Axiocam 208 camera

Nematodes were extracted from soil samples using the modified Cobb sieving and flotation-centrifugation method [62 ]. For preliminary examinations, fresh nematodes were transferred to the drop of distilled water, heat relaxed at 60 °C for 30–45 s, and observed under the Zeiss Axioskope 40 microscope. Permanent mounts were prepared as described in Seinhorst [63 (link)] and De Grisse [64 ]. Light micrographs of the mounted specimens were acquired using a Zeiss Axioskope 40 microscope equipped with a Zeiss Axiocam 208 camera (Carl Zeiss Microscopy, Jena, Germany). Standard morphometrical characters were selected based on previously published studies [25 (link),28 (link),57 (link),65 ]. Measurements were made using ZEN blue 3.1 imaging software (Carl Zeiss Microscopy).

Soil samples were collected from different cultivated areas of southern Alberta. Nematodes were extracted from soil samples using the modified Cobb sieving and flotation-centrifugation method [14 ]. Mixed populations of Boleodorus species were detected in the samples from four different fields. Boleodorus members were collected individually from the mixture of soil nematodes and assigned the population numbers 40, 50, 61, and 62. For preliminary examinations, fresh Boleodorus adults were transferred to a drop of distilled water, heat relaxed at 60 °C and observed under a Zeiss Axioskope 40 microscope. Nematodes were fixed and permanent slides were prepared according to the methods of Seinhorst [15 (link)] and De Grisse [16 ]. Photo documentation of each specimen was carried out using a Zeiss Axioskope 40 microscope equipped with a Zeiss Axiocam 208 camera (Carl Zeiss Microscopy, Jena, Germany). Measurements on images were performed using ZEN blue 3.1 imaging software (Carl Zeiss Microscopy).

To further investigate if the melanized deposits observed in the cockroach gut do in fact contain melanin, we carried out two additional experiments. In the first experiment, we adapted a protocol to quantify melanin concentration in cells based on absorbance of light at 405 nm (39 (link), 40 (link)). Colony derived adult male cockroaches were starved for 3 d. Whole guts were dissected and scored based on the presence or absence of melanized deposits in the foregut by stereomicroscopy. The guts were then homogenized using a pestle in 500 µL of 1 N NaOH/10% DMSO solution. One hundred microliters of each sample in triplicate was loaded into a 96-well plate and absorbance at 405 nm was measured on a plate reader. Synthetic melanin (Sigma Aldrich, St. Louis, MO, USA) dissolved in the same solution was used as a positive control. A non-parametric one-tailed Mann–Whitney test was used to compare absorbance values in guts with or without melanin deposits as the assumption of normality was not met.
In a second experiment, we followed a process for detecting melanin autofluorescence by fixation in Carnoy’s solution (41 (link)). A solution of ethanol-chloroform-acetic acid, 6:3:1 [vol/vol] was used to fix three individual whole guts from colony derived males overnight. Autofluorescence was then observed using an Axioskop 2 fluorescence microscope with an Axiocam 208 camera (Zeiss).