Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.
Strengths:
Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.
Weaknesses:
I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).
Thank you for the suggestion. We have added the discussion in the text: "CAD96CA and FGFR1 have similar functions in JH signaling, including transmitting JH signal for Kr-h1 expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of transcription factors MET1 and TAI, and high affinity to JH III. CAD96CA and FGFR1 are essential in the JH signal pathway, and loss-of-function for each is sufficient to trigger strong effects on pupation. The difference is that CAD96CA expression has no tissue specificity, and the Fgfr1 gene is highly expressed in the midgut; possibly, it plays a significant role in the midgut. Other possibility is that they play roles by forming heterodimer with each other or other RTKs, which needs to be addressed in future study. CAD96CA and FGFR1 transmit JH III signals in three different insect cell lines, suggesting their conserved roles in other insects.".
In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.
Thank you for your question. We have modified the figure and the legends to make the conception clear.
Reviewer #2 (Public Review):
Summary:
Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects. While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identification and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives. In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.
Strengths:
Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.
Weaknesses:
Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.
If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.
Thank you for your question and suggestion. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and in cell lines, and overexpression of them in mammal HEK-293T heterogeneously. Figure 6 is drafted by these solidate evidences.
Cad96ca and Fgfr1 G0 mutants caused slight acceleration of pupation is one of the types of evidence of JH signaling-deficient. Othe evidences include a set of gene expression and the block of JH induced-rapid intracellular calcium increase.
Kr-h1 is a typical indicator gene at the downstream of Jhamt and in JH signaling, so we used it as an indicator to examine JH signaling. Jhamt and Met or other genes might be affected in Cad96ca and Fgfr1 G0 mutants, which can be examined in future study.
We have discussed the question that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".
Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.
Thank you for your question. For Figure 5, our goal was to demonstrate that JH can induce calcium mobilization through CAD96CA and FGFR1. Controls have been established between different experimental groups within the same cell, as well as between different cells. Increasing the positive experimental group would make the results more complex.
Reviewer #3 (Public Review):
Summary:
In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.
Strengths:
The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.
Weaknesses:
The study has several weaknesses that need to be addressed. Firstly, it is not clear what criteria were used by the authors to discard several other RTKs that were identified as repressors of JH signaling. For example, while NRK and Wsck may not fulfill all the requirements to become JH receptors, other evidence, such as depletion analysis and target gene expression, suggests they are involved in proper JH signaling activation.
Thank you for your question. We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. WSCK was not involved in the phosphorylation of MET and TAI and was discarded during subsequent screening. NRK did not bind to JH III, did not meet the screening strategy, and was discarded.
We increased the information in the Introduction: "We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously.".
We increased discussion: "This study found six RTKs that respond to JH induction by participating in JH induced-gene expression and intracellular calcium increase, however; they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH induced-phosphorylation of MET and TAI and binding to JH III. We screen the RTKs transmitting JH signal primarily by examining some of JH induced-gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential.".
Secondly, the expression of the six RTKs, which, when knocked down, were able to revert JH signaling activation, was mainly detected in the last larval stage of H. amigera. However, since JH signaling is active throughout larval development, it is unclear whether these RTKs are completely required for pathway activation or only needed for high activation levels at the last larval stage.
Thank you for the question. We knocked down the genes at last larval stage to observe pupation, which is a relatively simple and easily to be observed target to examine the role of the gene in JH-maintained larval status. The results from CRISPR/Cas9 experiments showed: "Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".
Additionally, the mechanism by which different RTKs exert their functions in a specific manner is not clear. According to the expression profile of the different RTKs, one might expect some redundant role of those receptors. In fact the no reversion of phosphorilation of tai and met upon depletion of Wsck in cells with overactivated JH signalling seems to support this idea.
Nevertheless, and despite the overlapping expression of the different receptors, all RTKs seem to be required for proper pathway activation, even in the case of FGF1 which seems to be only expressed in the midgut. This is an intriguing point unresolved in the study.
Thank you for your comments. Yes, from our study, different RTKs exert their functions in a specific manner. We have increased discussion: "This study found six RTKs that respond to JH induction by participating in JH induced-gene expression and intracellular calcium increase, however; they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH induced-phosphorylation of MET and TAI and binding to JH III. We screen the RTKs transmitting JH signal primarily by examining some of JH induced-gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential.".
Finally, the study does not explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. in Drosophila, FGF1 is activated by pyramus and thisbe for mesoderm development, while CAD96CA is activated by collagen during wound healing. Now the authors claim that in addition to these ligands, the receptors also bind to JH. However, it is unclear whether these RTKs are activated by JH independently of their known ligands, suggesting a specific binding site for JH, or if they are only induced by JH activation when those ligands are present in a synergistic manner. Alternatively, another explanation could be that the RTK pathways by their known ligands activation may induce certain levels of JH transducer phosphorylation, which, in the presence of JH, contributes to the full pathway activation without JH-RTK binding being necessary.
Thank you for your professional questions. It is an exciting and challenging to explore the molecular mechanism by which multiple ligands transmit signals through the same receptor. It requires a long-term research plan and in-depth studies. We added discussion in the text: "CAD96CA (also known as Stitcher, Ret-like receptor tyrosine kinase) activates upon epidermal wounding in Drosophila embryos (Tsarouhas et al., 2014) and promotes growth and suppresses autophagy in the Drosophila epithelial imaginal wing discs (O'Farrell et al., 2013). There is a CAD96CA in the genome of the H. armigera, which is without function study. Here, we reported that CAD96CA prevents pupation by transmitting JH signal as a JH cell membrane receptor. We also showed that CAD96CA of other insects has a universal function of transmitting JH signal to trigger Ca2+ mobilization, as demonstrated by the study in Sf9 cell lines of S. frugiperda and S2 cell lines of D. melanogaster.
FGFRs control cell migration and differentiation in the developing embryo of D. melanogaster (Muha and Muller, 2013). The ligand of FGFR is FGF in _D. melanogaste_r (Du et al., 2018). FGF binds FGFR and triggers cell proliferation, differentiation, migration, and survival (Beenken and Mohammadi, 2009; Lemmon and Schlessinger, 2010). Three FGF ligands and two FGF receptors (FGFRs) are identified in Drosophila (Huang and Stern, 2005). The Drosophila FGF-FGFR interaction is specific. Different ligands have different functions. The activation of FGFRs by specific ligands can affect specific biological processes (Kadam et al., 2009). The FGFR in the membrane of Sf9 cells can bind to Vip3Aa (Jiang et al., 2018). One FGF and one FGFR are in the H. armigera genome, which has yet to be studied functionally. The study found that FGFR prevents insect pupation by transmitting JH signal as a JH cell membrane receptor. Exploring the molecular mechanism and output by which multiple ligands transmit signals through the same receptor is exciting and challenging.".
Reviewer #1 (Recommendations For The Authors):
As an experimental suggestion, I will only propose that authors test the double knock-down/knock-out or overexpression of CAD96CA and FGFR1 to give some hints into how redundant/independent the two receptors are.
Thank you very much for your professional advice. We agree with your point of view that double knockout of CAD96CA and FGFR1 is very important to resolve the redundant/independent of the two receptors, which can make our research more complete. Unfortunately, due to experimental difficulty and time constraints, we did not provide supplementary experiments. In this study, we aim to screen the cell membrane receptors of JH. Therefore, we focused on which RTKs can function as receptors. This article is a preliminary study to identify the cell membrane receptors of JH. To further understand the relationship between the two membrane receptors, we will conduct in-depth research in future work.
Apart from that, here are some minor points about the manuscript:
Figure 2A: changing the scale on the y-axis would help to better see the different genotypes (similar to the way it is presented in Figure 5).
Thanks for your reminding, we have changed the scale in Figure 2A.
Figure 4J: image settings could be improved to better highlight the green fluorescence.
Thank you for your advice, we have improved the imaged in Figure 4J.
In general, the manuscript would benefit from some proofreading since a number of sentences are incorrect.
Thanks for your reminding, we have carefully revised the manuscript.
Reviewer #2 (Recommendations For The Authors):
(1) Although the authors note that there are 21 RTK genes in Drosophila (line 55), I can only see 16 Drosophila RTKs in Figure 1 - Figure Supplement 1. Some important Drosophila RTKs such as breathless are missing. The authors need to redraw the phylogenetic tree.
Thanks for your reminding, we have presented the new phylogenetic tree in Figure 1-figure supplement 1.
(2) The accelerated pupation phenotype in Cad96ca and Fgfr1 G0 mutants needs to be better described. In particular, it is critical to examine which developmental stage(s) are shortened in these mutant larvae. Refer to a similar study on a JH biosynthetic enzyme in Bombyx (PMID: 22412378) regarding how to describe the developmental timing phenotype.
Thank you for your advice. We have re-shown Figure 4E and added the explanation in the text: "In 61 survivors of Cas9 protein plus Cad96ca-gRNA injection, 30 mutants were sequenced, and a mutation efficiency was 49.2%. Similarly, in the 65 survivors of Cas9 protein plus Fgfr1-gRNA injection, 35 mutants were sequenced, and a mutation efficiency was 53.8% (Figure 4C). The DNA sequences, deduced amino acids and off–target were analyzed (Figure 4—figure supplement 1). Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".
(3) The editing efficiency described in lines 211-213 is obscure. Does this indicate the percentage of animals with noisy sequencing spectra or the percentage of mutation rates analyzed by TA cloning?
Thanks for your reminder. We have revised the description in the text: "In 61 survivors of Cas9 protein plus Cad96ca-gRNA injection, 30 mutants were sequenced, and a mutation efficiency was 49.2%. Similarly, in the 65 survivors of Cas9 protein plus Fgfr1-gRNA injection, 35 mutants were sequenced, and a mutation efficiency was 53.8% (Figure 4C). The DNA sequences, deduced amino acids and off–target were analyzed (Figure 4—figure supplement 1). Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".
(4) In Figures 4F and G, the authors examined expression levels of some JH/ecdysone responsive genes only at 0 hr-old 6th instar larvae. This single developmental stage is not enough for this analysis. In particular, the expression level of Fgfr1 only goes up in the mid-6th instar according to their own data (Figure 1-Figure Supplement 4), so it is critical to examine expression levels of these genes at least throughout the 6th larval instar.
Thank you for your advice. Indeed, it is essential to detect the expression levels of JH/ecdysone response genes in the whole sixth instar larvae. Because we observed that the mutation has a shorter feeding stage at the sixth instar, we examined the expression level of the JH/ecdysone response gene at the early sixth instar. Due to the number of mutants obtained in the experiment was small and non-destructive sampling could not be performed in sixth instar period, there were no enough samples to test. In the future, we will generate Cad96ca Fgfr1 double mutations to carry out studies and detect the expression level of JH/ecdysone response genes in the whole sixth instar.
(5) As mentioned above, some important Drosophila RTKs such as breathless are missing in their analyses. As breathless is a close paralog of heartless (Htl), I am sure that Drosophila breathless is also orthologous to Helicoverpa FGFR1. The authors therefore need to analyze breathless in Figure 5B in addition to Htl.
Thank you for your advice. We added experiments and the results are shown in Figure 5B and Figure 5—figure supplement 1.
(6) More discussion about the reason why dsNrk and dsWsck can provide resistance to JHIII in Figure 1 is required.
Thank you for your advice. We added explanation in the discussion: "It is generally believed that the primary role of JH is to antagonize 20E during larval molting (Riddiford, 2008). The knockdown of Cad96ca, Nrk, Fgfr1, and Wsck showed phenotypes resistant to JH III induction and the decrease of Kr-h1 and increase of Br-z7 expression, but knockdown of Vegfr and Drl only decrease Kr-h1, without increase of Br-z7. Br-z7 is involved in 20E-induced metamorphosis in H. armigera (Cai et al., 2014), whereas, Kr-h1 is a JH early response gene that mediates JH action (Minakuchi et al., 2009) and represses Br expression (Riddiford et al., 2010). The high expression of Br-z7 is possible due to the down-regulation of Kr-h1 in Cad96ca, Nrk, Fgfr1 and Wsck knockdown larvae. The different expression profiles of Br-z7 in Vegfr and Drl knockdown larvae suggest other roles of Vegfr and Drl in JH signaling, which need further study."
Reviewer #3 (Recommendations For The Authors):
(1) The authors should consider optimizing their experimental approach by depleting the six candidate RTKs in an early larval stage rather than using a sensitized background with JH application in the last larval stage.
Thank you for your precious suggestion. We knocked down the genes at last larval stage to observe pupation, which is a relatively simple and easily to be observed target to examine the role of the gene in JH-maintained larval status. The results from CRISPR/Cas9 experiments showed: "Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.". To know the roles of other RTKs in the whole larval development needs future work since a lot of experiments are needed.
(2) Including a positive control for JH signaling, such as met or tai, would strengthen the assays and provide a benchmark for evaluating the downregulation of target genes and phenotype reversion upon JH application. This addition, especially in Figure 1, would enhance the interpretability of the results.
Thank you for your suggestion. We agree with your point of view that adding the detection of Met or Tai as a positive control. Our laboratory has reported in previous studies that knockdown of Met leads to decreased expression of genes in the JH signaling pathway and precocious pupation (PMID: 24872508), so we did not repeat this related experiment in this study. In the future, when performg Cad96ca and Fgfr1 double mutant experiments, Met mutant can be generated as a control to provide more references for the interpretation of the results.
(3) I recommend revising the manuscript to improve readability, particularly in the Results section, where descriptions of the binding part are particularly dense.
Thank you for your advice. We have carefully revised the manuscript.
(4) In line 122, please add the reference Wang et al., 2016.
Thank you for your reminding, we have added the reference in line 125 of the new manuscript.
(5) The authors should clarify why they chose to test the possible binding to JH of only Cad96CA, FGFR1, and NRK after conducting various assays while including OTK in the study as a negative control. This explanation should be included in the text.
Thank you for the suggestion. We added the explanation, as described in the text: "We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously.".
"Since Cad96CA, FGFR1, and NRK were not only involved in JH-regulated Kr-h1 expression, JH III-induced delayed pupation, and calcium levels increase, but also involved in MET and TAI phosphorylation, we further analyzed their binding affinity to JH III. OTK did not respond to JH III, so we used it as a control protein on the cell membrane to exclude the possibility of nonspecific binding.".
(6) The observed embryonic lethality of cad96ca and FGF1 mutants in Drosophila contrasts with the ability of the respective mutants in H. armigera to reach the pupal stage. The authors should discuss this significant difference.
Thank you for the suggestion. We added the explanation in the discussion, as described in the text: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".
(7) Building upon the previous point, it is noteworthy that the cad96ca and FGF1 mutants exhibit only a 24-hour early pupation phenotype, contrasting with the 48-hour early pupation induced by Kr-h1 depletion. This discrepancy suggests that while the function of these RTKs is necessary, it may not be sufficient to fully activate JH signaling. The expression profile of these receptors, primarily observed in the last larval stage, supports this hypothesis.
Thank you for your suggestion. We added the explanation in the discussion, as described in the text: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".
(8) The expression profile of the RTK hits described in Supplementary Figure 4A appears to be limited to the last larval stage until pupation. The authors should clarify whether these receptors are expressed earlier, and the meaning of the letters in the plot should be described in the figure legend.
Thank you for the suggestion. We added the explanation in the Figure 1—figure supplement 4 legend, as described in the text: "The expression profiles of Vegfr1, Drl, Cad96ca, Nrk, Fgfr1, and Wsck during development. 5F: fifth instar feeding larvae; 5M: fifth instar molting larvae; 6th-6 h to 6th-120 h: sixth instar at 6 h to sixth instar 120 h larvae; P0 d to P8 d: pupal stage at 0-day to pupal stage at 8-day F: feeding stage; M: molting stage; MM: metamorphic molting stage; P: pupae.".
We are very sorry, but due to time limitations, we will investigate the expression profile of RTK throughout the larval stage in future work.
(9) In Figure 4, panels F and G, the levels of Kr-h1 are shown in cad96ca and FGF1 mutants in the last larval stage. The authors should indicate whether Kr-h1 levels are also low in earlier larval stages or only detected in the last larval stage, as this would imply that these RTKs are only required at this stage.
Thank you for your suggestion. In this study, the Cad96ca and Fgfr1 mutants' feeding stage was shortened in the sixth instar, and they entered the metamorphic molting stage earlier. So, we detected the expression of Kr-h1 in the sixth instar. It is an excellent idea to detect the expression of Kr-h1 at various larvae stages to analyze the stages in which CAD96CA and FGFR1 play a role and to study the relationship between CAD96CA and FGFR1 in future.
(10) While Figure 5 demonstrates JH-triggered calcium ion mobilization in Sf9 cells and S2 cells, the authors should also include data on JH signaling target genes, such as Kr-h1, for a more comprehensive analysis.
Thank you for your advice. We added experiments, as described in the text: "To demonstrate the universality of CAD96CA and FGFR1 in JH signaling in different insect cells, we investigated JH-triggered calcium ion mobilization and Kr-h1 expression in Sf9 cells developed from S. frugiperda and S2 cells developed from D. melanogaster. Knockdown of Cad96ca and Fgfr1 (named Htl or Btl in D. melanogaster), respectively, significantly decreased JH III-induced intracellular Ca2+ release and extracellular Ca2+ influx, and Kr-h1 expression (Figure 5A, B, Figure 5—figure supplement 1A and B). The efficacy of RNAi of Cad96ca and Fgfr1 was confirmed in the cells (Figure 5—figure supplement 1C and D), suggesting that CAD96CA and FGFR1 had a general function to transmit JH signal in S. frugiperda and D. melanogaster.".
(11) The authors should consider improving the quality of images and some plots, particularly enlarging panels showing larval and pupal phenotypes, such as Figure 1B and Supplementary Figure C. Additionally, adding a plot showing the statistical analysis of the phenotype in Supplementary Figure C would enhance clarity. Some plots are overly busy and difficult to read due to small size, such as Figure 1C, Figure 2A, and all the plots in Figure 3. Figure 4E also requires improvement for better readability.
Thank you for your suggestion. We have adjusted Figure 1B, Figure 1C, Figure 1—figure supplement 1C, Figure 2A and Figure 4E. However, for Figure 3, we have not found a better way to arrange and adapt them, considering the overall arrangement of the results and the page space, so we keep them in their original state.
