Observation on Hemicriconemoides brachyurus (Loos, 1949) Chitwood & Birchfield, 1957 associated with grass in South Africa

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 Hemicriconemoides. Pictures were taken with a Nikon Eclipse 80i light microscope provided with differential interference contrast optics (DIC) and a Nikon Digital Sight DS-U1 camera (Nikon, Tokyo, Japan). The measurements and morphological analysis were done using microscopic facilities of Aquaculture Research Unit, University of Limpopo. Micrographs were edited using Adobe® Photoshop® CS.

DNA extraction was done using the Chelex method (Straube & Juen, 2013). Two specimens of the 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. Thirty 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 two hours and then incubated at 95 °C for 10 minutes to deactivate the proteinase K and finally spun for 2 min at 16000 rpm (Shokoohi, 2021). The supernatant was then extracted from each of the tubes and stored at –20 °C. Following this step, the forward and reverse primers, 988F (5^ʹ-CTCAAAGATTAAGCCATGC-3^ʹ) and 1912R (5^ʹ-TTTACGGTCAGAACTAGGG-3^ʹ) (according to Carta & Li, 2018), were used in the PCR reactions for partial amplification of the 18S rDNA region. PCR was conducted with eight μl of the DNA template, 12.5 μl of 2X PCR Master Mix Red (Promega, USA), one 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 s at 94°C; 54 °C annealing temperatures for 18S rDNA; extension for 45 s 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, four μl of product from each tube was loaded on a 1 % agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and one mM EDTA) for evaluation of the DNA bands. The bands were stained with RedGel and visualized and photographed on a UV transilluminator. The amplicons 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 (Guindon & Gascuel, 2003; Darriba et al., 2012). Analysis using the GTR+G+I model was initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 10⁶ generations for 18S rDNA. The trees were visualized with the TreeView program. Also, as outgroups, Criconemoides myungsugae (MH444644) was selected based on Nguyen et al. (2020). The original partial 18S rDNA sequence of H. brachyurus was deposited in GenBank under the accession numbers OM914954.

To evaluate the morphological variations between the nematodes isolated in this study and the nematodes species, including H. brachyurus and H. promissus Vovlas, 1980 as the close species of the genus Hemicriconemoides, principal component analyses (PCA) and Discriminant Analysis (DA) with different morphological traits were conducted. PCA analyses were carried out in XLSTAT (Addinsoft, 2007). Various morphometric features were obtained from fixed nematodes, including an average of body length, a, b, c, V, stylet length, total body annuli (R), Rex (annuli number from anterior end to the excretory pore), RV (annuli number from anterior end to the vulva), Rst (annuli number from anterior end to stylet knobs), Rph (annuli number from anterior end to pharynx), Ran (annuli number from tail terminus to anus), VL/VB (distance from terminus to vulva divided by body diameter at vulval position) were included in the PCA and DA analyses. The morphometric measurements of other species were taken from their original descriptions (Van den Berg & Heyns, 1977; Geraert, 2010; Vovlas, 1980; Vovlas et al., 2006). The measures were normalized using XL-STAT software before their analysis (Addinsoft, 2007). The scores values were determined for each species based on each of the principal components, and the scores for the first two components were used to form a two-dimensional plot (F1 and F2) of each isolate based on the eigenvalues given by the software XLSTAT.

The morphological analyses confirmed that the species was H. brachyurus. Measurements of H. brachyurus in this study are in agreement with the measurement of H. brachyurus in Geraert (2010), and Van den Berg and Heyns (1977) (Table 1). Females of H. brachyurus are characterized by having body slightly curved ventrally (Fig. 1E); lip region with two annuli, slightly flattened labial disc (Fig. 1B); vulva having flap (Fig. 1C), stylet robust, knobs 5 – 10 μm wide, and 3 – 5 μm high (Fig. 1A); vulva located near the posterior end; tail conical with small rounded lobe, in all specimens (Fig. 1D). Male not found.

Fig. 1

The forward 988 and reverse 1912 primers of 18S rDNA for H. brachyurus isolated 940 base pairs long. The nBlast test of 18S rDNA showed 98 % similarity of the test population with the Chinese population of H. fujianensis (MH444620). Compared with H. kanayaensis (MG029559) from China, it shows 15 (98 % identity) base pairs differences.

Our phylogenetic analysis using 18S rDNA, placed the South African H. brachyurus population in a clade together with other H. fujianensis, H. kanayaensis, and H. paracamelliae populations (Fig. 2). Findings in the current study were in agreement with the phylogenies of Hemicriconemoides species studied using 28S rDNA (Nguyen et al., 2020). Hemicriconemoides brachyurus and H. fujianensis are placed together in a group. This is also supported by their morphological similarities. They are similar in terms of the stylet of the female (48 – 69 vs 56 – 70 μm), VL/VB (0.8 – 1.4 vs 1.2 – 1.9), and presence of male (as both have sperm-filled spermatheca) (see Geraert, 2010; Maria et al., 2019; Mwamula et al., 2020). However, these species can be distinguished appropriately by the absence of a lateral vulval flap and a blunt tail terminus in H. fujianensis vs lateral vulval flap present and terminus ending in a small rounded lobe in H. brachyurus. Two permanent microscope slides containing the females of H. brachyurus were deposited in the Nematology collection of the University of Limpopo, South Africa. According to the literature, this is the first record of 18S rDNA sequence for H. brachyurus in South Africa. In conclusion, the ecological behavior of this species needs to be studied to find out the economic importance of the pest.

Fig. 2

Principal component analysis using morphometric features of H. brachyurus and H. promissus based on the female analysis showed that H. brachyurus has a morphometric variation (Fig. 3). The analyzed morphological characters allowed a clear separation between H. brachyurus and H. promissus.

Fig. 3

An accumulated variability of 51.23 % was observed in female-based PCA, specifically, 28.11 % in the F1 and 23.12 % in the F2 (Fig. 3).

We observed in the PCAs that some populations of H. brachyurus were placed together. The previously reported South African H. brachyurus (Heyns, 1970; Van den Berg & Heyns, 1977) showed similar morphological characters. The PCA result also indicated that the Japanese population of H. brachyurus (Gotoh, 1965) differ morphometrically from the rest of the populations (Fig. 3). The results indicated that there is intraspecific morphological variation across H. brachyurus, and it depends on the nematode’s geographical location (Fig. 3). Additionally, based on the DA, the populations of H. brachyurus and H. promissus grouped separately (Fig. 4). These two species are very similar in terms of morphology. However, they differ only on the basis of the vulval flap, which is present in the H. brachyurus and absent in the H. promissus (Geraert, 2010). Hemicriconemoides promissus was described by Vovlas in 1980. Then its validity was studied using a population from Spain by Vovlas et al. (2006). The DA analysis revealed that these two species are valid and distinguished by their morphology. The result obtained agrees with the result obtained by Vovlas et al. (2006).

Fig. 4

In conclusion, the vulval flap is a valuable taxonomic character in the species diagnosis of Hemicriconemoides. Besides, the ecological role of H. brachyurus needs to be investigated in the grassland quality of South Africa.