Entomological surveillance of mosquitoes (Diptera: Culicidae), vectors of arboviruses, in an ecotourism park in Cachoeiras de Macacu, state of Rio de Janeiro-RJ, Brazil.

Arboviruses are arthropod-dependent viruses to complete their zoonotic cycle. Among the transmitting arthropods, culicids stand out, which participate in the cycle of several arboviruses that can affect humans. The present study aimed to identify species of culicidae and to point out the risk of circulation, emergency, or reemergence of pathogenic arboviruses to humans in the region of the Jequitibá headquarters of the Parque Estadual dos Três Picos (PETP), in Cachoeiras de Macacu, state of Rio de Janeiro, Brazil. Sampling was carried out at five Sample Points (SP) demarcated on trails from the headquarters, with CDC light traps, HP model with dry ice attached to the side, for 48 hours of activity each month. Additionally, active catches were made with a castro catcher in the period of one hour per day in the field, from six to eleven o'clock in the morning, in each PM. After the captures, thematic map was assembled using the ArcGIS 10 software and performing a multidimensional scaling (MDS). A total of 1151 specimens were captured and the presence of culicids already incriminated as vectors of arboviruses circulating in the region was observed: Aedes fluviatilis Lutz, 1904 (71 specimens); Aedes scapularis Rondani, 1848 (55 specimens); Haemagogus leococelaenus Dyar and Shannon, 1924 (29 specimens). In addition to the subgenus Culex (culex) spp. (163 specimens). In this sense, we highlight the importance of strengthening the actions of continuous entomological surveillance of the emergence and re-emergence of new arboviruses in ecotourism visitation parks.

Introduction

Mosquitoes have a cosmopolitan distribution and are vectors of human and animal pathogens of global occurrence. In Brazil, they are incriminated as transmitters of four main arboviruses with mandatory notification in: dengue, zika, chikungunya and yellow fever [1-3]. Among the culicids already incriminated as transmitters of arboviruses to man, in urban and peri-urban areas, mosquitoes belonging to the genus Aedes stands out, in view of the direct incrimination of the species Aedes aegypti Linnaeus, 1762 and Aedes albopictus Skuse, 1894 as transmitters of these mandatory reporting arboviruses [4-7]

According to the epidemiological bulletin of arboviroses N°001 / 2018, the incidence rates of probable cases per 100 thousand inhabitants, for dengue, zika and chukungunya decreased, when comparing the years 2016 with the year 2017 in the state of Rio de Janeiro [8]. The yellow fever virus, on the other hand, showed great highlights between the years 2016 to 2017, with the expressive increase of cases and the occurrence in places where for a long time it was not notified [9, 10]. In the state of Rio de Janeiro there were 779 confirmed cases and 262 deaths for the period from July / 2016 to June / 2017 according to Epidemiological Report 084/2017 [11]. Presenting in the period from 2017 to 2018 a dispersion along the Brazilian east coast of the Atlantic Forest biome, where the characteristics of the fauna of vectors and primates favored the dispersion of the virus where there were no cases recorded for decades [12].

In nature, arboviruses maintain a complete and restricted zoonotic cycle between wild mosquitoes and wild animals [1, 2]. However, there may be human insertion in this dynamic due to exposure caused by anthropic changes in the environment, the frequency in protected areas for ecotourism activities or other profit-making activities, and even the presence of homes, providing contact with females of infected wild mosquitoes. In this contact with the wild cycles, man can become infected, becoming a source of infection for urban mosquitoes, favoring the emergence of urban cycles of these diseases [2].

Understanding the ecology of vector mosquito species is of paramount importance for later understand the dynamics of the transmission of these arboviruses to man [13]. In addition, the identification of culicidae fauna in ecotourism regions allows speculation about the possible transmission cycles that may occur in these areas or even be introduced or reintroduced, and to seek alternatives for the prevention of the emergence of these diseases to the local and visiting human community.

Thus, the present study aims to understand the distribution of the fauna of culecidae present in the Três Picos State Park (PETP), located in the municipality of Cachoeiras de Macacu, state of Rio de Janeiro. To assess the possibility of circulation of some of these arboviruses within the limits of the PETP headquarters, in addition to alerting to the possible introductions or reemergence of any new arboviruses due to the presence of their possible incriminated vector.

Methods

The study was carried out at the Jequitibá headquarters of the Três Picos State Park, located in Cachoeiras de Macacu under the collection authorization (N° 058/2015) of the State Environmental Institute (INEA).

i. Study area

The park consists of a fragment of Atlantic Forest, characterized by a dense rain forest and a tropical climate [14]. For the present study, five different Sample Points (SP) were demarcated within the limits of the Jequitibá headquarters, intended for the practice of ecotourism activities, being designated as: Parque Trail (SP 1): S: 22°24.834 ’HO: 42°36 .825 ’; Cristáis Trail (SP 2): S: 22° 24,964 ‘HO: 42° 36,543’; Jequitibá trail (SP 3): S: 22° 25.067 ‘HO: 42° 36.610’; Lookout trail (SP 4): S: 22° 25.067 ‘HO: 42° 36.610’; Visitor trail (SP 5): S: 22° 25.067 ‘HO: 42° 36.610’ (Fig 1).

Fig 1

Sample Points in the area of the Três Picos State Park (PETP) Cachoeiras municipality of Macacu, state of Rio De Janeiro.

Where (EM1) monitoring station 1, (EM2) monitoring station 2, (EM3) monitoring station 3, (EM4) monitoring station 4 and (EM5) monitoring station 5. Geographical meshes of the Brazilian Open Data Portal and a map created by the main author Thamiris Balthazar.

The collections were divided into two climatic periods: dry period and rainy period, following the distribution of months proposed by Minuzzi et al. [15]. In which the first encompassed the months of March to August 2017 and the rainy period covered the months of November 2016 to February 2017, together with the months of September and October 2017. Thus, eight collection campaigns were carried out, with a durability of two consecutive months, and an interval of one month between each one.

ii. Capture methods

The specimens were captured with the aid of light traps type CDC, model HP, adapted with the addition of a styrofoam apparatus containing dry ice, attached to the side of the trap. In this way, the elimination of CO2 from the apparatus worked as an additional attraction. The CDCs were installed in each of the determined sample points, at a height of 1.5 meters from the ground, and kept running in 2 cycles of 24 hours per field campaign, where at the end of each cycle the cages were changed. In addition, active captures were made with the help of a Castro catcher during the period of 1 hour at each demarcated sampling point of collection. Totaling 58 hours of sampling effort per field campaign.

iii. Taxonomic identification

All specimens were preserved at low temperatures for proper assembly on entomological pins, identification, and storage in the entomological collection of the Diptera Laboratory, Oswaldo Cruz Institute—IOC. And, the identification of the species was carried out through direct observation of the morphological characters evidenced by the stereomicroscope microscope (Zeiss®), and using dichotomous keys elaborated by Lane [16, 17], Faran & Linthicum [18], Consoli & Lourenço-de -Oliveira [19] and Forattini [20].

iv. Spatial analysis

To assist in visualizing the distribution of the collected specimens, thematic maps were generated. Therefore, forest coverage rates were estimated manually using ArcGIS 10 software. A Landsat 7 satellite image (from 2002) obtained from the United States Geological Survey platform "LandsatLook" (http://landsatlook.usgs.gov/viewer.html) was used to outline the forest cover polygons. The image resolution was 30 meters for bands 1 to 7 and 15 meters for band eight, with the strips merged resulting in a final image of 15 meters in resolution. For the composition of mosquito communities it was evaluated using a multidimensional scale (MDS). MDS is a method to measure the similarity between data sets, which in this study refers to the composition of Culicidae populations (data sets) in each sampling unit [21, 22]. For the structuring and analysis of the databases, the programs Microsoft Excel and SPSS 23 were used.

Results

At the end of the collections, a total of 1149 specimens were captured in their adult form, with 840 specimens collected in the rainy season (Table 1) and 309 specimens in the dry period (Table 2). Eight genera were identified: Culex (210 specimens); Aedes (163 specimens); Haemagogus (32 specimens); Limatus (338 specimens) Runchomyia (52 specimens); Trichoprosopon (165 specimens); Wyeomyia (166 specimens); Anopheles (17 specimens); Uranotaenia (6 specimens); Sabethes (2 specimens). Noteworthy are the most frequent species, among those considered of medical and veterinary importance: Aedes fluviatilis Lutz, 1904 (71 specimens); Aedes scapularis Rondani, 1848 (55 specimens); Haemagogus leococelaenus Dyar and Shannon, 1924 (29 specimens). In addition to the subgenus Culex (culex) spp. (163 specimens);

Table 1

Total specimens collected by Sample Point (SP) during the rainy period.

	Rainy season
	SP 1	SP 2	SP 3	SP 4	SP 5	TOTAL
Culex (carrollia) spp.	0	1	5	0	3	9
Culex (culex) spp.	13	2	40	68	36	159
Culex (Lutzia) spp.	10	3	4	1	5	23
Culex (melanoconion) spp.	0	2	2	0	0	4
Culex (microculex) spp.	1	0	0	2	6	9
Aedes (Stegomyia) albopictus	6	0	0	0	1	7
Aedes (Georgecraigius) fluviatilis	1	2	15	26	23	67
Aedes (Ochlerotatus) rhyacophilus	3	0	0	1	0	4
Aedes (Ochlerotatus) scapularis	1	5	1	43	4	54
Aedes (Ochlerotatus) serratus	3	1	0	1	0	5
Aedes (Protomacleaya) terrens	0	2	4	2	3	11
Haemagogus janthinomys/capricornii	0	0	0	1	2	3
Haemagogus leucocelaenus	1	1	5	13	6	26
Sabethes (sabethes) chloropterus	0	0	1	0	0	1
Sabethes (sabethes) belisarioi	0	0	1	0	0	1
Limatus durhamii	25	38	16	41	25	145
Limatus flavisetosus	9	10	5	10	8	42
Limatus paraensis	2	10	1	11	7	31
Runchomyia frontosus	2	0	2	5	5	14
Runchomyia lunatus	0	0	10	2	4	16
Runchomyia reversus	0	1	0	3	0	4
Trichoprosopon pallidiventer	0	0	1	0	1	2
Trichoprosopon digitatum digitatum	0	3	2	9	6	20
Trichoprosopon digitatum townsendi	0	0	0	0	0	0
Trichoprosopon obscurum	2	3	3	2	3	13
Trichoprosopon soaresi	4	12	15	18	18	67
Uranotaenia calosomata	1	0	0	0	4	5
Wyeomyia (phoniomyia) davisi	0	0	0	0	0	0
Wyeomyia aporonoma	1	0	0	3	5	9
Wyeomyia argenteorostris	0	1	13	5	8	27
Wyeomyia celaenocephala	0	0	0	0	1	1
Wyeomyia confusa	0	0	1	1	0	2
Wyeomyia flavifascies	1	0	0	2	0	3
Wyeomyia negrensis	2	1	16	9	13	41
Wyeomyia serratoria	0	0	0	1	0	1
Wyeomyia sp.	0	1	0	0	0	1
Anopheles (kerteszia) cruzii	0	4	4	0	7	15
Total:	88	103	165	280	204	840

Table 2

Total specimens collected by Sample Point (SP) during the dry period.

	Dry season
	SP 1	SP 2	SP 3	SP 4	SP 5	TOTAL
Culex (carrollia) spp.	0	0	0	1	0	1
Culex (culex) spp.	0	1	0	3	0	4
Culex (Lutzia) spp.	0	0	0	1	0	1
Culex (melanoconion) spp.	0	0	0	0	0	0
Culex (microculex) spp.	0	0	0	0	0	0
Aedes (Stegomyia) albopictus	2	0	0	0	1	3
Aedes (Georgecraigius)fluviatilis	0	1	1	2	0	4
Aedes (Ochlerotatus) rhyacophilus	0	0	0	0	0	0
Aedes (Ochlerotatus) scapularis	0	0	0	1	0	1
Aedes (Ochlerotatus) serratus	0	0	0	0	0	0
Aedes (Protomacleaya) terrens	0	0	4	3	0	7
Haemagogus janthinomys/capricornii	0	0	0	0	0	0
Haemagogus leucocelaenus	0	0	0	3	0	3
Sabethes (sabethes) chloropterus	0	0	0	0	0	0
Sabethes (sabethes) belisarioi	0	0	0	0	0	0
Limatus durhamii	19	7	10	4	2	42
Limatus flavisetosus	0	0	10	3	0	13
Limatus paraensis	13	4	40	6	2	65
Runchomyia frontosus	0	0	1	2	1	4
Runchomyia lunatus	0	4	2	1	0	7
Runchomyia reversus	0	0	0	6	1	7
Trichoprosopon pallidiventer	0	0	1	0	0	1
Trichoprosopon digitatum digitatum	0	0	4	7	2	13
Trichoprosopon digitatum townsendi	0	0	1	0	0	1
Trichoprosopon obscurum	0	0	4	1	0	5
Trichoprosopon soaresi	1	1	28	9	4	43
Uranotaenia calosomata	1	0	0	0	0	1
Wyeomyia (phoniomyia) davisi	0	3	0	10	0	13
Wyeomyia aporonoma	0	1	4	1	0	6
Wyeomyia argenteorostris	1	0	12	2	2	17
Wyeomyia celaenocephala	0	0	0	0	0	0
Wyeomyia confusa	1	0	10	2	0	13
Wyeomyia flavifascies	0	0	5	0	0	5
Wyeomyia negrensis	0	1	14	4	3	22
Wyeomyia serratoria	0	0	2	1	2	5
Wyeomyia sp.	0	0	0	0	0	0
Anopheles (kerteszia) cruzii	0	0	2	0	0	2
Total:	38	23	155	73	20	309

Two thematic maps were made, one for the rainy season (Fig 2) and another for the dry period (Fig 3) where it is possible to visualize the distribution of species of medical and veterinary interest in each MS, and these were correlated with the arboviruses of according to the literature.

Fig 2

Thematic map with the distribution of total species collected at each Sample Point during the rainy period.

Landsat 7 satellite image (from 2002) obtained from the United States Geological Survey platform "LandsatLook".

Fig 3

Thematic map with the distribution of total species collected at each Sample Point during the dry period.

Landsat 7 satellite image (from 2002) obtained from the United States Geological Survey platform "LandsatLook".

Among the species captured during the study, Limatus durhamii Theobald, 1901 was present in all SP in both climatic periods, totaling 145 specimens in the rainy season and 42 specimens in the dry period. The subgenus Culex (culex) spp was widely distributed throughout SP, in different proportions, during the rainy season, with 159 specimens in the total period (Fig 2; Table 1), however it was more sporadic for the dry period, which was present only in SP2 (1 specimen) and SP 4 (3 specimens).

Among the species belonging to the genus Aedes, Aedes terrens Walker, 1856 was identified comprising 1.31% (11 specimens) of the culicidian fauna of the PETP headquarters in the rainy season and 2.26% (7 specimens) in the dry season. In addition, other species of this genus have also been captured, such as, Ae. fluviatilis, with 67 specimens in the rainy season and only 4 in the dry season; Aedes rhyacophilus Costa-Lima, 1933 with only 4 specimens in the rainy season and Aedes serratus Theobald, 1901, also present only in the rainy season with 5 specimens.

Another species of medical and epidemiological importance present in the study was Ae. albopictus, captured in the two SPs with the greatest anthropic changes, these being SP1 and SP5, totaling 7 specimens in the rainy season and 3 specimens in the dry season. Aedes scapularis showed a high density, which was identified in the Mirante trail (SP4) during the rainy season, 43 captured adults. This species was present in this rainy period, albeit in a low proportion, in the SP2 (Cristáis Trail) with 5 captured specimens, the visitor’s trail (SP5) with 4 captured specimens, the Parque Trail (SP1) with only 1 specimen and the Jequitibá Trail (SP3) also with only one specimen collected at the site, throughout the rainy season. During the dry period, Ae. scapularis was captured only in SP4 (Lookout Trail).

We can also highlight two important species incriminated as vectors, Sabethes chloropterus Von Humboldt, 1819 and Sabethes belisarioi Neiva, 1908 collected in SP3 (Jequitibá Trail) during the rainy season, where only one specimen was collected decade. In addition to species of the Haemagogus genus, such as Haemagogus leucocelaenus Dyar and Shannon, 1924, which showed a density of 2.5% (29 specimens) of the total specimens collected in their adult form, in which 26 specimens of this species were captured during the period rainy season, and 3 caught in the dry season. And the species Haemagogus janthinomys Dyar, 1921, with only 3 specimens captured in the rainy season.

Discussion

In a recent study, Abreu et al. (2019) confirmed the incrimination of the species Haemagogus janthinomys and Haemagogus leucocelaenus in the recent cases of yellow fever that occurred in the reemergence of the disease in the Atlantic Forest regions in southeastern Brazil. In this same study, the species Sabethes chloropterus and Aedes scapularis were incriminated as secondary vectors or local primary vectors of yellow fever [23]. In the present study, we point out the importance of constant entomological surveillance in areas of natural protection, such as PETP, which have ecotourism activities, considering that we observe the presence of the species Haemagogus leucocelaenus and Haemagogus janthinomys, in addition to the capture of a Sabethes chloropterus and Sabethes belisarioi.

Aedes scapularis was found with significant density on the trails, in the two collection periods. According to the literature, among the arboviruses in which this vector is incriminated, Forattini et al. [24-26] pointed out in their studies a strong correlation between this mosquito and the Rócio virus, a Flavivirus of great importance for the risk of reemergence in Brazil [27], in addition to its association with the Venezuelan equine encephalitis virus [28, 29].

Among the species of the genus Aedes, we highlight Ae. terrens, which has great importance in the transmission of arboviroses circulating in Brazil, with emphasis on its vectorial capacity for the Chikungunya virus, for example [30, 31]. Ae. fluviatilis, Ae. rhyacophilus and Ae. serratus that have also been observed, have already been incriminated by other authors as vectors or possible vectors of most arboviruses of the Togaviridae family and Flaviviridae family, in addition to some arboviruses belonging to the Bunyaviridae, Reoviridae and Rhabdoviridae families [32].

The study records the presence of Ae. albopictus, which is already known for its ability to be incorporated into urban and peri-urban environments, which are increasingly present, being this species of great vector importance incriminated for viruses already circulating around the park, such as Zika viruses, Dengue viruses and viruses. Chikungunya [4, 5]

In view of this, the presence of these vectors warns of the possible risk of circulation of certain arboviruses within the limits of PETP, since in the municipality of Cachoeiras de Macacu confirmed cases of arboviruses with compulsory notification in Brazil, as stated in the Epidemiological Report 045/2017 (SUS, 2017). From January to November 2017, the period in which the present study was being carried out, in parallel to these epidemiological surveys, the municipality of Cachoeira de Macacu presented 1 case of yellow fever notified on 6/2/2017, according to the epidemiological report 045/2017 [33], where species of the subgenus Sabethes (Sabethes) spp. captured in September, three months after this data. These factors point to the ability of this arbovirus to circulate in the PETP area, since the vector and its respective viral agent are present in this region.

Other arboviruses were also confirmed to be circulating in the municipality of Cachoeiras de Macacu, such as the Dengue virus, with an incidence of 5.4 cases per 1000 inhabitants, only two of which were confirmed; and chikungunya virus with an incidence of 1.8 cases per 1000 inhabitants, but without any confirmation [33]. The presence of the circulation of the Zika virus in the municipality was not observed, since, according to the epidemiological bulletin [33] in this period, the incidence of cases presented was zero. However, the presence of the vector and the presence of cases in neighboring municipalities warn of possible underreported circulation.

Limatus duhramii was found more frequently and in all SP, indicating a risk of introducing the Orthobunyavirus virus, since it has been incriminated in previous studies as a species of suspected transmission of this virus [34]. However, this evidence would require further studies on the vectorial capacity of this species.

In addition, the presence of species of the subgenus Culex (culex) spp. also, points to the possibility of introducing other arboviruses, since this subgenus encompasses important species of mosquitoes already incriminated as vectors of several arboviruses in the world, such as Japanese Encephalitis in Southeast Asia [35], West Nile Virus in South Africa [36] and viruses of the family Bunyaviridae already isolated [37]. Within this subgenus is Culex (Culex) quinquefasciatus Say, 1923, which is described with evidence and vector capacity for the Zika [38, 39]. However, a study carried out with mosquitoes of this species in the city of Rio de Janeiro did not show the same transmission capacity as Zika virus and proved that Zika virus does not replicate in mosquitoes of the species Cx (Cux) quinquefasciatus [40, 41]. Thus, these studies refute the hypothesis that this species is a vector of Zika virus.

The presence of incriminated or suspicious vectors in the transmission of arboviruses that have not yet been reported or considered to have been eradicated in Brazil, raises hypotheses of possible introduction, or reintroduction of these viruses, through the frequency of visitors from different regions of the world in areas of natural protection, as observed at PETP. In addition, with the notification of arboviruses already circulating in the municipality of study and the meeting of the respective vector species, they alert to the fact that PETP is a place of possible active circulation of mandatory notification arboviruses: dengue, zika, chikungunya and yellow fever.

Thus, the importance of strengthening entomological surveillance actions is highlighted. And, in addition, it is important to carry out health education actions to teach preventive measures to the local population and visitors to the PETP, aiming at blocking the circulation of arboviruses already present in the local human population, in addition to avoiding the insertion of new arboviruses in this population. Being the ecotourism visitation parks characterized as important regions for the continuous surveillance for the emergence and reemergence of new arboviruses.

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23 Jun 2021

PONE-D-21-16082

Entomological surveillance of mosquitoes (Diptera: Culicidae), vectors of arboviruses, in an ecotourism park in Cachoeiras de Macacu, state of Rio de Janeiro-RJ, Brazil.

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1. 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

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

Reviewer #1: Yes

Reviewer #2: N/A

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3. 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

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4. 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

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5. 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: overall it is a simple but useful paper dealing with field research and interesting results on Yellow Fever and other important arboviruses. Since data were collected in Brazil, specifically in an eco-touristic park, it is always a surveillance monitor or geographical point of their movement or seasonal activity. Individually, most of culicidae species found are importants, but to find all of them in a small geographical area is really scary for public health reasons!! I would realy like to have lab virological study of the collected mosquitoes. I would not be surpraised findings of Yellow Fever, Dengue or other lethal pathogens!

Reviewer #2: The genesis of this manuscript is to emphasis the importance of entomological surveillance of mosquitoes in sylvatic habitats with high incursion of humans such as Natural Parks located in Atlantic Forest. This manuscript does an excellent job demonstrating significant of mosquito species surveillance in this sylvatic habitat.

The title and abstract are appropriate for the content of the text. Furthermore, the article is well constructed, the surveillances were well conducted, and analysis was well performed. However strong conclusions such as “favor the circulation of different arbovirus” (line 37 and 38 in the abstract) are not warranted, since no direct viral surveillance was carried out in the study were presented.

Thus I suggested some minor alterations to the manuscript:

Line 37 “favor the circulation of different arbovirus” This conclusion is not warranted by the study since no viral surveillance as performed. Please amend the conclusions to scope of the results presented.

Line 129 “All specimens were sacrificed”, I think that the word preserved will be more appropriated than sacrificed.

Line 269 Culex quinquefasciatus and Zika virus transmission, the authors should also mention the others published studies suggesting that Culex quinquefasciatus do not transmit Zika virus.

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Reviewer #1: No

Reviewer #2: No

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9 Oct 2021

Figures 1, 2 and 3 were produced by lead author Thamiris Balthazar, using layers of open and public databases: Brazilian Portal for Open Data (https://dados.gov.br/dataset/malha-geometrica-dos-municipios -Brazilians) and the United States Geological Survey's "LandsatLook" platform (http://landsatlook.usgs.gov/viewer.html).

There was a change in the order of the figures. Thus, the legend named figure 3 referred to figure 2, and the legend in figure 4 referred to figure 3. Therefore, there is no fourth figure to be inserted in this article.

In line 37, the conclusion was changed, pointing to the importance of entomological surveillance in ecotourism parks.

On line 133, the word "sacrificed" has been replaced by the word "preserved".

In line 279, a continuation of the paragraph was included pointing to the articles that refute the vectorial capacity of Culex quinquefasciatus in Zika virus transmission.

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

29 Nov 2021

Entomological surveillance of mosquitoes (Diptera: Culicidae), vectors of arboviruses, in an ecotourism park in Cachoeiras de Macacu, state of Rio de Janeiro-RJ, Brazil.

PONE-D-21-16082R1

Dear Dr. Balthazar,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Humberto Lanz-Mendoza

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: All comments have been addressed

Reviewer #2: All comments have been addressed

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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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: It is very amazing the amount of mosquito species found during the sampling period. Just with the reports of Sabethes species is enough to alert park visitors to have Yellow fever vaccine. Great Culicidae taxonomy work!

Reviewer #2: Previously the reviewers presented some suggestions to the manuscript. In my opinion, I observed that the issues raised were addressed in this new version. Concerning the minor comments, the authors fully addressed all points raised by the reviewers.

In conclusion I recommend the publication of the presented review article.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Alvaro Gil Araujo Ferreira

15 Dec 2021

PONE-D-21-16082R1

Entomological surveillance of mosquitoes (Diptera: Culicidae), vectors of arboviruses, in an ecotourism park in Cachoeiras de Macacu, state of Rio de Janeiro-RJ, Brazil.

Dear Dr. Balthazar:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Humberto Lanz-Mendoza

Academic Editor

PLOS ONE