Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state of Minas Gerais, Brazil.

Caves are extreme and inhospitable environments that can harbor several species of vertebrate and invertebrate animals. Among these animals are phlebotomine sand flies, vectors of parasites of the genus Leishmania that cause leishmaniasis. This study aimed to evaluate the species composition of sand flies of four caves, a cave located at Moeda Sul (MS) and three at Parque Estadual Serra do Rola Moça (PESRM), in the region of the Quadrilátero Ferrífero in the state of Minas Gerais, southeastern Brazil. Sand flies were collected with automatic light traps. Non-Metric Multidimensional Scaling, using a dissimilarity matrix calculated with the Jaccard index, and Multivariate Permutation Analysis were used to evaluate sand fly species composition among entrance, interior, and the surrounding environments of each sampled cave and to infer biological mechanisms from patterns of distribution among these different cave environments. A total of 375 phlebotomine sand flies representing 14 species and six genera were collected. The most abundant species were Evandromyia tupynambai (54.7%), Brumptomyia troglodytes (25.6%), Evandromyia edwardsi (6.1%), Psathyromyia brasiliensis (4.8%) and Lutzomyia longipalpis (4.3%). Thirty individuals were collected at MS, 16 inside the cave and 14 from its surroundings. At PESRM, five individuals were collected from the surroundings of cave RM38, 190 individuals from cave RM39 (48 in the cave and 142 from its surroundings) and 150 individuals from cave RM40 (42 in the cave and 108 from its surroundings). The results revealed a rich sand fly fauna with similar species compositions among the entrance, interior, and surrounding environments of each sampled cave, suggesting that both caves and their surroundings are important for maintaining sand fly communities.

Introduction

Phlebotomines sand flies are insects of the family Psychodidae, subfamily Phlebotominae (Diptera). They are invertebrate hosts for protozoan parasites of the genus Leishmania, which cause leishmaniases in humans and other mammals. These insects can be found in urban, rural and wild environments, including caves [1-4].

Cave environments, though seemingly inhospitable due to the lack of light in areas furthest from the cave entrance and the low availability and variety of resources [5, 6], can harbor many species of vertebrates and invertebrates, including phlebotomine sand flies [7–13, 4].

Studies conducted inside caves have produced interesting findings, such as greater, minor or equal diversity of sand flies inside caves compared to their surrounding environments, and differences in the species composition of sand fly communities of caves and their surrounding areas, with species unique to each environment and some common to both [14, 15, 11]. Some species have been described in caves [16, 17, 8, 10], and differences in activity periods and diversity among several caves of the same region have been reported [18, 19, 20]. Studying five caves and their surroundings over two seasons of the year, Campos et al. [4] showed that sand flies exhibit differences in the rhythm of their activity among the interior, entrance and surrounding areas of caves due to differences in the presence of sunlight, which suppresses the flight activity of these mainly nocturnal insects. Despite all these investigations, however, little is known about the distribution patterns of sand flies within caves and in their surroundings even though species composition is known to differ.

Sand fly communities structure between caves and their surroundings have systematically been compared by few studies, namely Galati et al. [14, 15] and Carvalho et al. [11]. Other studies were restricted to caves, either evaluating few months of the year or recording new species. Therefore, no ecological studies have been carried out in ferruginous environments evaluating the environmental gradient of caves and its surrounding phlebotomine communities.

The present study aimed to use statistical analysis to evaluate similarity on sand fly species composition based on species presence or absence along an environmental gradient. This type of analysis is particularly well suited to detect whether the local biological communities are structured by ecological variables or environmental gradients. Dissimilarity measures have a long-standing history and have been widely used to evaluate different biotas distribution [21-23].

Thus, the study evaluated the similarity in species composition of sand flies among the entrance, interior, and surrounding environments of caves at two different locations in the region of the Quadrilátero Ferrífero, state of Minas Gerais. The use of dissimilarity indexes is important for inferring biological mechanisms from sand fly distribution.

Materials and methods

Study area

The present study investigated caves and their surroundings at two different locations in the state of Minas Gerais, Brazil: Moeda Sul (MS) and Parque Estadual Serra do Rola Moça (PESRM) (Fig 1). These locations are within the region of the Quadrilátero Ferrífero (Iron Quadrangle) of Minas Gerais, a region with a great number of recorded caves, intense mining activity, real estate exploitation and urbanization [24, 25]. The region possesses abundant ferruginous fields called “cangas”, a rare geological formation found only in the Quadrilátero Ferrífero of Minas Gerais and in Carajás in the state of Pará [26, 27], as well as high altitude fields beyond areas of “capoeira” (secondary formations) [27, 28].

Fig 1

Location of the study area in Moeda Sul (MS) and Parque Estadual Serra do Rola Moça (PESRM), municipality of Nova Lima, MG.

The study focused on four caves and their surroundings, all named by Centro Nacional de Pesquisa e Conservação de Cavernas (CECAV): cave MS at MS, and caves RM38, RM39 and RM40 at PESRM (Figs 2–5 and Table 1). The caves at PESRM are located about 500 m from each other and about 20 km from cave MS. Caves RM39 and RM40 do not have an aphotic zone, whereas caves MS and RM38 have aphotic zone that reaches about 5 m into the interior from the entrance.

Fig 2

Cave MS and its surroundings in Moeda Sul (MS), Minas Gerais, Brazil.

Fig 5

Cave RM40 and its surroundings in Parque Estadual Serra do Rola Moça (PESRM), Minas Gerais, Brazil.

Table 1

Description of the studied caves and their surroundings at Moeda Sul (MS) and Parque Estadual Serra do Rola Moça (PESRM), municipality of Nova Lima, MG, in the region of the Quadrilátero Ferrífero.

Caves were sampled from February 2014 to March 2015.

Description of caves
Location	Name	Coordinates	Elevation (m a.s.l)	Horizontal extension (m)
MS	MS	20°12'7"S, 43°58'2"W	1,440	70
PESRM	RM38	20°00'48"S, 43°58'43"W	1,363	50
	RM39	20°00'56"S, 43°58'38"W	1,272	30
	RM40	20°00'59"S, 43°58'40"W	1,264	20

All caves located at MS and PESRM are “canga” caves with no running water. They are located in the municipality of Nova Lima in a transition zone between the Cerrado and Atlantic Forest biomes. Cave MS and its surroundings are located in a region of intense exploitation for real estate and urbanization [25], while the caves and their surroundings of PESRM are located in a largely conserved forest area.

Collection of sand flies

Sand flies were collected under license of Ministério do Meio Ambiente do Brasil (Nº 45636–1) and Instituto Estadual de Florestas de Minas Gerais (Nº 082/2014) (S1 and S2 Files).

Systematic collections were performed using HP-model automatic light traps [29], throughout an entire year: April 2014 to March 2015 at MS, and February 2014 to January 2015 at PESRM. Sampling took place monthly for three consecutive days for a total of 72 h of sampling effort per trap per month.

At MS, four traps were installed in cave MS and eight in its surroundings for a total of 12 traps. At PESRM, three traps were placed in cave RM38 and six traps in its surroundings for a total of nine traps, and two traps were placed in caves RM39 and RM40 and four in their surroundings for a total of six traps each. Thus, a total of 33 traps were used with 11 traps inside of caves and 22 in cave surroundings. For all the caves, the first interior trap was set at the entrance and the others every 10 m into the cave. The first trap in the surroundings was set 10 m from the cave entrance in the forest and the others every 10 m further. All traps were installed approximately 1 m above the ground.

The number of traps installed inside a given cave was the maximum numbered possible based the aforementioned spacing, the horizontal extension of the cave and the difficulty of moving inside. Thus, for a better sampling of the sand fly fauna of the forest fragments surrounding these caves twice the number of interior traps was placed in the surroundings of each cave.

After sampling, the traps were removed and the sand flies killed in glycerinated alcohol, sorted and sexed. Males and females were then placed in test tubes containing 70% alcohol.

Sand fly identification

Male sand flies were prepared and mounted in Canada Balsam. Female sand flies had their head, thorax and abdomen separated and mounted in Berlese medium, with the head positioned ventrally. All sand flies were identified following the key and classification of Galati [30]. Specimens will be deposited in the Coleção de Flebotomíneos of the Centro de Pesquisas René Rachou/Fiocruz (FIOCRUZ/COLFLEB).

Statistical analysis

Similarity of species composition among the entrance, interior, and surrounding environments of each sampled cave (caves MS, RM39 and RM40) was determined using Non-Metric Multidimensional Scaling (NMDS) based on a dissimilarity matrix calculated with the Jaccard index (species presence or absence). This method is considered useful for identifying patterns in ecological data. Non-parametric PERMANOVA (Multivariate Permutation Analysis) with 999 replications across the distance matrices [31] was used to test if species composition differed among the environments (entrance, interior, and surroundings) of each sampled cave (MS cave, RM39 cave and RM40 cave). All analyses were performed using R 3.2.4 software [32] (Fig 2).

Analyses by NMDS and Non-parametric PERMANOVA considered one sampling point at the entrance, one in the interior and one in the surroundings of each studied cave. No phlebotomine sand flies were caught inside cave RM38, and so it was removed from the NMDS analysis.

Results

A total of 375 phlebotomine sand flies of 14 species and six genera were collected. The number of specimens and species in all environments (cave and surroundings) are shown in Table 2. The most abundant species was Evandromyia tupynambai (54.7%), followed by Brumptomyia troglodytes (25.6%), Evandromyia edwardsi (6.1%), Psathyromyia brasiliensis (4.8%) and Lutzomyia longipalpis (4.3%). All other species accounted for 4.5% of the total (Table 2).

Table 2

Phlebotomine sand flies collected in caves and in their respective surroundings in Moeda Sul (MS) and Parque Estadual Serra do Rola Moça (PESRM), municipality of Nova Lima, MG, from February 2014 to March 2015.

“S” after the cave name indicates samples collected from cave surroundings; “CAV” indicates samples collected from within the cave.

Species Study locations	Moeda Sul (MS)			Parque Estadual Serra do Rola Moça (PESRM)							TG (%)
Collection environments	MS_S	MS_CAV	TL	RM38_S	RM38_CAV	RM39_S	RM39_CAV	RM40_S	RM40_CAV	TL
Individuals/environments	TE (%)	TE (%)		TE (%)	TE (%)	TE (%)	TE (%)	TE (%)	TE (%)
Brumptomyia troglodytes	-	-	-	4 (80.0)	-	39 (81.2)	9 (6.3)	36 (85.7)	8 (7.4)	96	96 (25.6)
Evandromyia edwardsi	1 (6.2)	-	1	-	-	1 (2.1)	21 (14.8)	-	-	22	23 (6.1)
Evandromyia evandroi	-	1 (7.1)	1	-	-	-	-	-	-	-	1 (0.3)
Evandromyia termitophila	-	1 (7.1)	1	-	-	-	-	-	-	-	1 (0.3)
Evandromyia tupynambai	1 (6.2)	-	1	-	-	3 (6.3)	108 (76.1)	-	93 (86.1)	204	205 (54.7)
Lutzomyia ischyracantha	1 (6.2)	-	1	-	-	-	-	-	-	-	1 (0.3)
Lutzomyia longipalpis	1 (6.2)	5 (35.7)	6	-	-	-	2 (1.4)	1 (2.4)	7 (6.5)	10	16 (4.3)
Lutzomyia renei	-	-	-	1 (20.0)	-	-	-	-	-	1	1 (0.3)
Nyssomyia whitmani	-	-	-	-	-	-	-	1 (2.4)	-	1	1 (0.3)
Psathyromyia brasiliensis	11 (69.0)	7 (50.0)	18	-	-	-	-	-	-	-	18 (4.8)
Psathyromyia limai	-	-	-	-	-	1 (2.1)	-	-	-	1	1 (0.3)
Pintomyia fischeri	-	-	-	-	-	-	-	1 (2.4)	-	1	1 (0.3)
Pintomyia misionensis	1 (6.2)	-	1	-	-	-	-	-	-	-	1 (0.3)
Pintomyia monticola	-	-	-	-	-	4 (8.3)	2 (1.4)	3 (7.1)	-	9	9 (2.4)
Individuals/environments	16 (100.0)	14 (100.0)	30	5 (100.0)	-	48 (100.0)	142 (100.0)	42 (100.0)	108 (100.0)	345	375 (100.0)
Total species	6	4	8	2	0	5	5	5	3	9	14

Environments: MS_S and MS_CAV = Moeda Sul surroundings and cave; RM38_S and RM38_CAV = Rola Moça 38 surroundings and cave; RM39_S and RM39_CAV = Rola Moça 39 surroundings and cave, RM40_S and RM40_CAV = Rola Moça 40 surroundings and cave. TE, TL and TG = total sandflies per environment, location and general.

Thirty individuals were collected at cave MS, with 16 coming from inside the cave and 14 from its surroundings. The most abundant species in cave MS was Pa. brasiliensis (50%) followed by Lu. longipalpis (35.7%), whereas the most abundant species in the surroundings was Pa. brasiliensis (69%). Only five individuals, belonging to Br. troglodytes (80.0%) and Lutzomyia renei (20.0%), were collected from the surroundings of cave RM38 (none from inside the cave). In Cave RM39, a total of 190 individuals were collected: 48 inside and 142 outside. Ev. tupynambai (76.1%) was the most abundant species inside whereas Br. troglodytes (81.2%) was the dominant species outside. A total of 150 individuals were collected at cave RM40, with 42 coming from inside the cave and 108 in its surroundings, with Ev. tupynambai (86.1%) being the most abundant species inside the cave and Br. troglodytes (85.7%) the most abundant in its surroundings (Table 2).

Non-Metric Multidimensional Scaling (NMDS), calculated with species presence or absence data, explained 80% of the variation in the data on two axes (Fig 6). The permutation test revealed that sand fly species composition did not differ among entrance, interior, and surroundings of each of the sampled caves (caves MS, RM39 and RM40) (PERMANOVA: F1 = 0.49; R = 0.06; P = 0.87).

Fig 6

Non-Metric Multidimensional Scaling (NMDS) using a Jaccard dissimilarity matrix calculated with species presence or absence data among the entrance, interior, and surroundings of each sampled cave (caves MS, RM39 and RM40).

Discussion

The results of the present study showed that sand fly species composition did not differ among the entrance, interior, and surroundings of each sampled cave of the locations MS and PESRM, indicating that many species appear to use the caves as places for rest, protection, shelter, breeding, and feeding, as reported by Carvalho et al. [11]. According to Galati et al. [20], sand flies commonly use cave environments for shelter or as breeding sites, or as preferred habitat. The latter was also observed by Alves et al. [18], who recorded the presence of Deanemyia maruaga, a species restricted to the interior of caves, in the municipality of Manaus, state of Amazonas. In this study also was observed a low number of sand flies collected, which may be explained by the remarkable severity of the external environment in ferruginous systems [33].

The phlebotomine sand fly fauna inside and outside of the studied caves was found to be very diverse, with some species restricted to caves, others restricted to cave surroundings and others common to both environments. Cave environments with rich sand fly faunas have been reported previously by Carvalho et al. [11] for caves in the municipality of Lassance, Minas Gerais; Campos et al. [4] in the municipality of Pains, Minas Gerais; and Galati et al. [14, 15] for caves in the state of São Paulo.

The most abundant species in our study—Br. troglodytes, Ev. edwardsi and Lu. longipalpis—are commonly found both inside and outside of caves [20, 14, 15, 19, 11, 4]. Other abundant species were Pa. braziliensis and Ev. tupynambai, which have few and no records for caves, respectively.

The fact that the caves and their surroundings of PESRM are in a conserved forest fragment can explain the occurrence of Ev. tupynambai and Br. trogodytes as the most abundant species inside and outside of the caves, respectively, at that location. These species are very common in conserved forest environments, although they have also been found in shelters for domestic animals, marginal areas and human habitations [34, 14, 35]. Finding E. tupynambai to be the most abundant species inside the studied caves was a surprise since, despite its wide distribution, there are no records in the literature of this species occurring in cave environments, although a very closely related species, E. petropolitana, was reported from a stone slot [36]. On the other hand, the finding of Br. trogodytes as the most abundant species in cave surroundings was expected since it is a characteristic species of forest areas [35].

The cave and its surroundings at MS are situated in a very anthropized region surrounded by a residential area in a periurban environment and a Brazilian federal highway. It was not surprising, therefore, to find the most abundant species to be Pa. brasiliensis and Lu. longipalpis since they are very common species in areas of secondary forest, urban areas and domestic animal shelters [37, 3, 4]. There is no record in the literature of Pa. brasiliensis being the most abundant species inside a cave environment, as was found here, but it being the most abundant in cave surroundings is not unexpected since it is known to occur in anthropized areas [37]. Lutzomyia longipalpis was also one of the most abundant species inside cave MS, which corroborates Campos et al. [4], who found this species to be one of the most abundant species inside caves in another area of Minas Gerais.

The species incriminated in the transmission of leishmaniasis to human found in the present study—Lu. longipalpis and Nyssomyia whitmani—have been previously recorded from cave environments by other authors [20, 15, 11, 4]. The other species found are not implicated in transmission of Leishmania parasites to human.

The majority of the sand flies collected in the present study were trogloxenes and Ev. tupynambai and Ev. edwardsi, the two most abundant species. These two species can be considered troglophiles since they were recorded in almost every month of collection with more than 90% of the individuals occurring inside the cave. This result corroborates Carvalho et al. [11] who reported the presence of trogloxenes and troglophile phlebotomines.

An interesting finding of the present study was the lack of any record of sand flies at the entrance or in the interior of cave RM38 of PESRM, only in its surroundings. One possible explanation is the very small entrance of this cave in relation to the other caves of PESRM. Studying several caves in Serra da Bodoquena, state of Mato Grosso do Sul, Galati et al. [20] found three caves with low densities of sand flies, with the one with the highest density having a wide entrance, which may be more attractive to several species of trogloxenes.

In summary, a rich phlebotomine fauna was recorded for MS and PESRM, with similar sand fly communities among the entrance, interior, and surroundings of each sampled cave. Furthermore, species incriminated in Leishmania transmission were recorded, including the presence of Lu. L. longipalpis in the surroundings of cave MS. Even though this species occurred in low abundance at this cave, it deserves special attention because the cave is close to a residential area and it is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis [38]. Therefore, we conclude that both caves and their surroundings are important for the maintenance of sand fly communities. Considering the low frequencies of most species of sand flies and their collection in the surroundings, it seems that these insects are using caves as shelters and resting places for the species that reproduce in the surroundings, which would therefore classify them as trogloxenes. In addition, some species may be practicing hematophagia inside and outside of the caves—because bats and guano were observed inside the caves—and getting carbohydrate energy from around the caves, which would identify them as troglophiles.

In the present study, some limitations were found, such as: at the beginning of the project our idea was to measure humidity and temperature in caves versus their surroundings, however we had several problems with the devices, from thermometer breakage to loss (probable theft), so we did not measure the climate variables; the caves studied were difficult to access, which made a longer collection period impossible and last the Leishmania detection study was not performed in the studied sites, because we had methodological problems when performing sample extraction which made this part of the study impossible.

We believe that the limitations mentioned did not compromise the conclusions of study. The proposed purpose was to evaluate the spatial distribution of the species of sand flies at different cave environments and their surroundings, therefore, it does not depend necessarily of seasonality studies, environmental descriptors and molecular biology, although important, to obtain an appropriate and reliable conclusion. For this purpose, we consider the collection of sand flies in different sampled locations to be more appropriate than over a two-year period, since the composition of sand flies from one year to the next varies more in terms of abundance distribution than in terms of presence or absence. A study carried out in an urban environment in the state of Minas Gerais found a similarity in the occurrence of sand flies between collection years, but with variation only in the distribution of abundance of these species (unpublished data). Thus, we consider a collection year as a satisfactory time to assess the presence or absence of sand fly species at a given location.

The low number of sand flies collected is a characteristic of the studied region (Quadrilátero Ferrífero) and given that, we prefer not to add the sand fly number per night and traps, as it would be a very low number. The fact that it has few sand flies compared, for example, to a limestone cave [1, 4] should not be an impediment to studying it. It is important to point out that the low number of sand flies collected may be explained by the high altitude of the studied sites, environmental degradation due to anthropic activities in the study region and the environmental severity of ferruginous grasslands. Despite the low number of sand flies collected over the one-year period, the statistical analysis chosen for the data were adequate for drawing reliable conclusions, since it was based on presence or absence species data in the community (not species abundance), which was very diverse.

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27 Aug 2019

PONE-D-19-18993

Similarity of species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state Minas Gerais, Brasil

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Reviewer #1: General comment: Please ask for English revision by a native speaker, the quality of English is not sufficient.

Abstract:

Number of caves should be mentioned.

Introduction:

Page1: Reference 1 is obsolete and looks like a Brazilian book which is hardly available in other countries. Consider to replace it by some current review or leave this general statement without any reference.

Page2: I think that nine references cited for a single simple statement are too much. Is there any review to be referred instead?

15 references mentioned (numbers 2-16) deal with sand fly fauna in caves and this fact puts in doubt the following statement "However, despite all these investigation, little is known of the patterns of distribution of sand flies in caves..." Try to explain more clearly what is the novelty of your work.

Materials and Methods

Explain why different numbers of traps were used in different cages and what was the distance between traps within one cage. All these details may affect catches per trap.

Add which keys were used for morphological identification of sand flies.

Are there any males or rest of female bodies available in ethanol for eventual molecular studies (confirmation of species identification or population studies) in the future?

Results

Did you trap any males with noninverted genitalia or do you have any other evidence that caves serve as breeding sites for sand flies?

In Table 2, explain what is T. Do you mean "n"?

I am missing the information about sand fly "density", for example the sand fly number per night and trap.

Do you have any data about temperature and humidity in caves versus their surroundings?

Discussion

You are frequently using term "condominium". Do you mean village or single house?

I suggest to improve/change discussion why caves are important for "the maintenance of their sand fly communities". Could you specify if these caves are serving as resting sites, breeding sites, and/or what else?

Reviewer #2: The manuscript entitled “Similarity of species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state Minas Gerais, Brasil“ described an interesting study concerning the sand fly fauna in caves and its surroundings. Several major points need to be addressed before publishing this work, and therefore, this manuscript cannot be published in its actual form.

The authors reported a similarity of sand fly communities among the entrance, interior, and surroundings environments of each sampled cave at places, MS and PESRM. My major concern with this study is that only the analysis was based on a total of 375 sand flies caught during a whole year period. With such low number, no conclusion can be drawn. By using other traps, the authors may increase the number of sand flies needed for an objective statistical analysis. On the other hand, the authors should compare the similarity on a monthly basis and NOT during the whole period because it is well known that phenology differ among species. In addition, the authors should report Leishmania infection in both sites.

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9 Oct 2019

Dear editor,

Below are the responses to the reviewers' comments.

We are available for clarification.

Reviewer #1: General comment: Please ask for English revision by a native speaker, the quality of English is not sufficient.

Answer: The English was revision by a native speaker.

Abstract: Number of caves should be mentioned.

Answer: The number of caves was mentioned.

Introduction: Page1: Reference 1 is obsolete and looks like a Brazilian book which is hardly available in other countries. Consider to replace it by some current review or leave this general statement without any reference.

Answer: The reference 1 was taken from the page 1 of the introduction.

Page2: I think that nine references cited for a single simple statement are too much. Is there any review to be referred instead?

Answer: Only one reference was left for each environment studied, urban, rural, wild and cave (1, 2, 3, 4). And there is no any review to be referred instead.

15 references mentioned (numbers 2-16) deal with sand fly fauna in caves and this fact puts in doubt the following statement "However, despite all these investigation, little is known of the patterns of distribution of sand flies in caves..." Try to explain more clearly what is the novelty of your work.

Answer: It appears that many projects have been carried out in Brazil's caves, but given the size of the country and the number of existing caves, this number becomes small. In addition, this is the first systematic work carried out in Brazil's iron caves.

Materials and Methods: Explain why different numbers of traps were used in different cages and what was the distance between traps within one cage. All these details may affect catches per trap.

Answer: The caves sizes were different. Was installed the maximum possible number of traps that fit within each cave, however were made some modifications in text to improve understanding. The distance between traps within one cave is in the text “For all caves and their surroundings sampled, the first trap inside a cave was at its entrance and the other was set every 10 m further into the cave. In the surroundings, the first trap was set in the forest 10 m from the cave entrance and the others were set every 10 m further away from the cave into the forest.” Modifications were done in the topics Collection of sand flies, Sand fly identification and Statistical analysis of the Materials and Methods.

Add which keys were used for morphological identification of sand flies.

Answer: This information was added to the text with some modifications.

Are there any males or rest of female bodies available in ethanol for eventual molecular studies (confirmation of species identification or population studies) in the future?

Answer: No. The information in line 152-153 “and females were placed in test tubes containing 6% DMSO for future study of Leishmania DNA detection” was taken from the text due to a misunderstanding, since the study was only ecological. In the future we will do studies of this kind.

Results: Did you trap any males with noninverted genitalia or do you have any other evidence that caves serve as breeding sites for sand flies?

Answer: Was not found any males with noninverted genitalia, as it is very difficult to collect sand flies at this stage of development. An evidence that caves serve as breeding sites for sand flies was the records of presence of troglophils species.

In Table 2, explain what is T. Do you mean "n"?

Answer: Was explain in table caption 2 what T means. We also added other information to table 2.

I am missing the information about sand fly "density", for example the sand fly number per night and trap.

Answer: The number of sand flies collected was low, a fact common in this type of environment (Quadrilátero Ferrífero), so we prefer not to do these analyzes of sand flies collected by trap and per night.

Do you have any data about temperature and humidity in caves versus their surroundings?

Answer: We do not have this information. At the beginning of the project our idea was to measure humidity and temperature, however we had several problems with the devices, from thermometer breakage to loss (probable theft), so we did not measure the climate variables.

Discussion

You are frequently using term "condominium". Do you mean village or single house?

Answer: Condominiums are houses in a periurban environment, surrounded by walls. The entry of people is controlled. It is common in the metropolitan region of Belo Horizonte and presents preserved vegetation and wildlife present near the residents.

I suggest to improve/change discussion why caves are important for "the maintenance of their sand fly communities". Could you specify if these caves are serving as resting sites, breeding sites, and/or what else?

Answer: Information was added in this part of the discussion to improve understanding of the conclusions reached.

Reviewer #2: The manuscript entitled “Similarity of species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state Minas Gerais, Brasil“ described an interesting study concerning the sand fly fauna in caves and its surroundings. Several major points need to be addressed before publishing this work, and therefore, this manuscript cannot be published in its actual form.

The authors reported a similarity of sand fly communities among the entrance, interior, and surroundings environments of each sampled cave at places, MS and PESRM. My major concern with this study is that only the analysis was based on a total of 375 sand flies caught during a whole year period. With such low number, no conclusion can be drawn. By using other traps, the authors may increase the number of sand flies needed for an objective statistical analysis. On the other hand, the authors should compare the similarity on a monthly basis and NOT during the whole period because it is well known that phenology differ among species. In addition, the authors should report Leishmania infection in both sites.

Answer: The low number of sand flies collected is a characteristic of the studied region (Quadrilátero Ferrífero) and is not a problem of the methodology used. The fact that it has few sand flies compared to, for example, a limestone cave should not be an impediment to studying it. Information was added in the discussion topic for a better understanding of the overall conclusions obtained as well as for the clarification of the statistical analysis used to arrive at an appropriate and reliable conclusion. The comparison of sand flies fauna on a monthly basis will not be possible, since there have been many months with only one species or few species of sand flies collected, which made it impossible to compare the similarity monthly. The NMDS analysis can be considered useful in trying to find out if there are patterns in the ecological data, which was the purpose of the study. Thus, we believe that analyzing the similarity of the phlebotomine community by caves and their surroundings during the entire collection period is the best way for what was proposed in the present study, since the NMDS analysis was to investigate the composition of sand fly species based on the presence and absence of species inside and outside the caves regardless of the sampling period, in other words, was an analysis to evaluate the spatial distribution of the community and not temporal distribution. The Leishmania detection study was not performed in the studied sites, because we had methodological problems when performing sample extraction which made this part of the study impossible.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

30 Dec 2019

PONE-D-19-18993R1

Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state of Minas Gerais, Brazil

PLOS ONE

Dear Dr. Andrade Filho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Given the conflicting judgements in the original reviews, two novel reviewers were invited. Language has been thoroughly improved, but all four reviewers raised points that still need your attention. Please, be generous in incorporating the suggestions. Below, I also made some minor comments in order to further contribute with the report. When responding to the reviewers, make sure to clearly state the line were the comment / suggestion was addressed / incorporated in the revised manuscript's text.

We would appreciate receiving your revised manuscript by Feb 13 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Albert Schriefer, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

1- Add one or two paragraphs in discussion dealing with the limitations faced by the study, and to what extent they may have influenced conclusions. Please, make sure to include the following points raised by more than one reviewer, but also expand to other possible limitations: (1) the analyses were based on a limited sample; (2) seasonality was not approached; (3) the study should have encompassed more than a single year.

2- I also suggest adding a paragraph to discussion indicating the existence and overall findings of similar studies conducted in other New World and Old World countries, in order to improve contextualization of the current study.

3- State in discussion which of the species described in Table 2 were implicated in transmission of Leishmania parasites to human beings, and which were not.

4- Discussion, line 273. Please correct Leismania infatum with Leismania infantum.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Some comments were handled satisfactorily, some were answered in the reply to reviewer only.

It is necessary to reflect at least two following comments also in the text of the manuscript.

: Explain in Discussion why the sand fly number per night and trap is not given.

: The term "condominium" needs to be explained in the text, please understand that you are writing also for non-Brazilian audience.

Finally, it is good that you indicated which taxonomy (Galati, 2003) was used to classify sand fly species, however, I am still missing the information about keys used for morphological identification.

Reviewer #2: The revised manuscript entitled “Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state Minas Gerais, Brasil“ described a study concerning the sand fly fauna in caves and its surroundings. In this revision, authors did not address major points raised previously. In addition, arguments advanced by authors were not convincing.

My major concern with this study is that the analysis was based only on a total of 375 sand flies caught during a whole year period. With such low number, no conclusion can be drawn. To be accepted, this entomological survey should be performed over a 2-year period.

In conclusion, this manuscript in its revised form should not be accepted for publication

Reviewer #3: This study aimed to address the phlebotomine fauna in caves in a mining region of Minas Gerais state Brazil. The study is very preliminary/descriptive and did not ascertain sandfly infection by Leishmania. This is of importance since several hosts can also use the caves and hence, be potential blood sources for the vectors. I think this manuscript is more suitable for a short communication elsewhere. Although the authors claim that the revision was made by a native English speaker, I myself found several spelling and grammar mistakes in the text. I will indicate some. I therefor recommend rejection of the manuscript in its current form.

Abstract

Lines 16-18 - "Caves, though seemingly inhospitable due to the lack of light in areas furthest from the

entrance and the low availability and variety of resources, can harbor many species of

vertebrates and invertebrates."

Although I understand what the authors meant the sentence would be much easier to understand as follows:

Caves are extreme and inhospitable environments that can harbour several species of vertebrate and invertebrate animals.

Lines 20-22 - The authors shouls state that one cave in is MS and the other three in PNSRL, by reading the abstract we have the idea that four caves in each area.

Introduction

Lines 44-47 - sentences identical to abstract. The authors should avoid this type of thing.

Lines 53-54 - "differences in the period of activity or in its diversity among several caves of the same region have been shown". I would suggest as follows: differences in activity periods and diversity among several caves of the same region have been reported.

Line 62 - "Given the above" - please remove this and start the sentence with "Few studies ...

Lines 66-69 - Very long and confusing sentence, please consider shortening and reviewing the English.

Line 69 - "then" is unnecessary.

Line 70 - "to use ordination to structure local communities along an environmental gradient" - although I have worked with sandflies collection, I have no idea what the authors meant with this sentence. Considering revising the text.

Lines 79-83 - The authors claim that knowing the vector could help to avoid transmition of Leishmaniasis to humans. However, they did not perform PCR for parasite detection. This is the main weakness of this manuscript.

Line 179 - "whereas in the most abundant" replace for whereas the most abundant.

Lines 182 a 185 - consider english revision.

Suggestion: In Cave RM39, a total of 190 individuals were collected: 48 inside and 142 outside. Ev. tupynambai (76.1%) was the most abundant species inside whereas Br. troglodytes (81.2%) was the dominant species outside.

Lines 204-205 - "as was observed for" - this sentence should be completely rewritten.

Line 240, 270 - longipalpis not Longipalpis;

Line 273 - infantum not infatum;

Line 273 - although L. longipalpis is an incriminated vector for VL, in Lapinha Cave there is also this species and no transmission of VL in that area. The authors cannot conclude anything about the status of L. longipalpis in this cave without at least performing PCR to detect L. infantum.

Reviewer #4: Dear Editor,

Below is my summary opinion regard to the article sent to me for reviewing.

The authors reported a similarity of sand fly communities among the entrance, interior, and surroundings environments of each sampled cave located at, MS and PESRM. However, the insuficiente information to reach any firm conclusion due to a lack of season records of the collected species may weaken the reality of species composition or patterns of distribution. This means that the overall analysis obtained, although assured by the statistical tests, may not represent a similarity of sand fly communities in specific periods at places MS and PESRM.

The authors properly exhibited the study area, including trap distribution mode, sample collection and period. On the other hand, they did not include data on environmental descriptors (vegetation, soil type), temperature and humidity at the traps attachment points in caves and its surroundings, impairing the discussion on stratifications of the sandflies.

The manuscript entitled “Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state Minas Gerais, Brasil“ described an interesting study of sandflies fauna, including the finding of Evandromyia tupynambai notably predominating inside PESRM caves.

In my opinion, the presentation of the paper could be conducted more as a survey of sand flies fauna in the Quadrilátero Ferrífero, state of Minas Gerais, Brazil than as a study that responds patterns of distribution of sand fly communities among the entrance, interior, and surroundings environments of each sampled cave at places, MS and PESRM.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

10 Feb 2020

Dear editor,

Below are the responses to the editor and reviewers' comments.

We are available for clarification.

Responses to editor comments:

1- Add one or two paragraphs in discussion dealing with the limitations faced by the study, and to what extent they may have influenced conclusions. Please, make sure to include the following points raised by more than one reviewer, but also expand to other possible limitations: (1) the analyses were based on a limited sample; (2) seasonality was not approached; (3) the study should have encompassed more than a single year.

Answer: All requested information has been added to the text from line 306-337.

2- I also suggest adding a paragraph to discussion indicating the existence and overall findings of similar studies conducted in other New World and Old World countries, in order to improve contextualization of the current study.

Answer: We have included a paragraph about some studies of sand flies in caves. If the reviewer deems it necessary, we can include other data and include the necessary references.

3- State in discussion which of the species described in Table 2 were implicated in transmission of Leishmania parasites to human beings, and which were not.

Answer: This information was added to the text at line 262, 264-265.

4- Discussion, line 273. Please correct Leismania infatum with Leismania infantum.

Answer: The name of this species has been corrected in line 292.

Responses to reviewers comments:

Reviewer #1:

Explain in Discussion why the sand fly number per night and trap is not given.

Answer: This information was added from line 327-329.

The term "condominium" needs to be explained in the text, please understand that you are writing also for non-Brazilian audience.

Answer: To facilitate understanding, the term residential condominium was replaced by residential area in a periurban environment in line 252.

Finally, it is good that you indicated which taxonomy (Galati, 2003) was used to classify sand fly species, however, I am still missing the information about keys used for morphological identification.

Answer: This information was added in line 160.

Reviewer #2:

The revised manuscript entitled “Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state Minas Gerais, Brasil“ described a study concerning the sand fly fauna in caves and its surroundings. In this revision, authors did not address major points raised previously. In addition, arguments advanced by authors were not convincing.

Answer: Information regarding these questions has been added from line 306-337 for better clarification regarding the proposed study.

My major concern with this study is that the analysis was based only on a total of 375 sand flies caught during a whole year period. With such low number, no conclusion can be drawn. To be accepted, this entomological survey should be performed over a 2-year period.

Answer: We made some clarifications regarding these questions from line 306-337.

Reviewer #3:

This study aimed to address the phlebotomine fauna in caves in a mining region of Minas Gerais state Brazil. The study is very preliminary/descriptive and did not ascertain sandfly infection by Leishmania. This is of importance since several hosts can also use the caves and hence, be potential blood sources for the vectors. I think this manuscript is more suitable for a short communication elsewhere. Although the authors claim that the revision was made by a native English speaker, I myself found several spelling and grammar mistakes in the text. I will indicate some. I therefor recommend rejection of the manuscript in its current form.

Answer: Modifications were made throughout the entire text of the article, with emphasis on line 306-337, in order to clarify all these questions. In addition, English has been revised.

Line: 16-18 - "Caves, though seemingly inhospitable due to the lack of light in areas furthest from the entrance and the low availability and variety of resources, can harbor many species of vertebrates and invertebrates."

Answer: Subject accepts in line 16-19.

Lines 20-22 - The authors shouls state that one cave in is MS and the other three in PNSRL, by reading the abstract we have the idea that four caves in each area.

Answer: The line 22 has been rewritten in this part of the abstract to improve understanding.

Lines 44-47 - sentences identical to abstract. The authors should avoid this type of thing.

Answer: The Line 22 has been rewritten, so there are no more sentences identical to abstract.

Lines 53-54 - "differences in the period of activity or in its diversity among several caves of the same region have been shown". I would suggest as follows: differences in activity periods and diversity among several caves of the same region have been reported.

Answer: Subject accepts in line 54-56.

Line 62 - "Given the above" - please remove this and start the sentence with "Few studies ...

Answer: This whole paragraph, from line 64-76, has been replaced by the sentence from line 77-88. The English has also been revised.

Lines 66-69 - Very long and confusing sentence, please consider shortening and reviewing the English.

Answer: This whole paragraph, from line 64-76, has been replaced by the sentence from line 77-88. The English has also been revised.

Line 69 - "then" is unnecessary.

Answer: This whole paragraph, from line 64-76, has been replaced by the sentence from line 77-88. The English has also been revised.

Line 70 - "to use ordination to structure local communities along an environmental gradient" - although I have worked with sandflies collection, I have no idea what the authors meant with this sentence. Considering revising the text.

Answer: This paragraph has been restructured from line 83-88 to better understanding the context. English has also been revised.

Lines 79-83 - The authors claim that knowing the vector could help to avoid transmition of Leishmaniasis to humans. However, they did not perform PCR for parasite detection. This is the main weakness of this manuscript.

Answer: This paragraph has been removed from the line 93-97, because we consider it unnecessary for the study.

Line 179 - "whereas in the most abundant" replace for whereas the most abundant.

Answer: Sentence has been corrected in line 194.

Lines 182 a 185 - consider english revision.

Answer: The English was revision by a native speaker.

Suggestion: In Cave RM39, a total of 190 individuals were collected: 48 inside and 142 outside. Ev. tupynambai (76.1%) was the most abundant species inside whereas Br. troglodytes (81.2%) was the dominant species outside.

Answer: Suggestion was accepted from line 197-203.

Lines 204-205 - "as was observed for" - this sentence should be completely rewritten.

Answer: We agreed that this sentence was really misspelled with out of order affirmations, so this paragraph has been restructured from line 220-232.

Line 240, 270 - longipalpis not Longipalpis;

Answer: The name of this species has been corrected in line 259, 289.

Line 273 - infantum not infatum;

Answer: The name of this species has been corrected in line 292.

Line 273 - although L. longipalpis is an incriminated vector for VL, in Lapinha Cave there is also this species and no transmission of VL in that area. The authors cannot conclude anything about the status of L. longipalpis in this cave without at least performing PCR to detect L. infantum.

Answer: The reviewer is partly right. Lutzomyia longipalpis is known to be a species complex and probably with variable vector capacity between populations / species within the complex (see Casanova et al. 2015), for this reason we do not claim that Lu. longipalpis is the vector in the studied area, even though we do not know if there is Leishmania circulation in the studied environment. We only mention two species cited to Leishmania in other places

Regarding the population of Lu. longipalpis from Lapinha cave, we cannot say that there is no transmission of VL, as there are no systematic studies on the natural infection / detection of Leishmania DNA in that population. Several studies on experimental infection have already been carried out in colonies from the Lapinha cave (Secundino et al. 2012; Freitas et al 2012; Laurenti et al. 2009). A curious fact in relation to this area, is that one of the creators of the studies with Lu. longipalpis in this area, researcher Alberto Falcão, was infected with visceral leishmaniasis in Gruta da Lapinha in the 60s

Reviewer #4:

The authors reported a similarity of sand fly communities among the entrance, interior, and surroundings environments of each sampled cave located at, MS and PESRM. However, the insuficiente information to reach any firm conclusion due to a lack of season records of the collected species may weaken the reality of species composition or patterns of distribution. This means that the overall analysis obtained, although assured by the statistical tests, may not represent a similarity of sand fly communities in specific periods at places MS and PESRM.

Answer: Modifications were made throughout the entire text of the article, with emphasis on line 306-337, in order to clarify all these questions.

The authors properly exhibited the study area, including trap distribution mode, sample collection and period. On the other hand, they did not include data on environmental descriptors (vegetation, soil type), temperature and humidity at the traps attachment points in caves and its surroundings, impairing the discussion on stratifications of the sandflies.

Answer: Modifications were made throughout the entire text of the article, with emphasis on line 306-337, in order to clarify all these questions.

In my opinion, the presentation of the paper could be conducted more as a survey of sand flies fauna in the Quadrilátero Ferrífero, state of Minas Gerais, Brazil than as a study that responds patterns of distribution of sand fly communities among the entrance, interior, and surroundings environments of each sampled cave at places, MS and PESRM.

Answer: Modifications from line 89-93 were made to better understanding the proposed study.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

19 Feb 2020

PONE-D-19-18993R2

Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state of Minas Gerais, Brazil

PLOS ONE

Dear Dr. Andrade Filho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please, before we can proceed with your manuscript, fully incorporate the few additional editor comments at the end of this letter.

We would appreciate receiving your revised manuscript by Apr 04 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Albert Schriefer, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

1- As repeatedly requested by reviewer 1, please, completely disclose the missing the information about keys used for morphological identification.

2- Lines 64 to 66. Correct to "Therefore, no ecological studies have been carried out in ferruginous environments evaluating the environmental gradient of caves and its surrounding phebotomine communities.

3- Line 70. Correct from "structure" to "structured".

4- Line 293. add a 'to' in 'for example, to a limestone cave [1, 4]'.

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Feb 2020

Dear Academic Editor,

Below the answers to the questions

1- As repeatedly requested by reviewer 1, please, completely disclose the missing the information about keys used for morphological identification.

Answer: In the last version, the phrase on identification and classification of sandflies used in the article was modified (All sand flies were identified following the key and classification of Galati (2003) [30], lines 140 and 141. In case it is not clear we can change in order to facilitate the understanding of readers

2- Lines 64 to 66. Correct to "Therefore, no ecological studies have been carried out in ferruginous environments evaluating the environmental gradient of caves and its surrounding phlebotomine communities.

Answer: Change accepted

3- Line 70. Correct from "structure" to "structured".

Answer: Change accepted

4- Line 293. add a 'to' in 'for example, to a limestone cave [1, 4]'.

Answer: Change accepted

Submitted filename: Rebuttal Letter.doc

Click here for additional data file.

20 Feb 2020

Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state of Minas Gerais, Brazil

PONE-D-19-18993R3

Dear Dr. Andrade Filho,

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24 Feb 2020

PONE-D-19-18993R3

Species composition of sand flies (Diptera: Psychodidae) in caves of Quadrilátero Ferrífero, state of Minas Gerais, Brazil

Dear Dr. Andrade Filho:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Albert Schriefer

Academic Editor

PLOS ONE