Mediterranean Fruit Fly Ceratitis capitata (Diptera: Tephritidae) Eggs and Larvae Responses to a Low-Oxygen/High-Nitrogen Atmosphere
Round 1
Reviewer 1 Report
No suggested changes, so no file attached: This article was very well written, so I found nothing that needs changing.
Objective: Mediterranean fruit fly is a horticultural products pest worldwide that needs non-chemical post-harvest control technique. High CO2 storage of fruit can cause fruit damage but N2 does not. These experiments exposed C. capitate eggs and larvae in carrot diet to two low-oxygen/high N2 treatments (0.5% O2 + 99.5 N2 or 5% O2 + 95% N2). Complete control of C. capitata eggs and larvae occurred after eight days and nine days, respectively, of exposure to an atmosphere of 0.5% O2 at 25°C and 70 -75% RH. This study demonstrates and confirms the mortalities of C. capitata caused by low-oxygen treatment using nitrogen, which may help develop new postharvest strategies to control this destructive fruit fly pest.
Author Response
Thank you
Reviewer 2 Report
General comments
This manuscript presents results on the effect of variable time exposition to two Low oxygen-High nitrogen atmospheres on the survival of egg and larval instars of medfly. The aim of the work is relevant for the control of medfly in postharvest, an important issue in this quarantine pest in some countries.
The main concern about the manuscript is the lack of a criteria to compare the LTs values estimated, which leads to discuss the results using the actual estimated values, which may or not be significantly different. A suggestion is given in the detailed comments section.
The methodology has been designed and the experiments have been carried out very carefully.
I am not an English-speaking person, but I find the manuscript well written. I have only signalled some sentences whose meaning is not clear to me.
Detailed comments
Introduction
Line 60. Reference 26 is not suitable to support the point raised by the authors, as it does not deal with low-oxygen treatments, but with wind tunnel bioassays.
Material and Methods
Line 109. I do not understand why an air cylinder was used instead of an oxygen cylinder. Air also contains CO2 and other gases. In fact, O2 cylinders are mention in Line 200
Line 185. “h”, not “hr”.
Lines 208-210. I suggest substituting “… and counted. The emerged adults were investigated to determine if the low-oxygen treatment leads to unbalanced sex ratios of the surviving flies.” by “… when they were counted and sexed”.
Statistical analysis
Lethal Time is estimated for each (Low O2 High N2 treatment * medfly stage instar) combination using a probit or logit analysis, but no information on the goodness-of-fit of the data to the model is given (the authors only state that the probit or the logit model is selected according the Chi-square value, but they do not give their values, their significance or the values of the Heterogeneity Index), and this information is necessary. Using two different models difficult the results of the comparisons (for example among stages exposed to 5% O2 concentration). If the valued of the Chi-square tests are good enough, using only one model could be sufficient.
To stay which medfly stage is more or less tolerant to the exposition to low O2 concentration, the authors use the values of the LTs, but they do not state which criteria they used to decide whether to LTs are significantly different or not. In the comments that follow afterwards, I use the criteria based on the overlapping of the LT confidential limits (two LT are not significantly different if their confidence limits overlap (Robertson et al., 2002, for example).
I suggest reordering the sentences of the first two paragraphs. State first the criteria you used to decide when an individual was alive. State afterwards the formula used to correct (not to normalize) the mortality data. Finally, state the statistical analysis used.
Please, reason why LT values are calculated with the pupariation and with the adult emergence data. Do you hypothesize that low O2 concentration on egg and larval stage could influence pupal mortality?
Lines 232 – 233. I guess the sentence “Experiments were analysed using one-way ANOVA, followed by Tukey’s difference (HSD)” applies to the sex ratio results. Please, state it. Were data on percentage transformed prior to analysis? (arc sin square root).
Results
Line 235. Delete “To”
Lines 235 – 236. The sentence “Pupariation and emerged adult rates after treatment under both low-oxygen concentrations (0.5% and 5.0%) were significantly lower than the control for all tested stages (Figure 2 and 3).” is not supported by the statistical analyses carried out, as the percentage of pupariation and the percentage of adult emergence at each exposure time are not statistically compared to those of the control. Looking at Figures 2B and 3B, one can guess that some values from day 0 (control) and day 1 are not significantly different. I think this sentence is not necessary, as a probit or logit analyses has been carried out and the important results are the LTs, which can be used to answer the question on how many days at low O2 concentrations are needed to kill 99% of the individuals.
Lines 240-241. Rewrite the sentence “This result showed that 0.5% O2 treatment at 25.0 ºC for nine days offers complete control of all immature Medfly stages than at 5.0% O2 (Figure 2)”. I guess the words “than at 5.0% O2” must be deleted.
Lines 242-245. The comparison of the tolerance of the different medlfy stages to low O2 concentrations must be done using the calculated LTs, not the lines drawn in Figure 2 and Figure 3, which are drawn joining the observed mortality without any statistical analysis. For example, using the abovementioned criteria to compare LTs, L3 is significantly more tolerant to 0.5% O2 concentration than L2, L1 and egg for LT50, but not for LT90.
Lines 260-261. The sentence “We modeled the duration of low-oxygen treatment to induce 50, 90 and 99% mortality at four immature stages of C. capitata fed on a lab diet by using selected model” must be deleted from here, as it has been already stated in the Material and Methods section.
Lines 262-266. In my opinion, the sentence “under 0.5% O2 treatments, LT99 was 8.5, 8.8, 9.5 and 9.6 days for eggs, 1st instar, 2nd instar and 3rd instar, respectively (Table 1), further confirming that 3rd instar was the most low-oxygen tolerant stage (LT99=9.6) (Table 1).” is not supported from the results, as the confidence limits of all the mentioned LT99 overlap.
Line 298. “whether”, instead of “if”.
Line 299-300. I do not understand the sentence “we analysed the female adult ratios from treated flies at Day 0, Day one, Day two, Day three and Day four 4”. I guess the authors mean “flies treated 0, 1, 2, 3 or 4 days”, not at day 0, 1, 2, 3 or 4.
There are not significant differences when comparing the LTs estimated using the pupariation criteria and the LTs calculated the adult emergence criteria, in any case.
Discussion
Line 308. Delete “The”.
Line 308. Although it is stated in the Material and Methods section, it would useful for the reader to state again that N2, and not CO2, is the other component of the atmosphere.
The Discussion has to be rewritten taking into account the significant differences among the LTs, not the actual estimated values.
Line 346 onwards. In my opinion, the discussion on the sex ratio results is too long, as the comparison with the results observed on irradiated medflies are nor relevant to this manuscript.
Line 353. “Cydia pomonella”, instead of “Laspeyresia pomonella”, as it is the valid name at present.
Conclusions
Line 375. “and”, not “at”, probably.
Author Response
please find the file attached.
Author Response File: 
Author Response.docx
Reviewer 3 Report
The paper deals with a lab study carried out in order to evaluate the influence of low-oxygen/high-nitrogen against the immatures of Ceratitis capitata. The paper is interesting and give useful informations for future applications in developing postharvest control strategies against the pestiferous fruit fly. I would suggest that the introduction might be improved in the light of others similar control procedures set for the medlfy (in a separate file I am going to indicate to you 3-4 papers that could be discussed and cited in this chapter. Concering M&M I would prefere if the bioassays woud be conducted on fruits and instead of carrot diet, but I suppose that the little chamber was't suitable for these experiments..
Anyway I really appreciate the experimental setup and as a consequence also the results that are well presented and clear. There are also some little mistakes in the text that I put into the file. In my opinion, the present paper could be accepted for pubblication after some few revision
Comments for author File: 
Comments.pdf
Author Response
please find the file attached
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The author answers to my comments and suggestions are ok to me. Thank very much to the authors.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
