Vertical Distribution of Culicoides Biting Midges in Temperate Forests
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI have reviewed the manuscript titled “Vertical distribution of Culicoides biting midges in temperate forests” reporting on a study investigating the entomological fauna at height in a European forest with specific discussion on the implications of Culicoides surveillance. The paper and study design is reasonably sound but requires great detail in methodology and discussion.
Firstly, while I understand why Culicoides may be the focus of this paper, there was also a range of other insects collected and, as a consequence, may be of interest to a range of different entomological specialists in search of sampling/monitoring considerations in similar circumstances.
It would be useful to have a description of the study site. Where, relative to the forest environment, are the immature habitats of Culicoides? Are there waterbodies through the forest or adjacent to forest?
With regard to methodology, I would like to see authors be very specific in describing how the traps were deployed in each tree. My understanding was that only one trap was used per tree (i.e. each individual tree only had a single trap operated at one of the three designated heights) rather than three traps used per tree (i.e. each of the three designated heights had a trap deployed). However, how were the individual trees and trap deployments determined? With only three individual traps available, there does not seem to have been able to undertake any rotation/replication of trap deployment at different heights on the same individual tree. This may have identified a “tree specific” influence on movement of midges at different heights?
It would also be useful to have a comment in the methodology to explain why only UV traps were used and not any carbon dioxide baited traps that may have further enhanced collections. I appreciate that UV light alone is used to sample Culicoides of human and veterinary health concerns but it would make for a useful clarification of methodology here.
I’m also not 100% sure what is meant by this statement “In total insects were collected 9 nights of 3 each month from June to August”. It would be better to provide more detail, for example, Insects were collected on a total of 9 occasions each month over the study period, June to August (or whatever the actual amount of individual trap nights was). This would also assist in determining the averages used in the results section. In the statistical analysis sections, it is stated “We calculated the average numbers of insects collected using traps of the same height and provided them with standard errors” – as the results section contains the presentation of results for each month, it would be useful to be clear as to the number of trap nights used here.
Culicoides, like many other insects, are highly depended on temperature and rainfall. It would be useful to include climatic data here to determine any potential influence on the abundance of insects due to wind, rain, temperature change, humidity beyond the influence of trap height operation alone.
It seems that, from the results presented in Figure 3 on the average monthly abundance for the three heights, that the overall conclusion that more Culicoides were collected at the medium height category was substantially influenced by the outliner result for female specimens of Culicoides pictipennis in June. How should this result be considered when recommending the need to consider entomological sampling beyond ground height?
Finally, the authors should include some recommendations or assessment on whether there is a net benefit to the additional cost, time, and operational inconvenience of setting traps at height. While it is clear that there may be greater interaction between bloodsucking insects, such as Culicoides, and birds and other vertebrates in mid- to high-canopy, does this represent a significant deficiency in formal surveillance programs based on the outcomes of this investigation.
Comments on the Quality of English Language
Satisfactory.
Author Response
Reviewer 1
I have reviewed the manuscript titled “Vertical distribution of Culicoides biting midges in temperate forests” reporting on a study investigating the entomological fauna at height in a European forest with specific discussion on the implications of Culicoides surveillance. The paper and study design is reasonably sound but requires great detail in methodology and discussion.
Firstly, while I understand why Culicoides may be the focus of this paper, there was also a range of other insects collected and, as a consequence, may be of interest to a range of different entomological specialists in search of sampling/monitoring considerations in similar circumstances.
We thank the reviewer for all comments and suggestions. Our research was focused only on blood-sucking insects, and the BG-Pro traps (https://research-shop.biogents.com/products/collection-set-for-biting-midges-bg-pro) designed for collecting biting midges were used for that purpose. We added information about the collection method and specified the type of traps in the article. (L 72-73, 87).
When writing about other insects, we wanted to emphasize that when studying biting midges using the same methods, it is possible to successfully study other groups of insects and we determined which groups they belong to. This information may be useful to entomologists looking for other traps suitable for trapping insects, BG-Pro traps which were designed for collecting Culicoides, may be adapted for studies of other insect groups. We have shorten information about other insect groups, deleted Fig 2 and moved this information to the end of Results (L 229-238).
It would be useful to have a description of the study site. Where, relative to the forest environment, are the immature habitats of Culicoides? Are there waterbodies through the forest or adjacent to forest?
Thank You for this suggestion, information was added (L 90-92).
With regard to methodology, I would like to see authors be very specific in describing how the traps were deployed in each tree. My understanding was that only one trap was used per tree (i.e. each individual tree only had a single trap operated at one of the three designated heights) rather than three traps used per tree (i.e. each of the three designated heights had a trap deployed). However, how were the individual trees and trap deployments determined? With only three individual traps available, there does not seem to have been able to undertake any rotation/replication of trap deployment at different heights on the same individual tree. This may have identified a “tree specific” influence on movement of midges at different heights?
We corrected the method chapter to make it clearer (L 90-95, 107-111).
We used three traps on the same night. Only one trap was used per tree. It would be difficult to find a tree that could successfully hang traps at all three heights (the trap needs to be hung on a branch; the tree needs to be suitable for climbing it). We also tried to hang the traps not too close to each other to avoid interference between traps, that is why a distance of 20 meters was used between trees. All three trees grew in the same habitat and were not far away from each other. Knowing that biting midges fly short distances (up to few hundred meters, doi:10.1111/1365-2664.12875), we think that the traps reflect the overall abundance of biting midges in the habitat, with only main differences in heights.
It would also be useful to have a comment in the methodology to explain why only UV traps were used and not any carbon dioxide baited traps that may have further enhanced collections. I appreciate that UV light alone is used to sample Culicoides of human and veterinary health concerns but it would make for a useful clarification of methodology here.
We used BG-Pro UV traps which were designed for collecting Culicoides, as this type of traps was also used in our previous research of biting midges, and it worked effectively without carbon dioxide bait (https://doi.org/10.3390/insects15030157 ‚ https://doi.org/10.3390/d15050692 ,
https://doi.org/10.3390/microorganisms10050898).
We supplemented information about the traps used in methods (L 72-73, 87), we noted that these traps have been used successfully before. We are collecting material (not in this investigation) with and without dry ice (the source of carbon dioxide) and we will try to know how this affects the abundance and diversity of collected biting midges. However, these data are still being analyzed.
We have not used dry ice during this investigation, as this would cause additional difficulties in hanging the traps especially to the highest strata, at different heights gas has potential to be dispersed differently because of wind blow difference as this increase from the ground to tree tops. UV traps are free of that effect. We think that the use of dry ice could skew the results, as the carbon dioxide flow rate to different traps can vary depending on how close the carbon dioxide source is to the trap, or how intense the gas is being emitted from the dry ice container. We compared the abundance of biting midges by trapping under the same conditions at all heights, which is why the same type of trap was used everywhere.
I’m also not 100% sure what is meant by this statement “In total insects were collected 9 nights of 3 each month from June to August”. It would be better to provide more detail, for example, Insects were collected on a total of 9 occasions each month over the study period, June to August (or whatever the actual amount of individual trap nights was). This would also assist in determining the averages used in the results section.
We corrected the text making it clearer (L 90-95, 107-111). We made investigations in three forest areas. Each area was investigated three times per year (in June, July and August). Each investigation was conducted using three traps hung at three different heights the same night. We corrected the text and provided supplementary material with row data and believed that this is now clearer.
In the statistical analysis sections, it is stated “We calculated the average numbers of insects collected using traps of the same height and provided them with standard errors” – as the results section contains the presentation of results for each month, it would be useful to be clear as to the number of trap nights used here.
We corrected the sentence in the Statistical analysis (L 123).
Culicoides, like many other insects, are highly depended on temperature and rainfall. It would be useful to include climatic data here to determine any potential influence on the abundance of insects due to wind, rain, temperature change, humidity beyond the influence of trap height operation alone.
We provided information on meteorological conditions based on the data obtained from the Meteorological station of Lithuania, Utena department, which was the closest to all three study sites (L 108-110). We collected biting midges at the time (June – August) when the activity of Culicoides in our country is the highest – this information was added (L 73-74). All collection nights were without precipitation, the average wind speed varied between 0.9 and 3 m/s and average day air temperature varied between 10.9oC (August, Dusetos) and 26.4oC (June, Tauragnai). This data was also provided also as supplementary material. We agree that the activity of biting midges is temperature dependent as it was noted in our previous publication (https://doi.org/10.1007/s00436-021-07147-2). During this investigation we did not find correlation between air temperature and abundance of Culicoides and this can be related to the range of temperature favorable for the activity of biting midges (it is known for our country that the highest activity of Cuicoides has been recorded that air temperature was 19.7 ± 3.1 °C, (https://doi.org/10.1007/s00436-021-07147-2)).
It seems that, from the results presented in Figure 3 on the average monthly abundance for the three heights, that the overall conclusion that more Culicoides were collected at the medium height category was substantially influenced by the outliner result for female specimens of Culicoides pictipennis in June. How should this result be considered when recommending the need to consider entomological sampling beyond ground height?
Yes, we agree that Culicoides pictipennis collected in June very abundantly influenced the results. We discussed this in the text (L 304-305). But we would like to note, that our results decribed not only the abundance of C. pictipennis, as more than 80% of all collected Culicoides were caught at the mid-canopy and high-canopy. We think that this result is important for the future investigations especially if looking for certain Culicoides species in the wild, which prefer to live in canopy.
Finally, the authors should include some recommendations or assessment on whether there is a net benefit to the additional cost, time, and operational inconvenience of setting traps at height. While it is clear that there may be greater interaction between bloodsucking insects, such as Culicoides, and birds and other vertebrates in mid- to high-canopy, does this represent a significant deficiency in formal surveillance programs based on the outcomes of this investigation.
We added some recommendations (L 368-370) which were mostly related to the scientists investigating biodiversity (it can be neglected if collecting insects only at the ground level), bird – vector interactions or investigating biology or certain Culicoides species which prefer to live in canopy.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
thank you for your work presented for publication. The presented work “Vertical distribution of Culicoides biting midges in temperate forests” is devoted to the study of the distribution structure of midges in the forest. Despite the obvious practical importance of studying any features associated with bloodsuckers, the presented work leaves a feeling of insufficient thought of both the objectives of the study itself and the conclusions obtained. When reading the materials, a number of questions arose, some of which are given below.
1. Why was this particular method of catching, using UV lamps, chosen to count midges? Why was the standard method of mowing with a net not used, although it shows the greatest efficiency?
3. In chapter «3.1. Insect distribution across vertical strata in the forest 101
In total, 11277 insects have been collected. The most abundant insects were dipterous, 102 followed by Lepidoptera (Fig. 2). Among dipterous insects, the most abundant were 103 Cecidomyidae (45.7%), Chironomidae (22.3%), Ceratopogonidae (15.7%), and 104 Psychodidae (10.3%). Only 6.0% of the collected dipterous insects belonged to families 105 Sciaridae, Culicidae, Mycetophilidae, and to other families (Fig. 2): Figure 2. The part of collected insects from different orders (A) and the most abundantly 109 collected families of dipterous insects (B)» the variety of insects caught in traps is shown: from butterflies to fungus gnats, without giving the names of the caught species. Why is this in an article devoted to the study of the vertical distribution of midges?
4. It is known that midges successfully copulate in swarms that gather in open space near poles, a river bank, a bush, etc. It is also known that only female midges feed on blood for the successful maturation of eggs. Why should males be present in the forest near the nests of their hosts if they cannot wait for food or copulation there? (See Figure 3. The average numbers (± SE) of Culicoides males and females collected at different heights in June, July and August. G – ground level (~1.5 m above the ground), M – mid-canopy (~13 m above the ground), H – high - canopy (~ 23 m above the ground).)
5. Finally, the conclusions are based on the fact that: «Summarizing the results of our research, we can state that the abundance of Culicoides was higher in the tree canopy compared with the ground level in temperate forest». But the authors at the very beginning stated that the traps “many species of biting midges are more prevalent in tree canopy close to bird nests.” Don't the authors think they have proven an obvious conclusion? The small number of males is also quite understandable without research. What new things did the authors actually get as a result of their research?
Paying tribute to the research carried out, I would like to see greater argumentation of the conclusions and more clearly defined tasks and purpose of the work.
Author Response
Reviewer 2
Dear authors,
thank you for your work presented for publication. The presented work “Vertical distribution of Culicoides biting midges in temperate forests” is devoted to the study of the distribution structure of midges in the forest. Despite the obvious practical importance of studying any features associated with bloodsuckers, the presented work leaves a feeling of insufficient thought of both the objectives of the study itself and the conclusions obtained. When reading the materials, a number of questions arose, some of which are given below.
Why was this particular method of catching, using UV lamps, chosen to count midges? Why was the standard method of mowing with a net not used, although it shows the greatest efficiency?
We used BG-Pro traps which were designed for collecting Culicoides. This information was added to the manuscript (L 72-73, 87). This type of traps was used in our previous research of biting midges, and it worked effectively (see https://doi.org/10.3390/insects15030157 , https://doi.org/10.3390/d15050692 , https://doi.org/10.3390/microorganisms10050898). During investigation of Culicoides as vectors of bird parasites we were not sure about the best height of hanging traps. The main aim of this investigation was to get information about the best heights of collecting biting midges for research of avian blood parasites. We corrected introduction with more emphasis on this objective.
We did not use entomological net as we believe that traps are better for comparing the results and more biting midges can be collected using traps during the night. Unfortunately we expected to collect more biting midges when we collected, but we believe that some differences between results obtained at different heights can be seen based on our data.
In chapter «3.1. Insect distribution across vertical strata in the forest 101
In total, 11277 insects have been collected. The most abundant insects were dipterous, 102 followed by Lepidoptera (Fig. 2). Among dipterous insects, the most abundant were 103 Cecidomyidae (45.7%), Chironomidae (22.3%), Ceratopogonidae (15.7%), and 104 Psychodidae (10.3%). Only 6.0% of the collected dipterous insects belonged to families 105 Sciaridae, Culicidae, Mycetophilidae, and to other families (Fig. 2): Figure 2. The part of collected insects from different orders (A) and the most abundantly 109 collected families of dipterous insects (B)» the variety of insects caught in traps is shown: from butterflies to fungus gnats, without giving the names of the caught species. Why is this in an article devoted to the study of the vertical distribution of midges?
We wanted to point out that BG-Pro traps can be used not only for the collecting of biting midges, but also for the investigations of other insect groups. We changed the text and made it more succinctly, we removed it to the end of Results (L 229-238), we deleted Figure 2. We wanted to show that Chironomidae, Cecidomyidae and Psychodidae can be also traped using BG-Pro traps and the abundances of Cecidomyidae and Psychodidae likely are height dependent. Identification of Chironomidae and Cecidomyidae species needs a experienced specialist and this can be separate investigation not related to the vertical distribution of biting midges.
It is known that midges successfully copulate in swarms that gather in open space near poles, a river bank, a bush, etc. It is also known that only female midges feed on blood for the successful maturation of eggs. Why should males be present in the forest near the nests of their hosts if they cannot wait for food or copulation there? (See Figure 3. The average numbers (± SE) of Culicoides males and females collected at different heights in June, July and August. G – ground level (~1.5 m above the ground), M – mid-canopy (~13 m above the ground), H – high - canopy (~ 23 m above the ground).)
Thank You for pointing this. It is known that adult Culicoides are most abundant near breeding sites, but they disperse to mate and to feed. Some species can mate without swarming. Culicoides males are present in forests.
Sometimes it is very useful to catch males, because their identification is easier and more accurate, especially when we study morphologically close species (for example C. obsoletus and C. scoticus). We agree that usually more females when males are collected. For example only 1.4% of males was collected in https://doi.org/10.1186/1756-3305-7-34, only 2.2% of males was collected in https://doi.org/10.1016/j.actatropica.2022.106588, no male individual of some Culicoides species have been collected in https://doi.org/10.1186/s13071-018-3080-5 . It is known that adult males are attracted to and captured more frequently using UV traps, which is important for species that can only be identified based on male genitalia (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604845/). That is why we wanted to know when and where it would be better to set up traps in order to get more males. We think that our results will give new knowledge on where and when to catch males best. We supplemented the discussion with this point (L 338-341).
Finally, the conclusions are based on the fact that: «Summarizing the results of our research, we can state that the abundance of Culicoides was higher in the tree canopy compared with the ground level in temperate forest». But the authors at the very beginning stated that the traps “many species of biting midges are more prevalent in tree canopy close to bird nests.” Don't the authors think they have proven an obvious conclusion? The small number of males is also quite understandable without research. What new things did the authors actually get as a result of their research?
We cited two new publications (one about blackflies infected with Leucocytozoon parasites (Chakarov et al., 2020) and one about Culicoides biting midges (Kazak et al., 2023) collected nearby bird nests in tree canopy) and work of Braverman et al., 1993 in which it was determined that C. circumscriptus Kieffer and C. cataneii Clastrier showed a significant preference in height. Our traps were set up not close to the bird nests and this is the difference. To our knowledge there are not many studies investigating Culicoides abundance at big heights such as 22 meters in the forest in Europe. Hanging a trap at such a height requires efforts and skills. We found new preferences of some Culicoides species to live in canopy, such as C. festivipennis, C. punctatus, or the preference to live at ground level (C. kibunensis) (Braverman et al., 1993 investigation was made in Israel, Henry et al., 1975 worked in USA).
We detected a big number of males (almost half of all collected Culicoides at the medium height in July and August) and this information is useful for those searching to collect males.
Paying tribute to the research carried out, I would like to see greater argumentation of the conclusions and more clearly defined tasks and purpose of the work.
We corrected Introduction (L 68-74), Discussion (L 242-246) and Conclusion to make it clearer.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript examines the distribution of Culicoides biting midges among three different heights in temperate forests. Whilst the subject area remains underexplored and efforts to elucidate this distribution should be applauded, the dataset presented here is small allowing limited conclusions to be drawn. It reads as a preliminary study/proof of concept which should lead on to more intensive trapping to validate the observations. There are also some concerns in the methodology. For these reasons, I would recommend rejecting the manuscript in its current state but would encourage the authors to address the concerns here and resubmit as a short communication or letter.
Abstract
The authors state the C. festivipennis and C. punctatus were the most common species found. Their results show that C. pictipennis and C. festivipennis were the most common species.
Introduction
Line 50-51 state that many species of biting midges are more prevalent in the tree canopy close to bird nests. This makes it sound like the midges are ‘localised’ around nests. The referenced studies were just examining traps in the canopy and do not mention proximity to bird nests. Please remove the “close to bird nests” from this sentence.
Materials and methods
Lines 83-84: “In total insects were collected 9 nights of 3 each month from June to August.” This implies that insects were collected on 3 nights each month for a total of 9 nights. However, earlier in the section it is stated that insects were collected using three traps once a month in three forest areas. Could you clarify the exact trapping schedule and include the total number of trap nights? Were the three forest areas all trapped on the same night each month? How were the nights chosen for trapping (weather conditions etc)?
Results
There is too much detail on the bycatch that was caught during the study period. The manuscript is about the vertical distribution of Culicoides in temperate forests. The percentage of bycatch that belongs to other families along with details of the mosquitoes caught (section 3.2) does not add to the paper in a beneficial way, especially as one point of this is then only briefly touched upon at the end of the discussion, which feels like an afterthought.
It would be useful to include a table detailing the total number of each Culicoides species caught at each height, perhaps even stating the numbers of each which were parous, nulliparous etc.
Lines 151-154 detail the number of Forcipomyia caught. This is not relevant to the results section that it is included in, or indeed the subject of the manuscript.
Section 3.5 (lines 155-165) splits the Culicoides catch into males and parous and nulliparous females. However, there is no mention of bloodfed or gravid females throughout the results or manuscript. Were none caught in the traps? If so, this should be specified. If no gravid or bloodfed midges were caught, then this is an important observation, and it should be discussed.
Discussion
The main sections in the results describe the differences in parity status/sex between the three trapping heights but there is very little discussion around this, only taking up a paragraph of 10 lines towards the end of the discussion. The authors do not discuss possible hypotheses behind the differences or lack of, only compare them to results of two other studies.
The results also show a trend in the seasonal distribution of at least one of the species caught (albeit from a relatively small number of midges) which may provide important information for future studies. The authors describe this briefly in the results but do not draw further attention to this.
Line 217-219. The sentence regarding Culicoides being vectors of bluetongue virus etc belongs in the Introduction. It doesn’t continue on to make any point about Culicoides distribution and how this impacts upon the livestock viruses listed or how it ties in with the results of this study.
Line 238. The statement regarding the abundance of C. obsoletus group at different heights should be caveated that a low number of this group were caught making it difficult to draw firm conclusions.
Line 260-263. The sentence “Our data did not confirm Culicoides, which primarily feed on large mammals, would be…” is confusing. The authors hypothesise that species which feed on large mammals would be more abundant close to the ground but end by saying that C. kibunensis is known to feed on birds. The abundance of other mammal-feeding species in ground level traps is not discussed. It would be interesting for the authors to include information (if it is known) about the host mammal species which inhabit the forests as this may have influenced their results. If there are no significant populations of large mammals, then it may be expected that Culicoides species which prefer mammalian hosts are less abundant.
Line 265. There are missing words (presumably ‘height at’) in “between the (missing word here) which blood-engorged midges…”
Lines 263-266 state that the results are “in accordance with McGregor et al” regarding the association between where blood-engorged midges were collected and the class of vertebrate they had been feeding upon. The referenced study by McGregor et al examined midges which contained a bloodmeal and performed molecular analysis on them to determine the host thereby allowing the association to be determined. However, the present study does not make any mention of bloodfed midges that were caught or any bloodmeal analysis that was carried out so a conclusion like this cannot be made.
Lines 276-280 discuss the presence of other midges (one family of which is not known as a vector of avian parasites) and therefore outside the scope of this paper. It should be removed.
Comments on the Quality of English LanguageLine 35 Consider changing "...midges are investigated" to "...midges have been investigated"
Line 102 Consider changing "...insects have been collected" to "...insects were collected"
Line 159-160 Consider changing "...females have been determined" to "...females were determined"
Line 163 Consider changing "...differences have been detected" to "...differences were detected"
Line 196 Consider changing to "...biting midges are found not only at the ground level"
Line 214-216 Consider changing to "In the United States, field studies showed that two species of vector mosquitoes in canopy-level traps were much more likely to be infected with West Nile Virus than those in ground-level traps."
Author Response
Reviewer 3
This manuscript examines the distribution of Culicoides biting midges among three different heights in temperate forests. Whilst the subject area remains underexplored and efforts to elucidate this distribution should be applauded, the dataset presented here is small allowing limited conclusions to be drawn. It reads as a preliminary study/proof of concept which should lead on to more intensive trapping to validate the observations. There are also some concerns in the methodology. For these reasons, I would recommend rejecting the manuscript in its current state but would encourage the authors to address the concerns here and resubmit as a short communication or letter.
Thank You for Your comments and suggestions. We are resubmitting the revised manuscript as a communication.
Abstract
The authors state the C. festivipennis and C. punctatus were the most common species found. Their results show that C. pictipennis and C. festivipennis were the most common species.
Corrected (L 18-22).
Introduction
Line 50-51 state that many species of biting midges are more prevalent in the tree canopy close to bird nests. This makes it sound like the midges are ‘localised’ around nests. The referenced studies were just examining traps in the canopy and do not mention proximity to bird nests. Please remove the “close to bird nests” from this sentence.
Corrected (L 60).
Materials and methods
Lines 83-84: “In total insects were collected 9 nights of 3 each month from June to August.” This implies that insects were collected on 3 nights each month for a total of 9 nights. However, earlier in the section it is stated that insects were collected using three traps once a month in three forest areas. Could you clarify the exact trapping schedule and include the total number of trap nights? Were the three forest areas all trapped on the same night each month? How were the nights chosen for trapping (weather conditions etc)?
We corrected sentences making the text clearer. We also provided data as supplemented materials providing exact collecting dates in each locality. We collected biting midges in each study site once a month in June, July and August 2021 i.e. 9 trap nights. All three forest areas were trapped at different nights (we set up all three traps at the same time each trap night). We also made some notes on weather conditions. We choose nights without rain with low wind speed and collected biting midges from June to August as this is the time of the highest activity of Culicoides at our study area (based on https://doi.org/10.1007/s00436-021-07147-2 ). (L 90-95, 107-111).
Results
There is too much detail on the bycatch that was caught during the study period. The manuscript is about the vertical distribution of Culicoides in temperate forests. The percentage of bycatch that belongs to other families along with details of the mosquitoes caught (section 3.2) does not add to the paper in a beneficial way, especially as one point of this is then only briefly touched upon at the end of the discussion, which feels like an afterthought.
We changed the text and made it more succinctly, we mooved this part to the end of Results, we also deleted Figure 2. When writing about other insects, we wanted to emphasize that when studying biting midges, it is possible to successfully study other groups of insects and we determined which groups they belong to: Chironomidae, Cecidomyidae and Psychodidae.
It would be useful to include a table detailing the total number of each Culicoides species caught at each height, perhaps even stating the numbers of each which were parous, nulliparous etc.
This information was provided as supplemented material.
Lines 151-154 detail the number of Forcipomyia caught. This is not relevant to the results section that it is included in, or indeed the subject of the manuscript.
We have shorted this sentence a bit, but we would like to leave this information as Forcipomyia belong to the same family as Culicoides do, and this information can be interesting to research of these insects.
Section 3.5 (lines 155-165) splits the Culicoides catch into males and parous and nulliparous females. However, there is no mention of bloodfed or gravid females throughout the results or manuscript. Were none caught in the traps? If so, this should be specified. If no gravid or bloodfed midges were caught, then this is an important observation, and it should be discussed.
We have not investigated gravid females. The finding of parous females is important for parasitological investigations as only these females can harbor some blood pathogens transmitted by bloodsucking insects. We will address investigation of gravid females in future research as this is a part of insect biology. We did not detect blood-fed midges during this investigation and we added this information (L 199).
Discussion
The main sections in the results describe the differences in parity status/sex between the three trapping heights but there is very little discussion around this, only taking up a paragraph of 10 lines towards the end of the discussion. The authors do not discuss possible hypotheses behind the differences or lack of, only compare them to results of two other studies.
Thank You for Your comments. We supplemented the discussion (L 338-341).
The results also show a trend in the seasonal distribution of at least one of the species caught (albeit from a relatively small number of midges) which may provide important information for future studies. The authors describe this briefly in the results but do not draw further attention to this.
This information was added (L 251-254).
Line 217-219. The sentence regarding Culicoides being vectors of bluetongue virus etc belongs in the Introduction. It doesn’t continue on to make any point about Culicoides distribution and how this impacts upon the livestock viruses listed or how it ties in with the results of this study.
Corrected, the sentence was moved to the Introduction (L 31-34).
Line 238. The statement regarding the abundance of C. obsoletus group at different heights should be caveated that a low number of this group were caught making it difficult to draw firm conclusions.
Corrected (L 290-292).
Line 260-263. The sentence “Our data did not confirm Culicoides, which primarily feed on large mammals, would be…” is confusing. The authors hypothesise that species which feed on large mammals would be more abundant close to the ground but end by saying that C. kibunensis is known to feed on birds. The abundance of other mammal-feeding species in ground level traps is not discussed. It would be interesting for the authors to include information (if it is known) about the host mammal species which inhabit the forests as this may have influenced their results. If there are no significant populations of large mammals, then it may be expected that Culicoides species which prefer mammalian hosts are less abundant.
We decided not to discus the feeding preference of Culicoides on large mammals, as we have more information about ornithophilic Culicoides species. We think that Culicoides vertical distribution is not related to the host preferences of biting midge to the class level (birds – mammals), as birds can live both in big heights and on the ground level depending on a bird species.
Line 265. There are missing words (presumably ‘height at’) in “between the (missing word here) which blood-engorged midges…”
Corrected.
Lines 263-266 state that the results are “in accordance with McGregor et al” regarding the association between where blood-engorged midges were collected and the class of vertebrate they had been feeding upon. The referenced study by McGregor et al examined midges which contained a bloodmeal and performed molecular analysis on them to determine the host thereby allowing the association to be determined. However, the present study does not make any mention of bloodfed midges that were caught or any bloodmeal analysis that was carried out so a conclusion like this cannot be made.
Thank You for this, corrected (L 320-323).
Lines 276-280 discuss the presence of other midges (one family of which is not known as a vector of avian parasites) and therefore outside the scope of this paper. It should be removed.
Deleted.
Comments on the Quality of English Language
Line 35 Consider changing "...midges are investigated" to "...midges have been investigated"
Corrected.
Line 102 Consider changing "...insects have been collected" to "...insects were collected"
Corrected.
Line 159-160 Consider changing "...females have been determined" to "...females were determined"
Corrected
Line 163 Consider changing "...differences have been detected" to "...differences were detected"
Corrected
Line 196 Consider changing to "...biting midges are found not only at the ground level"
Corrected
Line 214-216 Consider changing to "In the United States, field studies showed that two species of vector mosquitoes in canopy-level traps were much more likely to be infected with West Nile
Corrected
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI thank the authors for addressing my comments and concerns in their revision of manuscript.
Author Response
Thank You very much for all Your comments.
Reviewer 3 Report
Comments and Suggestions for AuthorsAlthough the authors have made revisions to the manuscript, I feel that these have raised more questions about the methodology.
The inclusion of temperature from the trapping nights shows a wide range between locations during the same month (11°C difference in June, ~6°C difference in July and August). Are the temperatures representative of average daily temperatures for that time of year? These may have influenced the catches and only trapping once per month at each location may mean that local phenomena such as the temperature influenced the catch (For example, the lowest temperature of 10.9 in August resulted in a trap catch of 7). This would have biased results which can’t be evened out over time, as would be the case if a more intensive trapping schedule was used. There is no mention of these limitations in the discussion.
The authors state that they have not investigated gravid females in their response to my previous comment but do not include this in the manuscript. This would be of interest to the reader as one may wonder if Culicoides are using different heights during different parts of their life cycle. Counting the number of gravid females present would have been a simple process during the sorting of midges into parous/nulliparous and added valuable information to the manuscript.
Line 189-190 states n=74 for Culicoides kibunensis but table S1 shows n=65. It also states this species was only caught in June and July but table S1 shows that n=2 were caught in one locality in August.
Line 220-221 states C. pictipennis was only caught in June but table S1 and Figure 5 show this species was also caught in July at all heights.
Comments on the Quality of English LanguageLine 52 Change "to be able to have" to "may have"
Line 136 Change "males have been caught" to "males were caught"
Line 276 Change "can not be related" to "may not be related"
Author Response
Although the authors have made revisions to the manuscript, I feel that these have raised more questions about the methodology.
The inclusion of temperature from the trapping nights shows a wide range between locations during the same month (11°C difference in June, ~6°C difference in July and August). Are the temperatures representative of average daily temperatures for that time of year? These may have influenced the catches and only trapping once per month at each location may mean that local phenomena such as the temperature influenced the catch (For example, the lowest temperature of 10.9 in August resulted in a trap catch of 7). This would have biased results which can’t be evened out over time, as would be the case if a more intensive trapping schedule was used. There is no mention of these limitations in the discussion.
We provided information on meteorological conditions based on the data obtained from the Meteorological station of Lithuania, Utena department, which was the closest to all three study sites. We collected biting midges at the time (June – August) when the activity of Culicoides in our country is the highest.
We agree that the activity of biting midges is temperature dependent as it was noted in our previous (https://doi.org/10.1007/s00436-021-07147-2) and other publications (https://doi.org/10.1186/s13071-023-05920-z). During this investigation we did not find correlation between air temperature and abundance of Culicoides (r = -0.036) and this can be related to the range of temperature favorable for the activity of biting midges
Differences in temperatures are more related not to the locality, but to the collection date as air temperature differed day by day. During the research year, the average air temperature was different in different months: in June and July it was higher than the long-term average (19,2 oC and 22,1 oC respectively) and in August it was lower than long-term average (16,1 oC). July is usually the warmest month of the year in the country. Despite the fact that a positive relationship can be detected between air temperature and the total abundance of biting midges, although the highest abundances of biting midges in temperate zone is observed in June (https://doi.org/10.1007/s00436-021-07147-2). The same regularity was also observed during other studies (https://doi.org/10.3390/insects14120926) from the same country.
We agree that we would collect more biting midges if we would use a more intensive trapping schedule. In order to avoid the influence of temperature to our results we compare relative abundances of collected biting midges as all three traps in all three heights were used the same day.
We added this information to the discussion (L 238-253)
The authors state that they have not investigated gravid females in their response to my previous comment but do not include this in the manuscript. This would be of interest to the reader as one may wonder if Culicoides are using different heights during different parts of their life cycle. Counting the number of gravid females present would have been a simple process during the sorting of midges into parous/nulliparous and added valuable information to the manuscript.
Thank You for this comment, based on it we calculated number of gravid females and added these results to the manuscript (L 172-182). We hope that this will improve the manuscript. We also supplemented the discussion (320-330) and hope to investigate this issue deeper in the future.
Line 189-190 states n=74 for Culicoides kibunensis but table S1 shows n=65. It also states this species was only caught in June and July but table S1 shows that n=2 were caught in one locality in August.
Thank You, this was corrected (L 201-202).
Line 220-221 states C. pictipennis was only caught in June but table S1 and Figure 5 show this species was also caught in July at all heights.
Thank You, this was corrected (instead of “only” we wrote “mostly”.
Comments on the Quality of English Language
Line 52 Change "to be able to have" to "may have"
Corrected.
Line 136 Change "males have been caught" to "males were caught"
Corrected.
Line 276 Change "can not be related" to "may not be related"
Corrected.
