Morphological and molecular characterization of Xiphinemella esseri Chitwood, 1957 (Dorylaimida: Leptonchidae) from Florida, with the first molecular study of the genus

Nematode populations used in this study were obtained from four samples collected under the canopy of associated live oak (Quercus virginiana Mill.) trees in north Florida. Nematodes were extracted from soil using the centrifugal flotation method (Jenkins, 1964). All the extracted needle nematodes were used for morphological and molecular analyses. Nematodes were killed and fixed using the Golden’s method described in Southey (1970). Live specimens were hand-picked with an eye lash and transferred into 2 to 3 ml of distilled water in a watch glass and put in an oven at 43°C. After 15 min the watch glass was filled with fixative (a water solution of 3% formaldehyde and 2% glycerol) that was kept in the oven at the same temperature. The watch glass was then enclosed in a petri dish and kept in oven to allow a slow evaporation of the fixative and the infiltration of the nematode with glycerol for three to five days. Specimens were then mounted on permanent glass slides to allow handling and observation under LM.

Measurements of specimens were taken with an Olympus BX51 (Olympus, Tokyo, Japan) ocular micrometer. Morphometrics included de Man’s indices and standard measurements. Spicule terminology used follows Peña-Santiago et al. (2014). Some of the best-preserved specimens were photographed with the same microscope equipped with differential interference contrast and a Canon EOS 250D digital camera (Canon, Tokyo, Japan). Digital images were edited using Adobe^® Photoshop^® CS (Adobe Systems, San Jose, CA).

DNA was extracted from single individuals using the proteinase K protocol. DNA extraction, PCR, and cloning protocols were used as described by Tanha Maafi et al. (2003). The primers used for amplification of the D2-D3 expansion segments of 28S rRNA gene were the D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and the D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) (Subbotin et al., 2006). The 18S rRNA gene was amplified using two sets of primers (two overlapping fragments): (i) forward G18SU (5′-GCT TGT CTC AAA GAT TAA GCC-3′) and reverse R18Tyl1 (5′-GGT CCA AGA ATT TCA CCT CTC-3′) and (ii) forward F18Tyl2 (5′-CAG CCG CGG TAA TTC CAG C-3′) and reverse R18Tyl2 (5′-CGG TGT GTA CAA AGG GCA GG-3′)(Chizhov et al., 2006). PCR products were purified using the QIAquick PCR purification Kit (Qiagen) and used for direct sequencing. The sequencing was performed at GENEWIZ (San Francisco, CA, USA). The newly obtained sequences were submitted to the GenBank database under accession numbers: MW221076, MW221077, MW590305. The newly obtained sequences of the D2-D3 of 28S rRNA and 18S rRNA genes were aligned using ClustalX 1.83 (Thompson et al., 1997) (the D2-D3 of 28S rRNA alignment parameters: gap opening – 5, gap extension – 3; 18S rRNA alignment parameters: gap opening – 15, gap extension – 6.66) with published gene sequences (Holterman et al., 2008; Mullin, 2004; van Megen et al., 2009). All sequence alignments were analyzed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) under the GTR + G + I model. BI analysis was initiated with a random starting tree and was run with four chains for 8.0 × 10⁶ generations for the D2-D3 of 28S rRNA alignment and 3.0 × 10⁶ generations for the 18S rRNA alignment. Posterior probabilities (PP) in percentage are given on appropriate clades.

Xiphinemella esseri (Chitwood, 1957).

Figure 1.

Figure 1:

Nine females and seven males in good state of preservation.

See Table 1.

The new population of this species is characterized by its 2.30 to 3.32 mm long body, labial disc well developed, lip region offset by constriction and 16.5 to 17.5 μm broad, odontostyle 46 to 49 μm long with minute aperture, neck 288 to 296 μm long, pharyngeal expansion 80 to 87 μm long or 28 to 30% of total neck length, uterus a tripartite tube-like structure and 194 to 296 μm long or 3.6 to 5.9 times the corresponding body diameter, pars refringens vaginae absent, vulva transverse (V = 45.4-49.7), tail short and rounded (18-28 μm in length, c = 94-158, c′ = 0.6-0.9), spicules 41 to 45 μm long, and 8 to 10 irregularly spaced ventromedian supplements bearing hiatus.

The material herein examined fits well, morphologically and morphometrically, with the original description of Xiphinemella esseri by Chitwood (1957), and the subsequent re-description by De Bruin and Heyns (1991). Although some morphometrical differences were observed in the spicule length (41-45 and 40-45 μm in this study and original description by Chitwood (1957), respectively, vs 31 to 36 μm long in De Bruin and Heyns (1991). The differences found in the spicule length might be due to the fact that in our study, and also in the original description of the species, the measure of the arc of the spicule are provided, whereas in the De Bruin and Heyns’ description most likely (it is not specifically mentioned in the description) the measure of the chord of the spicule was taken.

The D2-D3 of 28S rRNA gene alignment generated with modified parameters (gap opening = 5 and gap extension = 3) was 907 bp in length and contained 64 sequences. Phylogenetic relationships of X. esseri within selected dorylaimid nematodes are given in Figure 2. Two sequences obtained from two specimens of X. esseri differed in 3 bp and clustered together (Clade XI). Relationships of X. esseri with other dorylaims were not resolved. This result is most likely due to the lack of 28S rDNA sequences of other leptonchid genera.

Figure 2:

The 18S rRNA gene alignment was 1754 bp in length and contained 66 sequences. Phylogenetic relationships of X. esseri within selected dorylaimid nematodes are given in Figure 3. Sequences obtained from X. esseri clustered with those of several other genera of Leptonchidae (Leptonchus, Tyleptus, Funaria, and Proleptonchus) in a well-supported clade (posterior probabilities BI: 96). This result supports and justifies the classification of Xiphinemella under the family Leptonchidae.

Figure 3:

Xiphinemella species have been reported associated in many geographical regions with forest trees and herbaceous plants such as cowpea (Vigna unguiculata (L.) Walp.), rice (Oryza sativa L.) and others (De Bruin and Heyns, 1991). However, there are no reports on the feeding habits of these species. So far, the species X. esseri has been found only in Florida, where it is often associated with oak trees such as live oak and Spanish oak or Southern red oak (Quercus falcata Michx.). The original population described by Chitwood was collected from a Spanish oak growing in the north west section of the city of Gainesville, an area now developed into residential subdivisions and apartment complexes. For their re-description, De Bruin and Heyns (1991) used specimens collected by R. P. Esser from weeds associated with Magnolia grandiflora L. in Brandon (about 200 miles south of Gainesville). The specimens used for this study were collected from live oaks in a tree farm operation located in the city of Alachua (latitude 29°88′00.63″ N, longitude 82°48′26.41″ W). The nematode records of the Florida Department of Agriculture and Consumer Services indicate that movement of this nematode with the trade of infested oak trees to other localities in and outside Florida has occurred for a long time. Many longidorids such as Longidorus longicaudatus (Siddiqi, 1962), species of Xiphinema americanum group and Xiphinema setariae (Luc, 1958) feed on oak roots and are associated with X. esseri. It seems X. esseri may also have plant parasitic habits as suggested by Chitwood (1957), although the parasitism of this species has not been scientifically demonstrated. For the convenience of taxonomists, we have deposited nine females and seven males mounted on glass slides in the nematode collection of the National Museum of Natural Sciences, Madrid, Spain; in addition, three females, two males and five juveniles were sent to the United States Department of Agriculture Nematode Collection, Beltsville, MD, USA.