Breeding Behaviors of the Endangered Prairie Butterfly Oarisma poweshiek (Lepidoptera: Hesperiidae) in Relation to Environmental Factors in an Ex Situ Conservation Setting

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This brief report examines how environmental conditions influence breeding behaviors of the endangered Poweshiek skipperling (Oarisma poweshiek) in an ex situ conservation setting. The study addresses an applied and conservation-relevant question using a valuable captive population and provides insights that could inform both husbandry practices and field survey timing. Overall, the manuscript is clearly written, well organized, and appropriate in scope for a Brief Report.

That said, several aspects of the statistical presentation, methodological justification, and interpretation would benefit from clarification or expansion. Addressing the points below would improve transparency, rigor, and ease of interpretation for readers.

Major Comments

1. Statistical Model Description Requires Clarification (Lines 92–103)

The description of the statistical model is difficult to follow in its current form. In particular, the model equation appears to contain formatting issues and internal inconsistencies, including duplicated temperature terms, unclear indexing, and missing operators, which obscure the intended model structure. It is also not clearly stated whether temperature was modeled solely as a linear predictor or whether a quadratic temperature term was included, nor is the rationale for such a choice explained if a nonlinear effect was intended. In addition, the manuscript does not indicate whether continuous predictors (sunlight intensity and temperature) were centered or standardized prior to analysis, which affects both model interpretation and reproducibility. Clarifying these points would substantially improve transparency and allow readers to better understand and evaluate the analytical approach.

2. Use of External Ambient Temperature Data (Lines 78–80)

Ambient temperature was obtained from a nearby Weather Underground station rather than measured directly within the breeding cages. Because the cages were exposed to direct sunlight, thermal conditions experienced by the butterflies may have differed substantially from ambient air temperature recorded at a fixed station, due to factors such as solar heating, limited airflow, and cage materials. This mismatch could reduce the ability to detect biologically meaningful relationships between temperature and breeding behavior, particularly given the emphasis later in the manuscript on heat stress and microclimatic effects. I recommend that this methodological choice be more explicitly acknowledged, and that its potential influence on the observed weak temperature effects be briefly discussed.

 

3. Repeated measures and temporal dependence (Lines 92–103).
   Behavioral observations were collected repeatedly from the same individuals at 5-minute intervals. While the inclusion of individual-level random intercepts is appropriate, it is not clear whether potential temporal autocorrelation among adjacent observation intervals was evaluated. Given the fine temporal resolution of the data, some degree of non-independence among successive observations is likely. Clarifying whether temporal autocorrelation was assessed during model diagnostics, and briefly explaining why the chosen random-effects structure is sufficient to account for repeated measurements, would strengthen the analytical transparency.
4. Rarity of breeding behaviors and statistical power (Lines 118–121).
   Only 66 of the 5,154 behavioral observations were classified as breeding behaviors. Although this likely reflects the natural rarity of these behaviors, such a low event frequency can limit statistical power and reduce sensitivity to detect weaker effects, such as those related to temperature. This is particularly relevant when interpreting non-significant results. A brief acknowledgment of this limitation in the Discussion would help contextualize the findings and clarify how low event frequency may influence inference.
5. Age Effects Are Descriptive Rather Than Inferential (Lines 111–116; Figure 2)

Age is later emphasized as an important factor in breeding behavior, yet no statistical model directly evaluates this relationship. Also, how many individuals contribute to each age category. In addition, Figure 2 presents error bars but does not indicate whether they represent standard deviation, standard error, or confidence intervals.

6. Table 1 clearly summarizes the total number of observed behaviors across the study. However, a brief clarification in the Methods or Discussion explaining how different breeding behaviors were aggregated at the 5-minute interval level, and how “breeding behavior frequency” should be interpreted biologically (e.g., courtship activity versus realized reproduction), would help readers better contextualize the results.

 

Minor Editorial Issues

Lines 75–76: Remove periods after “cm”.

Lines 95–97: Correct mathematical formatting errors.

Line 180: Remove the comma after “age”

 

>While sunlight intensity and ambient temperature are appropriately emphasized, other environmental variables such as relative humidity may also influence butterfly activity and breeding behaviors, particularly in outdoor ex situ settings. Although inclusion of additional variables is beyond the scope of the current study, briefly acknowledging humidity as a potential unmeasured factor in the Discussion would help contextualize the findings and guide future work.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This work is interesting, but it has a major flaw, namely your conception of ‘light’. Fortunately, you modify your definition in the discussion, but regardless, this makes your basic data set incorrect.

The intensity of sunlight that you measured is expressed in ‘lux’. This unit is directly related to the perception of the human eye at λ = 555 nm (corresponding to the colour green).

Insects, and butterflies in particular, have a completely different range of radiation perception than humans. Most insects have a wide range of perception in the UV spectrum (but of course, UV rays alone are not enough for butterflies to survive and reproduce).

In the hope that it will be possible to send it to you, I have prepared a graph representing the vision of Holarctic butterfly species (unfortunately, none of them fly in ecosystems quite similar to those of your model species, but rather in warmer ecosystems). Please note that these data have not yet been published, but similar data are available.

 

Another important factor for insects is the ‘warmth’ of light (colour temperature in Kelvin). In temperate countries, at maximum daylight, the temperature is around 6500 K (around 5000 K in Mediterranean and tropical regions), but in boreal areas (which may be closer to the biotopes of O. poweshiek), this temperature can reach 10,000 K. At high altitudes but lower latitudes, this value is also high. The higher the heat, the more UV rays can reach ground level.

Commercial light bulbs rarely offer a wide enough λ range to mimic sunlight (even the latest LEDs).

After this very theoretical introduction (sorry), let's get back to butterflies.

The genus Oarisma comprises two main groups: dark brown butterflies that fly in cool/cold places and all tropical species (which are mainly orange, with the exception of O. castanea from Brazil... which may belong to another genus?).

We can consider four taxa among the browns (we will not discuss here whether they are different species or subspecies).

The two Mexican taxa, O. era and O. calega, fly at very high altitudes (mostly above 2700 m in grassland areas extremely similar to those of the north-central United States at low altitudes).

The two North American species, O. garita and your model species O. poweshiek, fly in neighbouring areas, but O. garita above 500 m while O. poweshiek strictly (I think, please confirm) below 500 m.

The main question in terms of conservation, for me, is to understand why O. garita is still common and widespread while O. poweshiek is critically endangered. This can be done by comparing their ecosystems and life parameters, so that you can adapt your breeding conditions to optimise reproductive success.

• Have you checked whether O. poweshiek is a low-altitude vicariant of O. garita (Nick Grishin is sequencing many species, so perhaps the information is available)? Depending on how closely related they are, it may be possible to compare these two species.
• Have you compared the abiotic conditions (temperature, humidity) of the areas where garita flies with those where poweshiek flies?
• The four brown Oarisma species live in similar grasslands (but with considerable differences in altitude). Have you checked whether there are differences in terms of UV radiation/intensity/luminance/light heat?
• Is the fact that poweshiek flies at low altitude linked to greater destruction of its habitat? (Note that this point goes beyond the scope of your article).
• Many lepidoptera are polyandrous, with ‘older’ males (a few days old) being more successful at mating with newly hatched females. I know that this is almost a rule among Papilionidae and Nymphalidae. Have you checked this point? It does not seem obvious from your results. Is there any literature on Hesperiidae?

I believe your work is important and should be published, but you need to take at least some of my comments into account, particularly regarding the exact scope of your measures and those that are (or would be) necessary. So for now, I suggest ‘major revisions’.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Corrections and suggestions

 

1. Title: Reproductive behavior of the endangered prairie butterfly Oarisma poweshiek (Parker, 1870) (Lepidoptera: Hesperiidae) in relation to environmental factors in an ex situ conservation setting in the United States. ‘

The title was corrected by adding a scientific name, as the use of the common name ‘prairie butterfly’, according to the International Norms of Zoological Nomenclature, may vary among countries(1999, 4th edition)

2. Line 10: The Poweshiek skipperling (Oarisma poweshiek) - The poweshiek skipperling Oarisma poweshiek (Parker, 1870) (Lepidoptera: Hesperiidae).
3. Lines 17, 18, and 19: Sunlight was a “significant” predictor of breeding behavior frequency, but ambient temperature did “not significantly “impact breeding behavior. Null hypothesis tests were also conducted using the ANOVA function. The results were evaluated based on the F-value, degrees of freedom, and significance level (p). “There was a significant effect of the environmental factor on reproductive behavior (e. g., ANOVA, F2,18 = 5.34, p = 0.014).”
4. Line 48: Thymelicus lineola (Ochsenheimer, 1808) (Order? Hesperiidae). Norms of Zoological Nomenclature.
5. Lines 76-77: The nectar was provided in a plastic cup with flower cutouts and replaced once per day.
6. Lines 128-130: Males: χ² (chi-square test) = 16.77, p < 0.0001; Females: χ² = 3.95, p = 0.047 (Figure 1). Neither males nor females reacted strongly to ambient temperature (Males: χ² = 5.08, p = 0.079; Females: χ² = 1.08, p = 0.58).

Clarify in the text that this “χ²” refers to the chi-square test, since the design was not described in the Materials and Methods section.

7. Lines 135-137: Sunlight intensity was a “significant “predictor of breeding behavior frequency for males and females, while ambient temperature was not a “significant” predictor of breeding behavior frequency for either sex (Chi-square or ANOVA? insert as above).
8. Lines: 146-47: We observed a ‘’significant” (Chi-square or ANOVA? insert as above) positive association between sunlight intensity and breeding behavior frequency for both males and females (Figure 1).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the reviewer comments carefully and the revised manuscript is noticeably clearer and more transparent. The additional explanations of the statistical models, temperature effects, and residual diagnostics improve confidence in the analytical approach. Acknowledging the limitations associated with external temperature data and the low frequency of breeding behaviors appropriately contextualizes the results.

Overall, the revisions adequately address the concerns raised, and the manuscript now provides a clear and well-contextualized contribution to ex situ conservation research.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

I am well aware that many of my suggestions go beyond the scope of this preliminary article. However, your study system based on this genus is interesting, and as a lepidopterist, I would like to know more! You have taken some of my suggestions into account, and I hope that the others will be useful to you in your future studies. I therefore see no reason to hold up your article any longer and propose that it be accepted immediately.