A contribution to the knowledge of Quadraseta brasiliensis Goff and Gettinger, 1989 (Trombidiformes: Trombiculidae), with description of the deutonymph instar

In the Neotropical region the genus Quadraseta Brennan, 1970, includes 14 species, with ectoparasitic habits during the larval stage. Quadraseta brasiliensis Goff and Gettinger, 1989, was described from larvae collected on the rodentHylaeamys megacephalus (Fisher), cited as Oryzomys capito (Olfers). According to these authors, the holotype was deposited in the Museu de Zoologia da Universidade de São Paulo and the paratypes were deposited in three other collections: Bernice Pauahi BishopMuseum, SamNoble OklahomaMuseum of Natural History and United States National Museum of Natural History, however, no type specimens were found in any of these museums. Here we redescribe the larva, describe the deutonymph instar obtained from field-collected larvae, and report new hosts and localities for this species in Brazil. In addition we provide sequences of the 18S ribosomal RNA gene for this species.


Introduction
Around 3,000 species of chigger mites (Trombidiformes: Trombiculidae) have been described worldwide (Goff et al. 1982), and most of this diversity is known only by the type specimens. The life cycle of chiggers includes seven stages, namely egg, pre-larva, larva, protonymph, deutonymph, tritonymph and adult, yet active stages are larvae, deutonymphs and adults. While the majority of larvae from this group of mites are ectoparasites that feed on terrestrial vertebrates, other active stages are arthropod predators. Because of their parasitism in larval stage, the taxonomy of these mites has been based on this stage (Miyajima and Okumura 1917). The latest revision of chigger genera in the Neotropical and Nearctic regions was published in the 1970's accounting more than 100 genera, with 19 of these taxa documented for Brazil (Brennan and Goff 1977).
In the Neotropical region, the genus Quadraseta Brennan, 1970 is known only from larval stage and consists in 14 ectoparasitic species associated with small-mammals; however one species, Quadraseta flochi (Brennan and Jones, 1960), can also parasitize birds. Of these Neotropical representatives, Quadraseta brasiliensis Goff and Gettinger, 1989 and Quadraseta pazca (Brennan and Jones, 1964) have been reported from Brazil.
Quadraseta brasiliensis was described from larvae collected on the rodent Hylaeamys megacephalus (Fisher), cited as Oryzomys capito (Olfers), and from the marsupials Gracilinanus agilis (Burmeister), cited as Marmosa agilis, and Monodephis americana (Müller), all captured at an average of 25 km from Brasília (Federal District). In the original description of Q. brasiliensis, Goff and Gettinger (1989) designated the holotype based on larva collected from H. megacephalus, deposited in the MZUSP -Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil, and paratypes deposited in following three collections: BPBM -Bernice Pauahi Bishop Museum, Honolulu, Hawaii, USA (currently housed in the NMNH collection), OMNH -Sam Noble Oklahoma Museum of Natural History, Norman, Oklahoma, USA, and NMNH (earlier acronym USNM) -United States National Museum of Natural History, Smithsonian Institution, at United States Department of Agriculture, Beltsville, Maryland, USA. Unfortunately no type specimens of Q. brasiliensis were found while examining these collections, however, we found specimens mounted on slides, collected from the same host species and locality, but different date, that allowed us to confirm the identity of our material. Here we redescribe the larva, describe the deutonymph instar obtained from field-collected larvae, report new hosts and localities, and include genetic data for this species in Brazil.

Morphological study
Slide-mounted larvae of Quadraseta deposited in the NMNH, which are housed at the Systematic Entomology Laboratory (BARC-USDA-ARS), and in the Acari Collection from Butantan Institute, São Paulo, Brazil (IBSP), were examined and identified based on the original descriptions of the 14 known Quadraseta species.
Measurements and drawings were made using a Leica DFC 500 digital camera coupled to a Leica DM4000B optical microscope. Extended focal range images were composed using the Leica Application Suite version 2.5.0. Optical micrographs were prepared using Adobe Photoshop v. 13.0 and Inkscape V. 2. All measurements were made in micrometers (μm), followed by maximum, minimum, mean and standard deviation (SD). Micrographs of larvae and deutonymphs were made by the Low Temperature Scanning Electron Microscopy (LT-SEM) at the US Department of Agriculture, Electron and Confocal Microscopy Unit, Beltsville, Maryland as described in Dowling et al. (2010) and Bolton et al. (2014).
We followed the terminology of Goff et al. (1982), with adaptations proposed by Stekolnikov (2008) and Stekolnikov and Daniel (2012) concerning general nomenclature of larval stages. To standardize the nomenclature of the specialized setae of the Prostigmata group, we adopted the terminology used by Wohltmann (2006Wohltmann ( , 2007 and Kethley 1990. For the deutonymphs, we used the terminology proposed by Audy (1954) and Crossley (1960).
Chigger larvae collected on rodents and marsupials from the municipality of Morro Grande, Cotia, São Paulo were reared until deutonymph stage under laboratory conditions. To achieve this, part of these larvae were placed in Petri dishes, with a mixture of plaster of Paris and activated charcoal with different proportions (3:1, 2:1 and 1:1). The Petri dishes were kept in darkness at room temperature (range 26°C ± 1ºC), and the substrate was daily moistened with distilled water to provide (98% ± 1%) humidity. Once deutonymphs emerged, corresponding skins left on the dish surface were mounted on slides in Hoyer's medium for identification following Walter and Krantz (2009). Eggs of Onychiurus sp. (Insecta: Collembola) and Aedes aegypti (Insecta: Diptera) were offered as food (Lipovsky 1954). One part of the field-collected larvae and laboratory-reared deutonymphs was preserved in 100% ethanol for molecular analyses. Old larval skins and the rest of the material were deposited as voucher at the IBSP collection.

Molecular analyses
DNA extraction using the Guanidine Isothiocyanate lysis protocol (Chomkzynski 1993) was individually performed to 19 larvae (IBSP12557B) collected from Akodon montensis and to 5 deutonymphs reared from larvae hosted by the same rodent. Each mite was placed in an Eppendorf microtube, and punctured in the idiosomal region with a sterile needle (1.20 * 40 -18G). After the DNA extraction all exuviae were recovered and mounted in slides, as a voucher.

Quadraseta brasiliensis
Deutonymph. (Figures 4 and 5, Table 2) Sternal area with five branched setae on one side and four branched setae on the other side between coxae I; 8 distinct but minute dorsal teeth in cheliceral each blade, palp tibia with 5 setae branched and 1 dorsal nude seta; base of tibial claw flanked by 2 spoon-shaped accessory setae, palp tarsus with 7 branched setae, 2 nude apical sensory setae, a leaf-shaped tectum, with a single tectal seta, parascutal setae number approximately six on either side of crista, typically arranged at 2-4; three pairs of branched epivalval setae and six pairs of branched centrovalval setae.
Description -Deutonymph. Dorsal idiosoma -color whitish yellow, eyes absent; The entire body with branched setae on plates, shown in Figure 4B; a leaf-shaped tectum, with a single tectal seta; six parascutal setae on either side of crista, typically arranged at 2-4 ( Figure  4C); trichobothria long and slender, proximal portion with numerous short branches, and the distal portion with numerous long thin branches ( Figure 4C and E). Ventral idiosoma -Sternal area: with five branched setae on one side and four branched setae on the other side between coxae; six pairs of branched epivalval setae, 2 pairs of oval acetabula present, posterior acetabulum smaller than anterior and three pairs of branched centrovalval setae ( Figure 4D); a pair of anal plates, each one with eight branched setae ( Figure 4D). Gnathosoma -Cheliceral blade straight, V-shaped, with 8 distinct but minute dorsal teeth, along each. Entire hypostome not divided into lobes, with 4 pairs of nude apical setae; palp femur with 8 branched setae; palp genu with 7 branched setae; palp tibia with 5 setae branched and 1 dorsal nude seta; base of tibial claw flanked by 2 spoon-shaped accessory setae; palp tarsus with 8 branched setae, 2 nude apical sensory setae (ζ) and 1 basal sensory rod (ω) ( Figure 4F). Legs -seven-segmented, femur divided in basifemur and telofemur, tarsi with a pair of claws, onychotriches absent; leg I and IV longer than legs II and III; tarsus I oblong in shape, inflated, always longer than tibia I ( Figure 4A). All leg segments densely covered with normal setulated setae and the specialized setae present on leg I (tFe-Ta), II (tFe-Ta), III (tFe-Ta), IV (Ge-Ta) ( Figure 5A-F). The morphometric data are shown in Table 2.
Remarks -The species Q. brasiliensis resembles the species Q. pazca and Q. mackenziei (Yunker and Brennan, 1964), but is has the first row of dorsal idiosomal setae in lines and the all palpal tibia setae branched. The species Q. brasiliensis differs from the other species in presents 8 setae in the first row of idiosomal dorsal setae, while the other two species have 10 setae. Q.  Biology -In laboratory conditions, engorged larvae, recently collected from the hosts, kept in Petri dishes with substrate (1:1), remained active for 1-3 days. After this period the larvae enter in the process of quiescence (protonymph), at this stage there is an extrusion of the legs. This process lasted 7-11 days and a deutonymph emerged from the anterior dorsal portion of protonymph, the deutonymphs remained alive for up to 20 days without feeding.

Molecular analyses
Although expected size amplicons were obtained for 14/24 of the samples submitted to PCR, only three larvae and two deutonymphs yielded bands strong enough for sequencing. After an alignment of 421-bp including these five sequences, two haplotypes differing in one nucleotide were observed: one belonging to the deutonymphs and two larvae (GenBank accession numbers: KY934462, KY934464, MF113412 and MF113413), and another to one larva (KY934463). By BLAST analyses, both haplotypes of Q. brasiliensis were 96 -99% similar to the sequences available in GenBank for Allothrombium sp. (KP276493) and Eutrombicula splendens (Ewing, 1913) (KP325057) respectively. For the gene COI, none of the tested samples amplified.

Discussion
According to the original description of this species by Goff and Gettinger (1989), the holotype and paratypes of Q. brasiliensis were deposited in the BPBM, MZUSP, OMNH and NMNH collections. However, a search of these collections revealed no type specimens. Nevertheless, in the NMNH we found additional slide-mounted material coinciding with the same data of collection for the type specimens, except for the collecting date. After a review of all of the additional material, we found variation only in the position of the dorsal, preanal and postanal setae, depending on the degree of engorgement of the mites, as well as in the total number of idiosomal setae.
We have succeeded in obtaining deutonymphs, by rearing larvae on activated charcoal as substrate in order to remove plaster of Paris toxicity (Shatrov 2003). A great variety of substrate types have been tested successfully by other researchers (Wharton 1946;Wharton and Carver 1946;Michener 1946;Jenkins 1947;Farrel and Wharton 1949;Hyland 1951Hyland , 1961Lipovsky 1953Lipovsky , 1954Kaufman and Traub 1966;Nadchatram 1968;Everett et al. 1973;Mallow and Crossley 1984;Takahashi et al. 1993;Shatrov 1996;Tuegel and Wrenn 1998), including modifications in the proportion of activated charcoal, density of those substrates and sizes or kind of the culture containers.
In the present study, the deutonymphs of Q. brasiliensis were obtained under temperatures of 26°C (± 1°C), as well as the substrate with activated charcoal (1:1) demonstrated to be more efficient than in other proportion. Thus, the following parameters proved to be determinants of successful acquisition of deutonymph stages of Q. brasiliensis in laboratory conditions. The relative humidity inside the dish must be near the saturation level without being completely saturated in order to avoid the formation of fungi colonies that would need to be removed immediately. Lipovsky (1951) tested different types of food, such as eggs of Lepidoptera, Coleoptera, Collembola, and Diptera for Eutrombicula alfreddugesi (Oudemans, 1910) and Eutrombicula splendens (Ewing, 1913), noticing that the preferred food source varies depending of the trombiculid species. Specimens of T. alfreddugesi and T. splendens fed on eggs Collembola as well as of Diptera; whereas Hirsutiella zachvatkini (Schluger, 1948) (Shatrov 2003), Eutrombicula cinnabaris (Ewing, 1920) (Tuegel and Wrenn 1998), Neoschongastia americana (Hirst, 1921) (Everett et al. 1973), Leptotrombidium pallidum (Nagayo, Miyagawa, Mitamura and Tamiya, 1919) (Takahashi et al. 1993), and Hannemania hegeneri Hyland, 1956 fed almost exclusively on Sinella curviseta (Collembola) eggs, in laboratory rearing. Eggs of Onychiurus sp. (Collembola) were given daily (at least 10 eggs per day) presented pink-like color (by fungi infection), and this fact was previously recorded by Lipovsky (1951). This author verified that eggs were infected with Penicillium, caused progressive lethargy which led to death of the specimens of E. alfreddugesi and E. splendens.
Temperature control was not a high priority issue for colony maintenance of Eutrombicula batatas (Linnaeus, 1758) and Blankaartia alleei (Ewing, 1926), since temperature variations of ± 5°C (Jenkins, 1947) did not affect the success of the colony. Shatrov (2003) observed the same situation for H. zachvatkini.
The only record of Q. brasiliensis was the type locality described from the Brasília (Federal District), collected on rodents H. megacephalus (cited as Oryzomys capito) and on marsupials G. agilis (cited as Marmosa agilis) and M. americana (Goff and Gettinger 1989). In this study, the species was recorded in the southern region of Adrianópolis (state of Paraná), and E. russatus was a new host record. We also added the cities of São Paulo, Cotia, Itapevi, Peruíbe and Sete Barras as new occurrences (Figure 5), and rodents of the species: A. montensis, C. paca, O. nigripes and T. nigrita; and marsupials of the species D. aurita and M. iheringi as new hosts for Q. brasiliensis.
Partial sequences obtained for the 18S ribosomal RNA gene of two larvae and two deutonymphs were identical between each other, and one larva yielded a sequence differing only in one nucleotide. BLAST analyses sequences of Q. brasiliensis were highly similar (99%) to E. splendens, the comparison of the complete sequences of both species should be done in order to draw further conclusions. These sequences correspond to the first molecular identification of the genus Quadraseta. Kampen (2004) succeeded in amplifying the COI gene of Neotrombicula autumnalis (Shaw, 1790) larvae using primers for Tetranychidae mites designed by Navajas (1994); moreover, Moniuszko et al. (2015) were successful in amplifying the same gene, from larvae and deutonymphs of Hirsutiella zachvatkini (Schluger, 1948), using the primers by Dabert et al. (2008;. In the present study, both pairs of primers were tested and none of them amplified the COI gene for Q. brasilensis. This is the first description and record of Q. brasiliensis deutonymphs that developed from larvae maintained in laboratory conditions. The only previous description of the deutonymph instar of a chigger species recorded for Brazil was that of Eutrombicula ralphaudyi Vercammen-Grandjean and André, 1966.