Jenaval light microscope

Because of the difficulty in collecting and maintaining C. pilosa in insectariums, we only used one fifth-instar nymph that was collected in a cave in Marabá, PA, Brazil. This insect was dissected and its seminiferous tubules were placed in methanol:acetic acid (3:1). For slide preparation, each tubule was bathed twice in distilled water for 5 min. The tubules were then placed in a 45% acetic acid solution for 10 min, squashed, stained with lacto-aceto-orcein (De Vaio et al., 1985 , with modifications by Alevi et al., 2012) , and C-banding (Sumner, 1972) . The slides were examined (total increase of 1000X) under a Jenaval light microscope (Zeiss, Jena, Germany), and images were captured using AxioVision LE 4.8 (Zeiss).

To avoid the possible bias deriving from the different sampling methods, all samples were collected by the same author with sterile cotton swabs by rubbing the inside surface of the rubber door seals, as well as the drawers of washing powder and fabric softener. The samples were taken from the most contaminated surface determined by visual inspection. Swabs were transported to the laboratory in sterile tubes and processed within 24 h. Samples were inoculated to malt extract agar medium (MEA; 30 g l À1 malt extract, 5 g l À1 peptone, 15 g l À1 agar) containing 0.1 g l À1 chloramphenicol and incubated at 25 C for five days. Samples were also collected with scalpels from the deposited materials located on the surface of the washing machines. After culturing the samples, pure cultures of fungi were isolated. The composition of deposited materials was studied with a Carl Zeiss Jenaval light microscope at 300Â magnification.

Three adult males of each species (Table 1) were cytogenetically analyzed. The insects were donated by "Insetário de Triatominae" of the Biological Sciences Department of the Faculty of Pharmaceutical Sciences, State University of São Paulo, Campus Araraquara, São Paulo, Brazil. Microscope slides with the biological material (seminiferous tubules) were prepared by the crushing technique and stained with lacto-acetic orcein (De Vaio et al., 1985) (link) with modifications by Alevi et al. (2012) (link). The slides were analyzed using a Jenavallight microscope (Zeiss) coupled to a digital camera and an AxioVision LE 4.8 image analyzer (Zeiss). The images were magnified by 1000X.

At least three adult males each of T. pintodiasi, T. carcavalloi, and T. circummaculata were analyzed. The specimens of T. pintodiasi were provided by the National Laboratory and International Reference on Taxonomy of Triatominae, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, and the species of T. carcavalloi and T. circummaculata were provided by Insectarium of Triatominae, FCFAR/UNESP, Araraquara, São Paulo, Brazil. Seminiferous tubules of T. pintodiasi, T. carcavalloi, and T. circummaculata were isolated, shredded, smashed, and set on a slide in liquid nitrogen. They were then stained by the cytogenetic technique using lactoacetic orcein as outlined by De Vaio et al. (1985) (link), with modifications according to Alevi et al. (2012a) . The biological material was analyzed by a Jenaval light microscope (Zeiss) coupled to a digital camera and an image analyzer Axio Vision LE 4.8 (Copyright © 2006-2009 Carl Zeiss Imaging Solutions GmbH). The images were subjected to 1000X magnification. All karyotypes described in the literature are presented in Table 1.

Three adult males of N. bruneri were cytogenetically analyzed. The insects were donated by Insetário of Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil. Microscope slides containing biological material (seminiferous tubules) were prepared using the crushing technique and stained with lacto-acetic orcein (De Vaio et al., 1985) (link) with modifications as described by Alevi et al. (2012) (link). The slides were analyzed using a Jenavallight microscope (Zeiss) coupled to a digital camera and an AxioVision LE 4.8 image analyzer (Zeiss). The images were magnified 1000X.

At least two adult males from each species (T. arthurneivai and T. wygodzinskyi) were used, and the specimens were assigned by the insectariums of the National and International Laboratory of Reference for Triatominae Taxonomy, Oswaldo Cruz Institute (FIOCRUZ), Rio de Janeiro, Brazil (T. arthurneivai) and the Laboratory of Triatomines and Chagas Disease Epidemiology at the René Rachou Research Center (CPqRR/FICRUZ), Minas Gerais, Brazil (T. wygodzinskyi).
The biological material used to characterize cells was spermatocytes, which were easily obtained from testicular material by tearing the seminiferous tubules of adult males prior to fixation to a cover slip. The samples were then subjected to a cytogenetic technique that utilized lacto-acetic orcein (De Vaio et al., 1985 (link), with modifications based on Alevi et al., 2012) (link). At least 50 cells from each species were analyzed using a Jenaval light microscope (Zeiss) attached to a digital camera and an Axio Vision LE 4.8 image analyzer (Copyright 2006 (Copyright -2009 Carl Zeiss Imaging Solutions Gmb H), and the obtained images were magnified by a factor of 1000X.

In this study, we used 10 males of the species R. montenegrensis, assigned by the "Triatominae Insectarium" at the Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Araraquara campus. The seminiferous tubules of adult males, after being removed and fixated on a cover slip, were processed for cytogenetic analysis using lacto-acetic orcein technique (De Vaio et al., 1985 , with modifications described by Alevi et al., 2012a) . The biological material was analyzed using Jenaval light microscope (Zeiss) coupled to a digital camera and an image analyzer Axio Vision LE 4.8 (Copyright © 2006-2009 Carl Zeiss Imaging Solutions GmbH). The images were magnified by a factor of 1000.