Male-killer symbiont screening reveals novel associations in Adalia ladybirds
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While male-killing bacteria are known to infect across arthropods, ladybird beetles represent a hotspot for these symbioses. In some host species, there are multiple different symbionts that vary in presence and frequency between populations. To further our understanding of spatial and frequency variation, we tested for the presence of three male-killing bacteria: Wolbachia , Rickettsia and Spiroplasma , in two Adalia ladybird species from a previously unexplored UK population. The two-spot ladybird, A. bipunctata , is known to harbour all three male-killers, and we identified Spiroplasma infection in the Merseyside population for the first time. However, in contrast to previous studies on two-spot ladybirds from continental Europe, evidence from egg-hatch rates indicates the Spiroplasma strain present in the Merseyside population does not cause embryonic male-killing. In the related ten-spot ladybird, A. decempunctata , there is only one previous record of a male-killing symbiont, a Rickettsia , which we did not detect in the Merseyside sample. However, PCR assays indicated the presence of a Spiroplasma in a single A. decempunctata specimen. Marker sequence indicated that this Spiroplasma was divergent from that found in sympatric A. bipunctata . Genome sequencing of the Spiroplasma -infected A. decempunctata additionally revealed the presence of cobionts in the form of a Centistes parasitoid wasp and the parasitic fungi Beauveria . Further study of A. decempunctata from this population is needed to resolve whether it is the ladybird or wasp cobiont that harbours Spiroplasma , and to establish the phenotype of this strain. These data indicate first that microbial symbiont phenotype should not be assumed from past studies conducted in different locations, and second that cobiont presence may confound screening studies aimed to detect the frequency of a symbiont in field collected material from a focal host species.
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The revised version of the paper has addressed all concerns raised by the reviewers.
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Please make sure you address all the concerns raised by the reviewers, most importantly a more elaborate introduction is needed as raised by reviewer 1 and reviewer 2 raises an important concern, that details are lacking in the methods section- in particular around how and where the samples were taken and how they were fed in the lab.
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Comments to Author
The manuscript addresses an interesting topic. However, there are methodological ambiguities that need to be clarified. Otherwise, the replicability of the study would be unapproachable. Furthermore, the contribution of the study to the spatial component of the relationship between MK symbionts and Adalia species is not clear, if any. The description of insect sampling needs to be greatly improved. Where exactly are the sampling sites? Please provide complete information. How exactly was the sampling done? What method was used? Sweeping net, fall traps or any other? Are these localities representative of the male/female ratio of these species? Once again, the description of the sample collection is very poor. To what extent is this sample size representative of the biology of both Adalia species? In …
Comments to Author
The manuscript addresses an interesting topic. However, there are methodological ambiguities that need to be clarified. Otherwise, the replicability of the study would be unapproachable. Furthermore, the contribution of the study to the spatial component of the relationship between MK symbionts and Adalia species is not clear, if any. The description of insect sampling needs to be greatly improved. Where exactly are the sampling sites? Please provide complete information. How exactly was the sampling done? What method was used? Sweeping net, fall traps or any other? Are these localities representative of the male/female ratio of these species? Once again, the description of the sample collection is very poor. To what extent is this sample size representative of the biology of both Adalia species? In the section "Embryonic phenotype that kills males in A. bipunctata" it is mentioned that 20 females of A. bipunctata, after collection, were placed individually in Petri dishes together with filter paper on which they deposited eggs, provided with aphids to eat and a male of A. bipunctata to maintain fertility. It is not fully established in the manuscript if the presence of symbionts in the sampled individuals derives from other possible organisms (cobionts). Regarding this, it seems that the WGS detection was made only in specimens of A. decempunctata infected with Spiroplasma. Why was a female infected with A. bipunctata not sequenced? It seems to me that the study focused on two species of ladybugs, but that whole section of genomic sequence was apparently done on only one of the species. What aphids were used for food? where did the latter came from? From rearing stocks colonies or directly from the field? In this regard, it should be considered that aphids harbor several facultative bacterial endosymbionts, including Spiroplasma. How was the possibility that the presence of aphid-derived symbionts could be transferred to Adalia individuals excluded? The aphids used to feed the aphids may have eaten differently by Adalia individuals. Aphids free of facultative endosymbionts should have been used. That would needed a specific detection of endosymbionts in aphids used for food. On the other hand, in its first lines the manuscript highlighted the spatial variation in the distributions of symbionts, but the study itself does not shed new light on this issue. Instead, the manuscript attempts to link the presence of MK symbionts in two Adalia species, and that's it. Is the Merseyside region particularly relevant to these Adalia-MK symbiotes?
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Dear authors and editors, Thank you for the interesting paper, I enjoyed reading it. It presented novel research well with some intriguing conclusions and an abundance of future possibilities of study, including a very thorough experimental setup and write-up. My main criticisms of the article are mainly on style and the briefness of the introduction leading to a lack of background to the study, so I suggest minor revisions before accepting and publishing. 1. Methodological rigour, reproducibility and availability of underlying data The methodology of this article is very thorough and should make it easy to duplicate the entirety of the experiment for any researcher. The researchers not only screened the caught ladybirds for infections, they also investigated other strains of DNA found alongside the …
Comments to Author
Dear authors and editors, Thank you for the interesting paper, I enjoyed reading it. It presented novel research well with some intriguing conclusions and an abundance of future possibilities of study, including a very thorough experimental setup and write-up. My main criticisms of the article are mainly on style and the briefness of the introduction leading to a lack of background to the study, so I suggest minor revisions before accepting and publishing. 1. Methodological rigour, reproducibility and availability of underlying data The methodology of this article is very thorough and should make it easy to duplicate the entirety of the experiment for any researcher. The researchers not only screened the caught ladybirds for infections, they also investigated other strains of DNA found alongside the bacteria, leading to some of the more interesting conclusions: a braconid wasp's DNA was found, as well as an infectious fungus (Beauveria) which has not yet been described to infect Adalia spp. All the data was very easy to find, as it all has been submitted to databases online and have been directly linked in the article itself. 2. Presentation of results The results of the experiments have been presented clearly and succinctly. Some results can not be interpreted as statistically significant, which is stated upfront by the authors. As the incidence of these bacteria is low in these ladybirds, a weak statistical power is to be expected due to a relatively small number of data points (i.e. four infected females). However, this is typical for studies investigating these interactions (eg. von der Schulenberg et al., 2000) and still allows for comparison between populations. I am curious why "it was not possible to separate the two insect genomes satisfactorily" (line 276), as this can have a variety of reasons, but is not expanded upon. While the numbers of caught Adalia are small, the authors make no mention of the sex ratios of the wild-caught population which in a glance seem evenly divided - it might be worth mentioning as sex ratios of A. bipunctata can be extremely skewed (eg. Zakharov et al., 2006), as a result of male-killing endosymbionts. 3. How the style and organization of the paper communicates and represents key findings The paper has an odd balance of its organization: the introduction is extremely brief and fairly confusing, while the discussion is very thorough and intriguing. I was put off from reading the rest of the article from its introduction, which is a shame considering the results and discussion are well worth your time. The introduction would do well to introduce a few of the concepts outlined as results in the discussion: for example, the Scandinavian, Central European, and Scottish populations of Adalia spp. and their infection rates should be mentioned as they come in play later. Additionally, the urgency of this research is somewhat lost from its introduction as almost every citation is over 15 years old and some key points are argued in a confusing way. For example, line 73 is an odd sentence talking about a two-fold cost for the Adalia, which suddenly talks about a high reproductive success for the males without explaining why this is detrimental. Some other background mentioned in the discussion, such as the other associations (wasps, fungi), are not mentioned in the introduction at all. This also ties into the next point: 4. Literature analysis or discussion The background sketched in this article of the research subject is fairly poor. Most articles cited in the introduction are not recent, even though there is a wealth of recent research on Adalia spp. and other coccinellids with regards to their symbionts and how this affects their ecology. The introduction is much too brief and sometimes does not cite sources for important statements; for example, line 85 mentions "The disparity in the number, and prevalence, of different male-killers in A. bipunctata vs. A. decempunctata is poorly understood", but it is unclear if there have been investigations of both species and the sex ratios of their populations at all. There has been one mention in a German study of infections of A. decempunctata, but a lack of reports is not enough to state that there is such a disparity. The introduction could also expand on putting the trophic interactions between Adalia and their symbionts into a broader ecological context and their place in the variety of interactions of parasites in other coccinellids. Taking some more time to explain the background more in depth would significantly improve this paper. In comparison, the discussion is excellent, and raises several questions gained from the results of the study, as well as multiple avenues of interesting future research. It is worth mentioning with regards to novel Beauveria infection finding that infections of one species of parasite can sometimes result in susceptibility to other parasites; for example, in Harmonia axyridis, parasitic fungi, mites, and bacteria can affect other parasites, sometimes as competition, sometimes as enabler. 5. Any other relevant comments There are a few other minor comments or criticisms of the writing itself. Line 67 has a reference to Jaenike et al., but this is a very specific paper on defensive symbionts; a more general review on the effects of endosymbionts in insects would be better here. Line 70 mentions "selection", where selective pressure is meant. Line 78 has "Coccinelidae" in italics, as do the other mentions of families in rest of the paper; family names should not be italicized.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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