Involvement of Methoprene‐tolerant and Krüppel homolog 1 in juvenile hormone‐mediated vitellogenesis of female Liposcelis entomophila (End.) (Psocoptera: Liposcelididae)

Abstract Methoprene‐tolerant (Met) as an intracellular receptor of juvenile hormone (JH) and the Krüppel‐homolog 1 (Kr‐h1) as a JH‐inducible transcription factor had been proved to contribute to insect reproduction. Their functions vary in different insect orders, however, they are not clear in Psocoptera. In this study, LeMet and LeKr‐h1 were identified and their roles in vitellogenesis and ovarian development were investigated in Liposcelis entomophila (Enderlein). Treatment with exogenous JH III significantly induced the expression of LeKr‐h1, LeVg, and LeVgR. Furthermore, silencing LeMet and LeKr‐h1 remarkably reduced the transcription of LeVg and LeVgR, disrupted the production of Vg in fat body and the uptake of Vg by oocytes, and ultimately led to a decline in fecundity. The results indicated that the JH signaling pathway was essential to the reproductive process of this species. Interestingly, knockdown of LeMet or LeKr‐h1 also resulted in fluctuations in the expression of FoxO, indicating the complex regulatory interactions between different hormone factors. Besides, knockdown of both LeMet and LeKr‐h1 significantly increased L. entomophila mortality. Our study provides initial insight into the roles of JH signaling in the female reproduction of psocids and provided evidence that RNAi‐mediated knockdown of Met or Kr‐h1 is a potential pest control strategy.

The interaction of lower JH with higher 20E titers could induce molting behavior during the final instar larval or nymph growth stages (Kayukawa et al., 2017). In adults, JH is re-elevated and fulfills its other major functions during reproduction, such as vitellogenesis and oogenesis (Raikhel et al., 2005). Since JH occurs even in wingless hemimetabolous insects, prompting reproduction is thought to be the evolutionarily older of the JH roles (Truman, 2019). Therefore, exploring the effects of JH on reproduction is important to understanding the physiology of insect reproduction.
The JH signaling pathway begins with JH being secreted by the corpus allatum (CA) and then binds with its receptor, methoprene-tolerant (Met) (Jindra et al., 2013). Met subsequently recruits its partner Taiman (Tai) (also called SRC or FISC) to form the JH/Met/Tai complex, resulting in the activation of downstream signaling (Miyakawa et al., 2017;Roy et al., 2018;Santos et al., 2019). In hemimetabolous and holometabolous insects, Met conducts almost all physiological processes induced by JH, from the antimetamorphic action to the regulation of mating and sex pheromone production, Vg mRNA levels, and lipid metabolism, among others (Konopova et al., 2011;Lozano & Belles, 2011;Marchal et al., 2014;Minakuchi et al., 2008Minakuchi et al., , 2009Z. Zou et al., 2013). The realization of multiple functions of JH may be controlled through the different components of the Met cooperation (Marchal et al., 2014). In a previous study, Met mutation delayed ovarian development and decreased the fecundity of Drosophila melanogaster (Wilson & Fabian, 1986;Wilson et al., 2003).
JH is the principal gonadotropic hormone controlling female reproduction in hemimetabolous insects (Khalid et al., 2021;Roy et al., 2018;Santos et al., 2019;Wu, Yang, et al., 2020). Besides, relatively relative more research progress has been made on the function of nutrition pathways (insulin-like peptide [ILP] and target of rapamycin [TOR] signaling) on vitellogenesis in different species (Roy et al., 2018;Wu, Yang, et al., 2020). RNAi-mediated silencing of InR (ILPs receptor) causes significant inhibition of JH biosynthesis, Vg expression, and ovarian development in female B. germanica (Abrisqueta et al., 2014). The ILP pathway can interact with JH receptor complex in addition to controlling the synthesis and secretion of JH. In L. migratoria, ILP pathway can help JH to stimulate fat body cells polyploidization, which accelerates massive Vg synthesis for synchronous maturation of multiple eggs (Guo et al., 2014;Wu et al., 2016;. Therefore, the molecular basis of JH and nutritional pathway crosstalk in regulating insect reproduction needs to be further explored. The genus Liposcelis (Psocoptera: Liposcelididae) have become a major risk to global food security and safety (Ahmedani et al., 2010), especially Liposcelis entomophila (Enderlein) in tropical and subtropical regions (Nayak et al. 2014).
The rampancy of resistant psocids may be due to the unreasonable application of insecticides (Bai et al., 2020;Nayak et al., 2014). A recent study suggests that sublethal treatment of phosphine will promote the fecundity of L. entomophila (Lu et al., 2020). This phenomenon has also been found in the chemical control of N. lugens (Wu, Ge, et al., 2020). Our previous study has shown that JH III significantly induced the expression of the vitellogenin (Vg) gene and affected the oviposition of L. entomophila (S. Y. Miao et al., 2021). Insect reproduction has been a focus of research in pest control, but relevant information is limited in L. entomophila. In this study, cDNAs encoding LeMet and LeKr-h1 were identified and the relative expression of LeMet and LeKr-h1 was determined at different stages throughout development using RT-qPCR.
The role of LeMet and its downstream target LeKr-h1 in the reproduction of L. entomophila was investigated using RNA interference and exogenous JHIII treatment. This study confirmed that these transcription factors are key factors in the JH signaling pathway and suggest the important role of JH in vitellogenesis and ovarian development of L. entomophila.
It has laid the foundation for further exploration of the role of JH in the regulation of L. entomophila reproduction, which may help to effectively control the rampant of this pest.

| Insect rearing
The L. entomophila population used in this study was originally collected from a grain warehouse in Zhanjiang, Guangdong Province, China, in 2011. The colony was reared on an artificial diet consisting of wholewheat flour, skimmed milk, and yeast powder (10:10:1) (Leong & Ho, 1990) in a climatic chamber at 28 ± 1°C with 75 ± 5% relative humidity (RH) in continuous darkness. LeMet and LeKr-h1, individuals were collected from eggs and nymphs (1st, 2nd, 3rd, and 4th instars), females and males within 1 week after emergence, and adult females on different days after emergence. Tissues of adult females within 1 week after emergence (including head, thorax, midgut, ovary, and fat body) were dissected in phosphate-buffered saline (PBS). All samples were frozen immediately in liquid nitrogen and stored at −80°C until RNA extraction. The extracted RNA was instantly dissolved in TE buffer and checked for quality, concentration, and purity at an absorbance ratio of optical density (OD) 260/280 (1.8-2.1) on a BioPhotometer (Eppendorf). Each experiment contained 30 individuals and was performed in at least three biological replicates. Finally, first-strand cDNA was synthesized from 1 µg total RNA using a PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara), following the manufacturer's protocol. The synthesized first-strand cDNA was stored at -20°C until further processing.

| Amplification of the full-length mRNA of LeMet and LeKr-h1
The sequence of LeMet and LeKr-h1 was obtained from transcriptome datasets and confirmed by checking its homology against other family members using the BLAST tools of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Gene-specific primers for amplification of gene fulllength coding regions were designed using the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/ primerblast/) ( Table 1). The PCR program was 95°C for 3 min, 35 cycles of denaturing at 98°C for 10 s, annealing at 56°C-62°C (based on the primers' annealing temperature) for 5 s and extension at 72°C for 45 s, with a final extension at 72°C for 2 min using PrimeSTAR Max DNA polymerase (Takara) according to manufacturer's protocol.
The PCR amplification products were then electrophoresed on a 1% agarose gel, extracted, purified with a gel purification kit (Axygen), and cloned into a TA cloning vector pMD19-T (Takara), and transformed into Escherichia coli DH5a competent cells (Sangon Biotech Co., Ltd) according to the instructions of the manufacturer. Positive clones were confirmed using PCR for sequencing (Sangon Biotech Co., Ltd). The nucleotide and deduced amino acid sequences were reconfirmed using the BLAST server of NCBI. Sequences were predicted by the ORF finder (http:// www.ncbi.nlm.nih.gov/gorf/orfig.cgi). Tools from the ExPASy proteomics server (http://www.expasy. org) were used to deduce putative amino acid sequences from the corresponding sequences. The isoelectric point (pI) and molecular weight (Mw) of LeMet and LeKr-h1 were calculated using the Compute pI/Mw tool (http://web.expasy. org/protparam/). The modular domains of LeMet and LeKr-h1 were analyzed with the SMART program (http:// smart.embl-heidelberg.de/).

| Sequence comparisons and phylogenetic analysis
The BLASTx algorithm was employed to run the similarity searches. The amino acid sequences of LeMet and LeKr-h1 were aligned with those of other insects using the DNAMAN 6.0 software package (Lynnon Corporation), and the putative complete coding sequences were submitted to GenBank. Phylogenetic relationships between LeMet or LeKr-h1 and other insects were investigated by first aligning the respective amino acid sequences with ClustalW software, then using MEGA X software to construct two neighborjoining phylogenetic trees with a p-distance model and pairwise deletion of gaps, and each presented with a total of 1000 bootstrap replications were run to test topology (Kumar et al., 2018).

| Expression profiling analysis of LeMet and LeKr-h1
Quantitative real-time PCR (RT-qPCR) was used to measure the tissue-and stage-specific expression profile of LeMet and LeKr-h1. The β-actin gene of L. entomophila (GenBank accession no. MT603494.1) was used as a reference gene to normalize the target gene expression and to correct for sample-to-sample variation Specific qPCR primers were designed using the NCBI profile blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) for the experiment (Table 1) (S. Y. Miao et al., 2021). The reaction was performed in a 10 μl volume containing 0.4 μl of cDNA, 5 μl of 2 × TB Green™ Premix Ex Taq II (Tli RNaseH Plus) (Takara), 0.3 μl of each primer (10 mM), and 4 μl of RNase-free H 2 O and tested on a LightCycler 96 instrument (Roche Diagnostics, Basel) using the following protocol: 95°C for 2 min (for predenaturation), 40 cycles of 95°C for 15 s, and 60°C for 30 s (for fragment amplification and signal collection, respectively), and 60°C-95°C with 0.5°C increasing per 5 s (for dissociation and melting curve analysis on the specific amplification). Each sample had at least there technical replicates. The relative expression levels of LeMet, LeKr-h1, or LeVg were normalized by the mRNA abundance of β-actin using the 2 −ΔΔCT method (Schmittgen & Livak, 2008).
T A B L E 1 Primers used in this study for cloning, RT-qPCR, and dsRNA synthesis in Liposcelis entomophila

Purpose
Primer name Sequence (5′-3′) | 5 of 19 2.6 | RNA interference and JH III treatment RNAi was used to investigate the function of JH signaling pathway gene in L. entomophila ovarian development by knocking down LeMet and LeKr-h1. The primers for the RNAi experiment were listed in Table 1 and designed to amplify LeMet and LeKr-h1 fragments including a T7 promoter region in the sense and antisense strands. The green fluorescent protein gene (GFP) was used from the lab-owned as a heterologous control . The PCRamplified products were purified using a gel extraction kit (Omega bio-tek) as templates for dsRNA synthesis. The corresponding dsRNA was synthesized in vitro using the TranscriptAid™ T7 High Yield Transcription Kit (Thermo Fischer Scientific). The integrity of dsRNA was measured using agarose gel electrophoresis, and the concentration was determined using a BioPhotometer. The final dsRNA products were stored at −80°C and used within 1 week.
The RNAi bioassay comprised a treatment group (dsLeMet-fed or dsLeKrh1-fed) and a control group (dsGFP-fed).
RNAi was performed as described (S. Y. Miao et al., 2021;Wei et al., 2020) and was further optimized as follows: (1)  bioassays were performed at 28 ± 1°C, 75 ± 5% RH in complete darkness in an incubator. After 2 days of continuous feeding, the silencing efficiencies of the target genes were tested using RT-qPCR.
To examine the effect of LeMet and LeKr-h1 RNAi on survivability and oviposition was detected by calculating the survival rate, total number of eggs produced, and hatching rate by each group within 5 days after continuous feeding of dsRNA. In addition, in each treatment, the ovarian phenotypes of females were observed. The images of dissected ovaries of the remaining live psocids from the gene silencing experiment were taken under an Olympus CKX41 Microscope equipped with a DP27 digital camera (Olympus). The images were processed with Adobe Photoshop CC2019 (Adobe Systems Software Ireland Ltd).
JH treatments were performed to assess whether the expression of Lemet and Lekr-h1 were JH-dependent. A dose of 10 µg of JH III (Toronto Research Chemicals) was diluted in analytical grade acetone at a concentration of 10 µg/µl.
A volume of 23 nl of JH III solution was topically applied on the abdominal tergites of 7-day-old virgin female adults using a Nanoinjector (Drummond Scientific). Controls were equivalently treated with acetone. Dissections for mRNA measurements were carried out 4, 6, and 8 h later, respectively. Each of the above samples had three replicates.

| Statistical analysis
Statistical analyses of differences in mRNA expression and fecundity were performed using GraphPad Prism 8 software package. One-way analysis of variance followed by Tukey's HSD multiple comparison test was applied to test for significant differences among different developmental stages or tissues. An independent samples t-test (p < 0.05 and p < 0.01) was used to determine the significance of differences between the treatment and control in the dsRNA feeding assay. All data are expressed as mean ± standard error (SE). conserved than the other seven (Zn2-Zn8) which are highly conserved (Figure 1c).
Based on the predicted protein sequences of Met and Kr-h1 homologs from various insect species.
Phylogenetic analysis indicated that Mets and Kr-h1s from hemimetabolous were separately grouped (Figure 2).
The results were consistent with the classical taxonomic divergence. LeMet has the highest amino acid identity with D. punctata Met (48.10%), and LeKr-h1 shares high similarity to the Kr-h1s of other Blattaria; the highest sequence similarity was with L. migratoria Kr-h1(76.81%) (Figure 1b,c). These results suggest that Met and Kr-h1 sequences are evolutionarily more closely related to hemimetabolous insects than to other insects. with the highest expression in the fat body, followed by the head and thorax, and with the lowest expression in the ovary and midgut (Figure 3e). This suggests that JH stimulates the synthesis of Vg primarily in the fat body.

| Exogenous JH III treatment on the expression of LeMet and LeKr-h1
To examine whether LeMet and LeKr-h1 were regulated by JH, exogenous JH III was applied to the abdomen of L. entomophila. Overall, the induction effect of JH was progressively reduced with increasing time. LeMet expression was elevated but not significantly different at 4 and 6 h of treatment groups compared to that in the control group.
However, LeKr-h1 expression was 4.42-fold higher after 4 h of treatment ( Figure 4). Meanwhile, LeVg and LeVgR expression increased by 2.26 and 2.50 times at 4 h after treatment. These results indicate that LeKr-h1 is involved in the regulation of the expression of Vg in L. entomophila.

| Functional analysis of LeMet and LeKr-h1 by RNA interference
The LeMet and LeKr-h1 transcriptions were significantly inhibited after 2 days of feeding dsRNA-containing diet compared with the control groups (dsGFP) (54.45% and 44.36% less, respectively) ( Figure 5). Meanwhile, LeVg and LeVgR transcription were significantly reduced ( Figure 5). Yolk protein deposition was significantly reduced in both dsMet and dsKr-h1-treated females, whereas ovarian development was normal in dsGFPtreated females ( Figure 6). Survival of female adults gradually decreased after gene disturbance by dsMet or dsKr-h1 (Figure 6d). In addition, the cumulative egg production and hatching rates were significantly decreased in the dsMet-and dsKr-h1-fed groups compared with the dsGFP-fed group (Figure 6e

| DISCUSSION
The identification of Met and Kr-h1 has been reported in different insect orders (Wu, Yang, et al., 2020), this study is the first report on these two genes from Psocoptera insects. F I G U R E 2 (See caption on next page) the dynamic interactions between Met and its coactivators (Moglich et al., 2009;Partch & Gardner, 2010). Multiple alignments revealed that LeKr-h1 contains eight putative C 2 H 2 -type zinc-finger domains (Zn1-Zn8). Among that, the Zn1 domain was less conserved than the others, which was also found in S. furcifera (Hu et al., 2020), and B. dorsalis (Yue et al., 2018). Some crustaceans, such as Daphnia pulex, Hyalella azteca, and Portunus trituberculatus, have lost the Zn1 motif in their Kr-h1s (Xie et al., 2018). These suggest that Zn1 may have little effect on the DNA binding capacity of Kr-h1. The C-terminal sequence of LeKr-h1 contains two putative protein interaction motifs LPPRKR (a.a. 506-511) and SVIQFA (a.a. 534-539) (Figure 1c). It is shown that the unique structures of LeMet and LeKr-h1 are essential in achieving the function of JH signaling transduction.
In this study, LeMet and LeKr-h1 were expressed at all developmental stages of L. entomophila, which is consistent with most insects (Cheng et al., 2020;Han et al., 2022;Lin et al., 2015;Yue et al., 2018). The expression of LeMet was significantly higher at the embryonic stage than that in the nymphal stages of L. entomophila, which indicated that LeMet expression may be associated with embryonic development in L. entomophila. JH is involved in the regulation of insect development, including mating, reproduction, aging, and polymorphism in insects (Marchal et al., 2014). However, the role of JH in insect embryos development is mysterious (Fernandez-Nicolas & Belles, 2017). A recent study reported that Met was necessary for embryonic development in T. castaneum (Naruse et al., 2020). The involvement of Met in insect embryonic development remains to be investigated. Kr-h1 is the main effector of antideformation action of JH in holometabolous and hemimetabolous insects (Belles, 2020). The expression of LeKr-h1 decreased rapidly at the third and fourth nymphal instars indicating the antimetamorphic role of Kr-h1 in L. entomophila. This effect was also demonstrated in B. germanica (Lozano & Belles, 2011), P. apterus and Rhodnius prolixus (Konopova et al., 2011), where disturbance of Kr-h1 in the late nymphal stage triggers precocious metamorphosis (Belles, 2020). The temporal profile of LeKr-h1 transcripts after emergence was dynamic, with LeVg expression dependent on LeKr-h1, suggesting that Kr-h1 is an early-inducible gene of Vg. However, Met mRNA is relatively stable with few major fluctuations. Similar results have been reported in D. punctata, B. germanica, and S. gregaria (Gijbels et al., 2019;Lozano & Belles, 2014;Marchal et al., 2014). Besides, the high expression of Met, Kr-h1, and Vg in the fat body of adult females implies that the fat body is involved in the role of JH in regulating vitellogenesis (Roy et al., 2018;Wu, Yang, et al., 2020). Interestingly, LeMet and LeKr-h1 also had high expression levels in the head. A recent study showed that Vg was also detected in the thoraxes and heads of Plutella xylostella (M. M. Zou et al., 2020). It is speculated that the fat body cells of L. entomophila may be partially distributed in the thoraxes and heads (Hwangbo et al., 2004). JHs are synthesized and secreted by CA (Kotaki et al., 2009). Since the whole heads were sampled, rather than brain tissue exclusively, some contamination by the CA cannot be ruled out.
Although Met and Kr-h1 of the JH signaling pathway are involved in vitellogenesis in insects, their expression in adult males also indicates their involvement in other physiological processes. In Agrotis ipsilon adult males, Met and F I G U R E 2 The neighbor-joining phylogenetic tree analysis was built by MEGA X software for methoprenetolerant (Met) (a) and Kr-h1 were involved in the regulation of JH signaling in the maturation of the male accessory glands (Gassias et al., 2021), and the modulation of pheromone processing and sexual behavior (Duportets et al., 2012). These results indicate that Met and Kr-h1 are key response genes in the JH signaling pathway, and their expression at appropriate timing is essential for their correct function.
Exogenous hormone treatments are common methods to explore the physiological and biochemical effects of hormones on insects (Pener & Lazarovici, 1979). Exogenous JH or its analogs significantly stimulated the expression of Kr-h1 in B. germanica, T. castaneum, Bombyx mori, and A. aegypti (Cui et al., 2014;Kayukawa et al., 2014;Konopova et al., 2011;Naghdi et al., 2016;Zhang et al., 2018). In N. lugens and B. dorsalis, exogenous JHA application could greatly affect ovarian development, suggesting that JH played an important role in the female reproduction of these insects (Lin et al., 2015;Yue et al., 2018). JH III treatment increased the transcription of LeKr-h1, LeVg, and LeVgR within 4-6 h. This result confirmed that LeKr-h1 was an important transcription factor in vitellogenesis and female reproduction of L. entomophila. It is worth noting that Met was not dependent on the increase of JH, and the same phenomenon was shown in B. germanica (Naghdi et al., 2016). This is possible because of that Met, as a receptor of JH, is located upstream of Kr-h1 and may have been increased before the time of our observations. also inhibited the uptake of yolk protein and significantly reduced the egg production and hatching rate of L. entomophila. These results indicated that LeMet and LeKr-h1 in the JH signaling pathway were involved in the promotion of ovarian development and the egg-laying process. The knockdown of Mets reduced the transcription of Kr-h1s and Vgs, decreased yolk accumulation, and inhibited ovarian development in B. germanica, D. punctata, and Colaphellus bowringi (Liu et al., 2016;Marchal et al., 2014;Naghdi et al., 2016). In P. apterus and C. lectularius, Knockdown of Mets, rather than Kr-h1s, significantly reduced Vgs expression (Gujar & Palli, 2016;Smykal et al., 2014). These were probably due to incomplete silencing of Kr-h1s. Moreover, knockdown of Kr-h1 would seriously decrease the egg hatching in C. lectularius (Gujar & Palli, 2016), indicating the other role of Kr-h1 in embryonic development. The reproduction regulation in some Lepidopteran, Hymenoptera, and Diptera insects is more complex than in hemimetabolous insects (Khalid et al., 2021;Roy et al., 2018). This indicates that the regulation of reproduction in different insects varies from species. Anyhow, current evidence suggests that JH regulates LeKr-h1 through receptor LeMet-mediated signaling, which in turn regulates vitellogenesis and thus its fecundity in L. entomophila.
Nutrition-related pathways have important roles in controlling the biosynthesis and secretion of JHs in insects, thereby influencing their reproductive events (Roy et al., 2018). Although LeMet and LeKr-h1 in JH signaling are critical for vitellogenesis in L. entomophila, it seems to have interaction with other signaling pathways. FoxO plays a critical role in mediating the crosstalk between insulin and JH signaling pathways to coordinate insect vitellogenesis (Koyama et al., 2013;Roy et al., 2018;Santos et al., 2019;Smykal & Raikhel, 2015 the expression of JH methyltransferase (JHAMT), Met, and Kr-h1, further stimulating Vg expression and oocyte maturation (Abrisqueta et al., 2014;S. Zhu et al., 2020). In our study, knockdown of LeMet and LeKr-h1 genes significantly reduced FoxO mRNA expression but did not influence InR transcript levels. Similar results were also found in C. suppressalis . It was probably because LeMet and LeKr-h1 did not contribute directly to InR. LeMet or LeKr-h1 may interact with FoxO through certain indeterminate mediators to collaborate in regulating vitellogenesis in L. entomophila. Further study will be needed to investigate the role of the ILP/TOR pathway in the regulation of vitellogenesis in L. entomophila.
In conclusion, two related genes LeMet and LeKr-h1 were identified from L. entomophila. JHIII topical treatment induced the expression of JH signaling and Vg genes. Combined with the results of RNAi experiments, it was shown that JH regulated the expression of Kr-h1 through Met and promoted female ovary development. Our results revealed an important role of Kr-h1 in insect ovarian development and egg production. These two genes have the potential for application against this rampant pest based on RNAi or exogenous hormone analogs. Meanwhile, more in-depth studies of the role of JH and crosstalk with insulin signaling in insect reproduction regulation is well needed.