Paraffin histology

Opisthosomata of five virgin late-subadult, five virgin adult, five mated late-subadult and eight mated adult females were separated from the prosoma and the cuticle was punctured several times to allow penetration of the fixative Duboscq-Brasil (picric acid, 80% ethanol, 40% formol and acetic acid) [43]. After a minimum of one week in the fixative, the samples were dehydrated and washed in a graded series of ethanol concentrations as follows: 80% ethanol for 2 h, 96% ethanol for 30 min, 96% ethanol for 30 min, 96% ethanol and ≥ 99.5% tetrahydrofuran for 2 h, tetrahydrofuran for 18 h. The samples were then transferred into 1:1 solution of heated paraffin and tetrahydrofuran and placed in a heating cabinet (63 °C) for 24 h. Finally, the solution was exchanged with heated paraffin and stored in a heating cabinet (63 °C) for at least 1 day [34, 44]. The sample was then placed into an embedding mold filled with paraffin where it was left to harden for 2 days before sectioning. The block was trimmed and transversal sections were produced with a microtome (Microm HM 360) filled with distilled water heated up to 40 °C. Sections were 5 μm thick and cutting speed was 30 mm/s. The section ribbons were placed on object slides coated thinly with protein-glycerin to increase the adherence of sections. The object slides were then transferred onto a heating plate (40 °C) for at least 20 min.

To remove the paraffin, the object slides were immersed into Roti-Histol for 10 min and were transferred for 5 min into 2-Propanol, 96, 80 and 60% ethanol and finally into distilled water. For staining, the slides were transferred into nuclear fast red-aluminium sulphate for 30 min, which stains the cell nucleus red [45]. After washing with distilled water, a 10 min treatment with phosphotungstic acid (5%) followed to bleach and stain connective tissue. The sections were washed with distilled water and treated with aniline blue-orange G-acetic acid for 10 min, which stains e.g. cytoplasm and connective tissue [45, 46]. The sections were then immersed in distilled water and dehydrated in 60, 80, 96% ethanol and 2-Propanol, each for 5 min. The sections were transferred into Roti-Histol for 5 min, then coated with Roti-Histokitt II and protected by cover slips. Selected paraffin sections were photographed using a customised Visionary Digital BK Plus imaging system and the images were adjusted as described above.

In the genus Latrodectus, a spermatheca has a dumb-bell shape consisting of an anterior (AL) and posterior lobe (PL) connected by a narrow middle region [21]. Consequently, for females of different developmental stages (late-subadult, adult) and mating status (virgin, mated), we measured the area of the lumen, the thickness of the cuticle and the surrounding epithelium for each spermathecal lobe separately. For the measurements, we chose the mid histological section of each lobe of the right spermatheca. The sections were photographed with a light microscope (Olympus BX60 Fluorescence Microscope, software AxioVision 4.8). To assess the spermathecal lobe area, we used the contour line tool (AxioVision 4.8), tracing along the inner side of the spermathecal cuticle. To receive an estimate of the thickness of the cuticle and the surrounding epithelium, we defined a central point within each spermathecal lobe area as the crossing point of two perpendicular axes of maximum length. To assess the thickness of cuticle and epithelium, we attempted to take eight measurements around each lobe (at 45 degrees) with each measuring axes starting from the central point of the spermathecal lobe. Since some spermathecal lobes were partly ruptured from sectioning, the data are based on less than eight measurements per spermatheca on average (mean + SD for cuticle AL: 7.48 ± 1.16; cuticle PL: 7.52 ± 0.71; epithelium AL: 7.36 ± 1.58; epithelium PL: 7.24 ± 1.45) for both late-subadult and adult females.