A new species of the genus Eutrombicula Ewing, 1938 (Trombidiformes: Trombiculidae) and new records for the species Eutrombicula batatas (Linnaeus, 1758) in Brazil

The genus Eutrombicula consists of more than 80 known species worldwide. Species of this genus parasitize amphibians, reptiles, birds and mammals. In the present study, we describe Eutrombicula daemoni n. sp. collected on the social flycatcher, Myiozetetes similis (Passeriformes) from southeast region of Brazil and partial sequence of the 18S gene was obtained for this mite species. In addition, we provide new locality records and hosts for Eutrombicula batatas (L.) in Brazil.


Introduction
Currently, the genus Eutrombicula Ewing, 1938, is represented by more than 80 species worldwide (Stekolnikov and González-Acuña 2010). Species of this genus have been recorded as parasites of amphibians, reptiles, birds and mammals (Hoffmann 1990). Thirty-seven species of the genus have been recorded from South America; of these, five species were listed from Brazil (Jacinavicius et al. 2018). Brazil is divided into five administrative regions: north, northeast, central-west, southeast and south (Contel 2014). Eutrombicula bruyanti (Oudemans, 1910) was recorded from the southeast region of the country, while Eutrombicula goeldii (Oudemans, 1910) and Eutrombicula tinami (Oudemans, 1910) were described from Brazil without specific locality information (Oudemans 1910). However, Jacinavicius et al. (2018) recorded E. tinami from the state of São Paulo, in the southeast region. The other two species found in Brazil are Eutrombicula alfreddugesi (Oudemans, 1910) and Eutrombicula batatas (Linnaeus, 1758). Specimens of E. alfreddugesi were recorded from the central-west region in the states of Goiás (Carvalho et al. 2006) and Mato Grosso (Fonseca 1932); from the northeast region in the states of Bahia (Menezes et al., 2009;Delfino et al. 2011) and Maranhão (Faccini et al. 2017); and in the southeast region in the states of Minas Gerais (Carvalho et al. 2006) and São Paulo (Fonseca 1932). Eutrombicula batatas was recorded from the north region, in the states of Amazonas (Ewing 1925) and Pará (Ewing 1925;Bequaert 1926); from the northeast region in the state of Maranhão (Faccini et al. 2017); from the central-west region in the state of Mato Grosso (Confalonieri and Benez 1976); and from the southeast region in the state of Rio de Janeiro (Confalonieri and Carvalho 1973).
A new species of Eutrombicula is described based on a larva found on a social flycatcher, Myiozetetes similis (Aves: Passeriformes: Tyrannidae) collected in Fazenda Volta Grande, Santa Bárbara do Monte Verde Municipality, Minas Gerais state located in the southeast region of Brazil and a partial sequence of the 18S gene was obtained for this species. In addition, new records of localities and hosts for E. batatas in Brazil are provided.

Collection of mites and morphological study
Eight specimens (holotype and paratypes) of a chigger (Acari: Trombidiformes: Trombiculidae) were found on one specimen of a bird species, Myiozetetes similis (Passeriformes: Tyrannidae), collected with a mist net in Fazenda Volta Grande, Santa Bárbara do Monte Verde (21°58'S, 43°41'W, elevation 736 m), Minas Gerais State, Brazil, in September of 2014. The bird was identified using Ridgely and Tudor (2009). Of the material collected and stored in ethanol 100%, two specimens were mounted on slides in Hoyer's medium according to Walter and Krantz (2009) for identification and were deposited in the Acari Collection of the Instituto Butantan (IBSP), four specimens were prepared according to Walter and Krantz (2009) for scanning electron microscopy (SEM) which were coated with gold. After imaging, the specimens were removed from the stubs with acetone and stored in alcohol and deposited in the IBSP collection. The SEM micrographs were obtained with a Digital Scanning Microscope FEI, Quanta 250, located at the Laboratório de Biologia Celular, Instituto Butantan. In addition, two specimens were retained for molecular analysis.
Drawings and measurements of the new species were made with a Leica DFC 500 digital camera. Extended focal range images were composed with Leica Application Suite version 2.5.0. All the measurements are in micrometers (μm), followed by the mean and standard deviation. The holotype measurements are highlighted. The images were prepared with Adobe Photoshop v. 13.0, and Inkscape V.2.
Other species from the genus Eutrombicula deposited at the IBSP collection were examined for comparison with the new species. The specimens were identified using Brennan and Goff's (1977) key to the genera and Brennan and Reed's (1974) key to species of Venezuela, as well as original descriptions of the species of the genus Eutrombicula. For species with incomplete descriptions, such as Eutrombicula bruyanti (Oudemans, 1910), Eutrombicula batatas (Linneaus, 1790), Eutrombicula goeldii (Oudemans, 1910) and Eutrombicula tinami (Oudemans, 1910), we used redescriptions by Oudemans (1912) and Jenkins (1949) and illustrations by Brennan and Reed (1974).

Molecular analysis
In order to enrich genetic banks with chiggers sequencing and to provide future works, two fragment of DNA were tested. The DNA of two specimens of Eutrombicula daemoni n. sp., was extracted using the Guanidine Isothiocyanate lysis protocol (Chomczynski 1993). Each mite was placed in an Eppendorf microtube, and punctured in the idiosomal region with a sterile needle (1.20 * 40 -18G). After DNA extraction, the exuvia were recovered, mounted on slides, and kept as vouchers. A conventional PCR targeting a ≈500-pb fragment of the 18S ribosomal RNA gene was performed using primers Mite18S-1F (3'-ATATTGGAGGGCAAGTCTGG-5') and Mite18S-1R (3'-TGGCATCGTTTATGGTTAG-5'), as described by Otto and Wilson (2001). Positive samples were pooled and subsequently sujected to a second PCR analysis attempted to target ≈560-680-pb fragment of the cytochrome oxidase I gene (COI) using primers bcdF01 (CATTTTCHACTAAYCATAARGATATTGG) and bcdR04 (TATAAACYTCDGGATGNCCAAAAAA) following the protocol of Dabert et al. (2010) and Dabert et al. (2008), respectively, with modifications of Moniuszko et al. (2015). For each reaction, negative (Milli-Q water free of DNA), and positive controls. All PCRs were performed on a Mastercycler Gradient (Eppendorf® California, USA). PCR products with concentrations higher than 20 ng/µl, were selected and purified with ExoSap-IT (GE Healthcare Pittsburgh, PA). Sanger sequencing of the samples were performed in the "Centro de Pesquisa sobre Genoma Humano e Células Tronco do Instituto de Biociências da USP". Obtained sequences were assembled with Sequencing Analysis 5.3.1 software and submitted to BLAST analyses (Altschul et al. 1990) in order to infer similarities with that of other mites available in GenBank. Different haplotypes were visually discriminated after an alignment using CLUSTAL W algorithm (Thompson et al. 1994) implemented in Geneious R9 (Kearse et al. 2012).

Ethical approval
The bird host of the new species was caught and manipulated in accordance with the recommendations of the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethics Committee on Animal Experimentation (CEEA) of the Dean of the Universidade Federal de Juiz de Fora, MG (UFJF) -n°042/2012 (SISBio n°29268-6).

Morphological study
The description of Eutrombicula daemoni n. sp. is provided below and, in addition, the material previously deposited in the IBSP was identified as Eutrombicula batatas. These represent the first records of E. batatas parasitizing Nyctibius griseus (Nyctibiiformes: Nyctibiidae), Didelphis aurita (Didelphimorphia: Didelphidae) and Cavia aperea (Rodentia: Caviidae) from the following localities: São Paulo and Cotia municipality, São Paulo state; Wenceslau Braz municipality, Paraná state; Santa Cruz do Sul municipality, Rio Grande do Sul state and Tucuruí municipality, Pará state.

Differential diagnosis
The new species was compared with known Brazilian Eutrombicula species and with other South American species in the genus. Eutrombicula daemoni n. sp. can be separated from the other South American Eutrombicula species by the following combination of characters: six mastisetae on the leg III -two on the tibia and four on the tarsus (1F and 2B), and five pairs of dorsal opisthosomal C row setae. It is similar to Eutrombicula batatas, which has two mastisetae on the tibia of leg III and five pairs of setae (c1-c5) in C row on the dorsal opisthosoma, but differs in having four mastisetae on tarsus leg III compared to three in E. batatas. The new species, has a nude lateral palptibial seta, the inner prong smaller than the outer prong and four mastisetae on the tarsus of the leg III, whereas Eutrombicula bruyanti has a forked lateral palptibial seta the odontus with a long inner prong and a short outer prong arising in the middle part of shaft and bearing tarsus of the leg III with only one mastiseta. Eutrombicula daemoni n. sp. is similar to Eutrombicula alfreddugesi in having a branched ventral palptibial seta, but differs in having the inner prong of odontus arising in the middle part of shaft and four mastisetae on the tarsus of the leg III, whereas E. alfreddugesi has the bifurcation of outer prong of the odontus arises subapically to the inner prong, and only a single mastiseta on tarsus of leg III. The new species is also similar to Eutrombicula tinami in having branched ventral setae on the palptibia and the shape of the prongs of the odontus, but differs  in having two mastisetae on the tibia of leg III (no mastisetae on tibia leg III in E. tinami) and four mastiseta on the tarsus of leg III (one mastiseta in E. tinami). The shape of the odontus in Eutrombicula goeldii is similar to that of the new species, but E. goeldii has nude ventral setae on the palptibia whereas in the new species this seta is branched. In addition, E. bruyanti, E. alfreduggesi, E. tinami and E. goeldii have 4 pairs (c1-c4) of setae in the C row, differing from Eutrombicula daemoni n. sp. which has 5 pairs (c1-c5) of setae in the C row. We compared the new species to other South American Eutrombicula using Brennan and Reed's (1974) key to Venezuelan Eutrombicula. The three species most similar to Eutrombicula daemoni n. sp. were Eutrombicula spipi Reed, 1974, Eutrombicula vacillata Brennan andReed, 1974 and Eutrombicula wolfenbargeri Brennan and Reed, 1974. These three species and the new species have mastisetae in the tibia of leg III, but only the new species and E. spipi have 5 pairs (c1-c5) of setae in the dorsal opisthosomal C row and three sigma on leg I [(E. vacillata has 6 pairs (c1-c6) of setae and two sigma and E. wolfenbargeri has 7 pairs (c1-c7) of setae and two sigma)]; however, E. spipi differs from the new species in having only two mastisetae on the tarsus of the leg III.
Recently, Stekolnikov & González-Acuña (2015) Stekolnikov & González-Acuña, 2015. We compared these with the new species. Eutrombicula nerudai and E. mistrali have one mastiseta on the tarsus of the leg III, a nude seta on the palpgenu, and the outer prong of odontus arising subapically from inner prong, while E. daemoni n. sp. has four mastisetae on the tarsus of the leg III, a branched seta on the palpgenu and the odontus arising in the middle part of shaft. Eutrombicula picunche and E. alfreddugesi differ from the new species by the same characters.

Molecular analysis
Two samples (paratypes) of the new species were submitted to PCR and were positive to the 18S gene PCR and amplified two identical fragments of 409-bp (GenBank accession numbers: MG707783 and MG707784). BLAST comparisons of these sequences showed 99.7% (408/409-bp) of identity with homologous sequences from Eutrombicula splendens (KP325057) and 99.5% (407/409-bp) from Quadraseta brasiliensis (MF113413; MF113412; KY934464). None of the samples amplified for the COI gene.
In addition, we obtained partial sequences for the 18S gene from the new species. These sequences are the first molecular identification of this genus for Brazil. At this time, it is still not possible to infer molecular differences between species that have a high similarity to Eutrombicula daemoni n. sp. based on an analysis of their 18S sequences, due to the slow rate of evolution of this gene, so different genera when compared have high similarity, which reflects that these species belong to a same family of mites.
Unfortunately, there are only these two species of Eutrombicula with 18S sequences deposited in Genbank. It is necessary to enrich the gene bank for species of this genus for future comparisons. Until now, we do not have recent material of E. batatas for molecular analysis and didn't possible comparisons with the new species.