Glycolmethacrylate

Samples of young and mature leaves, and galls at the stages of induction, growth and development, maturation and senescence [19 (link)] (n = 5 per developmental stage) were collected from different individuals, and fixed in Karnovsky’s solution in 0.1 M phosphate buffer (pH 7.2) [23 ]. The material was dehydrated in ethanol series [24 ], embedded in glycolmethacrylate (Leica, Wetzlar, Germany), sectioned (6–10 μm) with a rotary microtome Hyrax (Zeiss, Oberkochen, Germany), stained with 0.05% Toluidine O blue, pH 4.6 [25 ]. Part of the material was dehydrated in n-butyl series [24 ], embedded in Paraplast [26 ], sectioned (12–14 μm) with a rotary microtome (Jung biocut) and stained with 9:1 (v/v) Astra blue and safranin [27 ] (modified to 0.5%). Histological slides were observed and photographed under light microscope (Leica DM500) coupled with digital camera (Leica ICC50 HD).

Unstained material (n = 3; from different individuals) fixed in Karnovsky’s solution in 0.1 M phosphate buffer (pH 7.2) [23 ], dehydrated in ethanol series [24 ], and embedded in glycolmethacrylate (Leica) was incubated with the monoclonal antibodies JIM5, JIM7, LM1, LM2, LM5, LM6, LM19 and LM20 (Centre for Plant Sciences, University of Leeds, UK). These antibodies specifically bind epitopes of low methylesterified homogalacturonans (HGAs) (JIM5) [15 (link), 16 (link), 29 (link), 30 (link)], medium- high methylesterified HGAs (JIM7) [15 (link), 16 (link), 30 (link)], extensins (LM1) [31 (link)–34 (link)], arabinogalactan proteins (APGs) (LM2) [35 ], galactans (LM5) [36 (link)], arabinans (LM6) [37 (link)], unesterified HGAs (LM19), and high methylesterified HGAs (LM20) [38 (link)]. The sections were immersed in blocking solution with 3% (w/v) powdered milk in PBS for 30 min to avoid cross labelling, and incubated with primary antibodies in PBS for 2 h at room temperature. For the control tests, the primary antibodies were suppressed. Sections were washed in PBS, and incubated in the secondary antibody anti-rat IgG—FITC (Sigma, St. Louis, MO, USA) in PBS for 2 h in the dark. After washing in PBS, the sections were mounted in 50% glycerin and analyzed using a Confocal Zeiss 510 META microscope, with excitation wavelength of 488 ηm and 505–530 ηm emission filter.

Gonads of 10 juvenile individuals (Thuringia population) were dissected from the body cavity prior to gutting of the fish. A central gonadal section of > 1 cm length was immediately transferred into 4% Histofix (Carl Roth, Germany) for histological analyses. All specimens from Coari, Pimenta Bueno, Presidente Figueiredo and Senador La Rocque were anaesthetized with 0.01% benzocaine (Acros, Morris, NJ, USA) and euthanized by cerebral concussion. A small sample of the fin was clipped for DNA extraction and a piece of the gonad was dissected and immediately fixed in 5% glutaraldehyde in phosphate buffer (0.1 mol l⁻¹ at pH 7.2), dehydrated and embedded in glycol methacrylate (Leica, Heidelberg, Germany). Samples were cut in 5 μm thick sections and stained with hematoxylin–eosin staining as described^{82 (link)}. Animals were sexed according to da Costa Amaral et al.⁸³ .
The animals originated from Pentecoste were sexed by endoscopy^{84 (link)} or vitellogenin detection, using the enzyme immune assay kit (Acobiom, Montpellier, France) developed specifically for Arapaima gigas according to the supplier’s recommendation.