Diurnal rhythmicity in metabolism and salivary effector expression shapes aphid performance on host plants

Reviewer #2 (Public review):

Summary:

The authors conducted a time-course of whole-body transcriptional analysis of a pest aphid, Rhopalosiphum padi, and identified four major clusters of the genes that show diurnal rhythmicity in transcription. In addition, they have conducted the analysis of aphid feeding behaviour and showed that aphids salivate longer from the end of the day toward the beginning of the night while their phloem feeding time does not change throughout a day. The genes up-regulated at nighttime were enriched with the genes involved in metabolic activities, collaborating with the results showing higher number of honeydew excretion at night. The authors identified the list of candidate salivary genes that show diurnal rhythmicity in the transcription and silenced a salivary gene C002 and the candidate salivary gene E8696. Silencing of these genes reduced aphid fecundity and survival rate on the host plant but not on the artificial diet.

Strengths:

The time-course transcription study and its analysis will be of interest to researchers studying diurnal rhythms in insect biology. Also, the analysis of aphid feeding behaviour at different time of day is interesting. This study provides variable resources for those who study insect biology.

Weaknesses:

Without the knowledge of the functions of the salivary effectors, especially their targets, it is hard to conclude that the rhythmical expression is important for the aphid performance. In addition, it is not clear whether increase of gene expression is directly corelated with the increase of protein secretion into the saliva and the plant.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

This study presents valuable data on diurnal patterns in aphid (Rhopalosiphum padi) feeding behavior and transcriptome profiles. The authors measured honeydew production by the aphids on plants and artificial diet during the day and night and conducted a comprehensive feeding behavior study using EPG with many biological replicates at 6 time-points in 24 hours. They also conducted transcriptome analyses of three samples of each 30 aphids at these time points. Differentially expressed transcripts were grouped into four clusters with distinct expression patterns. The expression of two genes found to be diurnally rhythmic was knocked down with RNAi and these aphids did less well, especially at night. They also analyzed the differential expression of candidate effector genes and found rhythmic ones to be enriched for more expression in aphid heads versus bodies - this pattern is expected given that effectors are most likely expressed in the salivary glands. Knockdown of a known effector (C002) that is diurnally rhythmic, and a novel effector gene, was found to alter aphid feeding dynamics and performance.

Thank you for your thoughtful review and summary of our study. We would like to clarify one aspect of your summary regarding our clustering analysis. We did not cluster differentially expressed transcripts. Instead, our clustering analysis was performed exclusively on transcripts that were significantly diurnally rhythmic, as identified in our time-course transcriptomic analysis. This approach allowed us to reveal patterns of gene expression that exhibit robust rhythmicity over the 24-hour cycle, rather than grouping transcripts based solely on differential expression at individual time points.

Strengths:

The manuscript was highly accessible, with clear writing, and the figures provided were both comprehensive and of good quality. The datasets generated from this research are valuable to the research field, especially the findings for honeydew secretion, EPG analysis, and transcriptome experiments.

The datasets generated in this study will be useful to scientists working on aphids and aphidplant interactions and will inform similar studies on other insect species.

Weaknesses:

The weaknesses mainly relate to the (depth of) analyses and interpretation of the data. Also, some methods require more explanation, as follows:

In Figure 1, data show that aphids produce more honeydew at night than during the day. This suggests that the aphids ingest more phloem (E2 phase). However, in Figure 1d the duration of the E2 phase does not show obvious differences among the time points in the 24 hours. The authors contribute the explanation that the aphids may osmoregulate more during the night, leading to more honeydew secretion at night. This may be the case, but there could be other explanations. For example, the physiology, including regulation of water transport, of plants is known to change during night/day. The authors may focus this section more on the differences in the E1 phase, as this involves the delivery of aphid saliva and effectors into the plant phloem.

Thank you for your constructive feedback. As noted, aphids excreted more honeydew at night, although the duration of the E2 phase did not differ significantly across time points. We agree that host plant physiology, particularly the composition of the phloem and its osmotic quality, also influences the observed osmoregulatory patterns in R. padi. However, a similar diurnal pattern of honeydew excretion was also observed on artificial diets (Fig. 1b), in which host-derived cues have been eliminated. This strongly suggests that increased nighttime honeydew excretion is primarily driven by enhanced aphid osmoregulation rather than plant factors alone. Nonetheless, we acknowledge that plant-derived factors may also contribute and cannot be entirely ruled out. We have revised the text in the discussion of the revised manuscript to reflect this broader interpretation. As suggested, we have also added further details to highlight the important role of the E1 phase in aphid salivation.

Transcriptome data shown in Figure 2 (and the experimental procedure of Figure 5b) appears to be based on three biological replicates. However, these replicates appear to have been harvested at the same time in the experiment, and this makes them technical replicates, not biological replicates. The inclusion of true biological replicates that include samples from time series experiments done on different days should be considered.

Thank you for your concern regarding the biological replication in our transcriptome analysis. Our experimental design included multiple independent pools of aphids collected at each time point. Specifically, each replicate consisted of a unique group of aphids collected from different leaf positions across multiple host plants, such that no individuals were shared among replicates. As a result, these samples represent biologically independent populations rather than technical replicates, which are defined as repeated measurements of the same biological sample used to estimate technical noise. Although all samples were collected within the same experimental time course, this approach is commonly used in time-series transcriptomic studies to minimize confounding variation associated with differences in insect age, entrainment history, or environmental conditions, all of which can obscure rhythmic gene expression patterns. By maintaining tightly controlled and consistent conditions across the sampling period, we aimed to ensure that observed transcriptional differences primarily reflected diurnal regulation rather than uncontrolled day-to-day variability.

We acknowledge that conducting time-series experiments on different days could provide additional insight into biological variability. However, our approach aimed to reduce potential confounding effects caused by day-to-day environmental fluctuations – such as minor changes in temperature or humidity - which could significantly influence gene expression in insects. By maintaining consistent conditions, we sought to ensure that observed transcriptional differences were due to diurnal rhythms rather than uncontrolled variation. Similar designs or strategies have been employed in studies examining diurnal and circadian gene expression in both insects and plants. We have revised the Methods section to clarify our replication strategy.

The authors conducted knockdown experiments targeting aquaporin 1 and gut sucrase 1 in aphids, resulting in reduced nymph production and decreased honeydew secretion. It is concluded that these results indicate significant roles of aquaporin 1 and gut sucrase 1 in diurnal regulation. However, it is essential to consider that these genes likely play crucial roles in aphid physiology beyond diurnal rhythms. Consequently, reduced expression would naturally impair aphid performance. The dsAQP1 and dsSUC1 aphids consistently produced less honeydew, regardless of the time of day, indicating a broader impact of gene knockdown. The observed increase of the phenotype at night may not be attributable to the specific roles of these genes in diurnal regulation but rather due to heightened aphid activity during that time (as evidenced by increased honeydew secretion) that could magnify the impact of the knockdown effect, making it easier to observe. Therefore, the knockdown of aquaporin 1 and gut sucrase 1 may exert a general negative influence on aphid fitness, independently of diurnal factors.

We agree that these genes likely play fundamental roles in aphid physiology beyond diurnal rhythms, and that reduced expression may affect overall aphid performance. However, it is important to highlight that if the observed effects are solely due to general fitness impairments, we would more likely expect a comparable reduction across time points rather than a disproportionately stronger impact at night. We agree that the increased honeydew excretion at night is likely due to heightened aphid excretory activity. However, since this excretory behavior is downstream of osmoregulatory functions, such as water cycling to the midgut and digestion, polymerization, and excretion of oligosaccharides, the increased nighttime phenotype is likely a result of an increased nighttime regulation of osmoregulation in aphids. This hypothesis is further supported by our functional analysis, where the knockdown of AQP1 and SUC1 resulted in a loss of diurnal variation in honeydew production (Fig. 2h), indicating that the observed effects are not merely a general impact on aphid fitness but are likely tied to the genes' roles in regulating diurnal physiological processes. We have revised the discussion to clarify that our findings do not exclude general physiological roles for these genes but instead suggest that their functions intersect with diurnal rhythms to influence aphid feeding and excretion patterns.

To analyze the roles of genes in diurnal regulation, additional controls should be incorporated. This could involve the knockdown of genes with essential functions that are not influenced by diurnal rhythms, providing a baseline comparison. Furthermore, consider including genes known to be involved in diurnal regulation in other insects, as documented in the existing literature, in the experimental design.

We agree that incorporating appropriate controls in the RNAi experiments would provide a useful baseline for comparisons, helping to distinguish between general physiological effects and specific diurnal effects. Unfortunately, given the current limited knowledge on rhythmic genes in aphids, particularly in the context of aphid-plant interactions, it is challenging to identify appropriate rhythmic and non-rhythmic controls that can be definitively linked to or unaffected by diurnal regulation within aphids. We will ensure to consider this valuable suggestion in our future experiments.

The same arguments as for aquaporin 1 and gut sucrase 1 above may be made for knockdown of effector genes (Figure 4). It has already been shown that knockdown of C002 impacts aphid performance, and the data herein may be explained by a general lower performance of aphids rather than a specific function of these effectors in diurnal regulation. It is also expected that knockdown of the effectors has less impact on aphids feeding from artificial diets. This does not necessarily indicate the role of the effectors in diurnal regulation.

Our response to this comment mirrors that expressed in our earlier response regarding AQP1 and SUC1.

In the abstract and elsewhere, the authors assert priority by stating, "...the first evidence of...". However, it's important to note that priority claims are often challenging to verify across many fields. Instead of relying solely on claims of precedence, the evidence presented in the research could stand on its own merit.

We understand that priority claims can be difficult to substantiate across various fields, and we appreciate the importance of allowing the evidence to speak for itself. Considering this, we have revised the language in the abstract.

Conclusion:

The study presents intriguing new findings, particularly in the realms of honeydew analysis, EPG, and transcriptome analysis. However, the interpretation of subsequent studies employing gene knockdowns needs further consideration.

We thank the reviewer for the thoughtful and constructive feedback. We appreciate the positive assessment of our findings on honeydew analysis, EPG, and transcriptome profiling. We have carefully revised the section on gene knockdown experiments to provide clearer interpretation and additional context, and we hope the concerns raised have now been appropriately addressed.

Reviewer #2 (Public Review):

Summary:

The authors conducted a time-course of whole-body transcriptional analysis of a pest aphid, Rhopalosiphum padi, and identified four major clusters of the genes that show diurnal rhythmicity in transcription. In addition, they conducted the analysis of aphid feeding behaviour and showed that aphids salivate longer from the end of the day toward the beginning of the night while their phloem feeding time does not change throughout the day. The genes upregulated at night time were enriched with the genes involved in metabolic activities, collaborating with the results showing a higher number of honeydew excretion at night. The authors identified the list of candidate salivary genes that show diurnal rhythmicity in the transcription and silenced a salivary gene C002 and the candidate salivary gene E8696. Silencing of these genes reduced aphid fecundity and survival rate on the host plant but not on the artificial diet.

Thank you for your thoughtful review and valuable comments on our study.

Strengths:

The time-course transcription study and its analysis will be of interest to researchers studying diurnal rhythms in insect biology. Also, the analysis of aphid feeding behaviour at different times of day is interesting. This study provides variable resources for those who study insect biology.

Weaknesses:

It is not clear to me which data was used to define the putative salivary effectors for R. padi, but the candidate salivary gene list made by Thorpe et al consists of the aphid genes encoding secreted proteins that are up-regulated in the head samples compared to the body samples. Although some proteins were confirmed to be secreted into the aphid saliva, many genes in the list are not confirmed to be expressed in the aphid salivary glands, and their products are not confirmed to be secreted into the saliva and the plant. Is E8696 expressed in the aphid salivary glands and secreted into its host plant? Without the data confirming the expression of the gene in the salivary glands and its secretion into the saliva and into the host plant, we cannot call the protein a salivary protein. Furthermore, without the observation that E8696 has some effect on plant biology, we cannot call it an aphid effector. Therefore, I cannot agree with the parts of the manuscript that refer to E8686 as an aphid salivary effector.

We have revised the text in the Methods to clarify the database used for defining putative salivary effectors. We have also added a sentence in the discussion to indicate that these are putative effectors. The putative effector E8696 was confirmed to be expressed in the salivary glands; however, its secretion into saliva and the host plant remains undetermined due to the lack of E8696-specific antibodies. Over the past year and a half, we have been creating an antibody for E8696. However, the antibody we generated is non-specific, and as a result, we are still unable to demonstrate that E8696 is secreted into host tissue and functions as an effector. While our functional analysis provided strong evidence of E8696’s impact on aphid fecundity and mortality on host plants but not on artificial diets, we agree that without further confirmation of its secretion and effect on the host plant, E8696 should be considered only a putative salivary effector. We expect to address these important questions in future research. To prevent any confusion, we have revised our manuscript to reflect that E8696 is only a putative effector.

It is interesting to know that some candidate salivary gene expression showed a diurnal rhythm. However, without the knowledge of the functions of the salivary effectors, especially their targets, it is not possible to conclude that the rhythmical expression is important for the aphid performance. In addition, I wonder whether the increase in gene expression is directly correlated with the increase of protein secretion into the saliva and the plant.

The primary goal of this study was to determine whether aphid genes, particularly those associated with osmoregulation and salivary effectors, exhibit diurnal patterns of expression and whether disrupting these rhythms affects aphid performance. While we agree that the precise molecular targets of these effectors in host plants remain to be identified, our functional assays provide evidence that rhythmic expression is biologically relevant for aphid physiology. Our results demonstrate that silencing rhythmic effector genes resulted in increased aphid mortality, reduced fecundity on host plants, and, more importantly, the disruption of diurnal honeydew excretion patterns, especially for C002. As honeydew excretion is a critical physiological process for aphids, the alteration of this behavior suggests that the rhythmic expression of these genes is functionally important for aphid physiology. We believe our results provide compelling evidence that rhythmic expression plays a critical role in aphid biology. We agree that rhythmic transcript abundance does not necessarily imply proportional changes in protein secretion into saliva, and direct measurements of effector protein dynamics will be an important direction for future work. However, the observed physiological and performance consequences of disrupting rhythmic gene expression support the conclusion that temporal regulation of these salivary genes is functionally important for aphid biology, even in the absence of detailed target identification.

Finally, the authors examined aphid survival, fecundity, and feeding behaviour. Those are important for overall aphid performance, but they do not "shape" aphid colonization. Aphid colonisation is shaped by the mechanisms by which aphids find and select their host plant and start to feed on it. Therefore, I do not agree with the title of this manuscript and some parts of the discussion.

We agree with your perspective and have revised the title and discussion to more precisely reflect the scope of our findings, focusing on aphid performance rather than colonization. The revised title now reads “Diurnal rhythmicity in metabolism and salivary effector expression shapes aphid performance on host plants”.

I would like the authors to develop how the knowledge of the diurnal rhythm of aphid feeding can contribute to optimise pest management. I see that there are some differences in aphid metabolism and feeding behaviour between day and night, but I would like to hear how such knowledge can optimise pest management strategies.

We have expanded the Discussion to address how knowledge of diurnal rhythms in aphid physiology and feeding behavior could inform the optimization of pest management strategies. Specifically, we discuss how time-of-day variation in aphid feeding activity and metabolism may influence the efficacy of control measures and how chronobiological insights could be integrated into future pest management frameworks.

Recommendations for the authors:

Reviewing Editor:

Based on comments from two reviewers, here are the six key areas that need to be addressed to improve the manuscript.

Clarity and Specificity:

(1) Salivary effectors: The manuscript defines "salivary effector" loosely. The reviewer argues for stricter criteria - a protein can only be called a salivary effector if it's confirmed to be produced in the salivary glands and/or secreted into the plant with saliva and function in or around the plant.

We have addressed this comment and clarified the definition in the revised manuscript.

(2) Diurnal rhythm: The paper finds a daily rhythm in aphid gene expression, but doesn't explain how these genes affect the plant. The reviewer argues that without understanding the function of these genes, the significance of the rhythm is unclear.

We have addressed this comment and clarified that the scope of our study is to elucidate diurnal rhythmicity in aphid gene expression and to evaluate the functional importance of rhythmic genes for aphid performance. We agree that understanding how these genes interact with host plants is essential for fully elucidating their molecular functions under diurnal regulation; however, this is beyond the scope of the current study and will be pursued in future research.

(3) Knockdown experiments: The reviewer suggests the observed effects of knocking down certain genes (aquaporin, sucrase, effectors) might be due to their general importance, not necessarily their role in the day-night cycle. They recommend including control genes and genes known to be involved in circadian rhythms for a more robust comparison.

We have addressed this comment and clarified the interpretation of these experiments in the revised manuscript.

Technical Issues:

(4) Honeydew production: The explanation for nighttime honeydew production needs more exploration. Plant changes at night might also play a role, and the daytime saliva delivery phase deserves more attention in the analysis (Figure 1).

We expanded the description and interpretation of the salivation phase by incorporating additional detail in the revised manuscript.

(5) Gene expression data: The current data (Figure 2 & Figure 5b) lacks proper biological replicates. Replicates collected at different times are essential for stronger conclusions.

We have addressed this comment and clarified the experimental design and replication strategy in the manuscript.

(6) Priority claims: The reviewer advises against focusing on claiming novelty ("first evidence"). The research should be impactful based on its own merit, not just being the first to find something.

We revised the sentences to avoid making claims of priority throughout the manuscript.

Reviewer #2 (Recommendations For The Authors):

Figures 2 f,g, and h : according to the legend, these experiments seemed to have a low number of replicates (n=3-5). However, Figure 2h has many data points. I understood that here n means the number of experimental replications, but it may be better to show the number of aphid samples examined.

You are correct that the n refers to the number of experimental replicates, with each replicate comprising multiple individual aphids. Because our analyses were performed on replicate-level averages across multiple days, rather than on individual aphids, we believe this notation most accurately reflects the experimental design. To improve clarity, we have revised figure legends to explicitly state that each replicate includes several individual aphids.

Are the orthologous proteins of E8696 expressed in aphid salivary glands or detected in saliva? Such data will strengthen the claim that E8696 is a salivary protein of R.padi.

E8696 is expressed in aphid salivary glands, but it is not confirmed to be secreted into saliva or host plants due to the lack of specific antibodies. We have revised our manuscript to reflect that E8696 is only a putative effector. We will address this question in future research.