First Report of Nematodes in Lutzomyia Edwardsi (Diptera: Psychodidae

Home , Lutzomyia, Macuco, Phlebotominae, Sumidouro

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SCIENTIFIC NOTES

First report of nematodes in Lutzomyia edwardsi (Diptera: Psychodidae,

Phlebotominae) in cave, in the municipality of Sumidouro, State of Rio of Janeiro,

Brazil.

João Ricardo Carreira Alves1,2*, Cleber Nascimento do Carmo3, Rodrigo Caldas

Menezes4, Mauricio Luís Vilela5#, Jacenir Reis dos Santos-Mallet5.

1.Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Fundação

Oswaldo Cruz, Pavilhão Mourisco, Sala 212. Instituto Oswaldo Cruz, Fundação Oswaldo

Cruz - FIOCRUZ, Endereço Institucional: Av. Brasil, 4365, Manguinhos, Rio de Janeiro,

RJ, Brasil.-CEP: 21.040-900. Tel: (21) 998526734. ORCID:0000/0001-5885-6758. 2.

Programa de Pós-Graduação em Biologia Animal, Universidade Federal Rural do Rio de

Janeiro, Seropédica, Rio de Janeiro State, Brazil. 3. Escola Nacional de Saúde Pública

Sergio Arouca. Fundação Oswaldo Cruz, Rua Leopoldo Bulhões, 1480/807, Manguinhos,

Rio de Janeiro, RJ, Brasil. CEP: 21.041-210.ORCID:0000-0003-41652198 4.

Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos, Instituto

Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz. Av. Brasil 4365,

Manguinhos, Rio de Janeiro, Rio de Janeiro State, Brazil. CEP: 21.040-360.

ORCID:0000-0003-1853-3449.5. Laboratório Interdisciplinar de Vigilância

Entomológica em Diptera e Hemíptera. Instituto Oswaldo Cruz, Fundação Oswaldo Cruz,

Av. Brasil, 4365, Manguinhos, Rio de Janeiro, Rio de Janeiro State, Brazil. CEP: 21.040-

900. ORCID:5# 0000-0001-7610-0719/50000-0003-4728-7638

*Corresponding author: [email protected]

ABSTRACT

It is registered for the first time, the encounter of Lutzomyia edwardsi (Mangabeira)

species of sandfly considered as a probable vector of Leishmania (Viannia) braziliensis

(Vianna), main etiological agent of American cutaneous leishmaniasis in humans, for the

municipality of Sumidouro, State of Rio of Janeiro. Leishmaniasis is currently considered

a neglected tropical disease in the Americas. The objective of this study was to know the

phlebotomine fauna in an area of Atlantic forest, with a cave in this municipality. From

the sandflies collected, in 2009 and 2010, with a light trap, type CDC it is reported in a

unique way, the encounter of a female L. edwardsi, infected with nematodes in a cave in

the locality of São Caetano, in Sumidouro, state of Rio of Janeiro, Brazil. The description

of sandfly and nematodes was carried out. The presence of this species in areas of

leishmaniasis cases in the municipality of Sumidouro and its surroundings, requires the

adoption of relevant entomological and / or epidemiological surveillance, by the

Municipal Health Department of the region, when registering this disease.

KEY WORDS: entomoparasite, sandflies, American cutaneous leishmaniasis, visceral

leishmaniasis, vector.

A total of 1,008 species of sandflies are known worldwide, of which more than half

are registered in the American continents. In Brazil, 279 species are known, representing

31% of the species known worldwide and with 22 proven or suspected species of

leishmaniasis transmitting agents (Aguiar & Vieira 2003).

According to Young & Duncan (1994), in the New World, the subfamily

Phlebotominae consists of the genera Lutzomyia França, Brumptomyia França and Parrot

and Warileyia Hertig (Diptera: Psychodidae). The species of medical importance belong

to the genus Lutzomyia.

From the discovery that some species of sandflies were vectors in the transmission

of pathogens to humans and animals, studies on these insects have increased considerably,

aiming to investigate their life cycle and identify which vectors occur in certain regions,

considering the different species identified as important from an epidemiological point of

view (Forattini 1973).

Carvalho et al. (2014) concluded from a literature review, based on collections

made by the State Health Department of Rio de Janeiro, that the phlebotomine fauna of

this state is composed of 65 species, eight of which belong to the genus Brumptomyia and

57 to the Lutzomyia genus. This research related the species, their distribution by

municipality and reported that Lutzomyia edwardsi (Diptera: Psychodidae,

Phlebotominae) was described by Mangabeira Filho in 1941 in the municipality of Nova

Iguaçu, state of Rio de Janeiro. The specimens of this sandfly were collected in the forest,

in a paca den (Agouti paca L.), in which seven males and two females were captured. In

1995, De Souza et al., Registered L. edwardsi in the municipality of São José do Vale do

Rio Preto, later, Carvalho et al. (2014) pointed out, L. edwardsi in two municipalities in

the mountain region: Bom Jardim and Petrópolis. Peres-Dias et al. (2016) researched the

phlebotomine fauna of the municipality of Cantagalo, having collected 3,310 specimens bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

from 12 species, eleven of the genus Lutzomyia and one of Brumptomyia. In this research,

a male specimen of L. edwardsi was collected, being the first record of this species for

this municipality.

In the mountainous regions of the state of Rio de Janeiro, from 2007 to 2017, 95

cases of American cutaneous leishmaniasis (ATL) were recorded, the highest occurrence

being recorded in the municipality of Trajano de Moraes, with nineteen cases. Followed

by Bom Jardim with nine cases. In Cantagalo, there were six cases from 2007 to 2015. In

the municipalities of Duas Barras, Macuco, São Sebastião do Alto, Sumidouro and

Teresópolis, one case in each unit (SINAN / SVS / WEB, 2019). In this region, four cases

of visceral leishmaniasis (VL) were recorded, two in Petrópolis and two in Teresópolis

(SINAN / SVS / WEB, 2019).

In order to report the encounter of L. edwardsi with nematodes, in addition to

providing subsidies, which allowed the Health Departments of the above-mentioned

municipalities to carry out a plan for the control and combat of leishmaniasis vectors,

considering also that L. edwardsi has already been found infected with Leishmania

braziliensis (Kinetoplastida: Trypanosomatidae) in the state of São Paulo. This work is

justified and published.

The municipality of Sumidouro is located at Latitude 22º 2`43 ”S, Longitude 42 °

40`24” W, in the mountain region of the state of Rio de Janeiro, bordering the

municipalities of Nova Friburgo, Teresópolis, Carmo, São José do Vale do Rio Preto,

Sapucaia and Duas Barras (Fig 1). The study area was described in more detail by Alves

et al. (2020)

Poisson regression was performed to assess and prove that the collections of L.

edwardsi, between the São Caetano cave, its surroundings and the forest, showed

statistically significant differences. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In order to describe the phlebotomine and the entomoparasite, the Zess microscope,

with Axiolmager, equipped with Apo Tome, was used to perform the photos,

measurements and formatting of the nematodes and the specimen of L. edwardsi. (Fig 2

a, b, c, d)

Fig. 1. Geographic location of the neighboring municipalities of Sumidouro: Carmo (21º 55` 53. 40`` S / 42º 36` 18. 23`` W), 2 Duas Barras (22º 03` 15. 60`` S / 42º 31` 24. 24`` W), 3 Nova Friburgo (22º 17` 19. 58`` S / 42º 32` 02. 88`` W), 4 - Teresópolis (22º 25` 05. 35`` S / 42º 58` 15. 49`` W), 5 São José do Vale do Rio Preto (22º 13` 00`` S / 42º 51` 06`` W), 6 Sapucaia (21º 59` 40`` S / 42º 54` 57``W), 7 Sumidouro (22º 2`43 ”S / 42 ° 40`24” W). 8- Nova Iguaçu (22º 45`37.16” S / 43º 26`51.82” W) Locality type of L. edwardsi. The number in parentheses corresponds to the cases of leishmaniasis registered in the municipalities of Bom Jardim, Cantagalo and Petrópolis from 2007 to 2017, where L. edwardsi was found.

Fig 2. a- Cibary of the female of L. edwardsi, with vertical teeth, in detail shows a nematode found on the head. b - Abdomen with nematodes, L. edwardsi spermateca. c - Details of the spermatec in bright field, d - Individual duct, body and head. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The identification was made using the taxonomic key of Poinar et al. (2002), and

through the photos of the nematodes that were sent to Dr. Poinar Jr, Department of

Entomology; Oregon State University, United State of American.

1,756 sandflies belonging to 14 species were captured, 11 of which from the genus

Lutzomyia: Lutzomyia gasparviannai Martins, Godoy & Silva, L. edwardsi (Mangabeira),

L. tupynambai (Mangabeira), were the most prevalent and three species of the genus

Brumptomyia. The methodology, the material used and the results are described by Alves

et al. (2020).

A total of 290 specimens of L. edwardsi were collected, 67 males and 223 females.

In the cave, there were 37 males and 114 females, among these specimens, it was found

the existence of a female of L. edwardsi, infected with more than 11 nematodes in the

abdomen.

Poinar et al. (1993) found in a colony of L. longipalpis, in Colombia, specimens

infected with nematodes, which were taken to a laboratory in the United States. The

authors described Anandranema phlebotophaga n. gen., n. sp. (Allantonematidae:

Tylenchida).

The nematodes described by the authors are morphologically similar to the

nematodes found in our study. Poinar et al. (1993) also point out that these nematodes are

making the females of L. longipalpis infertile, leading the authors to consider them an

entomoparasite and suitable to be used as biological control for this species.

In 2001, in the municipality of Cotia, state of São Paulo, Brazil, a case of visceral

leishmaniasis occurred in a cat. During the research, infection of L. edwardsi by Le.

(Viannia) braziliensis was recorded, by PCR, etiological agent of American cutaneous

leishmaniasis. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Which led us to consider its relevance in the studied area, since two cases of ATL

were registered in the region. In addition, the species was collected at the three collection

sites, demonstrating a wide distribution in the studied location. It is noteworthy that the

region is frequented by farmers, residents, hunters, students, paleontologists, biologists.

In 2002, Poinar et al. describe a new nematode, Elaeolenchus parthenonema n.

gen., n. sp. (Nematoda: Sphaerularioidea: Anandranematidae n. Farm.) In an Elaeidobius

kamerunicus (Curculionidae: Coleóptera). The new genus is placed in Anandranematidae

n. farm., together with the genus Anandranema Poinar et al. (1993), as they are

characterized by being nematodes with a single auto generation.

The authors confirm the genus and species of Anadranema phlebotophaga, which

resembles the nematodes found by us.

In 2002, Secundino et al. detected and morphologically characterized a new

entomoparasite of L. longipalpis, using electron microscope, fluorescence and

differential. They point out that the adult forms of these nematodes are different from

those described by Poinar et al. (1993) and those found in Sumidouro. Studies are being

done to identify and describe it.

L. edwardsi were collected in São José do Vale do Rio Preto, Bom Jardim,

Cantagalo and Petrópolis, the last three municipalities were responsible for 17 cases of

ATL, between 2007 and 2016. In Petrópolis, two cases of VL were registered, in 2016.

In absolute terms, the highest number of L. edwardsi occurred in Sumidouro,

considering the cases of ATL in these four municipalities, the number of females was

more accentuated in our study. Catches with human bait were made in Petrópolis, with

negative results for this species. The methodology used in Bom Jardim differs from that

applied in the other three municipalities, in which light is used to attract sand flies. It is

evident that the CDC trap, with light bait, is more efficient, whether in the home, in the bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

woods or in the forest. However, inside and outside a house and / or shelter for domestic

animals in an area with human cases, the castro catcher is recommended. These works,

show us that L. edwardsi can be found in a cave environment, in its surroundings, in the

forest, and in the state of Rio de Janeiro.

The results of the Poisson regression showed that there was a significant difference

(p <0.01) between the collections of the cave and the forest, as well as, of the forest and

the surroundings for L. edwardsi. Which confirms the importance of the collections made

in Sumidouro.

Galati et al. (2010) found L. monticola infected with microfilarias in the

Speleological Province of Vale do Ribeira, São Paulo. In his article, he concludes that

this species is anthropophilic.

In this study, L. monticola was not found, which does not corroborate Galati et al.

(2010), nor microfilarias and there is no evidence that L. edwardsi is anthropophilic,

which differs from Souza et al. (2002). Which suggests that the nematodes found could

not be transmitted to man.

In 2011, Souza et al. recorded the encounter of L. fischeri infected by nematodes

belonging to the family Allantonematidae and the genus Anandranema. This material was

found in the municipality of Rio de Janeiro, state of Rio de Janeiro.

In this research L. fischeri was not found, however, Carreira-Alves (2008)

registered this species in the municipality of Carmo. According to the literature, this

species is anthropophilic, wild and is adapting to the anthropic environment. (Aguiar &

Vieira, 2018)

Preliminary studies on nematodes highlight the difference between the size of the

stylus, the position of the gonads in relation to that described by Poinar et al. (1993), as

well as the existence of the cuticle, in the anterior part, in young parasites (Fig 3 a, b, c) bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(Fig 4 d). It is also noted that some species are also hermaphrodites in agreement with the

others cited by the author (Fig 4 d). These observations were made according to the

taxonomic key of Poinar et al. (2002).

Fig 3. a and b - Anandranema phlebotophaga species described by Poinar et al, 1993. c and d - are the species that were collected in this work. The red circle and a thick black arrow mark the stylets (a and c), while the arrows (b and c) show the cuticles. Thin black arrows - indicate the sub - ventral opening of the gonads (a). Fig. 4 - According to Poinar, some of these species are hermaphrodites. (d). bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305151; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The catch of L. edwardsi, infected with nematodes, is registered for Brazil.

Considering that they are hermaphrodites, have stilettos, anterior gonads, and are

entomoparasites of sandflies, as the literature points out. We believe that they belong to

the Family Anandranematidae n. fam.

Because L. edwardsi and the nematodes were mounted between slide and cover slip

with berleze, it was not possible to identify even the species of the nematodes.

The fact that it was collected in a cave in areas with stones and forest of volcanic

origin, differentiates it from the reports of the aforementioned authors.

The existence of this species in the area of cutaneous leishmaniasis cases, must have

a more conscious and effective entomological and / or epidemiological surveillance, by

the Municipal Health Department of the region. Thus, allowing this information to be

used for the study and development of technical standards for the control and prevention

of vectors and leishmaniasis.

Acknowledgements

To Dr. Felipe Ferraz Figueiredo Moreira and Dr. Márcio Felix, Laboratory of

Entomological Biodiversity of Fundação Oswaldo Cruz for logistical support. Dr.

Francisco Gerson de Araújo, Federal Rural University of Rio de Janeiro for support and

learning. The Heloisa Maria Nogueira Diniz the production and processing of images.

Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) -

Funding Code 001.

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