Electroantennographic and Behavioral Response of Female Moroccan locusts [Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae)] to Male-Specific Aldehydes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript on "Electroantennographic and behavioral response of female Moroccan
locusts [Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae)] to
male-specific aldehydes." Provides data concerning the production of pheromones by males and responses to pheromone components by females. The paper provides some significant data on new findings that the authors have uncovered about the chemical ecology of this species. An additional possible component of the male pheromone blend, 15:Ald is revealed and the progression of emission over time by adult males is described, with SPME work determining the source of emission from the hind legs for three compounds. The dose-dependent physiological EAG responses to these three compounds are well described showing the differences in response profiles from virgin and mated females to each compound.

The above components of the study were well conceived, and the results clearly presented. The behavioral data however, relied on two choice assays that consisted of very few replications over many of the treatments (Table 4). This number was as low as 8 and 9 in two of the treatments. Furthermore the significant results were only seen for treatments that were replicated far more often than most of the other treatments (34 for both 14:Ald  and 15:Ald 10ug mated). This bias would be more understandable if the overall attempts were similar across all treatment including non-choice results, but those replication numbers also are heavily biased toward those treatments. 

Because of the strength of the data concerning pheromone emission and electrophysiological response and weakness of the behavioral response data, I would recommend gathering more behavioral data in a less biased fashion before publishing it. The portions of the manuscript without the behavioral data are worthy of publication on their own and could be published now as is if the authors did not want to wait to collect more behavioral data to include in this paper.

Other minor comments:

ln 90-93: The meaning of this sentence is unclear. Please rewrite for clarity and consider breaking into two sentences.

ln 132-135: The reason that SPME will allow for targeted collection from the hind legs will not be obvious to many readers without specialized knowledge of chemical ecology methods. Consider adding a short sentence explaining that the fibers of the SPME can be applied directly to the surface of the body.

Fig 2: The internal table and legend describing the lines are givin in numerical order from up to down. However, the highest actual line is 16:Ald, followed by 14:Ald, then 15:Ald. Consider that the reader may be able to process the information more clearly if the table and legend also used that order (ie 16, 14 15 from top to bottom) 

Fig 2: Also double check the letters in the table. Is week 3-4 for 16:Ald really ab or is it b? week > 5 would more likely be abin that row. Similarly, is c appropriate for week >5 for 15:Ald?

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled "Electroantennographic and behavioral response of female Moroccan locusts [Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae)] to male-specific aldehydes" investigates responses of female Moroccan locusts to male-specific aldehydes. The study employs electroantennography to detect olfactory signals and behavioral assays to observe female responses, aiming to identify pheromones involved in mating and sexual attraction in this economically significant pest. The primary objective is to characterize a specific aldehyde component that attracts females to males, which may be critical for developing locust control strategies.

The manuscript is likely to be of interest to the journal's readership. The following points require clarification or further detail.

Line 95: Is the behavioral activity defined according to the insect's physiological state? Are there existing studies that specify the age at which males release varying amounts of these compounds? Please clarify and, if possible, provide relevant references.

Line 130: Please provide the rationale for selecting fifth-instar nymphs for the experiments.

Lines 140 and 193: Please specify the materials and equipment used to measure airflow.

Line 149: Fiber saturation is managed by adjusting exposure time. Were preliminary extractions to ensure the exposure time was 8 hours? Please clarify this.

Line 189: Please indicate the age in days or weeks of the virgin and mated females used in the study.

Lines 198-199: Please explain the criteria for not changing the filter paper after each stimulus application. Additionally, clarify the rationale for not rotating the olfactometer after several repetitions and for only changing the stimulus position.

Lines 205-206: Please clarify whether all females advanced immediately after placement at the base of the main arm, or if a specific activation period was allowed before non-responding insects were excluded.

Line 260: You can include the letters indicating significant differences above each ng SEM point; I don't understand why you're putting them separately in a table. If it's due to scale and spacing, you can add a negative value to the Y-axis so that the 15:Ald line and the first 14:Ald values ​​aren't confused with the X-axis.

Line 283-284: I want to assume that in Figure 3b, in virgin females for 1 ug is "a" with no significant difference with 10 ug?. Furthermore, comparisons between virgin or paired samples per µg should use a specific font (e.g., lowercase), and comparisons between concentrations within each line of virgin or paired samples can use uppercase letters.  Review the letter assignment for the statistical difference.

Line 285: What is the value of n (replications) that you added to the statistical test? It shouldn't be an interval. The interpretation and selection of post-hoc comparison tests must account for differences in sample sizes to avoid erroneous conclusions.

Line 314: While the discussion provides valuable comparisons with previous studies, addresses limitations, and suggests future research, it is recommended to shorten this section and address the central research question more concisely and directly.

Comments on the Quality of English Language

The English could be improved to make the research clearer.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This submission reports on the identification of a further male-specific compound, a saturated fifteen aldehyde, in the Maroccan locust, Dociostaurus maroccanus, and demostrates that this compound elicits antennal and, in lab-tests, behavioral response from females. Electrophyiological, as well as behavioral activity of two chemically closely related aldehydes, identified and pubished earlier, were also shown. The main new result is, however, rather the age-dependence of the release of these aldehydes, as the newly identified compound has exerted only modest behavioral activity in lab-tests, as the Authors themselves admit this in the Discussion (lines 407-408). 

 

The paper is very well written, the experiments are well-designed, carefully conducted and evaluated. The overview of related literature background is impressive. Nevertheless, there are two constrains:

 

1. Why was 2E,7R,11R-phytal, the previously reported male-specific pheromone-candidate compound completely left out from the tests?
2. Why were not various mixtures of 14:Ald, 15:Ald and 16:Ald tested in the laboratory behavioral tests?

These should be treated in sufficient details, at least in the Discussion.

Minor comments:

L 112-115: Were virgin and mated locust specimens of the same age, when used in the biotests?

L 141: What was the amount and mesh of Super Q?

L 155-156: „oven temperature was initially set at 40 °C” Did you use on-col injection? Pls state it firmly.

Figure 2 / insert table/ 15:Ald: „a b b c” - should not it be „a b b a”?

Figure 2 / insert table/ 15:Ald: „a b ab b” - should not it be „a b ab a”?

L 330-331 – 14:Ald and 16:Ald are likely related to eachother in course of biosynthesis, however 15:Ald does not fit to this line. Maybe this should briefly discussed, or some explanations could be useful.

L 362 „pre-copulatory o copulatory courtship” – „o” ??

L 366-367 „Even though the number of responding individuals in behavioral tests was relatively low...” – An obvious reason for this could be that the so-far identified compounds still do not represent the full natural pheromone blend. I think this should be mentioned.

Supplementary material

Figure S1: - I encourage the Authors to transfer this figure to the main text. This figure is very convincing and supports very much the main message (i.e. male-specific compounds).

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all of the issues brought up by the reviewers in a serious manner. The paper is greatly improved as a result. The authors did address the issue of sparce replicates in the behavioral data and added context in the discussion. While they do not follow the recommendation to collect more data before publishing this work, the current rewrite is an improvement. However, it is still of questionable value to publish data that was obtained with a biased sampling protocol. (increasing replicates in treatments that seemed to be showing trends).