Review Reports - A Computational Analysis Based on Automatic Digitization of Movement Tracks Reveals the Altered Diurnal Behavior of the Western Flower Thrips, <i>Frankliniella occidentalis</i>, Suppressed in <i>PKG</i> Expression

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title: Constructed Excellently (If possible, to do short)

Introduction: The introduction is well-structured and effectively sets the stage for the content that follows.

Abstract: Well written.

Materials and Methods (M&M): The methodology section is exemplary, demonstrating a thorough and precise approach to the research process.

Results: The results section is flawless, with no errors or redundancies noted. The data presentation is clear and concise, ensuring the accuracy and reliability of the findings.

Discussion: The discussion is notably compelling, providing insightful analysis and vigorous interpretation of the results in relation to existing literature and future implications.

Comments on the Quality of English Language

English writing is good not required.

Author Response

Comment #1: Introduction: The introduction is well-structured and effectively sets the stage for the content that follows. Abstract: Well written. Materials and Methods (M&M): The methodology section is exemplary, demonstrating a thorough and precise approach to the research process. Results: The results section is flawless, with no errors or redundancies noted. The data presentation is clear and concise, ensuring the accuracy and reliability of the findings.

Discussion: The discussion is notably compelling, providing insightful analysis and vigorous interpretation of the results in relation to existing literature and future implications.

Response: We appreciate your encouragements. In addition, we revised the manuscript with valuable comments raised by other reviewers. After all revision, the corresponding authors edited the entire manuscript to keep consistency.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an impressive study. It uses sophisticated methods, takes an integrative approach from gene expression to behavior, provides important new data on the biology of a global crop pest, and demonstrates effective use of methods for expanding research on circadian rhythms from a small number of lab-based model organisms to field organisms and crop pests. This study also demonstrates the importance of taking time of day into account in any assays of gene expression. The experimental designs and statistical analyses are rigorous.

There is one important theoretical issue that needs clarification, and some minor grammatical suggestions.

The one critical suggestion I have is to clarify the difference between analysis of a diel rhythm (e.g. activity and transcription in this case) and analysis of a circadian rhythm. The entire analysis was conducted on rhythms in a light:dark photoperiod, which means that the diel rhythms could be driven by, or altered by the photoperiod and not by a circadian clock mechanism. The most direct way to demonstrate that the diel rhythms are circadian is to demonstrate that they persist under constant conditions, in the absence of the light:dark cycle. Another way to demonstrate that the diel rhythms are driven by a circadian clock is to show that altering the circadian clock mechanism alters the rhythm: the authors do show that PKG RNAi alters the expression of two circadian clock genes and the activity rhythm, but these could be independent effects- it's not clear that the RNAi effect on the clock genes is causing the effect on the behavioral rhythm- that could be directly mediated through PKG expression. This result does, however, demonstrate that PKG expression is regulating the expression of Per and Clock (assuming no collateral effects of the RNAi on other genes), and the authors show associated changes in both clock gene expression and the behavioral rhythm, which is consistent with circadian clock regulation of the behavior. I think the authors have clearly demonstrated the importance of PKG in regulating circadian activity rhythms here, but as they point out in the discussion, the mechanisms mediating clock, PKG, physiological mechanisms and behavioral mechanisms are not clear and require further study. The authors are in an excellent position to conduct further studies, and this research is an excellent and impressive demonstration that these mechanisms are functionally linked. So far, however, in the absence of direct manipulation of the circadian clock mechanism as an independent variable, or demonstration of a free-running circadian activity rhythm in the thrips, the authors need to be careful not to over-interpret their results with respect to whether the PKG effects on behavior are mediated through a circadian clock mechanism or some other mechanism, and whether the PKG effects on clock genes are causal or incidental with respect to the changes in behavior. Is PKG, for example, regulating a circadian clock which in turn alters the behavior, or is it coupling clock function to the behavior (like PDF in fruit flies), or both?
The only other non-grammatical point is that Figure 7 could be explained more clearly. Combining Kolmogorov-Smirnoff and Mann-Whitney statistical analysis is not a commonly used method, so the authors might include, in the Figure legend, a little more guidance on how one should interpret the figure. Also, the figure legend notes "A" and "B" type data, but there is no "A" or "B" on the figure itself so it's not clear which element in the graphs correspond to the A and B variables. The method itself is clever and well done but it's difficult to understand the figure as described.
Line 40: replace "the gene expression" with "PKG gene expression"?
Lines 56-57: replace "under the oscillating expressions of the" with "by interacting with the expression of circadian"?
Lines 61-62: replace "give a feeding" to "causes"?
Line 63: replace "virus" with "viruses" and causing a devastating" to " causing devastating"?
Lines 64-65: replace "hiding behavior into flowers" with "ability to hide in flowers and "to be effectively controlled by the" with effectively control"?
Line 67: replace "suppress the outbreaks" with "suppress outbreaks"?
Line 68: replace "to sticky trap" with "in sticky traps"?
Line 69: replace "the visual" with "visual" and "useful" to "used" and "of the thrips under the" with and facilitated"?
Line 78: replace "influence of" with "influences"?
Line 79: delete "around diet"?
Line 82: replace "depending on the onset of light signal by" with "by signals mediated through"?
Line 83: replace "and catalyzes" with "which catalyzes"?
Line 85: replace clock remained" with "clock function remained"?
Line 88: delete "this study predicted", replace "PKG gene in" with "PKG gene expression in, and replace "and its expression levels were" with "was"?
Line 89: replace "during 24-h period" with "over a 24-h period in a 12:12 LD cycle"?
Line 92-93: replace "automatically detected data of the movement tracks by a continuous 24 h-monitoring device" with "continuous automated 24-hour monitoring of movement tracks"?
Line 524: replace "exhibited" with "showed" and "by" with "with"?
Line 526: replace "kind of the" with "kind of" and replace "controlled by" with "consistent with" and replace "machinery equipped with" with "regulation mediated through"?
Line 527: replace Per/TIM" with "the Per/TIM"?
Line 536: replace "with the clock gene expressions" with "clock gene expression"?
Line 555: replace "study" with "studies"?
Line 612: replace "staying near" with "to stay near"?
Lines 630-631: replace "on reasonable guidance for" with "relating" and replace "molecular physiological approach to behavioral data" with "molecular and physiological mechanisms to behavior""?
Line 632: replace "in illustrating" with "to illustrate"?

Comments on the Quality of English Language

Minor grammatical edits

Author Response

Comment #2-1: This is an impressive study. It uses sophisticated methods, takes an integrative approach from gene expression to behavior, provides important new data on the biology of a global crop pest, and demonstrates effective use of methods for expanding research on circadian rhythms from a small number of lab-based model organisms to field organisms and crop pests. This study also demonstrates the importance of taking time of day into account in any assays of gene expression. The experimental designs and statistical analyses are rigorous.

There is one important theoretical issue that needs clarification, and some minor grammatical suggestions.

Response: We appreciate your clear understanding on the manuscript. Other comments are carefully reflected in the revised version.

Comment #2-2: The one critical suggestion I have is to clarify the difference between analysis of a diel rhythm (e.g. activity and transcription in this case) and analysis of a circadian rhythm. The entire analysis was conducted on rhythms in a light:dark photoperiod, which means that the diel rhythms could be driven by, or altered by the photoperiod and not by a circadian clock mechanism. The most direct way to demonstrate that the diel rhythms are circadian is to demonstrate that they persist under constant conditions, in the absence of the light:dark cycle. Another way to demonstrate that the diel rhythms are driven by a circadian clock is to show that altering the circadian clock mechanism alters the rhythm: the authors do show that PKG RNAi alters the expression of two circadian clock genes and the activity rhythm, but these could be independent effects- it's not clear that the RNAi effect on the clock genes is causing the effect on the behavioral rhythm- that could be directly mediated through PKG expression. This result does, however, demonstrate that PKG expression is regulating the expression of Per and Clock (assuming no collateral effects of the RNAi on other genes), and the authors show associated changes in both clock gene expression and the behavioral rhythm, which is consistent with circadian clock regulation of the behavior. I think the authors have clearly demonstrated the importance of PKG in regulating circadian activity rhythms here, but as they point out in the discussion, the mechanisms mediating clock, PKG, physiological mechanisms and behavioral mechanisms are not clear and require further study. The authors are in an excellent position to conduct further studies, and this research is an excellent and impressive demonstration that these mechanisms are functionally linked. So far, however, in the absence of direct manipulation of the circadian clock mechanism as an independent variable, or demonstration of a free-running circadian activity rhythm in the thrips, the authors need to be careful not to over-interpret their results with respect to whether the PKG effects on behavior are mediated through a circadian clock mechanism or some other mechanism, and whether the PKG effects on clock genes are causal or incidental with respect to the changes in behavior. Is PKG, for example, regulating a circadian clock which in turn alters the behavior, or is it coupling clock function to the behavior (like PDF in fruit flies), or both?

Response: This comment is helpful to explain the limitation of this study. Thus we rephrased the discussion related with this issue as follows: “The continuous monitoring of the thrips behavior showed a diurnal rhythmicity with relatively high activity during photophase and relatively low activity during scotophase. This kind of diel pattern is consistent with its circadian regulation mediated through two oscillatory loops: the Per/TIM oscillatory loop and the CRY loop [27,28]. In each of these loops, CLK/CYC acts as a transcriptional activator that promotes Per and TIM, or CRY transcription. The product proteins Per and TIM, or CRY are thought to provide negative feedback to inhibit the transcriptional activator. In D. melanogaster, CRY is expressed in specific clock neurons in the brain [29]. CRY is then activated by blue light and catalyzes TIM degradation through its protease activity, at which point the circadian clock is reset [30,31]. In F. occidentalis, Per and CLK genes exhibited the diel patterns with relatively high expressions during photophase and relatively low expressions during scotophase. This supports the circadian rhythmicity of the thrips behaviors controlled by clock genes be-cause any interruption of the clock gene expressions significantly altered the diel rhyth-micity [8]. Interestingly, this current study showed that PKG expression followed this diel pattern, suggesting a functional association of its expression with clock gene expression.

RNAi specific to PKG expression led to alterations in the clock gene expressions dur-ing photophase by preventing up-regulation of the clock genes. PKG expression is associ-ated with division of labor in honey bee, Apis mellifera, in which specific expression in the mushroom bodies of the brain, suboesophageal ganglion, and corpora allata is associated with foraging behavior to collect pollens and nectars [9]. The role of PKG expression in the labor division in social insects was also found in a fire ant, Solenopsis invicta, in which RNAi specific to PKG reduced the locomotory activity and facilitated the behavioral change from foragers to nurses [32]. PKG activity and locomotory activity has been well established in Drosophila, in which flies are discriminated into sitters with low PKG activ-ity or rovers with high PKG activity [33]. These suggest that PKG expression is associated with high locomotory activities of F. occidentalis during photophase. Thus, the RNAi spe-cific to PKG expression resulted in reduced immature development and adult fecundity presumably by inhibiting feeding and ovipositional behavior. In addition, the suppression of PKG expression led to the suppression of the clock gene expressions during photophase, supporting the functional association of the PKG expression with the circadian rhythmic-ity. In mammals, the suppression of clock gene expressions led to the suppression of PKG protein level [34]. This suggests that the alterations of the clock gene expressions may be indirect influence of the reduced PKG mRNA level caused by RNAi in F. occidentalis. The control of PKG level by the clock gene expressions needs to be addressed in future studies. It is also noteworthy that there are at least four isoforms of PKG in F. occidentalis genome: 176 (XP_052119469.1), 417 (XP_052132331.1), 694 (XP_026272945.1), and 1,010 (XP_026272942.1) amino acid residues. In this study, we analyzed only PKG with 694 amino acid length isoform. Thus, the other isoforms should be analyzed in their inde-pendent roles. In addition, our current study showed the altering expressions of two cir-cadian genes by manipulating PKG expression but did not show the control of the PKG gene expression by the circadian gene expression. Thus, it is still unclear whether the PKG effects on clock genes are causal or incidental with respect to the changes in behavior. This needs to be clarified in subsequent study.”

Comment #2-3: The only other non-grammatical point is that Figure 7 could be explained more clearly. Combining Kolmogorov-Smirnoff and Mann-Whitney statistical analysis is not a commonly used method, so the authors might include, in the Figure legend, a little more guidance on how one should interpret the figure. Also, the figure legend notes "A" and "B" type data, but there is no "A" or "B" on the figure itself so it's not clear which element in the graphs correspond to the A and B variables. The method itself is clever and well done but it's difficult to understand the figure as described.

Response: Explanations on interpreting statistical results are added in the legend of Figure 7, and ‘(A) and (B)’ in Figure 7 are eliminated, as advised. Also additional statements regarding assumptions of statistical differences are provided in ‘Materials and Methods’ and ‘Discussion’: [Figure 7] ‘Statistical differentiation between dsCON and dsPKG in each hour phase in 24-h cycle (14-hour photophase (P1-P14) and 10-hour scotophase (S1-S10)) according to combined results from Mann-Whitney U and Kolmogorov-Smirnov tests with higher shade presenting stronger statistical differentiation (see text), in which movement parameters and duration rates (%) in different micro-areas were compared among different light phases (see green arrows indicating S4 with significant difference from other light phases.)’ [Materials and Methods] ‘Two aspects of statistical difference were considered in this study: difference between dsCON and dsPKG at each light phase, and difference among light phases within each strain. Behavior data in this study had a property of measurement dependence. Since observations were continuously conducted throughout the whole observation period, movement across light phases are dependently observed (i.e., observed continuously). In this study measurements in different light phases were considered as independent since this study is an initial phase of behavior study to confirm physiological effects, and an extensive experimental design would be required for analyzing repeated measurements possibly with large number of trials.’ [Discussion] ‘In this study measurements in different light phases were considered as independent for statistical analyses (see Section 2.8. Statistical analysis). When one-way re-peated ANOVA was applied to dependent measurements of parameters and durations (%), statistical differences were not obtained between light phases. This would be partly due to low number of trials and partly due to heterogenous conditions of tested females. In the future a more extensive experimental design will be needed including an increase in trial numbers and increase in physiological homogeneity of test females (e.g., precise range in age) as well.’

Comment #2-4: Line 40: replace "the gene expression" with "PKG gene expression"?