The genus
The specimens were extracted using the tray method and were fixed with a hot 4% formaldehyde solution and transferred to anhydrous glycerin using the De Grisse (1969) method. The classification provided by Geraert (2010) was used for the taxonomical study of
DNA extraction was done using the Chelex method (Straube and Juen, 2013). Five specimens of each species were hand-picked with a fine tip needle and transferred to a 1.5 ml Eppendorf tube containing 20 μl double distilled water. The nematodes in the tube were crushed with the tip of a fine needle and vortexed. In total, 30 microliters of 5% Chelex® 50 and 2 µL of proteinase K were added to each of the microcentrifuge tubes that contained the crushed nematodes and mixed. These separate microcentrifuge tubes with the nematode lysate were incubated at 56°C for 2 hr and then incubated at 95°C for 10 min to deactivate the proteinase K and finally spin for 2 min at 16,000 rpm (Shokoohi et al., 2020). The supernatant was then extracted from each of the tubes and stored at −20°C. Following this step, the forward and reverse primers, SSU F04 (5′-GCTTGTCTCAAAGATTAAGCC-3′) and SSU R26 (5′-CATTCTTGGCAAATGCTTTCG-3′) (Blaxter et al., 1998); D2A (5″-ACAAGTACCGTGAGGGAAAGTTG-3″) and D3B (5″-TCGGAAGGAACCAGCTACTA-3″) (De Ley et al., 1999), were used in the PCR reactions for partial amplification of the 18S and 28S rDNA region, respectively. PCR was conducted with 8 μl of the DNA template, 12.5 μl of 2X PCR Master Mix Red (Promega, USA) for the Botswanan specimens, 1 μl of each primer (10 pmol μl−1), and ddH2O for a final volume of 30 μl. The amplification was processed using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), with the following program: initial denaturation for 3 min at 94°C, 37 cycles of denaturation for 45 sec at 94°C; 54°C and 56°C annealing temperatures for 18S and 28S rDNA; extension for 45 sec to 1 min at 72°C, and finally an extension step of 6 min at 72°C followed by a temperature on hold at 4°C. After DNA amplification, 4 μl of product from each tube was loaded on a 1% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with RedGel and visualized and photographed on a UV transilluminator. The amplicons of each gene were stored at −20°C. Finally, the PCR products were purified for sequencing by Inqaba Biotech (South Africa). The ribosomal DNA sequences were analyzed and edited with BioEdit (Hall, 1999) and aligned using CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The HKY + Γ (gamma distribution of rate variation with a proportion of invariable sites) model was selected using jModeltest 2.1.10 (Darriba et al., 2012; Guindon and Gascuel, 2003). Analysis using the HKY + Γ model was initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 106 generations for 18S and 28S rDNA. The trees were visualized with the TreeView program. Also, as outgroups,
The morphological and molecular analyses confirmed that the species was
Measurements of females of
|
10 ♀♀ |
|
318 ± 24.8 (275-342) |
|
9.7 ± 0.4 (9.2-10.2) |
|
3.4 ± 0.1 (3.2-3.6) |
|
40.6 ± 4.5 (34.1-44.7) |
|
0.5 ± 0.02 (0.4-0.5) |
VL/VB | 0.6 ± 0.4 (0.7-0.9) |
|
91 ± 3.5 (88-96) |
VL | 24 ± 0.5 (23-24) |
Stylet | 48 ± 2.3 (45-51) |
Nerve ring | 71 ± 4.9 (67-74) |
Pharynx | 96 ± 5.7 (87-103) |
Excretory pore | 102 ± 8.8 (96-115) |
Neck base diameter | 33 ± 1.4 (31-34) |
Vulval-body diameter | 28 ± 3.1 (25-32) |
Anal body diameter | 17 ± 1.8 (16-20) |
Tail length | 8 ± 0.8 (7-9) |
Rst | 12 ± 1.0 (11-13) |
Rph | 22 ± 0.5 (21-22) |
Rex | 21 ± 1.2 (20-22) |
Rv | 4.6 ± 0.5 (4-5) |
Ran | 2.3 ± 0.5 (2-3) |
Rvan | 2.6 ± 0.5 (2-3) |
|
66 ± 1.4 (65-67) |
The forward SSU F04 and reverse SSU R26 primers of 18S rDNA (Blaxter et al., 1998); forward D2A and the reverse D3B primers of 28S rDNA for
The phylogenetic analysis using 18S and 28S rDNA placed the Botswanan