Calcium Accumulation and Loss and Vitamin D3 Content of Feeder Black Field Crickets (Gryllus bimaculatus) Fed on a High Calcium Diet with and without UVB Irradiation
Round 1
Reviewer 1 Report
In the introduction maybe if the study Oonincx et al 2018 does not necessarily align with the logistics needs of institutions working with insectivores in captivity, could be used for institutions raising these insects, because as demonstrated by this note, the nutritional status of an organism can not be changed in short periods of time. And this is one of the observations for this study, that all the time of 72 hours is too short to complete all the vitamin D cycle which involves different organs such as skin, liver and kidneys and different molecules such as steroid cholesterol, cholecalciferol, calcidiol, and calcitriol, maybe in this period of 72 hours these intermediate products have an impact but not vitamin D3. for the posterior process of calcium absorption, the time required to have an impact would be much longer as reported by Oonincx et al 2018.
For that reason, the fasting period of 24 hours would affect just the gut content that will be probably empty, but not in the composition of the cricket itself, so the 72 hours of UV treatment would not be enough to have an effect on the synthesis of vitamin D3 or Calcium accumulation in the organism and I think this study is just measuring the calcium variation in the gastrointestinal tract that is not affected by UV light.
figure 1 is not clear what the colours mean, please provide more information, text under the figure should be enough to understand the figure without the document.
In lines 140 to 143, the authors talk about melanin and its possible effect in results, the fact that is proved to have an impact on vitamin D3 synthesis but also it would be interesting, if possible to have information about keratine impact in UV light blocking the pass of the light to lower levels of tissues.
Another aspect that would be good to know more is about the calcium variation and how Phosphorus stays stable. Why this pattern? could be that Calcium is not the limiting component but Phosphorus? that the organism has mechanisms to limit phosphorus levels that have consequences in the Calcium concentrations due to the C:P ratio? that normally in this kind of insects is negative?
Author Response
Reviewer 1:
Thank you for your review of our MS. We have addressed each point raised below.
Rev. Comment: In the introduction maybe if the study Oonincx et al 2018 does not necessarily align with the logistics needs of institutions working with insectivores in captivity, could be used for institutions raising these insects, because as demonstrated by this note, the nutritional status of an organism can not be changed in short periods of time. And this is one of the observations for this study, that all the time of 72 hours is too short to complete all the vitamin D cycle which involves different organs such as skin, liver and kidneys and different molecules such as steroid cholesterol, cholecalciferol, calcidiol, and calcitriol, maybe in this period of 72 hours these intermediate products have an impact but not vitamin D3. for the posterior process of calcium absorption, the time required to have an impact would be much longer as reported by Oonincx et al 2018. For that reason, the fasting period of 24 hours would affect just the gut content that will be probably empty, but not in the composition of the cricket itself, so the 72 hours of UV treatment would not be enough to have an effect on the synthesis of vitamin D3 or Calcium accumulation in the organism and I think this study is just measuring the calcium variation in the gastrointestinal tract that is not affected by UV light.
Response: Thank you for these comments. Evidence from mammals (cited in the MS) shows that vitamin D3 can be synthesised within the timeframe of the experiment in that group. In tetrapods, the string of organs and molecules involved in d3 synthesis, which the referee refers to, are well known, but insects lack these organs and the only recently discovered capability of these animals to synthesise vitamin D3 is poorly understood. We feel that our Discussion explores these points adequately, as we state that D3 was apparently not produced over the short time period by the crickets, that this may be due to the short timeframe and/or the physiology of the species in question, and that although mammals can synthesise the hormone in this sort of timeframe, this is unknown for crickets.
Rev. Comment: figure 1 is not clear what the colours mean, please provide more information, text under the figure should be enough to understand the figure without the document.
Response: Thank you for this comment – we have amended the figure legend to clarify the colour groups.
Rev. Comment: In lines 140 to 143, the authors talk about melanin and its possible effect in results, the fact that is proved to have an impact on vitamin D3 synthesis but also it would be interesting, if possible to have information about keratine impact in UV light blocking the pass of the light to lower levels of tissues.
Response: Thank you for the comment. We assume that the reviewer means chitin, rather than keratin, as the former is the main component of insect exoskeletons and the latter is not present in arthropods. We do not feel that this is a relevant discussion point, as other research has shown that insects with chitin rich exoskeletons synthesise vitamin D3, so it is does not prevent this process, and there is no evidence that our study species has more chitin than other species.
Rev. Comment: Another aspect that would be good to know more is about the calcium variation and how Phosphorus stays stable. Why this pattern? could be that Calcium is not the limiting component but Phosphorus? that the organism has mechanisms to limit phosphorus levels that have consequences in the Calcium concentrations due to the C:P ratio? that normally in this kind of insects is negative?
Response: Thank you for these thoughts. Phosphorus remains stable because we did not provide a supplement containing non-negligible quantities of this element; calcium increased and then decreased because it was present in the insects’ diet, and then the animals were starved. Crickets, as the referee points out, have a low Ca:P ratio and so their homeostasis prevented additional calcium being stored in tissues and therefore after fasting this element concentration returned to baseline. Fasting was not long enough for the insects to change the elemental composition of their actual tissues, only their gut contents. Phosphorus homeostasis influencing calcium levels may or may not be the mechanism behind maintaining baseline Ca;P ratios in insects, but this is outside the scope of this work and our experimental design was not formulated to address this. Nevertheless, we have now included a short paragraph discussing phosphorus results in the Discussion.
Reviewer 2 Report
The manuscript reports on topic mainly relevant for insectivorous pet owners and exotic animal research. But considering the growing field of edible insects in the world, the ability to manipulate captive insect’s nutritional values is important also. There is little literature available on this topic and it should be considered very novel research. I find the scientific methods and interpretation of results appropriate.
It’s is obvious the manuscript is written by native English speaker and is clear and concise, and therefore I’m not qualified to comment on the language. It was a pleasure to read.
I was not able to find the supplementary materials therefore I can’t comment on these extra figures and tables listed in the end of the manuscript.
The introduction and discussion focus to large extent on comparing this study against Oonincx et al, 2018 article. Unfortunately the authors were not able to provide conclusive explanations why their results were so much different. However, this is not authors fault as such questions have not been studied yet. I believe the provided speculations are correct given the existing knowledge on insect metabolism and the biological differences between insect’s species.
General comments:
- First concern is that the cricket’s exposure to UV light was not accurately measured. Since the UV lights were used only for 48 hours, it would have been more scientifically valuable to make sure the crickets really were exposed to UV light. Crickets were routinely observed to be basking in the light, but with egg cartons in the enclosures, it’s impossible to know how long all crickets were directly exposed to UV light. However, I understand that removing hiding material could increase mortality and would not represent practical rearing conditions.
- the age of the crickets used in the study was not mentioned nor discussed. In Oonincx et al, 2018, they had used fourth instar nymphs house crickets. Metabolism and endogenous cutaneous photosynthesis can differ during nymph stage and in adults. Authors should provide information about the approximate development stage of the crickets and explain if this could be of importance in design of future studies.
Author Response
Thank you for reviewing our work. We have responded to each comment, below; our response are in bold.
The manuscript reports on topic mainly relevant for insectivorous pet owners and exotic animal research. But considering the growing field of edible insects in the world, the ability to manipulate captive insect’s nutritional values is important also. There is little literature available on this topic and it should be considered very novel research. I find the scientific methods and interpretation of results appropriate.
We are delighted by these comments, thank you!
It’s is obvious the manuscript is written by native English speaker and is clear and concise, and therefore I’m not qualified to comment on the language. It was a pleasure to read.
Thank you for the kind words.
I was not able to find the supplementary materials therefore I can’t comment on these extra figures and tables listed in the end of the manuscript.
Thank you for noticing this. There are no Supplementary Materials, this was a part of the MS template that we neglected to remove. It has now been deleted.
The introduction and discussion focus to large extent on comparing this study against Oonincx et al, 2018 article. Unfortunately the authors were not able to provide conclusive explanations why their results were so much different. However, this is not authors fault as such questions have not been studied yet. I believe the provided speculations are correct given the existing knowledge on insect metabolism and the biological differences between insect’s species.
Thank you for these comments. We are painfully aware of the paucity of knowledge in this area and plan to undertake further work to investigate it.
General comments:
- First concern is that the cricket’s exposure to UV light was not accurately measured. Since the UV lights were used only for 48 hours, it would have been more scientifically valuable to make sure the crickets really were exposed to UV light. Crickets were routinely observed to be basking in the light, but with egg cartons in the enclosures, it’s impossible to know how long all crickets were directly exposed to UV light. However, I understand that removing hiding material could increase mortality and would not represent practical rearing conditions.
Thank you for this comment. As well as reflecting practical rearing conditions, we were also required to work within the ethical framework of our organisation. We have now included a paragraph in the Discussion to reflect this.
- the age of the crickets used in the study was not mentioned nor discussed. In Oonincx et al, 2018, they had used fourth instar nymphs house crickets. Metabolism and endogenous cutaneous photosynthesis can differ during nymph stage and in adults. Authors should provide information about the approximate development stage of the crickets and explain if this could be of importance in design of future studies.
Thank you for these thoughts. We did already say ‘adult’ crickets in our Methods, but we have clarified that this specifically means final instar crickets. We have also included a paragraph in the Discussion highlighting the potential difference between life-stages in interaction with UVB radiation and highlight the need for future research on this topic.
