Review Reports - The Phenology of <i>Aphalara itadori</i> in Canada: Timing of Spring Activity and the Onset of Oviposition

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript provides valuable insights into the phenology and spring activity of Aphalara itadori as a biological control agent for invasive Fallopia japonica in Canada. The study design and methodology are generally sound, and the findings offer useful data on the timing of psyllid activity and oviposition. However, there are areas that could benefit from further clarification and refinement.

Major Comments:

Introduction Section:

The introduction provides a solid background on the invasive nature of Fallopia japonica and the role of Aphalara itadori in biological control. However, I recommend briefly mentioning how Aphalara itadori has already been deployed in other regions outside Canada, and summarize the successes/failures to set the context for the Canadian study.

Methods Section:

The description of the experimental setup (e.g., container design, soil composition, and leaf exposure) is well-detailed. However, the methodology for sexing psyllids could be further clarified to ensure reproducibility. Was there any pre-selection done for sex ratio?
The rationale behind using only 50 psyllids per container could be elaborated. It would be useful to justify if this number is statistically sufficient to capture natural variations in psyllid activity and survival.

Results Section:

The data presentation is clear, but some figures (e.g., Figures 4, 5, 6) require more detailed captions to make them more understandable to readers unfamiliar with the experimental setup.
A more explicit explanation of the relationship between the freezing events and psyllid mortality could enhance the results section. How do the authors interpret the low psyllid recovery in light of the freezing events, especially in early spring?

Discussion Section:

The discussion of the results is generally robust, but there seems to be a missed opportunity to explore in more depth how local environmental factors, such as humidity or microclimates within the experimental containers, may have influenced psyllid survival.
The low psyllid recovery in the experiment is a critical point. The authors speculate that container design may have influenced survival, but further discussion of potential methodological adjustments (larger containers, different materials, etc.) would be beneficial for future studies.

Statistical Analysis:

The use of generalized linear models with a negative binomial distribution is appropriate given the count data. However, it would be helpful to include some clarification on model assumptions. Did the authors check for overdispersion in the count data before applying the model?
It would also strengthen the analysis if the authors could mention any post-hoc tests used to interpret significant results, especially in cases where differences in psyllid activity across groups were found.

Minor Comments:

There are some typographical errors in the paper, such as the incorrect spelling of "Aphalara" as "Apahlara" in multiple locations (e.g., page 7, line 236). Please correct these typos for consistency.
The references section is generally well-organized, but I noticed that a few citations, such as for Bourchier's unpublished data, might need to be clarified further in terms of how these were sourced or corroborated.

Author Response

Reviewer 1

Major Comments:

Introduction Section:

The introduction provides a solid background on the invasive nature of Fallopia japonica and the role of Aphalara itadori in biological control. However, I recommend briefly mentioning how Aphalara itadori has already been deployed in other regions outside Canada, and summarize the successes/failures to set the context for the Canadian study.

This is a good suggestion. We have adapted the following passage, and added a new citation, to improve this area of the introductions:

‘Aphalara itadori releases have been conducted in the United Kingdom (UK) in 2010, in Canada in late 2014, and most recently in the U.S and the Netherlands. in 2020. The largest releases, totaling over 200,000 individuals, have been conducted in British Columbia, Alberta, and Ontario, Canada (Bourchier, unpublished data). Across all of these release locations, diapausing adults, released in the fall, have been observed to overwinter successfully and to lay large numbers of eggs in spring. The resulting populations have generally failed to persist for longer than two generations, and have not reached densities capable of exerting any control over the weed [19].’

Methods Section:

The description of the experimental setup (e.g., container design, soil composition, and leaf exposure) is well-detailed. However, the methodology for sexing psyllids could be further clarified to ensure reproducibility. Was there any pre-selection done for sex ratio?

Thank you. We have included two passages in the methods section explaining how sex ration was treated in the psyllids:

‘Psyllids were then shipped to the University of Toronto on cut plants secured inside cardboard containers. On arrival, 100 psyllids were aspirated and sexed to determine an approximate sex ratio (42 males and 58 females). Psyllids were then placed into rearing cages prior to the initiation of the experiment. Psyllids were kept in mixed sex populations prior to the onset of diapause, so were likely mated prior to the start of the experiment.’

‘Psyllids were not sexed prior to their addition to experimental cages, however, a roughly even sex ration was assumed based on an overall assessment of the population (see above).’

The rationale behind using only 50 psyllids per container could be elaborated. It would be useful to justify if this number is statistically sufficient to capture natural variations in psyllid activity and survival.

This is a good point. We have added a sentence (below) explaining our rationale for using 50 psyllids per container:

‘We used 50 psyllids per container as we felt that this would be enough individuals to capture natural variations in psyllid behavior, while not being so many as to create competition for preferable overwintering locations within the containers.’

Results Section:

The data presentation is clear, but some figures (e.g., Figures 4, 5, 6) require more detailed captions to make them more understandable to readers unfamiliar with the experimental setup.

This is a good suggestion. We have added futher details to the captions for figures 4, 5, and 6 to make these figures easier to interpret for readers who have not read the methods section.

A more explicit explanation of the relationship between the freezing events and psyllid mortality could enhance the results section. How do the authors interpret the low psyllid recovery in light of the freezing events, especially in early spring?

We agree that it would be nice to be able to attribute psyllid mortality of lack thereof) to specific freezing events. However, because of the overall low psyllid survival, we were not able to make strong conclusions about psyllid mortality factors, and we don’t want to make the discussion overly speculative. We feel that it is useful to point out these freezing events, which we have done in figure 4. What we can say is that some psyllids seemed to survive freezing events after having access to foliage, suggesting that feeding in early spring does not affect the psyllids ability to survive periods below freezing. We have addressed this carefully in the discussion, but would not want to speculate any further.

Discussion Section:

The discussion of the results is generally robust, but there seems to be a missed opportunity to explore in more depth how local environmental factors, such as humidity or microclimates within the experimental containers, may have influenced psyllid survival.

We include the following passage in the discussion, addressing the low overall psyllid survival. We believe this provides adequate discussion of the factors that might have caused high overall mortality, without being too speculative:

‘The number of psyllids recovered from experimental containers was low across all treatments. This low recovery rate was surprising because laboratory populations of A. itadori in Canada have previously been overwintered using a similar methodology with high levels of success (Bourchier, unpublished data). The minimum winter temperature observed in the experimental containers was -9°C. This represents fairly typical conditions for the region. Mean minimum winter soil temperatures (at a depth of 2.5cm) between 2019 and 2021 were approximately -10°C (NicheMapR version 3.3.2 [27]). It is possible that the small overwintering containers may have trapped excessive moisture, which could have affected the psyllids directly, or impacted their ability to survive extreme cold [28]. Future research should explore the effects of soil moisture on the winter cold tolerance of A. itadori. An alternative explanation for the low survival of psyllids in our experiment is that spring temperatures inside the containers may have been too high. Although the maximum temperature recorded inside the containers was 28.5°C, which is below the estimated maximum development threshold for the species [2], it is possible that the clear inverted containers could have trapped heat and elevated temperatures during the monitoring periods. Finally, it is possible that the small experimental containers did not provide enough suitable overwintering sites for the psyllids. A lack of deep bark crevices is thought to have contributed to low overwinter survival of A. itadori in a previous container study in the UK [24].’

The low psyllid recovery in the experiment is a critical point. The authors speculate that container design may have influenced survival, but further discussion of potential methodological adjustments (larger containers, different materials, etc.) would be beneficial for future studies.

This is a good point. We now include the following passage at the end of the paragraph in the above response:

‘Future overwintering studies should be conducted in wider containers to ensure that ample overwintering locations, at optimum litter depths, are available to the psyllids. Future work should seek to explore the effects of abiotic factors such as humidity, soil moisture, and temperature fluctuations on overwinter survival of A. itadori.’

Statistical Analysis:

The use of generalized linear models with a negative binomial distribution is appropriate given the count data. However, it would be helpful to include some clarification on model assumptions. Did the authors check for overdispersion in the count data before applying the model?

It would also strengthen the analysis if the authors could mention any post-hoc tests used to interpret significant results, especially in cases where differences in psyllid activity across groups were found.

These are good points. The descriptions of statistical analyses in the methods have been changed to include these details for each analysis. For example:

‘Insect count data were tested for overdispesion and were not over dispersed (χ2/df = 0.36). Pairwise comparisons between monitoring periods were made using Wald χ2 comparisons with Bonferroni-adjusted p values.’

Minor Comments:

There are some typographical errors in the paper, such as the incorrect spelling of "Aphalara" as "Apahlara" in multiple locations (e.g., page 7, line 236). Please correct these typos for consistency.

The paper has been gone through carefully to remove any typographical errors. Thanks you for your thorough review.

The references section is generally well-organized, but I noticed that a few citations, such as for Bourchier's unpublished data, might need to be clarified further in terms of how these were sourced or corroborated.

The Bourchier unpublished data citation has been replaced with a recent paper from 2025.

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The revised version look acceptable to me.

Author Response

Reviewer 2

The revised version looks acceptable to me.

Thank you

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Main Concerns

Undefined Response Variables

The manuscript does not clearly define psyllid activity or oviposition. Before line 195, psyllid activity should be operationally defined (e.g., live adults, eggs, or adults + eggs per leaf, per container, or per adult). Instead, survival results, observed psyllids, and recovered psyllids are presented without a prior methodological definition, rendering the study irreproducible.

Discrepancy Between Title, Objectives, and Analyses

Although the title emphasizes the onset of activity and oviposition, the analyses focus on other measurements such as psyllid survival and abundance. Oviposition is only mentioned once (line 256) and is not statistically analyzed by treatment or time. This discrepancy undermines the stated objective.

Unclear Experimental Design and Factorial Structure
The factors, levels, and observation periods must be explicitly described to allow for the evaluation of the statistical model, as well as the dependent variables.

Undefined Time Frame
It is unclear whether the onset of oviposition occurred synchronously across replicates. The observation periods should be explicitly defined (e.g., 1, 2, and 3 months) to support temporal comparisons.

Lack of Methodological Rigor and Consistency
Key outcomes (survival, psyllids observed, psyllids recovered) are reported without being defined in the methods. Furthermore, the only statistically significant outcome appears to be the number of psyllids observed, not psyllid activity or the number of adults recovered, which further weakens the coherence between the objectives and the results.

Minor Concerns
Information on pre-trial diet is lacking. After line 104, the authors should indicate the diet the aphids received before the trial.

Ambiguity in the coverage of the observations: It is unclear whether the observations were made on every consecutive day in March, April, and May, as the temperature data appears to cover only a few days of the trial in each month.
Context of the environmental data: The temperature data are valuable, but their temporal alignment with the biological observations needs clarification.

Author Response

Undefined Response Variables

We feel that these metrics are very clearly defined in the methods section. See the passage below:

‘To monitor psyllid activity, soil and leaf litter was removed to expose the container lids. Container lids were removed and replaced with an inverted 0.93L plastic container (Figure 2C). Any active psyllids were able to be viewed through this transparent container. Supported within the inverted containers was a single F. japonica leaf in florist foam (Oasis Floral Products, Kent, Ohio, USA) (Figure 3). The number of psyllids observed on these sentinel leaves was recorded over a period of three days during each monitoring period. Leaves were monitored at 10am, 1pm, and 4pm on each day. To avoid double counting the same psyllids, the maximum number of psyllids observed on the sentinel leaf at any one time was recorded as a measure of psyllid activity for each container.’

Discrepancy Between Title, Objectives, and Analyses

Our stated objective was to observe the earliest incidence of viable egg laying in Aphalara itadori, and we did this. We do not feel that the number of times the word oviposition appears in the results section is relevant. Although we do address measurements such as psyllid survival, the core purpose of the study was to understand A. itadori phenology in part of its introduced range. We feel that the title, objectives and analyses reflect this.

We made edits to the methods section to make it clearer exactly how our factors and monitoring periods are defined. We have expanded out description of our analyses to make our design as clear as possible. See this expanded section here:

‘To compare psyllid activity among monitoring periods (March, April, and May) we conducted a generalized linear model with negative binomial distribution and log link function. The model had one independent variable (monitoring period) with three levels (March, April, and May), and one dependent variable which was psyllid counts (the number of psyllids observed in containers during the monitoring periods) We included only the first observation period from each treatment group (A, B, and C) such that psyllid activity was not affected by previous foliage exposure and replication was equal among treatment groups (n = 20). Insect count data were tested for overdispesion and were not over dispersed (χ2/df = 0.35). Pairwise comparisons between monitoring periods were made using Wald χ2 comparisons with Bonferroni-adjusted p values.

To explore the effects of early spring feeding on psyllid survival to late spring, we compared psyllid activity among treatment groups (A, B, and C) during the May observation period using a generalized linear model with negative binomial distribution and log link function. The model had one independent variable (prior feeding) with three levels (two, one, and zero prior exposures to spring foliage), and one dependent variable which was psyllid counts (the number of psyllids observed in containers during the monitoring periods) Insect count data were tested for overdispesion and were not over dispersed (χ2/df = 0.36). Pairwise comparisons between monitoring periods were made using Wald χ2 comparisons with Bonferroni-adjusted p values. During the May observation period, containers in treatment groups A, B, and C, had been given access to foliage twice previously, once previously, and never before respectively.

After the final observation period in May, all living psyllids were harvested from the experimental containers. The number of psyllids was compared among treatment groups (A, B, and C) and control containers using a generalized linear model with negative binomial distribution and log link function. The model had one independent variable (treatment group) with four levels (treatment groups A, B, and C, and control containers), and one dependent variable which was psyllid counts (the number of psyllids observed in containers during the monitoring periods) Insect count data were tested for overdispesion and were not over dispersed (χ2/df = 0.37). Pairwise comparisons between monitoring periods were made using Wald χ2 comparisons with Bonferroni-adjusted p values.’

The observation periods have been explicitly defined as March, April, and May. Their exact dates are outlined in the methods. We feel that it makes sense to define the monitoring periods using a named month, as it comunicates clearer phenological information than, for example, defining monitoring periods as month 1, 2, and 3.

Lack of Methodological Rigor and Consistency
Key outcomes (survival, psyllids observed, psyllids recovered) are reported without being defined in the methods.

We believe that these terms are very clearly explained in the methods section. However, some passages in the methods have been edited to ensure clarity. For example:

‘At the conclusion of the final foliage exposure on 16 May 2021, the number of surviving psyllids in each container was assessed. Any active psyllids were aspirated from each container. Leaves were then replaced in each container every two days, and active psyllids were removed, periodically, keeping a record of the total number of psyllids recovered from each container. This process was repeated until two days passed without any further psyllid activity being observed.’

Furthermore, the only statistically significant outcome appears to be the number of psyllids observed, not psyllid activity or the number of adults recovered, which further weakens the coherence between the objectives and the results.

The number of psyllids observed during a monitoring period was used as a measure of psyllid activity. We have made some edits to the text to clarify this. It is true that the one statistically significant relationship we observed was between psyllid activity (the number of psyllids observed in the containers) and monitoring period, with higher psyllid activity observed in March and May than in April. It is not clear to us how a lack of other statistically significant relationships undermines the coherence between our objectives and our results.

Minor Concerns
Information on pre-trial diet is lacking. After line 104, the authors should indicate the diet the aphids received before the trial.

Thank you. We have adapted the following sentence to include this information:

‘Psyllids used in the experiment were reared on potted F. japonica plants and held at 15°C and a 12h/12h light/dark cycle at AAFC for several weeks in early fall in order to stimulate the onset of diapause.’

Thank you. The following sentence has been added to the methods section to clarify this point:

‘Spring psyllid activity was monitored in the experimental containers during three-day monitoring periods in March, April, and May.’

Context of the environmental data: The temperature data are valuable, but their temporal alignment with the biological observations needs clarification.

Figure 4 shows the temperature data throughout the experiment. As described in the figure legend, the transparent blue bars indicate the periods during which psyllid observations were made.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents useful early-spring phenology data for Aphalara itadori in Canada. The work is relevant to biological control programs, and the study design has merit. However, several technical aspects require clarification, and the novelty should be articulated more clearly.

Major Comments

Clarify the experimental design.

The description of the overwintering containers and the movement of psyllids between the buried container and the upper observation chamber is difficult to follow. Figures are helpful, but the text should explain more clearly how psyllids access the foliage during observations.

Extremely low overwinter survival requires explanation.

Only a small fraction of the 50 introduced adults per container survived to spring. Please discuss potential causes (shipping stress, diapause induction, microclimate, container humidity, etc.) and compare survival with previous studies.

Statistical reporting needs correction.

Several F-values are reported as negative (e.g., F = –7.185), which is not possible. Please recheck all statistical results and ensure consistent presentation of sample sizes for each test.

Justify the use of repeated-measures ANOVA.

Earlier you mention that data did not meet assumptions of normality, yet you apply a parametric repeated-measures ANOVA for Treatment A. Please justify this decision or consider a non-parametric alternative.

Interpretation of feeding vs. non-feeding treatments is overstated.

Because recapture numbers were very low, conclusions about survival being unaffected by the presence or absence of knotweed foliage should be presented more cautiously. Acknowledge this limitation.

Strengthen the section on photoperiod and reproduction.

The manuscript states that spring oviposition is “not limited by photoperiod,” but day length near the first oviposition event is close to reported diapause-termination thresholds. This deserves a clearer comparison with previous laboratory thresholds and discussion of potential temperature or host-plant influences.

Improve the discussion of novelty.

Laboratory studies have already established photoperiod and thermal thresholds for A. itadori. The Introduction or Discussion should explicitly state that the novel contribution here is field-based documentation of overwintering emergence and first oviposition under Canadian spring conditions.

Environmental data need better presentation.

Instead of only presenting mean and minimum temperatures, a small figure showing the temperature profile (especially freeze events) would help readers understand the spring conditions.

Clarify methodological details.

Was the sex ratio of adults known or controlled?
Were the adults mated before diapause?
How long did psyllids remain in diapause before burial?
Why were egg viability checks only conducted for the earliest oviposition event?

Consider reorganizing parts of the Discussion.

The first paragraphs repeat the Results closely. Streamlining this section and focusing more on interpretation would improve readability.

Minor Comments

The Abstract repeats several points already stated in the main text; consider tightening it.
Some background sections in the Introduction are longer than necessary; focusing more directly on knowledge gaps would improve flow.
Add sample sizes directly to Figures 4 and 5.
Figures would benefit from clearer labels identifying Treatment A, B, and C.
A few sentences could be edited for clarity (e.g., long multi-clause sentences in the Discussion).

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study looks at effects of periodicity and survival of the Japanese knotweed psyllid given intermittent resource availability and other abiotic factors, concluding effectiveness as a biological control would not be adversely affected under conditions tested.

Overall the paper is well constructed and well written.
The Introduction is excellent and informative.
The methods and experimental protocol seem appropriate to the questions asked (although this is not my expertise).
I have only a couple of minor criticisms:
Summary: I would use "this biological control agent" as otherwise, to those unfamiliar with the system, it sounds like you are talking about a biological control of A. itadori if you use "the biological control agent". Similarly, I would add "plants" after "on invasive knotweed" to help make absolutely clear what is what at start.
The 2nd and 3rd paragraphs of the Discussion seem to include Results that are not necessary to report again there. I would keep the Discussion more general.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Insufficient replication in treatment O (leaves were not provided). The experiment only included five replicates, well below the minimum of twenty required to ensure statistical robustness.

Lack of temporal replication because the experiment was conducted in a single season, preventing verification of results under varying environmental conditions.

Lack of a negative control for psyllid presence. There is no treatment without added psyllids under the same experimental conditions, preventing the evaluation of the natural incidence of psyllids.

Author Response

Please see the attachment

Author Response File: Author Response.pdf