protein genes that control larval-to-pupal metamorphosis in Bombyx mori, and silencing of Abd-A has demonstrated its role in patterning the third to sixth abdominal segments during embryonic development [41]. In Spodoptera litura, Abd-A is essential for larval segmentation, and mutation can lead to ectopic pigmentation during embryonic development [32]. Phenotypic impacts of Abd-A mutants observed in O. furnacalis ( Figure 6) are consistent with McGinnis et al. (1992), which are that Abd-A modulates the development of posterior abdominal segment 1 to anterior abdominal segment 7 in Drosophila [19].
Ubx carries similar functions for wing development and scale morphology in different insect species [38,65]. It modulates the transformation of wings to halteres in third thoracic segment in Dipterans through the repression of wing-specific genes [65]. Ubx, in beetle, Tribolium, regulates the development of elytra in larvae, while RNAi on both Ubx and Abd-A results in the formation of elytron primordia in all abdominal segments [66]. The short-andlong-wing switch in rice planthoppers is also regulated by Ubx gene, and nutritional status of the brown planthopper affects the expression of Ubx, thereby influencing the development between short and long wing forms [22]. This observation is in accordance with our result that shows that knockdown of Ubx disrupts the wing development in O. furnacalis (Figure 7). In our study, knockout of OfUbx gene induced disabled mating behavior and decreased the fecundity of adults ( Figure 7B). Our research on OfUbx mutagenesis verifies the functional role of Ubx gene in mediating wing development, from pupal to the adult stages, in O. furnacalis ( Figure 7A).
Hox genes play important roles in thoracic and abdominal segmentation, but the genetic relationship among genes in the Hox family is still controversial [1]. Hox genes encode functional transcription factors that modulate distinct identities of thoracic and segmental structures through interactions with a series of downstream genes [67]. To further characterize the role of OfAbd-A and OfUbx in the Hox family, we examined the relative transcript level of Hox genes after mutagenesis ( Figure S1). Abd-A control the expression of Lab, which is localized in gut endothelial cells in the visceral mesoderm [20]. Given that the relative transcript level of Lab dramatically increased after OfAbd-A mutagenesis in the pupal stage, the role of Lab may be to compensate for the lost expression of OfAbd-A, and to replicate its function ( Figure S1). In addition, lab was reported to specify the intercalary and posterior head segments, while pb, together with Scr, regulates the development of proboscis [68]. Dfd and Scr act downstream of Abd-A to define antennal identity in head segments [69]. Dfd is responsible for the development of eye-antennal discs [70]. In our study, increased expression pattern of Dfd in the OfAbd-A mutants agrees with the report that states that Dfd is involved in the homeotic transformation of thoracic epidermis formation, in addition to being responsible for the ventral and labial epidermis formation on head [49]. As Antp is involved in the development of thoracic segments in B. mori [71], we also examined its relative expression level in OfAbd-A and OfUbx mutants ( Figure S1). Our study shows that Antp was highly expressed after OfAbd-A mutation, which suggests that it may act as an essential factor in controlling thoracic and abdominal development ( Figure S1A). Abd-A could activate the wingless gene to induce the extra formation of the abdominal segment [72]. In our study, the expression of Abd-B was dramatically increased, while the expression of Wnt1 was significantly decreased in the OfAbd-A mutant ( Figure S1A), which further confirms the role of Abd-B, which suppresses the wingless gene through suppression of Abd-A in the A7 region for the elimination of abdomen in Drosophila [72].
Analysis of the downstream gene in OfUbx mutant found that Dfd was upregulated after OfUbx was knocked out ( Figure S1), which suggests that Dfd could be suppressed by Ubx. A previous study on Drosophila and B. mori shows that Ubx regulates a series of downstream genes, such as Scr and wingless [38]. Our result found that the expression of Abd-A was significantly downregulated, while the expressions of Abd-B and Wnt1 were highly elevated in the OfUbx mutants ( Figure S1B).
Ostrinia furnacalis is one of the most destructive pests of maize, especially in China and northeast Asia [73]. The extensive use of chemical pesticides has led to adverse environmental and non-target impacts, and the development of resistance has also become a serious concern for O. furnacalis management [73]. Therefore, novel pest management approaches, such as RNA-based control alternatives, are urgently needed [74,75].
Our study demonstrates that disruption of OfAbd-A can lead to embryonic lethality, and that OfUbx mutation induces adult sterility (Figure 8), which suggest their potential as targets of genetic tools for pest management. Since the transgenic line in O. furnacalis has been constructed for genetic control by using a piggybac transposon [76], the transgenic insect technique can be carried out by targeting Hox genes, such as OfAbd-A and OfUbx, in order to exert female-specific interruption ( Figure S2). By releasing transgenic adult males that carry a female-specific promoter that initiates the Cas9 protein and the U6 promoter to drive the targeted gene-specific sgRNA expression, males that mate with females in the field can induce female-specific lethality and sterility in the next generation as a form of pest control ( Figure S2). Future research regarding how to target OfAbd-A and OfUbx to induce the abnormal development of pupae and adults in the field is highly warranted. The two Hox genes that are identified and characterized in this study provide fundamental knowledge regarding the development of O. furnacalis and show potential as targets for genetic pest control that would be beneficial to maize growers, and that could also extend to other lepidopteran pests ( Figure S2).

Conclusions
In this study, we hypothesized that OfAbd-A and OfUbx modulate morphological identity and development of thorax and abdomen in O. furnacalis. By using a newly developed CRISPR/Cas9 genome editing system in O. furnacalis, we knocked out OfAbd-A and OfUbx respectively, and subsequently observed substantial phenotypic impacts during insect development. Specifically, after OfAbd-A mutagenesis, abnormal development of abdominal segments was observed in larvae ( Figure 6). The abnormal folding of the wings in pupae and adults was observed after OfUbx mutagenesis, which prevented the wings from completely covering the thoracic region of the pupae ( Figure 7A), or limited the ability of adults to fly for the purposes of mating or reproducing ( Figure 7B). Interestingly, the disruption of OfAbd-A led to embryonic lethality, while OfUbx mutation induced adult sterility. These combined results not only support our hypothesis, but they also provide a potential molecular target in homeotic genes for the genetic control of this global insect pest.