Knockout of cryptochrome 1 disturbs the locomotor circadian rhythm and development of Plutella xylostella

Cryptochrome 1 (CRY1) functions as a light‐responsive photoreceptor, which is crucial for circadian rhythms. The identity and function of CRY1 in Plutella xylostella remain unknown. In this study, cry1 was cloned and identified in P. xylostella. Then, a cry1‐knockout strain (Cry1‐KO) of P. xylostella with a 2‐bp deletion was established from the strain Geneva 88 (G88) using the CRISPR/Cas9 technology. No daily temporal oscillation of cry1 was observed in G88 and Cry1‐KO, and cry1 mean daily transcription of Cry1‐KO was lower than that of G88. Both G88 and Cry1‐KO demonstrated rhythmic locomotion under the light/dark condition with Cry1‐KO being more active than G88 in the daytime, whereas Cry1‐KO completely lost rhythmicity under constant darkness. The developmental period of pre‐adult of Cry1‐KO was longer than that of G88; the lifespan of the Cry1‐KO male adult was shorter than that of G88; the fecundity of Cry1‐KO was lower than that of G88; and Cry1‐KO showed lower intrinsic rate of increase (r), net reproduction rate (R0), finite increase rate (λ), and longer mean generation time (T) than G88. Our results indicate that cry1 is involved in the regulation of locomotor circadian rhythm and development in P. xylostella, providing a potential target gene for controlling the pest and a basis for further investigation on circadian rhythms in lepidopterans.


Introduction
The circadian clock drives rhythms in animal behavior, physiology, and metabolism with a photoperiod of a 24-h light/dark cycle. Circadian rhythms are driven Correspondence: Guang Yang, State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China. Email: yxg@fafu.edu.cn * Shao-Ping Chen and Dan-Feng Wang are the authors who contributed equally to this work. by a molecular oscillator that contains several conserved clock genes, such as period (per), timeless (tim), clock (clk), cycle (cyc), and cryptochrome (cry), acting in transcription/translation feedback loops (Young & Kay, 2001;Hardin, 2006;Chaves et al., 2011;Tomioka & Matsumoto, 2015).
Light is a major zeitgeber responsible for the circadian clock to synchronize with the daily environmental light/dark cycle Helfrich-Förster et al., 2001). The CRY1 acts as a blue-light photoreceptor for entrainment of the circadian clock, and the transcription of cry1 is affected by light Yan et al., 2013;Chang et al., 2017Chang et al., , 2019Tokuoka et al., 2017;Kourti & Kontogiannatos, 2018;Ueda et al., 2018). The Drosophila mutant for cry1 still displays a circadian locomotor rhythm but manifests a reduced entrainment to light/dark cycles (Stanewsky et al., 1998;Helfrich-Förster et al., 2001). In L. striatellus, suppression of cry1 still maintains a circadian behavioral rhythm under light/dark cycles but results in a reduced locomotor rhythmicity under constant darkness (Jiang et al., 2018). Cyclical locomotor activity coincides with rhythmic feeding behavior in insects (Eckel-Mahan & Sassone-Corsi, 2013), and some other insect behaviors, such as egg hatching (Tao et al., 2017;Nartey et al., 2021), larval growth (Suszczynska et al., 2017), adult emergence (De et al., 2012;Nartey et al., 2021), and reproductive capacity (Tobback et al., 2011;Xu et al., 2019) are influenced by the circadian clock, indicating that growth and development of insects are regulated by the circadian clock. CRY1 is also critical in mediating light regulation of growth and development in plants (Lin & Shalitin, 2003;Wang & Lin, 2020).
Plutella xylostella (diamondback moth, Lepidoptera: Plutellidae) is one of the most destructive pests in crucifers worldwide, and the molecular mechanism underlying circadian rhythms of P. xylostella remains unknown. CRY1 is hypothesized to contribute to the maintenance of circadian rhythms and to play an essential role for mediating behavior, growth, and development in P. xylostella. To verify this hypothesis, we first identified and cloned the cry1 of P. xylostella, and then analyzed its daily tran-scriptional profile. Finally, the cry1 mutant was generated by using the CRISPR/Cas9 technique, and the assays of locomotor activity and life table of the mutant were conducted to clarify the function of cry1 in locomotor circadian rhythm and development, respectively.

Insect strain
Geneva 88 (G88) strain was used in the study. The larvae were reared on an artificial diet (Frontier Scientific Services, Newark, DE, USA), and adults were fed with 10% honey solution for the addition of nutrition. The insects were maintained under 26 ± 1°C, 60% ± 10% relative humidity, and 14 : 10 (light : dark [L : D]) photoperiod.
The total RNA was isolated from the heads of 10 adults using the Eastep ® Super Total RNA Extraction Kit as per the manufacturer's protocol (Promega, Madison, WI, USA). The complementary DNA (cDNA) was synthesized using the Reverse-Transcription System Kit (Promega) with 500 ng of total RNA, following the manufacturer's protocol.
The PCR was carried out using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) under the following conditions: 95°C for 5 min, and then 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 120 s, and a final elongation at 72°C for 5 min. The PCR product was detected by 1.2% agarose gel electrophoresis and purified using the gel extraction kit (Omega, Morgan Hill, GA, USA), and then cloned into the PESI-Blunt simple vector (Yeasen, Shanghai, China) for sequencing (Biosune Biotech Company, Fuzhou, China).
The conserved domains were predicted with Pfam 35.0 (http://pfam.xfam.org/). Full-length amino acid sequences of CRY1 and CRY2 of some insects (Supporting Information Table S1), and P. xylostella CRY1 were used to construct the phylogenetic tree by using the maximum likelihood method with 1000 bootstrap replications in MEGA-X.

Determination of daily transcriptional pattern of cry1 by RT-qPCR
About 15 heads of 2-to 3-d-old adults were collected from zeitgeber times (ZT) 1, 5, 9, 13, 17, and 21 h under a 14 h : 10 h (L : D) photoperiod. About 15 heads of adults were collected from circadian times (CT) 1, 5, 9, 13, 17, and 21 h on the first day of constant darkness condition. The total RNA isolation is described in the Cloning and analysis of cry1 section, and cDNA was synthesized by 5× FastKing-RT SuperMix (Promega) with 1 μg total RNA, following the manufacturer's protocol. The reverse transcription quantitative PCR (RT-qPCR) was performed by using the Eastep qPCR Master Mix Kit (Promega) under the following conditions: 95°C for 2 min; 39 cycles of 95°C for 15 s and 60°C for 40 s with the Bio-Rad CFZ96 Real-Time System (Bio-Rad, Hercules, CA, USA). Primers (q-Pxcry1-F: 5 -TAGAAGTGCAAGAGCGAGCC-3 ; q-Pxcry1-R: 5 -CAGACGGAGCTCCTGCATAG-3 ) were used for RT-qPCR. The gene EF1 (PxEF1-F: 5 -TATTCGCCCCCGC TAACATC-3 ; PxEF1-R: 5 -TTGTTCTTGGAGTCTCC GGC-3 ) of P. xylostella was used as the reference gene for cry1 expression analysis. All experiments were repeated three or four times, and three technical replicates were performed. The comparative Ct method (2 − Ct ) was used to calculate the transcript level (Livak & Schmittgen, 2001). Kruskal-Wallis test was performed to examine the differences in relative mRNA expression among the different times. Mann-Whitney U test was performed to examine the significant differences in cry1 transcription between G88 and cry1-knockout strain (Cry1-KO) at the same ZT or CT. The Wilcoxon signedrank test was employed to detect the significant difference in cry1 daily mean transcription between G88 and cry1 mutant.

Knockout of cry1 by CRISPR/Cas9 technology
The small guide RNA (sgRNA) sequence was designed using the principle of GG(18 N)NGG (with the protospacer adjacent motif sequence underlined). The sgRNA was synthesized in vitro by using the HiScribe TM T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. The mixture of sgRNA (300 ng/μL) and Cas 9 protein (100 ng/μL, GenCrispr, Nanjing, China) was microinjected to develop the cry1 mutant line. The mutation screening strategy has been described in detail elsewhere . Briefly, the microinjected individuals were crossed with G88 strain individually for generating progeny. The mutated adults were identified by DNA extraction, PCR amplification and sequencing using specific primers (t-Pxcry1-F: 5 -ACAAAAATAGTCGGCTACAAC-3 /t-Pxcry1-R: 5 -GAAGCAGAGCTTGCGGAT-3 ). Sib-crossing in single pairs was performed until the homozygous mutant line was established.

Bioassays of locomotion
The circadian rhythms of moths were monitored using the Trikinetics Drosophila locomotor activity monitor (Trikinetics, Waltham, MA, USA) under L : D for 4 d and then in constant dark for 6 d at 20°C. The procedures of locomotor bioassay have been described in detail previously . In brief, moths were placed into tubes with 10% honey solution for the addition of nutrition, and locomotor activity was monitored by using the LAM16 activity monitors. Activity records were collected in 1-min bins by the DAMSystem3 (Promega), and then 1-and 30-min bins channel files were produced by the FileScan (Promega). The data were analyzed with MATLAB toolboxes (Levine et al., 2002) and FaasX (Klarsfeld et al., 2003).

Life table
One hundred newly laid eggs (<30 min) were maintained individually in plastic cups (38 × 29 × 32 mm). Fresh artificial diets were provided. After emergence, each moth pair was transferred into a new plastic cup and fed with 10% honey solution, and all deposited eggs were recorded daily. The survival of each individual was checked and recorded daily until death.
The life-history parameters, including the duration of different developmental stages, fecundity, age-specific survival rate (l x ), net reproductive rate (R 0 ), intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T) were obtained using the TWOSEX-MSChar (version 2018.08.10). The variances and standard errors of these parameters were calculated with 100 000 bootstrap replications.
The paired bootstrap tests were performed to detect the significant differences of each parameter between G88 and cry1 mutant. The Wilcoxon signed-rank test was employed to detect the significant difference in age-specific survival rate (l x ) between G88 and cry1 mutant.

Identification and characterization of P. xylostella cry1
In P. xylostella, the size of coding sequence of cry1 was 1614 bp, and the amino acid sequence analysis revealed that P. xylostella CRY1 contained a DNA photolyase domain and a flavin-adenine-dinucleotide-(FAD-) binding domain (Fig. 1). The phylogenetic tree revealed that the total of 41 insect CRY proteins were divided into 2 clades of CRY1 and CRY2, and CRY1 of P. xylostella was only clustered with other lepidopteran CRY1s (Fig. 1).

Establishment of homozygous P. xylostella cry1-knockout strain
The sgRNA was designed to target exon 2 of cry1 (Fig. 2). The sequence-specific sgRNAs were mixed with Cas9 protein, and then microinjected into 263 fresh preblastoderm eggs of P. xylostella. After this, 22.1% (58/263) of the microinjected eggs successfully developed into adults (G 0 ). The sequencing analysis of these G 0 individuals showed mutation efficiencies of 20.7% (12/58) in exon 2 of cry1. The Cry1-KO mutant with a 2-bp deletion was obtained (Fig. 2) after sib-crossing up to 6 generations (G 1 -G 6 ). In the Cry1-KO, the mutation site was located in the DNA photolyase domain, with the amino acid sequence changed from residue 77 and the translation terminated at residue 193 (Supporting Information Fig. S1), indicating that Cry1-KO was an effective mutant.
G88 (the rate of rhythmic G88, 13/30 = 43.3%) showed a stronger locomotor activity rhythm than Cry1-KO (the rate of rhythmic Cry1-KO, 0/20 = 0%) under DD  The light : dark ratio of activity in G88 and Cry1-KO. The light : dark ratio of activity was calculated with "total activity in light/total activity in dark." Two-tailed unpaired Student's t test was performed to examine the significant differences in light : dark ratio of activity between G88 and Cry1-KO. Cry1-KO, cry1-knockout strain; G88, Geneva 88; ZT, zeitgeber time.

Effects of cry1 mutation on developmental duration, fecundity, and population parameters in P. xylostella
The durations of the 2nd instar, 3rd instar, and pupa of Cry1-KO were significantly longer than those of G88 (Table 1). The duration of pre-adult, including the durations of egg, larva, and pupa, of Cry1-KO was significantly longer than that of G88 (Table 1). The male longevity of Cry1-KO was shorter than that of G88, while the female longevity of Cry1-KO was not different from that of G88 (Table 1). The fecundity of Cry1-KO was significantly lower than that of G88 (Table 1).
The age-specific survival rate (l x ) of Cry1-KO was significantly lower than that of G88 (Z = −4.931, P < 0.001) (Fig. 6). The r, λ, and R 0 of Cry1-KO were significantly lower than those of G88, while T of Cry1-KO was significantly longer than that of G88 (Table 2).

Discussion
A DNA photolyase domain and an FAD-binding domain which have been demonstrated to be sufficient for light detection (Cashmore et al., 1999;Busza et al., 2004;Chaves et al., 2011), were found in the putative amino acid sequence of cry1 gene cloned in the study. Similar structural motifs were also present in CRY2 of other insects. Therefore, it was impossible to distinguish CRY1 and CRY2 according to the structure alone, indicating that distinct roles in circadian clock might be attributable to minor structural perturbations between CRY1 and CRY2.
The P. xylostella cry1 mRNA levels did not exhibit a significant circadian cycling in either L : D or DD conditions, which was consistent with some other insects, such as D. plexippus (Zhu et al., 2008) and L. striatellus (Jiang et al., 2018). In Drosophila, dcry mRNA manifests with a peak at ZT 5 and a trough at ZT 17 under a 12 : 12 (L : D) photoperiod, and the cycling persists, just with a lower transcript under a constant darkness condition . In Agrotis segetum (turnip moth, Lepidoptera: Noctuidae), cry1 mRNA manifests with a peak at ZT 8 and a trough at ZT 16 under a 14 : 10 (L : D) photoperiod, but with a peak at CT 12 and a trough at CT 24 under a constant darkness condition (Chang et al., 2019). In Helicoverpa armigera (cotton bollworm, Lepidoptera: Noctuidae), cry1 mRNA expression peaks at ZT 4 and reaches a trough at ZT 20 under a 16 : 8 (L : D) photoperiod, but the cycling disappears under a constant darkness condition (Yan et al., 2013). Therefore, the daily expression pattern of cry1 was different among species. Plutella xylostella cry1 mutant caused a lower mRNA expression of mutant cry1, and whether cry1 mutation af-fects the expression of other clock genes, such as per and tim, needs to be studied.
A nocturnal locomotor pattern of P. xylostella was further confirmed, consistent with the previous study that P. xylostella adults were active at night (Pivnick et al., 1990;Wang et al., 2021). The cry1 mutants showed more activities than G88 in daytime, in other words, the mutant of cry1 caused a certain defect in P. xylostella to distinguish day and night, which indicated that CRY1 was Note: Significant differences were values of P < 0.05 and marginal significant differences were 0.10 < P < 0.05. Abbreviations: Cry1-KO, cry1-knockout strain; G88, Geneva 88; SE, standard error. Note: Significant differences were values of P < 0.05 . Abbreviations: r, intrinsic rate of increase; λ, finite rate of increase; R 0 , net reproductive rate; T, mean generation time. Cry1-KO, cry1-knockout strain; G88, Geneva 88.

Fig. 6
Age-specific survival rates (l x ) of G88 and Cry1-KO. One hundred newly laid eggs were used in the experiment. Cry1-KO, cry1-knockout strain; G88, Geneva 88. a photoreceptor. Plutella xylostella cry1 mutants maintained the rhythmicity under the light/dark condition, which might be due to CRY1 was not the only photore-ceptor participated in response to light. Some opsins in retina or brain, such as Rh5, Rh6, and Rh7 in Drosophila (Szular et al., 2012;Ni et al., 2017), and green-sensitive OpLW in Gryllus bimaculatus (two-spotted cricket, Orthoptera: Gryllidae) (Komada et al., 2015), are also essential for circadian photoentrainment. The Drosophila glass 60j cry b double mutants (which lack all opsin-based external photoreceptors in addition to being devoid of dcry) are completely circadian-blind (Helfrich-Förster et al., 2001). Further study will be needed to explore whether any other photoreceptor is involved in the circadian rhythm in P. xylostella. The mutation of cry1 causes rhythm resetting defects in Drosophila Stanewsky et al., 1998). The suppression of cry1 reduces the rhythmicity of L. striatellus in constant dark conditions (Jiang et al., 2018). In P. xylostella, the cry1 mutant completely lost the rhythmicity in constant darkness. However, the mutation of cry1 keeps Drosophila rhythmic in constant darkness (Stanewsky et al., 1998). Therefore, CRY1 was indispensable for regulating circadian locomotion of P. xylostella, and the mechanisms of circadian rhythm might be different among species.
The developmental duration of the pre-adult of the Cry1-KO mutant was longer than that of the G88, which might be due to the disturbed rhythm of feeding behavior. Cyclical locomotor activity is associated with rhythmic feeding behavior, which is also observed in the larvae of Spodoptera littoralis (cotton leafworm, Lepidoptera: Noctuidae) (Suszczynska et al., 2017), Trichoplusia ni (cabbage looper, Lepidoptera: Noctuidae) (Goodspeed et al., 2012), and Spodoptera exigua (beet armyworm, Lepidoptera: Noctuidae) (Kim & Hong, 2015). The reproductive competence of P. xylostella cry1 mutant was compromised, which might be because of the disturbed rhythm of sexual activities. Sexual activities of many moth species, such as female calling, male response to female sex pheromones, and mating, show specific circadian rhythms (Groot, 2014;Brady et al., 2021). The copulation behavior has a significant effect on cry1 mRNA expression in Spodoptera litura (tobacco cutworm, Lepidoptera: Noctuidae) (Xu et al., 2019), which indicates an important role of the circadian clock in reproduction. Cry1-KO showed a significantly lower intrinsic rate of increase (r), net reproduction rate (R 0 ), finite increase rate (λ), and longer mean generation time (T) than G88. These results substantiated the importance of the endogenous circadian clock to the growth and development of insects, which indicated that the gene can be potentially used in integrated pest management to disturb the growth and development of P. xylostella by suppressing cry1 expression through RNA interference.
In summary, CRY1 was indispensable for regulating circadian locomotor rhythm, growth, and development of P. xylostella. Our research contributes to the understanding of circadian rhythm mechanisms in P. xylostella.