Sm 300

The morphological study by OM and scanning electron microscopy (SEM) consisted of the observation of the head, thorax and abdomen of 30 adult females and 30 adult males, as well as 40 eggs of R.taquarussuensis sp. n. and the same number of specimens of R.neglectus, according to Barata (1981) , Quintero (2003), Obara et al. (2007) (link), Rosa et al. (2012) , Rosa et al. (2014) (link), Souza et al. (2016) (Figs 3–9).
Female external genitalia were observed from the dorsal, posterior, and ventral sides (Fig. 6) by SEM, according to Rosa et al. (2010) (link). The study of the male genitalia was carried out by OM (Figs 8, 9), following a technique developed by Jader de Oliveira based on Gallati (2016). The denominations used were those defined by Lent and Jurberg (1969) .
The Leica MZ APO stereoscope from the Faculty of Pharmaceutical Sciences, UNESP, Araraquara, and the scanning electron microscope Topcon SM-300 located in the Department of Physical Chemistry at the Chemistry Institute, UNESP, Araraquara, were used for observation and capture of images.

For the SEM analyses, ten eggs of each of the 12 parental species used in the crosses and of the 12 hybrids resulting from the interspecific crosses were prepared and analyzed in SEM, according to Mendonça et al.¹¹ : cleaned in an ultrasound machine, dehydrated in alcoholic series, dried in an oven at 45° for 20 min and then fixed in small aluminum cylinders “stubs” with colorless enamel. Subsequently, they were metalized by “sputtering” for 2 min with a power of 10 mA in a “Sputter” SCD 050 device and, finally, they were analyzed and photo-documented in SEM Topcon SM-300 (total magnification of 850 ×).

In order to perform the cell morphology analysis, the cells were seeded on glass slides placed at the bottom of 24-well plates. In each time interval of analysis (n = 2), the cells were fixed in 2.5% glutaraldehyde (Sigma Chemical Co.), and then post-fixed in 1% osmium tetroxide (Sigma Chemical Co.), dehydrated in increasing concentrations of alcohol (30, 50, 70, 90, and 100%) and submitted to chemical drying in HMDS (1,1,1,3,3,3-hexamethyldisilazane; Sigma Chemical Co.). Finally, the slides with the cells were fixed on metal stubs, kept in a desiccator for 72 h, sputter-coated with gold, and finally analyzed by scanning electron microscopy (SEM-FEG) (SM-300, Topcon, Tokyo, Japan).