Factors Influencing Cucurbitacin-E-Glycoside Content in Bitter Hawkesbury Watermelon as Potential Synergist in Cucurbit Pest Management
, Brent Short 5 and Donald C. Weber 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments are in manuscript.
Comments for author File: 
Comments.pdf
Author Response
In addition to accepting all suggested edits to grammar and formatting, we have provided a response to all reviewer comments and indicated where changes have been made in our manuscript.
Reviewer 1 (Comments cut and pasted from PDF)
Comment 1: In my opinion, the title does not correspond to the content, because only a small part of the research is based on the examination of synergy, and the larger part actually process different influencing factors on the content of CEG in plants. I suggest something like "Factors influencing to the content of cucurbitacin-E-glycoside in Hawkesbury watermelon as potential synergist in cucurbit pest management"
Response 1: We have changed the title of this manuscript to “Factors influencing cucurbitacin-E-glycoside content in bitter Hawkesbury watermelon as potential synergist in cucurbit pest management”, as we agree with the nuanced difference highlighted by the reviewer.
Comment 2: Line 208: What was the distance between different treatments? Did you have replication? How many? This field trial is quite unclear.
Comment 3: Line 215: Also, you did not write the period in which you did the observation (month, date, morning, afternoon....)
Comment 4: Line 216: Only in one place or you walked and checked different plants? Which part of the plant you checked? How did you observe, is not clear? One minute of observation was for all insects or for each species or stage one minute?
Comment 5: Line 217: You applied treatment twice, did you check also twice number of insects, after first and second application? Or just once? If you checked twice why you didn't show that in results?
Comment 6: Line 218: You didn't describe method, how many plants have you analyzed? In what way? Add a sentence about it.
Response 2-6 (Lines 204-228): To address all of the above recommendations for improvement, we’re re-written this paragraph. We also made additions to the data analysis section (Lines 245-248), to clarify that the two observations made after each of two treatments were incorporated into our model.
We conducted a field trial to evaluate a potentially reduced-risk approach for chemical control using a BHW-derived synergist at New Hampshire’s Agricultural Experiment Station’s research farm in Madbury, NH. We carried out a small plot spray trial in zucchini, Cucurbita pepo ‘Green Machine’, which were transplanted into black plastic mulch with [m] row spacing and [m] between plants. This field was subdivided into 4 quadrants, then treatments were applied in a randomized block design to 3 m long plots (5 plants/plot; n = 4). Treatments included foliar applications of spinosad (8 fl.oz./A Entrust; Corteva, corteva.us), acetamiprid (2.3 oz. Assail 30SG; United Phosphorus Inc., King of Prussia, PA) or lambda cyhalothrin (1.92 fl.oz./A LamCap II; Syngenta, syngenta-us.com) versus ground-application of the same rates plus the high rate (of an attractive feeding stimulant (10 gal/A; CIDETRAK L; Trécé Inc., Adair. OK). We used hand-pumped sprayers (1-Gallon Multi-Purpose Sprayer; Chapin International Inc., Batavia, NY) to apply all treatments using the equivalent of 100 gal/A and the addition of a spreader sticker (1% v/v; BondMax, Loveland Products, Loveland, CO). We removed the sprayer nozzle for ground applications to allow for a larger droplet size, and also added corn starch (1 g/L; Argo, Oakbrook, IL) in order to visually confirm that material was applied to the ground and not on crop plants.
We applied treatments twice, 7 days apart, and recorded the number of SCB, SpCB, and squash bug egg masses, nymphs, and adults observed on each plot within a 1 min timed observation, 3 days after each treatment. Each of these timed observations were conducted between 10 am and 1 pm, during clear conditions with no rain, and we examined the base of each plant and the undersides of leaves for all insect species and life stages. We also destructively sampled all plants at the end of the experiment to observe the number of squash vine borer larvae infesting stems (5 plants/plot).
Comment 7 (Line 281-282): If you observed number of alive insects 3 days after the treatment in this case, does that mean that you are attracted insects with CIDETRACK L but you didn't kill them all, the effectiveness of the insecticide is reduced? Unless the figure 4 shows the number of dead insects, but I don't see it written. Because you had a higher number of insects in ground treatment than in the foliar application? So the conclusion is not good? If I understood correctly, the goal was to attract more insects and also kill them after that? If so, you need data on the number of insects you attracted and also killed, and I don't see that here. Although you do not have statistically significant differences between foliar and ground applications of insecticides, usually, more insects mean more damage. It is good as long as you have a few insects per treatment, what if there are many more? Do you have some data about damage? Or bacterial wilt?
Response 7: This comment is a little hard to interpret but, if understood correctly, we should have made a better distinction between some of the terms we have used. We state in the introduction (Line 31) that cucurbitacins are known to be arrestants and gustatory stimulants (not attractants) for these specialist insects. We have clarified that our original goal was not to attract insects in our discussion of potential attractant effects of the crude extract (lines 339-352). We agree with this reviewer that we have not provided data to support strong conclusion regarding this effect, but that it is worthy of further investigation.
There were some indications that CIDETRAK L was attractive to pest insects in this system, as more SCBs were observed in CIDETRAK L control plots than water control plots (Fig. 4A). This was not expected because cucurbitacins are known to act as arrestants and feeding stimulants for insects after they have landed on a plant and are not known to attract insects from a distance [1-5]. Additional volatile organic compounds (VOCs) in the BHW juice used to manufacture CIDETRAK L were the likely cause of long-distance attraction in this field study.
Yes, Fig. 4 is a count of the living insects observed in plots. We have included this clarification in the figure description (line 307). No, we did not collect data to reflect damage, and we saw no signs of bacterial wilt.
Comment 8 (Line 281): Why did you observed this insect species and different insect stage if they are not in high population? Did you have some preliminary results for this pests?
Response 8: These are key insect pests that are commonly observed in cucurbit crops in this region, which is cited in our introduction (lines 59-77).
Comment 9 (Line 292): Do you have some data about SCB resistance on spinosad and acetamiprid? Because they are not different from the control treatment. Also do you have some date about pest population before treatment?
Response 9: We agree with this reviewer that this result is noteworthy, but we do not suspect that chemical resistance is the reason that these chemistries failed to control SCB. Personal experience and anecdotal evidence point to field degradation of active ingredient and insect behavior as the explanation here, however, we don’t have the data to support a strong conclusion, so we did not draw attention to this lack of significant difference in our discussion.
Comment 10 (Line 347): I don't see this in your results
We have omitted this statement “A selective feeding stimulus, such as CEG, can contribute to more sustainable pest management in cucurbit systems by increasing efficacy of chemical controls and reducing non-target impacts on beneficial insects.” from our conclusion section.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this MS, a series of experiments were carried out and the growing bitter HBW for a cucurbit pest management synergist was studied. The topic of this MS is very interesting and useful in the pest management in fields. While ther are some shortcomings which were following as:
Q1: Data Analysis: What about the contrast tests? Introduce the contrast tests in details, here.
Q2: Fig.1: This data figure is not clear that it can not distinguish the data of MD from NH.
Q3: Fig.2: No significance was signed on the data columns of different harvest-time treatments.
Q4: L228-229: No difference that P<0.0001?
Other comments are marked directly in the PDF file.
Comments for author File: Comments.pdf
Author Response
In addition to accepting all suggested edits to grammar and formatting, we have provided a response to all reviewer comments and indicated where changes have been made in our manuscript.
Comment 1: Data Analysis: What about the contrast tests? Introduce the contrast tests in details, here.
Response 1: We have provided details for post hoc analysis for means separation, now lines 250-251.
For each test (SCB/plot and SVB/plot) we conducted post hoc analysis using Bonferroni corrections to determine means separation between each treatment.
Comment 2: Fig.1: This data figure is not clear that it can not distinguish the data of MD from NH.
Response 2: I was not our intention to distinguish between data from MD and NH. We have included language in the figure description to clarify this, now lines 260-262.
Figure 1. Cucurbitacin-E-glycoside (CEG) concentration (mg/g) of bitter Hawkesbury watermelon fruit versus the concentration of its parent fruit, grown in all open-pollinated field experiments
Comment 3: Fig.2: No significance was signed on the data columns of different harvest-time treatments.
Response 3: Thanks for this catch! We have included an asterisk to indicate significant effect in figure 2.
Comment 4: L228-229: No difference that P<0.0001?
Response 4: We have adjusted our language to clarify the statistic here, now lines 291-299.
We observed fewer SCB adults in plots treated with the broad-spectrum pyrethroid, lambda-cyhalothrin, than in control plots (Fig. 4A; F7,30 = 52.3, p < 0.0001). We observed no significant differences between SCB observed in plots treated with foliar versus ground application for any of the chemistries evaluated. However, we did observe fewer SCB adults in acetamiprid + CIDETRAK L plots than in CIDETRAK L control plots, while there was no difference between the foliar application of acetamiprid and the water control. We also observed significantly more SCB in CIDETRAK L control plots than other treatments, with the exception of the spinosad + CIDETRAK L ground treatments, which were not different from its control.
Reviewer 3 Report
Comments and Suggestions for AuthorsVery applied research and interesting to read, but the manuscript requires more careful editing. A few questions and suggestions:
1. Injection Volume: Does overloading occur when using an injection volume of 50 µL? This seems like the maximal loading for the column.
2. Line 221: How is "the high rate" of an attractive feeding stimulant defined?
3. Line 212: Why wasn't corn starch applied to all treatments to eliminate its potential effect as a confounding variable?
4. Line 225: The phrase "mixed mixed-model analyses" seems incorrect. Did you mean "mixed-effects model analysis"?
5. Line 239: Please clarify the meaning of "DAT" in this context.
6. Figures 1 and 3: Statistical values should be included directly in the figures to make them more self-explanatory.
7. Lines 256-257: "We found a significant effect of harvest maturity on offspring CEG accumulation in our 2022 experiment." Is this result shown in Figure 2 or another figure?
8. Statistics for Figure 4: It appears that the Poisson model has "treatment" as the only main effect. Why not introduce "pesticide type" and "CideTrak L" as two major effects? Was this due to different application methods, making a two-way ANOVA inappropriate?
9. Field Experiment Discussion (Lines 335-345): The discussion of the field experiment is too brief. This is the only paragraph on the field experiment, and it should be expanded to discuss the results and implications in more detail, such as the fact that ground application treatments with the gustatory stimulant had more SCB visitors.
10. Expanding the Field Experiment Discussion: In addition to gustatory (feeding) stimuli, other behavior-modifying stimuli such as visual cues, sex pheromones, and oviposition stimulants are also commonly used in combination with pesticides in 'attract-and-kill' approaches. These are also key components of 'push-and-pull' strategies in integrated pest management. Please mention these to better justify the merit of the present study.
References:
Environmental entomology 45.6 (2016): 1480-1488.
Journal of Integrative Agriculture 22.4 (2023): 1093-1103.
Scientific reports 9.1 (2019): 13747.
Annu. Rev. Entomol. 52.1 (2007): 375-400.
11. The conclusion paragraph did not mention the field experiment at all.
Author Response
Comment 1: Injection Volume: Does overloading occur when using an injection volume of 50 µL? This seems like the maximal loading for the column.
Response 1: Overloading is likely not occurring in this case for a few reasons. The amount being injected on column ranges from around 100 ng (near LOD) up to about 250 microgram per injection. While the upper limit of that injection amount may seem high in some cases, we found no significant deviation in peak symmetry/shape, or in peak width. Gradient solvent was chosen to maximize solute solubility and resolution, injecting samples containing 20% methanol into a system containing 50% methanol at the beginning of each injection. This will have minimal effect on peak retention and should limit any overloading occurring from early peak elution.
Comment 2 (Line 221): How is "the high rate" of an attractive feeding stimulant defined?
Comment 2: Thanks for catching that we failed to include the label rate. We have added label rates for all products used in lines 212-221.
Comment 3 (Line 212): Why wasn't corn starch applied to all treatments to eliminate its potential effect as a confounding variable?
Comment 3: This is a good point and the addition of corn starch to a foliar application may have made this a more consistent experiment but would have influenced spray coverage by clogging spray nozzles, which we removed for ground applications. Our decision to distinguish between application strategies was made largely in an effort to demonstrate a “reduced risk” strategy by having fewer, larger droplets of pesticide on the plastic below the plants versus full coverage from a spray. We have included language on lines 212-221 to clarify this.
Treatments included foliar applications of spinosad (8 fl.oz./A Entrust; Corteva, corteva.us), acetamiprid (2.3 oz. Assail 30SG; United Phosphorus Inc., King of Prussia, PA) or lambda cyhalothrin (1.92 fl.oz./A LamCap II; Syngenta, syngenta-us.com) versus ground-application of the same rates plus the high rate (of an attractive feeding stimulant (10 gal/A; CIDETRAK L; Trécé Inc., Adair. OK). We used hand-pumped sprayers (1-Gallon Multi-Purpose Sprayer; Chapin International Inc., Batavia, NY) to apply all treatments using the equivalent of 100 gal/A and the addition of a spreader sticker (1% v/v; BondMax, Loveland Products, Loveland, CO). We removed the sprayer nozzle for ground applications to allow for a larger droplet size, and also added corn starch (1 g/L; Argo, Oakbrook, IL) in order to visually confirm that material was applied to the ground and not on crop plants.
Comment 4 (Line 225): The phrase "mixed mixed-model analyses" seems incorrect. Did you mean "mixed-effects model analysis"?
Response 4: Thanks for this catch! We’ve corrected this typo, now line 235.
Comment 5 (Line 239): Please clarify the meaning of "DAT" in this context.
Response 4: Done.
Comment 6 (Figures 1 and 3): Statistical values should be included directly in the figures to make them more self-explanatory.
Response 5: This is not a familiar convention but – never having published in this journal previously - we’ve included statistical values in figure descriptions and trust that this can be resolved with copy editing.
Comment 7 (Lines 256-257): "We found a significant effect of harvest maturity on offspring CEG accumulation in our 2022 experiment." Is this result shown in Figure 2 or another figure?
Response 7: Yes, we have indicated that we’re referring to results shown in Figure 2, now line 270.
Comment 8 (Statistics for Figure 4): It appears that the Poisson model has "treatment" as the only main effect. Why not introduce "pesticide type" and "CideTrak L" as two major effects? Was this due to different application methods, making a two-way ANOVA inappropriate?
Response 8: Yes, it’s a good suggestion. However, it may not have been appropriate to include Cidetrak as a main effect because the variability in application methods. We feel confident that this model is appropriate for the story we’re trying to tell.
Comment 9 (Field Experiment Discussion Lines 335-345): The discussion of the field experiment is too brief. This is the only paragraph on the field experiment, and it should be expanded to discuss the results and implications in more detail, such as the fact that ground application treatments with the gustatory stimulant had more SCB visitors.
Response 9: Yes, we have re-written this paragraph, now lines 204-230.
We conducted a field trial to evaluate a potentially reduced-risk approach for chemical control using a BHW-derived synergist at New Hampshire’s Agricultural Experiment Station’s research farm in Madbury, NH. We carried out a small plot spray trial in zucchini, Cucurbita pepo ‘Green Machine’, which were transplanted into black plastic mulch with [m] row spacing and [m] between plants. This field was subdivided into 4 quadrants, then treatments were applied in a randomized block design to 3 m long plots (5 plants/plot; n = 4). Treatments included foliar applications of spinosad (8 fl.oz./A Entrust; Corteva, corteva.us), acetamiprid (2.3 oz. Assail 30SG; United Phosphorus Inc., King of Prussia, PA) or lambda cyhalothrin (1.92 fl.oz./A LamCap II; Syngenta, syngenta-us.com) versus ground-application of the same rates plus the high rate (of an attractive feeding stimulant (10 gal/A; CIDETRAK L; Trécé Inc., Adair. OK). We used hand-pumped sprayers (1-Gallon Multi-Purpose Sprayer; Chapin International Inc., Batavia, NY) to apply all treatments using the equivalent of 100 gal/A and the addition of a spreader sticker (1% v/v; BondMax, Loveland Products, Loveland, CO). We removed the sprayer nozzle for ground applications to allow for a larger droplet size, and also added corn starch (1 g/L; Argo, Oakbrook, IL) in order to visually confirm that material was applied to the ground and not on crop plants.
We applied treatments twice, 7 days apart, and recorded the number of SCB, SpCB, and squash bug egg masses, nymphs, and adults observed on each plot within a 1 min timed observation, 3 days after each treatment. Each of these timed observations were conducted between 10 am and 1 pm, during clear conditions with no rain, and we examined the base of each plant and the undersides of leaves for all insect species and life stages. We also destructively sampled all plants at the end of the experiment to observe the number of squash vine borer larvae infesting stems (5 plants/plot).
Comment 10 (Expanding the Field Experiment Discussion): In addition to gustatory (feeding) stimuli, other behavior-modifying stimuli such as visual cues, sex pheromones, and oviposition stimulants are also commonly used in combination with pesticides in 'attract-and-kill' approaches. These are also key components of 'push-and-pull' strategies in integrated pest management. Please mention these to better justify the merit of the present study.
Response 10: Yes, although the focus of this manuscript is on gustatory cues, we agree that it’s well worth addressing the potential for attract-and-kill in this system. We have made additions to the discussion section on lines 353-364, and we have included recommended references that were pertinent to our discussion.
Attracting more insects to treated plots compared to control plots may be an undesirable quality for an insecticide synergist, but a desirable quality for use in other behavioral control approaches. The addition of attractive volatiles plus a feeding stimulant could induce pest insects to ingest toxic baits that might not otherwise be accepted as a food source in an attract & kill or push-pull approaches [46,47]. Attraction, aside from arrestment and feeding stimulation, may also have been an important factor in achieving the same efficacy in foliar versus ground-applied treatments (Fig. 4). Crawling insects (e.g. SCB) may have encountered and arrested on these discrete droplets of bait by chance, however, a flying insect (e.g. SVB) would likely need to respond to attractive VOCs to encounter a bait applied to the ground next to their host plant. Further investigations to optimize droplet density and efficacy of this approach with other pests in the complex are warranted.
Comment 11: The conclusion paragraph did not mention the field experiment at all.
Response 11 (Lines 371-373): We have included a concluding statement incorporating the results of the field experiment.
Our field experiment indicates that the addition of CIDETRAK L allowed for equivalent control of target pests while using a potentially reduced-risk application strategy.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI agree to publish the manuscript in this form.
Author Response
The "Comments and Suggestions for Authors" in this report states "I agree to publish the manuscript in this form".