Gills preparation
Specimens of L. fortunei were collected in February 2019 in the fish farming reservoir of the Volta Grande, border between the states of Minas Gerais and São Paulo, Brazil (VG; 20°01'54.0"S, 48°13'10.0"W) where measured water parameters were 29.2 ºC, pH 7.5, dissolved oxygen (DO) 3.2 mg L-1, and turbidity (NTU) 0.1. They were packed in cloth bags (to decrease overlapping individuals) and during transportation they were submerged in water at constant aeration. In our laboratory at the Centro de Bioengenharia de Espécies Invasoras de Hidrelétricas (CBEIH), the animals were acclimated for 3 weeks in aquarium with 36 L capacity containing artesian water well, pH 7.7, dissolved oxygen 6.8 mg L-1, and turbidity (NTU) 1.68, at constant aeration and temperature 18 ºC to minimize stress. After acclimation, the mollusks were kept under the same conditions and temperature of 22 ± 1 ºC. For the purpose of this work, one approximately 1.5 cm long bivalve was taken out of the aquarium so their valves could be kept partially open. Next, the specimen was submerged in a 1.5 mL Eppendorf® tube filled with modified Karnovsky fixative solution (paraformaldehyde 2.0% and glutaraldehyde 2.5%). The VG specimen was kept into the fixative solution for 3 days. The gills were collected afterwards to further process them for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The collected gills were then placed into 1.5 mL tubes filled with 0.1 M phosphate buffer solution (PBS). In this present work, we also used unreported TEM and SEM data of the gills from another individual that was collected in a different place, in the Paranaíba river (PR), downstream the confluence with Barreiro river, near the municipality of Paranaíba, Mato Grosso do Sul, Brazil. The sample preparation details about this latter specimen can be found in Andrade et al. (2015).
Scanning electron microscopy (SEM)
Right before the secondary fixation, the excess volume of PBS in the tube was first removed and replaced by an appropriate volume of a fresh 0.1 M PBS and incubated for 10 min. This washing process was performed three times. The PBS was then replaced by an appropriate volume of 1% osmium tetroxide (OsO4) in 0.1 M PBS (pH 7.3 ± 0.1) in the fume hood and incubated for 1 h in darkness and room temperature (RT). The sample was with 0.1 M PBS and incubated for 10 min, for three times. The excess PBS was removed and replaced by 1% tannic acid (C76H52O46) solution and incubated for 20 min at RT. After washing with PBS for three times, the solution was replaced by 1% OsO4 solution and incubated for 1 h in darkness and RT. The samples were then washed in distilled water three times and dehydrated in a sequence of alcohol solutions (35%, 50%, 70%, 85%, 95% and 100%), 10 min each. The last step with absolute alcohol was performed twice. The sample was critical point dried with CO2 (using a Leica EM CPD 030), placed on an aluminum SEM stub with a carbon tape and finally coated with gold nanoparticles (5 nm thickness) in a sputter coater (Bal-Tec MED 020). The VG sample was analyzed in a field emission scanning electron microscope FEI Quanta 200, operated at 5 kV and 15 kV. The SEM images of the PR sample were acquired using the same microscope but at 20 kV and low vacuum mode (0.30 Torr).
Transmission electron microscopy (TEM)
For TEM analysis, sample was firstly washed for three times in 0.1 M PBS, 10 min each. PBS was replaced by 2% OsO4 in 0.1 M PBS (pH 7.3 ± 0.1) in the fume-hood and incubated for 2 h in darkness and at RT. The sample was then washed with deionized water four times, 10 min each. Distilled water was then replaced by 2% uranyl acetate (C4H8O6U) solution in darkness and incubated overnight in darkness at 6 ºC. The samples were again washed with deionized water, three times, 5 min each. Samples were dehydrated as described erlier. Absolute alcohol was replaced by acetone and incubated 20 min. Acetone was then replaced by EponTM resin in three steps, using different mixtures of acetone/resin (2:1, 1:1, and 1:2). In each of these three steps, the tube was enclosed and homogeneously agitated (using a Norte Científica NH2200) for at least 2 h. After drying the excess acetone/resin with a filter paper, the samples were transferred to a new polypropylene tube containing EponTM resin prepared with DMP-3 (Sigma Aldrich) and incubated at 40 ºC for 1 h. The gills sample was carefully embedded so to section it longitudinally to the lateral surface of gills filament. The ultrathin (60 nm thick) sections were obtained using an ultramicrotome (Leica EM UC6) and a diamond knife Ultra 45º (Diatome), with the sections being transferred to C-film Cu-TEM grids. Sections were post-stained using a 2% uranyl acetate and lead citrate. TEM analysis was performed in a thermionic W-filament transmission electron microscope FEI Tecnai Spirit G2-12 BioTwin, operated at 120 kV. SEM and TEM sample preparations and their analysis were performed at the Center of Microscopy at the Universidade Federal de Minas Gerais (UFMG).
Structural analysis
The micro-morphological analysis of the L. fortunei gills structure was performed using SEM images. TEM was used to describe the ultrastructural morphology of the ciliary gill epithelium. When suitable, measurements of the gill filaments were performed using transmitted light microscopy images (Additional file 2), taken in a Leica DM4500 microscope, with the same plastic embedded blocks for TEM preparation. Filament width, interfilament space, and the filament linear density (number of filaments per linear distance) were obtained from SEM images. Cilia length was obtained in SEM images, and the cilia diameter and cilia linear density (number of cilia onto the filament over the linear distance along the filament) using the TEM images. Measurements were performed using the free softwares FIJI / ImageJ v. 1.52p (Wayne Rasband - National Institutes of Health, USA) and the DigitalMicrograph® v. 3.41.2916.1 (Gatan, Inc).