Effect of Headstarting Eggstrands of the Endangered Houston Toad (Bufo = [Anaxyrus] houstonensis) from a Captive Assurance Colony on Native Breeding Pond Microbiomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article focuses on the impact of captive breeding activities of the endangered Houston toad (Anaxyrus houstonensis) on the microbial communities of its natural breeding ponds, especially the potential transmission risk of Mycobacterium. The study employed Illumina 16S rRNA V3 amplicon sequencing technology to conduct a comprehensive analysis of microbial communities in water and sediment samples from both captive breeding facilities and natural ponds. Overall, the research design is rational, the data analysis methods are advanced, and the conclusions hold scientific value, providing important references for microbial ecology studies in the conservation of endangered species.

 

However, some improvement in the elaboration of the research background, supplementation of methodological details, depth of data analysis, and rigor of conclusions can be considered.

 

1.  Research Background and Problem Statement

The introduction mentions that habitat loss, climate change, environmental pollution, and pathogens and their interactions are key factors leading to the decline in amphibian populations (lines 35-37), but it does not elaborate on how these factors specifically affect the survival of the Houston toad. It is recommended to supplement with references to relevant studies or specific cases to enhance the persuasiveness of the background.

 

The article notes that the population of the Houston toad is continuously declining (lines 45-46), but it does not provide specific data or trend charts. It is suggested to supplement with historical data or charts on population changes to allow readers to more intuitively understand the severity of the problem.

 

2.  Methods Section

The sample collection section mentions "collecting water samples from 75-liter glass tanks at the Houston Zoo" (lines 85-95), but it does not specify the exact timing and frequency of sample collection. It is recommended to supplement with the schedule and frequency of sample collection to assess the impact of temporal factors on microbial communities.

 

The amplicon sequencing section mentions the use of "515f and 806r primers" (lines 119-121), but it does not explain the rationale for primer selection or the range of microorganisms they cover. It is suggested to supplement with background information on primer selection to help readers better understand the limitations of the sequencing results.

 

3. Data Analysis

The article states that "all tank samples were dominated by Proteobacteria" (lines 170-174), but it does not conduct statistical analysis on the differences between tanks. It is recommended to supplement with statistical analysis results to assess the significant differences in microbial communities between different tanks. For example, ANOVA or Kruskal-Wallis tests could be used to evaluate differences in the abundance of Proteobacteria among tanks.

 

The article mentions that "the proportion of Proteobacteria significantly increased during the release of eggstrands" (lines 195-198), but it does not indicate whether this change is statistically significant. It is suggested to supplement with statistical test results (such as t-tests or ANOVA) to support this conclusion. For example, specific p-values and effect sizes could be provided to illustrate the significance of this change.

 

4.  Discussion Section

The discussion mentions that "Mycobacterium is widely distributed in various water sources" (lines 275-279), but it does not discuss the specific distribution patterns of Mycobacterium in captive breeding facilities and natural ponds in conjunction with the data from this study.

 

The article states that "captive breeding activities did not significantly affect pond microbial communities" (lines 305-307), but it does not discuss the possible reasons. It is recommended to further explore why the impact of captive breeding activities on microbial communities is minimal, such as whether it is related to environmental conditions in the ponds (e.g., water temperature, pH levels).

 

The article mentions that "the differences in microbial communities in some ponds during the fall may be related to seasonal changes" (lines 313-314), but it does not provide specific evidence to support this view. It is suggested to supplement with relevant environmental data (such as water temperature, dissolved oxygen) to enhance the persuasiveness of the discussion. For example, seasonal changes in water temperature, pH levels, and dissolved oxygen could be provided to illustrate how these factors affect microbial communities.

 

5.  Conclusion Section

The conclusion clearly states that captive breeding activities have minimal impact on natural pond microbial communities (lines 336-338), but it does not discuss the limitations of this conclusion. It is recommended to supplement with a discussion of the study's limitations, such as whether the sample size is sufficient and whether long-term impacts were considered. For example, it could be explained whether the sample size is adequate to represent the entire breeding season and whether long-term tracking studies were conducted.

 

Author Response

Please see our replies to your review in the attached document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors presented an up-to-date study.

Note:

1. It is necessary to pay attention to the route of invasion and entry of microorganisms when releasing from artificial containment sites. Currently, artificial containment for the purpose of study or breeding can also serve as a route of entry for various pathogens, for example, the analysis of ranaviruses (Majji et al., 2006, Chen et al., 2013, doi: 10.1186/1297-9716-44-101; Lisachova et al., 2025, DOI: 10.1007/s10393-025-01703-3), showed both the route of infection and entry into natural populations, and higher pathogenicity. Another source of influx is aquaculture objects, during release or escape from holding facilities or transportation, in particular for the bullfrogs, a common species in aquaculture (Yang et al., 2022, doi: 10.1016/j.aqrep.2022.101037).

2. The main biotope of the species Anaxyrus houstonensis (Sanders, 1953) are temporary reservoirs; in permanent reservoirs, the population is suppressed by predators. The drying period may change the infection routes, for example, with the participation of aquatic organisms permanently living in the water.

3. In aquaculture, artificial maintenance, transportation and even temporary capture, the emergence and combination of different pathogens is possible, as was shown in a study for Pelophylax nigromaculatus (Hallowell, 1860) (Wei et al., 2024, doi: 10.3390/fishes9030091).

Reference:

Majji, S.; Lapatra, S; Long, S.M.; Sample, R.; Bryan, L.; Sinning. A.; Chinchar, V.G. Rana catesbeiana virus Z (RCV-Z): a novel pathogenic ranavirus. Disease of Aquatic Organisms 2006, 73, 1–11.

Author Response

Please see our replies to your review in the attached document

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript (and their 2021 publication) shows much of the authors' self-awareness - the possibility that by trying to conserve Houston Toads they might inadvertently introduce into their ecosystems pathogens that wipe them out. It's the physician's "Do No Harm" motto applied to conservation biology. My comments are minor.

First, in the Methods I'd like you to look though your terminology around egg strands. You are headstarting toads by releasing egg strands. In places it reads like you are headstarting egg strands.

The sentence beginning on line 308: Replace 'visibly' with a word like 'notably' or 'statistically significant' - something like that. Microbiota are hard to visualize.

In the Discussion, lines 310-312 you might want to add variations in nighttime anoxia/hypoxia influencing the composition of the microbiota. Wetlands with an abundance of macrophytes have wide daily swings in dissolved oxygen levels - highest in mid-afternoon, lowest at dawn. Some of the taxonomic variation you observe in the microbiota may be due to hypoxia tolerance.

Finally, in the amphibian chytrid fungus world, two models are in play. The first is that chytrid spread like a wave through Central into South America, and across Australia and wreaked havoc. The second is that fulminant chytrid strains have always been present, but weather extremes caused by climate change have stressed amphibians to the point where infections become the disease. Your results parallel the latter scenario.

Author Response

Please see our replies to your reviews in the attached document

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewer thanks the authors for the changes made. The article has been improved, I recommend it for publication.