Henosepilachna vigintioctopunctata adults were collected from Solanum melongena L. in Nanjing city, Jiangsu Province, China, in the summer of 2018. The beetles were routinely maintained in an insectary at 28 ± 1°C under a 14:10 h light-dark photoperiod and 50–60% relative humidity using potato (Solanum tuberosum) foliage at the vegetative growth or young tuber stages to assure sufficient nutrition. Under this feeding protocol, the larvae progressed through four distinct instars, with approximate periods of the first, second, third, and fourth instar stages of 3, 2, 2, and 3 days, respectively. Upon reaching full size, the fourth larval instars stopped feeding, fixed their abdomen ends to the substrate surface, and entered the prepupal stage. The prepupae spent approximately 2 days to pupate. The pupae lasted about 4 days and the adults emerged.

TRIzol reagent (Invitrogen, New York, NY, United States) was used to extract the total RNA following the protocols of the manufacturer. The NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, New York, NY, United States) was applied to perform the RNA quantification. RNA purity was determined by assessing optical density (OD) absorbance ratios at OD260/280 and OD260/230. The integrity of RNA was analyzed via 1% agarose gel electrophoresis with ethidium bromide staining. Reverse transcription was performed using a PrimeScript™ RT reagent Kit with a gDNA Eraser (TaKaRa Biotechnology Corporation Ltd., Dalian, China). Briefly, the reaction was incubated at 37°C for 15 min and then 85°C for 5 s. The synthesized cDNAs were preserved at −20°C for further use.

The putative adc, ebony, and tan genes were obtained from H. vigintioctopunctata transcriptome data (Zhang et al., 2018). The correctness of the sequences was substantiated by PCR using primers in Supplementary Table 1. The sequenced cDNAs were submitted to GenBank (accession numbers: adc, MW380963; ebony, MW380964; and tan, MW380968).

The protein sequences of ADC, Ebony, and Tan from other species were acquired from the NCBI.¹ Phylogenetic analysis was conducted using MEGA-X software² and the neighbor-joining method with 1,000 bootstrap replications.

The cDNA fragments derived from adc, ebony, tan, and enhanced green fluorescent protein (egfp) were, respectively, amplified by PCR using specific primers (Supplementary Table 1) conjugated with the T7 RNA polymerase promoter. These targeted regions were further BLAST (BLASTN) searched against H. vigintioctopunctata transcriptome data (Zhang et al., 2018) to identify any possible off-target sequences that had an identical match of 20 bp or more. The dsRNA was synthesized using the MEGAscript T7 High Yield Transcription Kit (Ambion, Austin, TX, United States) according to the instructions of the manufacturer. Subsequently, the synthesized dsRNA (at a concentration of 5–8 μg/μl) was determined by agarose gel electrophoresis and the NanoDrop 1,000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) (data not shown) and kept at −80°C until used in the subsequent experiment.

RNA interference of larvae was performed according to a previously described method (Xu et al., 2020; Ze et al., 2021). Briefly, an aliquot (0.1 μl) of the solution including 300 ng dsRNA was injected into the newly ecdysed third instar larvae. Blank and negative control larvae were injected with the same volume of PBS and dsegfp solutions, respectively. A group of 15 injected larvae was set as a replicate. Each dsRNA injection was repeated 8 times. Three replicates (each replicate contained at least six individuals) were sampled at 48 and 72 h after injection for qRT-PCR to test RNAi efficacy. Another two replicates were used to observe the phenotype.

Three biological independent experiments were carried out using the newly ecdysed third instar larvae and were planned to determine the RNAi effects of Hvadc, Hvebony, and Hvtan on the performances and cuticle tanning of the resultant larvae, pupae, and adults. Three treatments were set as follows: phosphate-buffered saline (PBS), 300 ng dsegfp and 300 ng dsadc; PBS, 300 ng dsegfp and 300 ng dsebony; or PBS, 300 ng dsegfp and 300 ng dstan.

For temporal expression analysis, cDNA templates were derived from 3-day-old eggs, the first, second, third, and fourth larval instar, the prepupae, pupae, and adults at an interval of 1 day (D0 indicates newly molted larvae, pupae, or newly emerged adults). For comparison of the expression levels in different portions, the heads and the remaining portions (bodies) were separately collected from 2-day-old third instar larvae, and 1-, 2-, and 3-day-old fourth instar larvae. Each sample contained 10 individuals and repeated three times. For analysis of the effects of treatments, total RNA was extracted from treated larvae. Each sample contained at least six individuals and repeated three times. The RNA was extracted using SV Total RNA Isolation System Kit (Promega, Madison, WI, United States). cDNA was prepared according to the instructions by using HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China). The qPCR performed on Applied Biosystems 7500 System (Life Technologies, Carlsbad, CA, United States) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the instructions of the manufacturer. Each 20 μl reaction solution containing 10 μl of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.4 μl of each primer (10 μM), 1 μl of cDNA template, and 8.2 μl of nuclease-free water. The cycling parameters were: 1 cycle of 95°C for 3 min; 40 cycles of 95°C and 60°C for 10 s and 30 s, respectively. Quantitative mRNA measurements were performed by qRT-PCR in technical triplicate, using two internal control genes (ribosomal protein S18, HvRPS18; ribosomal protein L13, HvRPL13; and the primers listed in Supplementary Table 1) according to the published results (Lü et al., 2018). An RT negative control (without reverse transcriptase) and a non-template negative control were included for each primer set to confirm the absence of genome DNA and to check for primer-dimer or contamination in the reactions, respectively.

According to a previously described method (Bustin et al., 2009), the generation of specific PCR products was confirmed by gel electrophoresis. The primer pair for each gene was tested with a 10-fold logarithmic dilution of a cDNA mixture to generate a linear standard curve [crossing point (CP) plotted vs. log of template concentration], which was used to calculate the primer pair efficiency. All primer pairs amplified a single PCR product with the expected sizes, showed a slope less than −3.0, and exhibited efficiency values ranging from 2.5 to 2.6. Data were analyzed by the 2^–ΔΔCT method, using the geometric mean of the four internal control genes for normalization.

Digital photographs of all the phenotypes were taken with Nikon SMZ 25 stereo microscopy (Nikon, Japan). Images of hindwings, dissected from 7-day-old adults whose third instar larvae were treated with dsRNA, were transferred to RGB stack images, and color intensity in equivalent regions was determined as mean grayscale values (average luminance) via ImageJ software. Under this measurement, the lower color intensity indicates the darker cuticle pigmentation (Noh et al., 2016). All the treated groups and the corresponding control groups were photographed under the same conditions.

Statistical significance of differences in the mRNA expression or other biological experiments among PBS, dsegfp, and dsRNA treatment groups was determined by one-way ANOVA and Turkey’s test in SPSS Statistics 20.0 software (at P < 0.05). In the color intensity measurement of hindwings, the statistical significance of differences in mean gray values between the dsRNA-treated groups was assessed by Student’s t-test.

By mining of H. vigintioctopunctata transcriptome data and constructing the phylogenetic trees, Hvadc, Hvebony, and Hvtan were identified (Supplementary Figures 1–3).

To detect the expression patterns of Hvadc, Hvebony, and Hvtan during different developmental stages, qRT-PCR was performed. The results revealed that Hvadc, Hvebony, and Hvtan were detectable from the embryo (egg) to adults. They peaked in the 4-day-old pupae or 0-day-old adults. Moreover, at the first, second, third, and fourth larval instar and pupal stages, the transcription levels of Hvadc, Hvebony, and Hvtan were high just before and/or right after the molt, and were low at the intermediate stages (Figures 1A–C).

The expression levels of Hvadc, Hvebony, and Hvtan in larval heads were compared to those in the bodies of 2-day-old third instar larvae, and 1-, 2-, and 3-day-old fourth instar larvae. Both Hvadc and Hvebony were abundantly transcribed in the larval heads of 2-day-old third instar larvae and 1-day-old four instar larvae (Figures 1D,E). Similarly, the levels of Hvtan in the head samples of 1- to 3-day-old fourth instar larvae were higher than those in the bodies (Figure 1F).

Three days after the introduction of dsadc into the newly ecdysed third instar larvae, the accumulated mRNA level of the target transcript was heavily suppressed (Figure 2A).

All the controls (PBS- and dsegfp-introduced) and dsadc-treated larvae normally molted to the fourth instar larvae. However, the color was darkened in the heads of the Hvadc RNAi larvae, compared with those of the PBS- and dsegfp-treated ones (Figures 2B,D vs. C,E), especially the mouthparts (Figures 2F vs. G) and the patches around larval ocelli and the antennae (Figures 2H vs. I). In addition, a wide cream band called epicranial suture (ES) was narrowed, and the V-shaped frontal arms and the U-shaped patch on the head top were widened in the Hvadc RNAi larvae (Figures 2F vs. G).

In the mouthparts of the control fourth instar larvae (PBS- and dsegfp-injected), the median part of the labrum is cream, while the lateral and anterior parts are brownish narrow bands. Other appendages of the mouthparts, namely, mandibles, maxilla, and labium were black in color (Figures 2F,J). By contrast, in the Hvadc RNAi larvae, the color of all the pigmented regions was deepened and blackened (Figures 2F,J vs. G,K). Moreover, the scoli, strumae, and legs were all darker-colored in the Hvadc silenced larvae (Figures 2L,N vs. M,O). In addition, small mastoids queuing up on the abdomen were darker pigmented in dsadc-injected larvae, compared with that of dsegfp-injected larvae (Figures 2D,P vs. E,Q).

Knockdown of Hvadc did not affect the pupal morphology (Figure 3A) and the pupation rate (Figure 3B), but the coloration.

The integument of the 2-day-old pupae developed from the third instar larvae treated with dsegfp or dsadc were generally pale yellow, especially on the ventral one (Figure 3A, left panel). Then, the overall color shifted to dull yellow in the 4-day-old pupae (Figure 3A, right panel). From the dorsal views, the paired black markings in the meso and meta thoraxes, and the dark patches in tergite I–IV were similar between the control (dsegfp-treated) and the Hvadc RNAi pupae (Figure 3A).

However, the antennae and mouthparts of the 2-day-old Hvadc-silenced pupae were blackened (Figure 3D), while those parts in 2-day-old pupae treated with dsegfp exhibited no obvious pigmentation (Figure 3C). Four days after pupation, the mouthparts of control displayed rufous color, and the color of antennae and legs did not obviously change (Figure 3E). By contrast, the cuticle color between two compound eyes of the Hvadc-silenced pupae was obviously darker, and obvious central black patches and several small dark spots were present (Figures 3E vs. F). The antennae, mouthparts, and legs became darker (Figure 3F).

The emergence rate of the pupae in the dsadc-treated group was 95% on average, showing no significant difference with the one in the PBS- or dsegfp-treated group (Figure 4A). For Hvadc RNAi adults, 28 black markings were distributed symmetrically on two hard elytra 1 day after emergence. Then, the black pigments were gradually deposited and finally covered the whole body from the dorsal view 7 days after emergence, while the pigmentation of the control (dsegfp-treated) adult remained the same (Figures 4B vs. D). Similarly, a pair of black markings on the sternum of the metathorax was seen in the control and 1-day-old Hvadc RNAi adults; while the whole body from the ventral view was blackened in the 7-day-old Hvadc RNAi adults (Figures 4C vs. E). Knockdown of Hvadc caused the overall darkening pigmentation in elytra, which seems to be due to the enlargement of the black spots (Figures 4F vs. G). Moreover, the view of the amplified elytra from Hvadc RNAi adults showed that the setae and circle pits were all dark-colored, in contrast to the reddish-brown color in the control beetles (Figures 4H vs. I). Further dissection of the head, pronotum, scutellum, hindlegs, and hindwing revealed that these regions all shifted to the darker pigmentation (Figure 4J and Supplementary Figure 4). The color intensity of the corresponding regions of hindwings showed a significant difference between dsadc and dsegfp treated groups (Figure 4K).

Since both the Adc and Ebony play central roles in the synthesis of the NBAD (Massey et al., 2019), we have knocked down the expression of Hvebony by injection of dsebony to the newly ecdysed third instar larvae (Figure 5A).

The pupation rate and the emergence rate of the Hvebony RNAi larvae were similar to those of the control larvae which were injected PBS or dsegfp (Figures 5B,C). The mandibles were darker-colored in the dsebony-introduced newly molted fourth instar larvae, compared with those in the control larvae treated with PBS or dsegfp (Figures 5D,E, 15 min). Eight hours postmolting, the scoli and strumae from the larval thorax to the eighth segment of the abdomen, along with head and leg, became brownish in the PBS- and dsegfp-introduced larvae (Figures 5D,E, above panel). By contrast, the color of the body parts, namely, head, scoli, strumae, and legs became dark brown in the dsebony-treated larvae (Figures 5D,E, lower panels).

Similarly, at the wandering stage, the cuticles of the larvae head capsules, scoli, strumae, legs, and spots on the abdomen were more blackened in Hvebony RNAi larvae than those in the control larvae (Figures 5F,H,J vs. G,I,K).

In the dsebony-introduced pupae, the black dorsal markings in the meso and meta thoraxes and the dark patches in tergite I–IV showed no significant difference with PBS or dsegfp-treated pupae (Figure 6A). However, the mandibles were darker in the 3-day/4-day-old Hvebony-silenced pupae, than those in control groups (Figure 6A). Similar to the phenotype of the Hvadc depleted pupae, several small dark spots appeared in the cuticle between two compound eyes of the dsebony-introduced 4-day-old pupae (Figures 6B vs. C). Furthermore, the cuticle of the head, legs, elytra, and pronotum in dsebony-introduced 4-day-old pupae became significantly darker than that of pupae injected with PBS or dsegfp (Figures 6B,D,F vs. C,E,G). The enlarged dorsal view displayed that many short black setae and dots appeared on the cuticle of the dsebony-injected 4-day-old pupae to make the cuticle blacker in color than the control (Figures 6F vs. G).

Thirty minutes after emergence, the elytra of the control adults treated with PBS or dsegfp still exhibited light yellow in color. Subsequently, the elytra turned copper yellow, and 28 spots appeared, gradually darkened (Figure 7A, dsegfp-dorsal view). By contrast, knockdown of Hvebony led to an overall gray pigmentation and enlarged 28 dark spots in elytra within 30 min after emergence. The elytra of the Hvebony RNAi adults also gradually darkened (Figure 7A, dsebony-dorsal view), but they looked completely black in color 7 days after emergence (Figures 7B vs. C). Further dissection of the head, pronotum, scutellum, and hindlegs of the Hvebony RNAi adults showed that these regions all turned to darker pigmentation than control adults (Supplementary Figure 4). Similar to the Hvadc-silenced adults, the 28 black spots on the elytra of the Hvebony-silenced adults became larger than those on the control elytra (Figures 7D vs. E), and the color of the setae and pits on the surface of the elytra was darker than that of the control elytra (Figures 7F vs. G). In addition, the pigment regions of the hindwing from the Hvebony RNAi adults were darker than controls (Figures 7H,I).

From the ventral view, six pairs of dark patches appeared along the lateral portions on the abdomen segments of the Hvebony RNAi adults, and the last two pairs extended and merged together. The pale-yellow color outside the dark patches was gradually darkened, until merging the dark patches 2 days after emergence (Figure 7A, dsebony-ventral view). The legs and the abdomen of the Hvebony-depleted adults were blacker in color than the control beetles (Figure 7A, dsebony-ventral view).

Two days and three days after injection of dstan to the third instar larvae, the expression of the target Hvtan transcript was significantly reduced, compared with the control larvae injected PBS or dsegfp (Supplementary Figure 5A).

Depletion of Hvtan exerted little influence on pupation and emergence rates (Supplementary Figure 7). The results of color intensity measurement (data not shown) indicated that knockdown of Hvtan had a very limited influence on the cuticle pigmentation at the larval, pupal, and adult stages, compared with the controls subjected to PBS or dsegfp injection (Supplementary Figures 5B,C, 6A–M). In addition, there was no significant difference in the color intensity of the hindwings between dsegfp- and dstan-treated ladybirds (Supplementary Figure 6N).