Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

Aline Tanure; Felipe Dutra Rêgo; Gabriel Barbosa Tonelli; Aldenise Martins Campos; Paloma Helena Fernandes Shimabukuro; Célia Maria Ferreira Gontijo; Gustavo Fontes Paz; José Dilermando Andrade-Filho

doi:10.1371/journal.pone.0234445

. 2020 Jun 24;15(6):e0234445. doi: 10.1371/journal.pone.0234445

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

Aline Tanure ¹, Felipe Dutra Rêgo ¹, Gabriel Barbosa Tonelli ¹, Aldenise Martins Campos ¹, Paloma Helena Fernandes Shimabukuro ¹, Célia Maria Ferreira Gontijo ¹, Gustavo Fontes Paz ¹, José Dilermando Andrade-Filho ^1,^*

Editor: Vyacheslav Yurchenko²

PMCID: PMC7314019 PMID: 32579586

Abstract

This study aimed to describe the sand fly fauna and detect trypanosomatids in these insects from Casa Branca, state of Minas Gerais, Brazil, an endemic area of both visceral (VL) and tegumentary leishmaniasis (TL). Sand flies were collected bimonthly from May 2013 to July 2014, using automatic light traps exposed for three consecutive nights in peridomiciliary areas of nine houses with previous reports of VL and TL. ITS1-PCR and DNA sequencing were performed for trypanosomatids identification. A total of 16,771 sand flies were collected belonging to 23 species. The most abundant species was Nyssomyia whitmani (Antunes & Coutinho, 1939) (70.9%), followed by Lutzomyia longipalpis (Lutz & Neiva, 1912) (15.2%) and Migonemyia migonei (França, 1920) (9.1%). Leishmania amazonensis DNA was detected in Ny. whitmani (four pools) and Le. braziliensis DNA was detected in Psychodopygus lloydi (one pool). In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for Leishmania DNA, the parasite species was not determined due to the low quality of the sequences. Moreover, DNA of Herpetomonas spp. was detected in Ny. whitmani (two pools) and Cortelezzii complex (one pool). DNA of Crithidia spp. was detected in Ny. whitmani and Ps. lloydi (both one pool). Our results suggest that Ny. whitmani may be involved in the transmission of Le. amazonensis in the study area. The molecular detection of Le. amazonensis suggests the presence of this species in a sylvatic cycle between vertebrate and invertebrate hosts in the region of Casa Branca. Our data also reveal the occurrence of other non-Leishmania trypanosomatids in sand flies in Casa Branca District.

Introduction

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are known to be natural hosts of etiological agents such as bacteria, viruses and protozoa [1,2]. Among these agents, are species of the genus Leishmania, which are transmitted during blood feeding by female sand flies [1]. Several sand flies species have been considered proven or suspected vectors of Leishmania parasites in Brazil, suggesting that multiple vectors may be involved in parasite cycles, making the epidemiological picture more complex than currently recognized [3,4].

Sand flies are also possible natural vectors of several trypanosome species, both dixenous (i.e. those with two hosts in their life cycle—Trypanosoma, Endotrypanum, and Phytomonas) and monoxenous (i.e. those having only one host—Leptomonas, Crithidia, Blastocrithidia and Herpetomonas) [5–10]. However, the identification of these protozoa has been considered irrelevant from the epidemiological perspective. Recent studies have discussed the evolution of trypanosome taxonomy and, despite improvements have been made, the resolution of evolutionary relationships within the Trypanosomatidae is confounded by our incomplete knowledge of its true diversity [11–15]. More recently, several monoxenous flagellates have been identified from human clinical isolates and some of them, similar to both TL and VL [16–21]. Several authors have discussed the capacity of these trypanosomatids to cause leishmaniasis in a possible co-infection with Leishmania or even in a single infection [19,20,22,23]. The dixenous parasite Endotrypanum colombiensis, until recently Leishmania colombiensis [12], also represent another relevant Trypanosomatidae related to leishmaniasis in South America [24–27]. Endotrypanum originally includes intra-erythrocytic trypanosomatids isolated from sloth Choloepus didactylus (Linnaeus) (Mesnil and Brimont 1908) and the transfer of Le. colombiensis to this genus is still debated, since their ability to infect erythrocytes has not been proven yet [28]. Furthermore, the role of sand flies as vector of these monoxenous and dixenous parasites to humans need to be investigated, although some sand flies known as permissive vectors seems able to sustain the infection with trypanosomatids under laboratory conditions [29–31].

Intense urbanization over the last decades has caused changes in natural habitats, especially those occupied by phlebotomine sand flies. Some species are able to withstand several environmental modifications that may occur in natural breeding sites and successfully adapt to urban environments [1]. In some regions, the presence of vector species in the human environment may be associated with the emergence of autochthonous cases of leishmaniases, as occurs in the state of Minas Gerais, where both VL and TL are widely spread [32,33]. In this context, the municipality of Brumadinho has reported an increasing number of VL and TL-cases over the last years, and some of them have been reported in the district of Casa Branca (Source: Municipal Secretary of Health of Brumadinho, MG). In this area, the presence of Leishmania spp. in wild and synanthropic mammals have been recorded [34]. Given this situation, this study aimed to describe the sand fly fauna, detect and identify by molecular assay the presence of trypanosomatids in females sand flies from Casa Branca.

Material and methods

Ethic statements

The collections were carried out with the authorization of the Sistema de Autorização e Informação em Biodiversidade—SISBIO (license number 15237–2). The owners of the residences verbally gave permission to the installation of the traps during the study. The sand flies were deposited in the Coleção de Flebotomíneos of the Instituto René Rachou (Fiocruz/COLFLEB).

Study area, collection and identification of phlebotomine sand flies

The study was conducted in the district of Casa Branca (20°6'2.58"S; 44°2'59.45" W), northern region of the municipality of Brumadinho (Fig 1). Casa Branca is bordered by the Parque Estadual Serra do Rola Moça (PESRM) conservation unit, the third largest urban park in Brazil, covering a transition area between Atlantic Forest and Cerrado. About eight kilometers west of Casa Branca is a village called Córrego do Feijão, where, in January 2019, an ore tailing dam ruptured, causing great environmental and social impact in all areas of Brumadinho municipality, as well as the death of 270 people.

Sand flies were collected from nine sampling points in Casa Branca from May 2013 to July 2014. Bimonthly, two automatic light traps (model HP) [35] were set at each sampling point, in the peridomiciliary environment for three consecutive nights. Collection sites with previous report of human or canine leishmaniasis were selected.

Collected sand flies were taken to the laboratory for further screening by sex, clarification process and dissection. All sand flies collected in July/2013, November/2013, March/2014 and July/2014 were mounted in Canada balsam on a glass slide for species identification. The female sand flies from May/2013, September/2013, January/2014 and May/2014 were dissected by removing the last three segments of the abdomen and head, mounted on a glass slide in Berlese liquid. The remainder of the body was individually stored dry at -20°C in a 1.5 mL tube for further DNA extraction.

Species identification was determined using the updated version of the classification proposed by Galati in 2003 [36]. The females of Evandromyia sallesi and Evandromyia cortelezzii, were identified as belonging to the “Cortelezzii complex” since they are undistinguishable by their morphology [37]. The abbreviation of the genera followed the proposal of Marcondes [38].

DNA extraction and identification of trypanosomatids

Females sand flies without blood in their abdomen were processed individually or grouped into pools of up to 20 individuals of the same species, date and site of collection. Females were subjected to DNA extraction using the Gentra Puregene® kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol. To control cross-contamination during DNA extraction, males sand flies were used as negative controls in all procedures [39,40]. In addition, all instruments and the entire work area were previously treated with DNAZap (Ambion, Life Technologies, Inc.).

The extracted DNA was used to investigate the presence of trypanosomatids by amplifying the internal transcribed spacer 1 region (ITS1), following the conditions described previously [41,42]. Positive controls were used in all PCR-reactions, as follows: Leishmania amazonensis (IFLA/BR/67/PH8), Le. braziliensis (MHOM/BR/75/M2903), Le. infantum (MHOM/BR/74/PP75), Le. guyanensis (MHOM/BR/75/M4147), Crithidia fasciculata (Fiocruz-COLPROT 048), Endotrypanum monterogeii (Fiocruz-COLPROT 151), Herpetomonas samuelpessoai (Fiocruz-COLPROT 067), Leptomonas collosoma (Fiocruz-COLPROT 073) and Phytomonas serpens (Fiocruz-COLPROT 189). The non-Leishmania trypanosomatids controls were kindly provided by the Protozoa Collection of the Laboratório de Biologia Molecular e Doenças Endêmicas (Fiocruz/COLPROT). The amplicons were visualized in a 2% agarose gel stained with ethidium bromide (10mg/mL) with a 100 bp DNA Step Ladder provided as molecular length size standard. The PCR-positive products were digested using HaeIII enzyme following the conditions previously described [41]. The amplicons were purified with QIAquick® PCR Purification Kit (Qiagen, Valencia, CA, USA) as described by the manufacturer. Once purified, the products were used for DNA sequencing following the conditions described by Rêgo et al. [43]. Geneious software (v. 9.1.3) was used to check the electropherograms, align sequences and to perform comparison with sequences deposited in the GenBank database through BLAST alignment algorithm, considering the two most similar organisms according to identity above 90% for genus level and 98% for species level. These sequences are available in GenBank (Accession numbers MN638721-MN638737).

The positive rate of each sand fly species was determined considering only one specimen per positive pool.

Statistical analyses

To evaluate species abundance the index of species abundance "ISA", and the standardized index of species abundance "SISA" were calculated [44]. The values of SISA range from 0 to 1, with values closer to 1 representing the most abundant species. The Shannon index (H) and evenness index (J) [45] were used to determine species diversity and evenness of species abundance, respectively.

Results

Sand fly fauna

During the study period, 16,771 sand flies of 23 species and eight genera were collected in Casa Branca. The most abundant species was Ny. whitmani (70.9%; SISA 0.74), followed by Lu. longipalpis (15.2%; SISA 0.53) and Mg. migonei (9.1%; SISA 0.51) (Table 1 and Fig 2). The Shannon (H) and evenness (J) indexes were low for the study area (H’ = 0.9946229; J’ = 0.308997).

Table 1. Sand flies collected according to month at the Casa Branca, Brumadinho, Minas Gerais, from May 2013 to July 2014.

Months of collection
Species	May/13		Jul/14		Sep/13		Nov/13		Jan/14		Mar/14		May/14		Jul/14		Total
Species	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	Total (%)
Brumptomyia sp.	0	0	0	1	4	4	0	0	0	0	0	2	0	0	0	0	4	7	11 (0.1)
Cortelezzii complex	0	7	0	2	0	12	0	5	0	1	0	1	0	9	0	1	0	38	38 (0.2)
Evandromyia cortelezzii	1	0	2	0	3	0	1	0	2	0	1	0	0	0	0	0	10	0	10 (0.1)
Evandromyia edwarsi	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	1	1 (0.0)
Evandromyia evandroi	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	1	0	1 (0.0)
Evandromyia lenti	1	0	0	1	2	2	10	5	39	29	0	2	2	3	0	1	54	43	97 (0.6)
Evandromyia sallesi	1	0	0	0	2	0	0	0	0	0	0	0	1	0	1	0	5	0	5 (0.0)
Evandromyia teratodes	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	1	1 (0.0)
Evandromyia termitophila	0	1	0	1	0	1	0	5	0	0	0	0	0	3	0	0	0	11	11 (0.1)
Evandromyia tupynambai	0	1	0	0	3	0	0	2	0	2	0	0	1	10	0	0	4	15	19 (0.1)
Lutzomyia amarali	0	0	0	0	0	0	0	0	0	0	0	2	0	1	0	0	0	3	3 (0.0)
Lutzomyia ischyracantha	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	1	0	1 (0.0)
Lutzomyia longipalpis	528	127	57	5	430	112	181	41	63	22	214	62	547	104	55	3	2,075	476	2,551 (15.2)
Migonemyia migonei	219	90	72	12	175	53	156	49	104	17	73	24	373	62	41	13	1213	320	1533 (9.1)
Nyssomyia intermedia	0	0	1	2	1	0	0	0	0	0	0	0	0	0	0	0	2	2	4 (0.0)
Nyssomyia whitmani	3,449	2,535	79	263	793	680	547	343	625	324	101	72	974	959	77	62	6,645	5,238	11,883 (70.9)
Pintomyia bianchigalatiae	0	13	0	0	0	10	1	2	0	3	0	17	0	30	0	0	1	75	76 (0.5)
Pintomyia fischeri	7	13	6	2	7	11	34	15	27	34	14	18	11	49	5	4	111	146	257 (1.5)
Pintomyia mamedei	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1 (0.0)
Pintomyia misionensis	0	0	0	3	0	0	0	0	0	1	0	0	0	4	0	1	0	9	9 (0.1)
Pintomyia monticola	1	4	0	2	0	1	2	0	0	1	0	6	2	21	1	0	6	35	41 (0.2)
Pintomyia pessoai	1	1	0	0	1	18	0	19	3	0	1	0	1	3	0	0	7	41	48 (0.3)
Psathyromyia pascalei	0	0	0	0	1	0	0	0	0	1	0	0	1	0	1	0	3	1	4 (0.0)
Psathyromyia sp.	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1 (0.0)
Psychodopygus lloydi	1	33	0	4	0	0	6	3	4	47	0	0	6	38	0	0	17	125	142 (0.9)
Total	4,209	2,845	217	299	1,423	908	938	489	868	482	404	206	1,919	1,298	181	85	10,159	6,612	16,771 (100)
Total	7,054		516		2,331		1427		1,350		610		3,217		266		16,771

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Evandromyia was the richest genera with eight species collected, followed by Pintomyia with six species (Table 1). May 2013 showed the greatest species abundance (7,054 sand flies) while July 2014 showed the lowest (266 sand flies). The highest species richness was observed in May 2014 with a total of 18 species (Table 1).

Molecular identification of trypanosomatids

Of a total of 4,913 females, 47 were individually tested and 4,866 grouped in 311 pools. Table 2 presents information about the species collected and their distributions for molecular analysis. No sample individually analyzed was PCR positive for trypanosomatids.

Table 2. Sand fly organized by species that were analyzed individually and in pools collected in Casa Branca, Brumadinho, Minas Gerais, from May 2013 to May 2014.

Species	Sand flies collected	Number of analyzed pools	Specimens analyzed individually
Brumptomyia sp^*	4	1	0
Cortelezzii complex	25	5	3
Evandromyia edwardsi	1	0	1
Evandromyia lenti	31	4	3
Evandromyia teratodes	1	0	1
Evandromyia termitophila	6	1	4
Evandromyia tupynambai	13	2	1
Lutzomyia amarali	1	0	1
Lutzomyia longipalpis	248	19	0
Migonemyia migonei	144	14	0
Nyssomyia whitmani	4128	220	0
Pintomyia bianchigalatiae	47	8	2
Pintomyia fischeri	80	13	0
Pintomyia misionensis	4	0	4
Pintomyia monticola	27	3	3
Pintomyia pessoai	16	3	1
Pintomyia sp.^*	21	0	21
Psathyromyia pascalei	1	0	1
Psychodopygus lloydi	115	18	1
Total	4913	311	47

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* sand flies damaged

Of the 311 pools tested, 13 (4.1%) were PCR-positive for Leishmania: Lu. longipalpis (1), Ny. whitmani (11) and Ps. lloydi (1) (Table 3). The positive rate obtained for Leishmania was 4.18% (13/311). Four pools of Ny. whitmani were positive for Le. amazonensis (4/311 = 1.2%), however, in other seven pools of this sand fly (7/311 = 2.2%) it was not possible to determinate the Leishmania species, due to low quality of the sequences, which contained a high number of ambiguous sites. Therefore, sequences were left as undetermined (Leishmania sp.). One pool of Ps. lloydi was positive for Le. braziliensis (1/311 = 0.3%) and Lutzomyia longipalpis was positive for Leishmania sp. (1/311 = 0.3%) (Table 3).

Table 3. Results of DNA detection and identification of species of Trypanosomatidae according to species of sand fly, number of pool and collection date in Casa Branca, Brumadinho from May 2013 to May 2014.

Sand fly species	Seq ID Blast	% similarity	Collection date	Accession numbers
Cortelezzii complex	Herpetomonas sp.	95,38	may-2013	MN638722
Lutzomyia longipalpis	Leishmania sp.	80,2	may-2013	MN638733
Nyssomyia whitmani	Leishmania amazonensis	100	may-2013	MN638726
Nyssomyia whitmani	Leishmania amazonensis	99,39	may-2013	MN638738
Nyssomyia whitmani	Leishmania amazonensis	98,01	sep-2013	MN638734
Nyssomyia whitmani	Leishmania amazonensis	100	may-2013	MN638727
Nyssomyia whitmani	Leishmania sp.	99,51	may-2013	MN638724
Nyssomyia whitmani	Leishmania sp.	99,6	may-2013	MN638725
Nyssomyia whitmani	Leishmania sp.	99,66	may-2013	MN638731
Nyssomyia whitmani	Leishmania sp.	99,32	may-2013	MN638732
Nyssomyia whitmani	Leishmania sp.	99,04	may-2013	MN638735
Nyssomyia whitmani	Leishmania sp.	98,45	may-2013	MN638736
Nyssomyia whitmani	Leishmania sp.	98,72	may-2013	MN638737
Nyssomyia whitmani	Herpetomonas sp.	90,21	may-2013	MN638723
Nyssomyia whitmani	Herpetomonas sp.	97	may-2013	MN638729
Nyssomyia whitmani	Crithidia sp.	97,31	may-2013	MN638721
Psychodopygus lloydii	Leishmania braziliensis	99,32	may-2014	MN638730
Psychodopygus lloydii	Crithidia sp.	96,5	may-2014	MN638728

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Seq ID Blast = hit provided by Blast search.

Trypanosomatid sequence obtained from strain A9 from the Protozoa Collection (Fiocruz/COLPROT)

A total of five pools (5/311 = 1.6%) presented single amplicons of inconsistent size (400–500 bp), distinct to that predicted for Leishmania parasites (300–350 bp). These results led us to investigate the identity of these fragments and trypanosomatid species were identified. Nyssomyia whitmani and Cortelezzii complex (both one pool) were positive for Herpetomonas spp., while Psychodopygus lloydi (one pool) and Ny. whitmani (two pools) were positive for Crithidia spp. (Table 3). Sequence similarity to GenBank sequences ranged between 90–97%. The five non-Leishmania sequences found in our study, Herpetomonas spp. and Crithidia spp., are sufficiently different between them and possibly represent three and two, respectively, different MOTUs (Molecular Operational Taxonomic Units).

Discussion

The sand fly diversity found in Casa Branca may be explained by the proximity to the PESRM conservation unit. The residences selected are also close to forest environments and presented breeding sites for phlebotomine sand flies, such as vegetation (fruit trees) and animal shelters (hennery, pigsty and dog kennel). The highest standardized index of species abundance (SISA) in this study was found for Ny. whitmani (0.74). Despite the quite diversity of the study area (H ' = 0.995), the species abundances were not similar, in which Ny. whitmani was a dominant species. This likely explains the low value for the evenness index (J ' = 0.309), since the great abundance of just one species tends to impact the uniformity of the other ones.

Here, Ny. whitmani was found in high density close to residences. This species frequently has been found in endemic areas for TL in southeastern Brazil, and there is no doubt about their role as vector of Le. braziliensis [46]. Furthermore, Ny. whitmani has also been found harboring Le. infantum DNA in the state of Minas Gerais [47,48]. The presence Le. amazonensis DNA within Ny. whitmani has been previously reported in the states of Tocantins and Maranhão [49,50]. Changes in the natural environment may triggered a new epidemiological profile of leishmaniases that could involve Leishmania amazonensis, an alternative vector (i.e. Ny. whitmani) and new reservoirs (domestic and wild). Fonteles et al. [51] reports the capacity of Ny. whitmani to sustain infections with Le. amazonensis, suggesting their role as vector of this parasite in the state of Maranhão. In this context, as we shown here, the same may be occurring in Casa Branca where this sand fly was found harboring Le. amazonensis DNA.

Lutzomyia longipalpis, the main vector of Le. infantum in Brazil, was the second most abundant species in Casa Branca (15.22%) and was also found to be frequent close to the residences. This finding reinforces the adaptation of this species to human dwellings and peridomiciliary sites [52]. Lu. longipalpis is able to sustain infections with several Leishmania species [53–56] and their role as the main vector of Le. infantum in Latin America is undeniable [2,4]. The finding of Leishmania DNA within Lu. longipalpis from Casa Branca draw attention specially about cases of both human and canine visceral leishmaniasis reports.

Migonemyia migonei was the third most captured species in the study area with 9.14% and is also extremely anthropophilic species and frequently found inhabiting peridomiciliary sites and feeding in domestic animals [57]. Recent studies have added information about the vector capacity of Mg. migonei. Guimarães et al. have reported their capacity to support Le. infantum development and late-stage infection [58] and moreover, natural infections by Le. infantum have been reported especially in the absence of the main vector, Lu. longipalpis [59–61]. Migonemyia migonei has also been implicated in the cycle of Le. braziliensis [62–64] and their capacity to support infections with this parasite have been confirmed by Alexandre et al [65].

The genus Evandromyia presented the highest number of species and although there are no proven vector species within this genus, natural infection or Leishmania DNA has been detected in some species [66,67]. In studies conducted in Minas Gerais, Carvalho et al. [68] detected Le. infantum DNA in Ev. cortelezzii and Saraiva et al. [69] found females of Ev. sallesi naturally infected by the same species of Leishmania. These findings may indicate that these species of the Cortelezzii complex (females of Ev. cortelezzii and Ev. sallesi do not differ morphologically) hold some epidemiological significance.

The species Ps. lloydi has been associated with the transmission of Le. braziliensis in wild areas of Minas Gerais [70,71] and was also found in this state with DNA of Le. infantum [72]. Psychodopygus lloydi was positive for DNA of Leishmania braziliensis, suggesting that this sand fly could maintain a wild cycle of TL in the District of Casa Branca.

The finding of Herpetomonas and Crithidia DNA within sand fly species from Casa Branca need to be investigated. Possibly, more than one species within each genera were detected, however, due to the small length of the amplicon it was not possible to confirm it. On the other hand, the molecular detections presented here, may represented different populations (different MOTUs), reinforcing the importance of further studies on these trypanosomatids. Although Herpetomonas seems not to be of epidemiological importance, it has been reported in Egyptian rat [73] and even in immunodepressed humans [18]. In their more common hosts, the Diptera, promastigotes live in the digestive tract, preferentially in the rectum, attached either to endothelian cells or as free-swimmers among fewer opisthomastigotes [74]. Crithidia-related parasites have been involved in an atypical manifestation similar to VL in Brazil [19]. The report of Crithidia causing clinical manifestations similar to VL and the detection of this parasites within sand flies from Brazil, as we found here and previously reported by Machado et al. [49] draw attention and need to be investigated.

Although in some positive pools were not possible to determinate the Leishmania species, due to the low quality of the sequences, it is noteworthy that there was no information about the sand fly fauna and putative vectors in Casa Branca. Here we provide consistent data that should be used for further investigations, such as trying to isolate Le. amazonensis from Ny. whitmani to confirm their role as a vector. Moreover, it is extremely important to investigate the presence of trypanosomatids in Casa Branca and their association with the sand fly fauna.

Conclusion

This is the first study of a sand fly fauna in the municipality of Brumadinho, Minas Gerais. Our findings show that the Casa Branca locality has a diverse sand fly fauna with species that have been previously reported in the state of Minas Gerais. The Ny. whitmani species is the probable vector of Le. braziliensis in Casa Branca and may also be involved in the transmission of Le. amazonensis. The knowledge of the interactions between sand flies and trypanosomatids reported in this study shows that the infection may be occurring in the peridomiciliary environment in the study area. In addition, these results help to understand the dynamics of the leishmaniases transmission cycle in Casa Branca providing support for disease control actions in the region. The presence of non-Leishmania trypanosomatids raises an issue that has been neglected and is of great importance, the circulation of these parasites within phlebotomine sand flies.

Acknowledgments

We thank Surveillance System in Health of Brumadinho for providing epidemiological data. We thank the residents of the collection sites for making the work possible.

Data Availability

Data are available within the manuscript.

Funding Statement

JDAF - 302701/2016-8 Conselho Nacional de Desenvolvimento Científico e Tecnológico (www.cnpq.br) JDAF - PPM-00792-18 Fundação de Amparo à Pesquisa de Minas Gerais (www.fapemig.br) AT - Finance Code 001 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (www.capes.gov.br). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Diversity of phlebotomine sand flies and molecular detection of trypanosomatids species in Brumadinho, Minas Gerais, Brazil

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PLOS ONE

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Reading the manuscript of Tanure at al I noticed the huge unbalance in the literature citations. Authors (almost exclusively) cite Brazilian papers effectively ignoring the huge amount of work on this topic, which has been done in other parts of the world. I invite authors to correct this discrepancy and submit a revised version for peer review. V, Yurchenko

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PLoS One. 2020 Jun 24;15(6):e0234445. doi: 10.1371/journal.pone.0234445.r003

Author response to Decision Letter 0

22 Apr 2020

PONE-D-20-05836

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids species in Brumadinho, Minas Gerais, Brazil

PLOS ONE

Dear Vyacheslav Yurchenko,

Below are our answers to requests from the Editor and the PLOS ONE technical team.

Thanks

Additional Editor Comments (if provided):

The Editor is right, there were many references in Portuguese that could be replaced by others, thanks for the suggestion.

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We understand that our article and files are based on the PLOS ONE style.

Grupo de Estudos em Leishmanioses is the author's affiliation, a name similar to "laboratory". All authors belong to the leishmaniasis study group (Grupo de Estudos em Leishmanioses)

All authors are listed and with their affiliation

4. Please ensure that you refer to Figure 2 in your text as, if accepted, production will need this reference to link the reader to the figure.

We have included the reference to figure 2 in the text, line 214

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We will provide copyright permission for figure 1 and include it in the submission system

Thank you!!!!

José Dilermando Andrade Filho

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PLoS One. doi: 10.1371/journal.pone.0234445.r004

Decision Letter 1

Vyacheslav Yurchenko

4 May 2020

PONE-D-20-05836R1

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids species in Brumadinho, Minas Gerais, Brazil

PLOS ONE

Dear Dr. Andrade Filho,

We would appreciate receiving your revised manuscript by Jun 18 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Vyacheslav Yurchenko

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please address all the concerns raised by 3 reviewers. In addition, I request: 1) to extend the introduction on trypanosomatids and discuss the following publications: Maslov et al, 2019 (Parasitology) and Lukes et al, 2018 (Trends Prasitol). 2) to spell all species names in Italic; iii) fix the references; iv) if you cite ref 60, please cite an accompanying paper- Kostygov et al, 2019 (Trop Med Int Health).

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: No

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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: This is a nice study of a sand fly fauna (in the Casa Branca district of Brumadinho municipality) demonstrating the high phlebotomine diversity and the presence of DNA of two Leishmania species as well as several (3-4) non-Leishmania (monoxenous) parasites. However, because the design of the study (bimonthly = visiting of the locality once per two months) is not sufficient for any “ecological” analysis, I would suggest deleting all parts connected with “seasonal” variation in sand fly abundance and climate (and other) data. The number of collections is really not sufficient for any similar analysis; but even without these unreliable and therefore misleading results, the study is fully reliable to be published. However, there are several less important unclarities to must be solved.

4 trypanosomatid species or trypanosomatids

32 HP – meaning

33 better trap/nights (to demonstrate the effort)

39-40 how many individuals and how many in pools (BTW, pools of 1 specimen is not a pool !!!)

(Of a total of 4,913 females, 47 were individually tested and 4,866 grouped in 311 pools).

42 instead of Leishmania sp. use … in seven pools of Ny. whitmani and one pool of Lu. longipalpis the leishmania species was not determined (to the low quality of the sequences) … or something like this, otherwise, it seems that some “new/unknown” leishmania species was detected

43 .

44-45 same type of result presentation as in the case of Leishmania spp. – it means: how many pools/individuals and in which sand fly species.

45 The presence of DNA is not proving of vector capacity! (… may be involved in the transmission of Le. amazonensis … etc. is much more accurate)

55-57 REFs 1-3 are more-or-less relevant, but there are several current reviews, e.g., Akhoundi et al. 2016 (PLoS Neglected Tropical Diseases 10(3): e0004349.), etc… that would fit here.

59 REF 4 – ok, but add also e.g., Rangel, Shaw (2018) Brazilian Sand Flies … (10.1007/978-3-319-75544-1) etc.

71-72 The genus Trypanosoma is missing! Sand flies are possible vectors of several trypanosome species – add a relevant reference(s)

(the sentence “Several authors have reported the presence of non-Leishmania trypanosomatids DNA in sand flies by means of molecular techniques [12, 13, 14, 15]” does not cover this issue clear enough)

73 REFs 10 and 11 – OK, but add some references on Leptomonas (e.g. Kraeva et al. 2015 (PLoS Pathog 11(8):e1005127)); and even more references would be appropriate here

71-73 differentiate between monoxenous and dixenous genera

77-81 The statement is controversial in some aspects – first of all, the situation with the genus “Endotrypanum” is rather complicated (see doi: 10.14411/fp.2017.020). And in addition to this, the sentence, which is articulated in this way and is given in the previous contexts, makes an impression on readers that the authors refer primarily to single-host (monoxenous) genera (such as Crithidia, Leptomonas, etc.).

124 CDC light traps (model HP) – I am sure that there is no official model HP within the CDC traps – it is an only modification based on the CDC trap design – which company produces these traps or is it just some home-made version?

125-6 not clear – it means that the traps run three nights (and two days)?

Sand flies were still alive (after three days???), How many sand flies survive till laboratory (approximately in % ???)

137-8 when the morphological identification was made? The key is from the end of 2018, the collection and slide preparation was made much earlier (as was mentioned in the previous response to reviewers)!!!

158 … of trypanosomatids (not Leishmania)

247 – the prevalence is right, but anyway it should be explained that the authors predict that only one specimen per pool was infected… (it corresponds with the low “pool” prevalence)

253/255 – why “it was not possible get to species level”? - due to the low quality of the sequences ??? or the sequences represent some new leishmania species ??? It must be explained!

258-9 “the presence of bands of inconsistent size (400-500 bp) with fragments for Leishmania (~350 bp)” – not clear. Does it mean that the double bands were detected = mixed infection of leishmania (350bp) + some monoxenous trypanosomatids ??? And if yes, it means that monoxenous trypanosomatids occur only in pools positive for leishmania parasites???? It is really very unusual and must be discussed!

260 - … three trypanosomatids … - must be clearly distinguished even in Tab. 3 – e.g., Herpetomonas sp. 1, Herpetomonas sp. 2 …

263/352 – Herpetomonas sp. means unidentified ONE species of the genus Herpetomonas, but it seems that two different Herpetomonas species were detected – thus Herpetomonas spp.!!!! Or better cortezzii – H. sp. 1, Ny.w. – H. sp. 2, to make it clear. The same for Crithidia – it is not clear if Crithidia parasites from Ps.l. and Ny.w. belong to one or two species – if the sequences represent two species, it would be better to use C. sp. 1 and C. sp. 2 to make it clear. You have to make it clear, especially if you are not present any phylogenetic tree etc.

Table 3 – sort it according to “Host” or according to “trypanosomatid parasites!, not according Acc.Nos., it makes no sense.

Seq ID Blast – the different unnamed Herpetomonas/Crithidia/Leishmania species (identified by BLAST) must be specified (by its voucher name/number etc., or even by their GB Acc.No.)!

Reviewer #2: I appreciate that authors improved the manuscript and add more international references. However, some of them were not chosen correctly. I suggest correct several references in the first half of the Introduction:

Line 55: Ref. 2 (Alkan et al) is not an optimal reference. Would be better to include the famous review by Maroli et al. 2013, see the pdf.

Line 57: Ref. 3 (Lestinova et al) is not suitable reference. Again, the review by Michele Maroli could be used.

Line 59: Ref. 4. There is more recent review on this topic: chapter by Dvorak et al, published in Bruschi Fabrizio, Gradoni Luigi (eds.), The Leishmaniases: Old Neglected Tropical Diseases. Springer 2018.

Lines 67-70: Please use some more recent reference. All this information is described in details by Jeffrey Shaw in the review by Dvorak et al.

In Discussion, I suggest to improve discussion about Mg. migonei vector competence to L. infantum (Lines 292-294). The vectorial status of Mg. migonei was recently proven experimentally by Fitipaldi Veloso Guimaraes et al, 2016. Please, add this reference (or replace the existing one, no. 45).

Finally, I suggest shorten the “Short title” as it is too long now.

For more details and some other minor corrections of typos, see the pdf file with my comments.

Reviewer #3: The authors shall be commended for the extremely high number of determined and examined insects, and the solid way they approached this task. Massive undertaking, the results of which are worth publishing, but this should happen in somewhat different form and format.

I gather that it is difficult to write a readable paper out of huge but relatively boring data, but the authors still shall try to do a better job. In general, the authors describe frequently and extensively things that can be found on Wikipedia (geography, how many people live in some district - the number is non-scientific because at the time of writing it was already incorrect), please remove all this, which unncessarily extends the manuscript and brings absolutely no novelty and has no relevance to the presented data (where is a correlation between the size of the district and phlebotomine sandflies?).

I would like to ask the authors to better balance the Results and the Discussion, the first being too short and the latter too long.

Discussing usefulness or not of PCR-RFLP and restriction enzymes is way outdated, this type of discussions were concluded 15 or rather 20 years ago and is irrelevant nowadays, when it is sequencing or nothing. At least as long as you want to publish in respected journals, such as PloS One. Hence, shorten this part of discussion by 90%. Again, sentences such as “DNA sequencing has generated good results when used for detection of 380 Leishmania in sand flies“ are funny and make no sense these days, sequencing when done properly always (!) generates good results, namely the letter AGCT.

Furthermore, the acknowledgements and technical details are extremely long and tedious, nobody says that gels were stained with EtBr anymore, this is a common knowledge - please avoid this South American stretching of the manuscripts, remove what is the capital etc. etc.

Give more attention to the really interesting stuff regarding the parasites found. There is tons of references, which are overlaping with their info (+ IBGE reference – accessed in 2016? What is this?) This and some others are not standard type references and has to be deleted. Refs are in different fonts, formats, excessively long list describing all the time the same thing. Yet there is absolutely no discussion and literature about the potentially interesting findings of non-Leishmania trypanosomatids, which is a mistake that has to be rectified.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Attachment

Submitted filename: PONE-D-20-05836_R1_reviewer1.pdf

Click here for additional data file.^{(2.2MB, pdf)}

PLoS One. 2020 Jun 24;15(6):e0234445. doi: 10.1371/journal.pone.0234445.r005

Author response to Decision Letter 1

15 May 2020

Manuscript ID: PONE-D-20-05836R1

Dear Dr. Yurchenko,

We would like to thank for your thoughtful review of the manuscript. You raised important issues and your inputs were well-considered and gratefully implemented. We agree with almost all your comments and we have revised our manuscript accordingly.

We have crafted a revised version of the paper stating the hypothesis and the implications of our work more clearly than before, including the suggestions. We are confident that the new version of the manuscript is greatly improved. We respond below in details to each of your comments. If we have slightly disagreed with something, we stated why. We hope that you will find our responses to your comments satisfactory, and we are willing to finish the revised version of the manuscript.

Please, find below your comments repeated in italics and our responses inserted after each comment. To facilitate the revision process, in some instances we refer to the plain text file indicating the page and the line (page-line).

Looking forward to hearing from you soon.

Sincerely,

José Dilermando Andrade Filho

Additional Editor Comments (if provided): Please address all the concerns raised by 3 reviewers. In addition, I request: 1) to extend the introduction on trypanosomatids and discuss the following publications: Maslov et al, 2019 (Parasitology) and Lukes et al, 2018 (Trends Prasitol). 2) to spell all species names in Italic; iii) fix the references; iv) if you cite ref 60, please cite an accompanying paper- Kostygov et al, 2019 (Trop Med Int Health).

Authors’ comment: We accept all your suggestions. The introduction has been improved with the mentioned papers; all species names and the references have been revised as well. Information about the paper Kostygov et al. 2019 was also added in the manuscript (now lines 58-59).

Authors’ comment: We totally agree with your comment. All data about the relationship between seasonal variation and sand fly abundance have been removed.

4 trypanosomatid species or trypanosomatids

Authors’ comment: The title has been changed (now line 3).

32 HP – meaning

Authors’ comment: The sentence has been changed (now line 20-22).

33 better trap/nights (to demonstrate the effort)

Authors’ comment: The sentence has been changed (now lines 20-22).

39-40 how many individuals and how many in pools (BTW, pools of 1 specimen is not a pool !!!) (Of a total of 4,913 females, 47 were individually tested and 4,866 grouped in 311 pools).

Authors’ comment: The sentence has been changed (now lines 20-24).

Authors’ comment: The sentence has been changed (now lines 28-30).

44-45 same type of result presentation as in the case of Leishmania spp. – it means: how many pools/individuals and in which sand fly species.

Authors’ comment: The sentence has been changed (now lines 27-33).

45 The presence of DNA is not proving of vector capacity! (… may be involved in the transmission of Le. amazonensis … etc. is much more accurate)

Authors’ comment: The sentence has been changed (now line 34-36).

55-57 REFs 1-3 are more-or-less relevant, but there are several current reviews, e.g., Akhoundi et al. 2016 (PLoS Neglected Tropical Diseases 10(3): e0004349.), etc… that would fit here.

Authors’ comment: The references have been changed (now lines 43-46).

59 REF 4 – ok, but add also e.g., Rangel, Shaw (2018) Brazilian Sand Flies … (10.1007/978-3-319-75544-1) etc.

Authors’ comment: The reference has been added (now line 49).

71-72 The genus Trypanosoma is missing! Sand flies are possible vectors of several trypanosome species – add a relevant reference(s) (the sentence “Several authors have reported the presence of non-Leishmania trypanosomatids DNA in sand flies by means of molecular techniques [12, 13, 14, 15]” does not cover this issue clear enough)

Authors’ comment: The sentence has been changed and references covering this issue have been added (now lines 50-70).

73 REFs 10 and 11 – OK, but add some references on Leptomonas (e.g. Kraeva et al. 2015 (PLoS Pathog 11(8):e1005127)); and even more references would be appropriate here

Authors’ comment: The reference was added (now line 70).

71-73 differentiate between monoxenous and dixenous genera

Authors’ comment: This information has been added (now lines 50-53).

Authors’ comment: The paragraph has been deeply revised. Now, we believe that the background context that we provided make clear our goal (now lines 50-70).

Authors’ comment: We agree with your comment. The automatic light trap (model HP) is an improved model of suction light trap for the capture of small insects, similar to CDC light traps. However, now in the manuscript text, we referred to those traps as “automatic light traps (model HP)” and not CDC light traps (model HP). This information could be check in the lines 99-101.

125-6 not clear – it means that the traps run three nights (and two days)?

Sand flies were still alive (after three days???), How many sand flies survive till laboratory (approximately in % ???)

Authors’ comment: The sentence has been improved (now lines 99-102). Many sand flies still alive after three days, but we could not provide the percentage since it was not evaluated. Furthermore, to carry out molecular techniques, living sand flies is unnecessary.

Authors’ comment: The reference Galati (2018) is a book chapter including updates of Galati (2003). In the previous review, the changing of the oldest reference for a newest one was suggested, especially because it is a Portuguese version, so we did it. Even so, we rewrite the sentence highlighting this information (now lines 110-111).

158 … of trypanosomatids (not Leishmania)

Authors’ comment: The sentence was been changed (now line 116).

247 – the prevalence is right, but anyway it should be explained that the authors predict that only one specimen per pool was infected… (it corresponds with the low “pool” prevalence)

Authors’ comment: We totally agree with your comment. However, in the new version we presented the prevalence per positive pool (now lines 183-198).

253/255 – why “it was not possible get to species level”? - due to the low quality of the sequences ??? or the sequences represent some new leishmania species ??? It must be explained!

Authors’ comment: All sequences without species level identification presented low quality, containing high number of ambiguous sites. We stated this information in the lines 185-188.

Authors’ comment: We observed that the non-Leishmania parasites shown amplicons of 400-500 pb, distinct to predicted for Leishmania parasites (300-350pb). And this fact, led us to investigate what was amplified. There was no double-bands/mixed infections. However, the sentence has been changed (192-195).

260 - … three trypanosomatids … - must be clearly distinguished even in Tab. 3 – e.g., Herpetomonas sp. 1, Herpetomonas sp. 2 …

Authors’ comment: The sentence has been changed (now lines 193-195).

Authors’ comment: The sentence was been changed (now lines 192-198). As the sequences have aligned with “Herpetomonas sp.” and “Crithidia sp.” on Blast tool, we are not sure about the presence of more than one species within both genera of trypanosomatids. So, we decide to keep “sp.” referring at least one species of each genera.

Table 3 – sort it according to “Host” or according to “trypanosomatid parasites!, not according Acc.Nos., it makes no sense.

Authors’ comment: We accepted your suggestion. The table 3 has been changed according hosts.

Seq ID Blast – the different unnamed Herpetomonas/Crithidia/Leishmania species (identified by BLAST) must be specified (by its voucher name/number etc., or even by their GB Acc.No.)!

Authors’ comment: We are not sure about your inquiry, since all species identified by Blast tool have already been identified by their accession numbers, as presented in Table 3.

Authors’ comment: We accept your suggestion. We add more international references when it was possible, especially in the first half of the introduction. However, for several aspects about sand fly fauna is desirable to use national references.

Line 55: Ref. 2 (Alkan et al) is not an optimal reference. Would be better to include the famous review by Maroli et al. 2013, see the pdf.

Authors’ comment: The reference has been added (now line 43-46).

Line 57: Ref. 3 (Lestinova et al) is not suitable reference. Again, the review by Michele Maroli could be used.

Authors’ comment: The sentence has been changed (now line 46).

Authors’ comment: The reference has been added (now line 49).

Lines 67-70: Please use some more recent reference. All this information is described in details by Jeffrey Shaw in the review by Dvorak et al.

Authors’ comment: The reference has been added (now line 49).

Authors’ comment: Discussion about Mg. migonei has been improved (now lines 230-238).

Finally, I suggest shorten the “Short title” as it is too long now.

Authors’ comment: The short title has been reviewed (now line 1).

For more details and some other minor corrections of typos, see the pdf file with my comments.

Authors’ comment: All suggestions marked in pdf file have been accepted.

Reviewer #3: I gather that it is difficult to write a readable paper out of huge but relatively boring data, but the authors still shall try to do a better job. In general, the authors describe frequently and extensively things that can be found on Wikipedia (geography, how many people live in some district - the number is non-scientific because at the time of writing it was already incorrect), please remove all this, which unncessarily extends the manuscript and brings absolutely no novelty and has no relevance to the presented data (where is a correlation between the size of the district and phlebotomine sandflies?).

Authors’ comment: We accepted your suggestion. We did a deeply revision in the manuscript file.

I would like to ask the authors to better balance the Results and the Discussion, the first being too short and the latter too long.

Authors’ comment: We accepted your suggestion. We did a deeply revision in the manuscript file.

Authors’ comment: We accepted your suggestion. We did a deeply revision in the discussion section.

Authors’ comment: We totally disagree with your comment. With all due respect, we do not understand how 2-3 lines of acknowledgments are quite boring. This is a personal section and we thank everyone who we would like. About gel electrophoresis, the most used staining for sure is EtBr however, it is not the only one. Alternative DNA dyes, such as SYBR Safe is also currently used. So, we decide to keep this information because it is not trivial, and it is not a "South American stretching" as you said. However, as we said in the last inquiries, we carefully revised the manuscript and we hope this version stated better our goal.

Authors’ comment: The discussion section has been improved. Now, we discuss about the finding of non-Leishmania trypanosomatids (now lines 252-261).

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PLoS One. doi: 10.1371/journal.pone.0234445.r006

Decision Letter 2

Vyacheslav Yurchenko

19 May 2020

PONE-D-20-05836R2

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

PLOS ONE

Dear Dr. Andrade Filho,

We would appreciate receiving your revised manuscript by Jul 03 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Vyacheslav Yurchenko

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please address the final comments.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: N/A

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: I am more-or less satisfied with the current version, but I do not fully agree with one specific explanation.

Based on the sequences, which are already available in the GenBank database, it is clear that the sequences of monoxenous trypanosomatids differ from each other (within the three Herpetomonas spp. as well as between the two Crithidia spp.). Therefore, this information should be mentioned somewhere in the text, i.e., that the found Herpetomonas spp. and Crithidia spp. probably represent three and two, respectively, species, or at least different populations (or the Molecular Operational Taxonomic Unit; MOTU, could be used in this case), to make it clear that these sequences do not represent the same herpetomonas or Crithidia species. And, accordingly, Herpetomonas spp. and Crithidia spp. must be used in the text.

I would also recommend changing the sentence in the abstract

Instead of

“In seven pools of Ny. whitmani and in one pool of Lu. longipalpis the Leishmania species was not determined due to the low quality of the sequences.”

change like:

“In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for leishmania DNA, the parasite species was not determined due to the low quality of the sequences.”

Reviewer #3: I find the way the authors responded acceptable and consider the paper much improved. I would still have some issues with the length of acknowledgements (theoretically you can have it as long as the discussion, there is no official limit, but it is simply not good), the way the gel is stained is irrelevant - it was stained somehow and that's all that matters, but I can live with the present version.

**********

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Reviewer #1: No

Reviewer #3: No

PLoS One. 2020 Jun 24;15(6):e0234445. doi: 10.1371/journal.pone.0234445.r007

Author response to Decision Letter 2

20 May 2020

Manuscript ID: PONE-D-20-05836R1

Dear Dr. Yurchenko,

Dear Editor,

below is our response to the reviewers' comments.

We are available for clarification.

Thank you

Sincerely,

José Dilermando Andrade Filho

Reviewer #1: I am more-or less satisfied with the current version, but I do not fully agree with one specific explanation.

I would also recommend changing the sentence in the abstract

Instead of

“In seven pools of Ny. whitmani and in one pool of Lu. longipalpis the Leishmania species was not determined due to the low quality of the sequences.”

change like:

“In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for leishmania DNA, the parasite species was not determined due to the low quality of the sequences.”

We agree with your comment. It is possible that are more than one monoxenous trypanosomatids within our molecular detection. However, based on the small fragment (~400 pb) it is not of the sequence it is not wise to assume this fact. We hope in the future we are able to isolate these trypanosomatids and confirm their identity as species level. We include the phrase in results "These five non-Leishmania sequences found in our study, Herpetomonas spp. and Crithidia spp., are sufficiently different between them and possibly represent two and three, respectively, different MOTUs (Molecular Operational Taxonomic Units)." In the discussion we include “Possibly, more than one species within each genera were detected, however, due to the small length of the amplicon it was not possible to confirm it. On the other hand, the molecular detections presented here, may represented different populations (different MOTUs), reinforcing the importance of further studies on these trypanosomatids.”

We changed that phrase in the abstract “In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for leishmania DNA, the parasite species was not determined due to the low quality of the sequences.”

We thank the reviewer for the comments

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PLoS One. doi: 10.1371/journal.pone.0234445.r008

Decision Letter 3

Vyacheslav Yurchenko

21 May 2020

PONE-D-20-05836R3

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

PLOS ONE

Dear Dr. Andrade Filho,

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

Please submit your revised manuscript by Jul 05 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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Vyacheslav Yurchenko

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Thanks for addressing all the comments.

A few final notes on formalities:

1) Make sure all species and generic names are in Italic (they are not in References)

2) Why all words in some titles in References are capitalized (f.e. 44, 52, and others)? Please fix.

3) Please check diacritics.

I am ready to accept the manuscript when these are corrected.

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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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jun 24;15(6):e0234445. doi: 10.1371/journal.pone.0234445.r009

Author response to Decision Letter 3

22 May 2020

Manuscript ID: PONE-D-20-05836R1

Dear Dr. Yurchenko,

We have reviewed all of your requests.

All references have been fixed. Now, the generic and species names are in italic; we have also removed the capitalized words in some reference titles and diacritics have been reviewed.

We are confident that the new version of the manuscript is greatly improved.

Looking forward to hearing from you soon.

José Dilermando Andrade Filho

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PLoS One. doi: 10.1371/journal.pone.0234445.r010

Decision Letter 4

Vyacheslav Yurchenko

27 May 2020

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

PONE-D-20-05836R4

Dear Dr. Andrade Filho,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0234445.r011

Acceptance letter

Vyacheslav Yurchenko

9 Jun 2020

PONE-D-20-05836R4

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

Dear Dr. Andrade-Filho:

I'm 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.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

Data are available within the manuscript.

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PERMALINK

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil

Aline Tanure

Felipe Dutra Rêgo

Gabriel Barbosa Tonelli

Aldenise Martins Campos

Paloma Helena Fernandes Shimabukuro

Célia Maria Ferreira Gontijo

Gustavo Fontes Paz

José Dilermando Andrade-Filho

Roles

Abstract

Introduction

Material and methods

Ethic statements

Study area, collection and identification of phlebotomine sand flies

Fig 1. Location of the study area in the district of Casa Branca, Brumadinho, Minas Gerais, Brazil.

DNA extraction and identification of trypanosomatids

Statistical analyses

Results

Sand fly fauna

Table 1. Sand flies collected according to month at the Casa Branca, Brumadinho, Minas Gerais, from May 2013 to July 2014.

Fig 2. Standardized index of species abundance of species collected at Casa Branca, Brumadinho, Minas Gerais from May 2013 to July 2014.

Molecular identification of trypanosomatids

Table 2. Sand fly organized by species that were analyzed individually and in pools collected in Casa Branca, Brumadinho, Minas Gerais, from May 2013 to May 2014.

Table 3. Results of DNA detection and identification of species of Trypanosomatidae according to species of sand fly, number of pool and collection date in Casa Branca, Brumadinho from May 2013 to May 2014.

Discussion

Conclusion

Acknowledgments

Data Availability

Funding Statement

References

Author response to previous submission

Transfer Alert

Decision Letter 0

Vyacheslav Yurchenko

Roles

Author response to Decision Letter 0

Decision Letter 1

Vyacheslav Yurchenko

Roles

Author response to Decision Letter 1

Decision Letter 2

Vyacheslav Yurchenko

Roles

Author response to Decision Letter 2

Decision Letter 3

Vyacheslav Yurchenko

Roles

Author response to Decision Letter 3

Decision Letter 4

Vyacheslav Yurchenko

Roles

Acceptance letter

Vyacheslav Yurchenko

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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