The Potential of Complementary Sex-Determiner Gene Allelic Diversity for Studying the Number of Patrilines within Honeybee Colonies

Round 1

Reviewer 1 Report

Mroczek et al. examined the variability of complementary sex determination (csd) gene to determine the genetic structure of honey bee colonies and paternities of workers derived from the multiple-mated queen in the honey bee. The results clearly showed that variability of csd was higher than that of microsatellite markers, suggesting that csd provides superior resolution in genotyping in the honey bee. The experiments were performed nicely and the manuscript has been well written. However, there are some questions and missing explanations that need to be answered and corrected.

The authors assumed that the csd sequence of the oocyte or spermatozoon is identical in each individual, but this needs to be proven. For example, the authors should indicate the number of alleles at the csd locus among drones in the colony and whether two alleles are always present among drones in a colony.

Also, it is possible that some of the male brood (pupae) may be derived from egg-laying workers in the colony. How can the authors exclude the possibility without examining colony queen samples? If the authors found only two maternal alleles in each colony, the results should be shown or mentioned in the Results section.

Line 39: High genetic diversity by polyandry in honey bee queens is based on the mixed sperm utilization in the spermatheca during oviposition. Several papers demonstrating the sperm mixed in the spermatheca and determination of patrilines by sequential egg-laying by using molecular markers have been published. These should be mentioned in the text with several citations.

Line 114: The authors stated that the specimens were stored at -200°C in 96% ethanol. Does this mean storing them in liquid nitrogen?

Table 1: Are the values in the table number of alleles in csd and microsatellite loci? I could not understand “the mean number and total number of distinct loci” in the table. Why are there so many csd loci?

Figure 3: Please indicate the units of the vertical axis with the axis labels in each figure. There were double peaks in the blue trace in the drone, but theoretically only one peak should be detected in the blue trace. Such a double-peak pattern was found in both the blue and green traces of the workers. How do the authors explain the double peaks?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

comments:

In the manuscript titled “The potential of complementary sex determining gene diversity for studying the genetic structure of honey bee colonies”, the authors investigated the potential of the complementary sex determining gene (csd) as a marker for genetic studies of honey bee colonies, comparing its efficacy with standard microsatellite markers, and conducted worker brood from five colonies was genotyped using both csd and microsatellite markers. After reviewing, I believe this article holds significant potential. However, substantial revisions are necessary before acceptance. These adjustments aim to ensure the article meets the journal's standards and elevates its overall quality.

It's crucial to note that these revisions involve substantial changes across various aspects, including structure, language, and content support. I believe these modifications will enhance the persuasiveness and value of your research.

For the forthcoming revisions, I suggest focusing on the following key areas:

In the whole Ms. csd should be “Csd”.

The title refers to genetic structure, when in fact there is no corresponding data of genetic structure. For genetic diversity, is it just allelic diversity? There are no other parameters?

1. Introduction

-line 43-44, "workers" can be changed to "worker bees” to avoid misunderstandings.

2. Materials and Methods

-line 110-111, Five microsatellite sites are a little too few.

-line 112, Please mark the coordinates at the sampling locations.

-line 113,  -200C prior to further processing??

-line 120-122, What sequence is the primer based on?

-line 143-144, The use of the software should be accompanied by references.

3. Results

-line 154-155, “from 50 to 60 worker bee…” Typically, specific numerical values, parameters, or coordinates in a research study are initially detailed in the "Materials and Methods" section.

-line 162…, T-RFLP?? Use full name the first time and short name the after second time.

Figures 2 and 3 do not make any sense and are recommended to be deleted.

4. Disscussion

-line 198-199, reference?.

-line 227-246, This paragraph is mostly about research results and is not part of the discussion.

How do RFLP and microsatellite results compare?

How to avoid errors caused by small number of alleles?

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have considered the previous comments and have improved the manuscript significantly by adding the explanation. Problems that I pointed out have been solved.

Reviewer 2 Report

The author has revised in accordance with the review comments.