Nematodes of the genus
During a nematode survey in Georgia, a nematode species was recovered from the body of a dead individual of
To obtain an isogenic population of this nematode species, one mature female and male were placed on water traps in the feeding area containing crushed
Nematode pathogenicity of
Measurements were conducted using a light biological research microscope (Motic®-DMB1). Nematodes were measured with an ocular micrometer and illustrated using a drawing (RA-4) tube.
Drawings and photomicrographs of nematodes fixed on glass slides were taken using a digital video camera Genius (G-Shot) DV 1110 (Figs. 1-3). In total, 20 specimens from each stage (adults and 22 specimens of dauer juveniles) were randomly collected from
Nematodes were fixed in triethanolamine formalin (TAF) (Courtney et al., 1955) and processed to anhydrous glycerol for mounting (Seinhorst, 1959). Specimens were mounted on glass slides, and the coverslip was supported by glass rods to avoid flattening. Morphometric characters were selected according to Hominick et al. (1997) and reported in Table 1.
Ratios and measurements (in µm) of heat relaxed specimens of
Character | Females | Males | 3rd juvenile stage |
|
---|---|---|---|---|
|
20 | 20 | 22 | 11 |
Body length | 1566 ± 120 (1353-1867) | 1280 ± 100 (1080-1494) | 663 ± 51 (566-772) | 1629 (1142-2149) |
Body width | 96 ± 10 (75-112 | 62 ± 4.8 (55-70) | 30 ± 2.4 (25-35) | 107 (80-132) |
Lip region diam | 19 ± 1.6 (16-22 | 17 ± 1.5 (15-20) | – | – |
Stoma length | 31 ± 1 (30-32) | 29 ± 2.2 (25-32) | 24 ± 1.7 (22-30) | 33 (30-38) |
Stoma diam | 5.1 ± 0.4 (5.0-6.2) | 4.7 ± 0.4 (3.7-5.0) | 2.6 ± 0.4 (2.5-3.7) | – |
Anterior end to nerve ring | 178 ± 6 (172-197) | 155 ± 10 (137-175) | 102 ± 6 (92-120) | – |
Stoma length as % pharynx length | 12.2 ± 0.4 (11-13) | 13 ± 1.2 (11-15) | 16 ± 0.8 (14-17) | – |
Pharynx length | 252 ± 5 (237-257) | 219 ± 17 (192-250) | 152 ± 6 (142-177) | – |
Corpus length | 144 ± 7 (135-162) | 122 ± 10 (107-137) | 87 ± 2 (80-92) | – |
Corpus as % pharynx length | 56 ± 2.3 (53-63) | 55 ± 2 (50-59) | 57 ± 2.5 (52-61) | – |
Median bulb (MB) diam | 27 ± 2.1 (22-30) | 30 ± 4.9 (20-37) | 11 ± 0.8 (10-12) | – |
Terminal bulb (TB) diam | 39 ± 4.1 (32-47) | 34 ± 2.7 (32-40) | 15 ± 0.6 (12-17) | 36-50 |
MB diam. as % TB diam | 77 ± 9.5 (54-88) | 79 ± 6.8 (62-93) | – | – |
Anterior end to excretory pore | 236 ± 14 (212-270) | 205 ± 13 (164-227) | 130 ± 10 (110-150) | 235 (183-295) |
Ex. pore posit. as % phar. length | 93 ± 5 (83-105) | 93 ± 8 (75-109) | 85 ± 6.7 (75-98) | – |
Tail length | 69 ± 11 (52-95) | 53 ± 5 (45-62) | 72 ± 4 (65-82) | – |
Anal (cloacal) body width (ABW) | 40 ± 3 (32-47) | 37 ± 3 (32-42) | 19 ± 1.8 (16-22) | – |
Gonad lengtha | 1285 ± 198 (995-1658) | 956 ± 87 (747-1268) | – | 890 (653-1157) |
Gonad length as % body length | 82 ± 15 (61-107) | 72 ± 5 (66-82) | – | 46-63 |
Gonad length. as % intestine leng.b | 108 ± 23 (72-143) | 93 ± 11 (69-110 ) | – | – |
Anterior gonad branch length | 800 ± 104 (555-928) | – | – | – |
Posterior branch length | 546 ± 128 (290-794) | – | – | – |
Testis flexure length | – | 201 ± 49 (123-260) | – | – |
Ant. flexure as % of ant. branch | 33 ± 3 (26-42) | 17 ± 4 (13-24) | – | – |
Post. flex. as % of post. branch | 27 ± 6 (13-34) | – | – | – |
Sperm diameter | n.d. | 5 ± 0.6 3. (7-7.5) | – | – |
Egg length | 60 ± 3.4 (55-67) | – | – | – |
Egg diam | 41 ± 4 (35-47) | – | – | – |
Rectum length | 38 ± 3.2 (30-42) | n.d. | 24 ± 1.5 (22-25) | – |
Rectum length/ABW | 0.9 ± 0.1 (0.8-1.3) | n.d. | 0.9 ± 0.1 (7-1.2) | – |
Anus to phasmid distance | 23 ± 1.8 (20-27) | n.d. | – | – |
Anus to phasmid distance/ABW | 0.6 ± 0.05 (0.4-0.7) | n.d. | – | – |
Posit. phasmid as % tail length | 35 ± 6 (27-48) | – | – | 32-61 |
Spicule length | – | 66 ± 3 (60-72) | – | 55-82 |
Gubernaculum length | – | 44 ± 2.2 (40-50) | – | – |
Gubern. leng. as % spic. length | – | 66 ± 4.5( 60-78) | – | – |
|
16.3 ± 2 (13.5-20.3) | 20.7 ± 1 (19-23.1) | 22.3 ± 1.5 (19.3-25.8) | 15.1 (12-18.7) |
|
6.1 ± 0.4 (5.4-7) | 5.8 ± 0.5 (5.1-7) | 4.2 ± 0.2 (3.7-4.7) | 6.2 (4.9-7.6) |
|
23 ± 3.4 (17.6-32.2) | 24.3 ± 2.8 (20.5-30.4) | 9.1 ± 0.5 (8-10) | 33 (26.3-37.9) |
|
1.6 ± 0.6 (1.3-2.2) | 1.3 ± 0.1 (1.2-1.7) | 3.7 ± 0.3 (3.2-4.5) | – |
V (vulva posit. in % body length) | 57 ± 1.9 (51.8-59.1) | – | – | 58 (55-64) |
Diagnostics characters (Table 2) were used to compare
Data matrix for cluster analysis of the species of
Characters | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
1 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 |
|
0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 2 | 1 | 0 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
|
1 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 |
|
0 | 2 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 2 | 1 | 0 | 2 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 |
|
0 | 2 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
|
0 | 0 | 0 | 0 | 1 | 1 | ? | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 2 | 0 | 0 | 0 | 0 |
|
1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 |
DNA was extracted from 15 individual specimens. Specimens were handpicked and singly placed on a glass slide in the lysis buffer (10 mM Tris-HCl, pH8.8, 50 mM KCl, 15 mM MgCl2, 0.1% Triton X100, 0.01% gelatine with 90 µg/ml proteinase K) and then cut into small pieces under a dissecting microscope, after which samples were incubated at 65°C for 1 hr, followed by deactivation of the proteinase K. PCR amplification, cloning, and sequencing protocols are described in detail by De Luca et al. (2004). The following primers were used for the ITS1-5.8S-ITS2 region using the forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and the reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (Joyce et al., 1994); for the D2 to D3 expansion segments of 28S rRNA using forward D2A (5′-ACAAGTACCGTGGGGAAAGTTG-3′) and reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (Nunn, 1992), the mitochondrial COI was amplified using COI-F1 (5′-CCTACTATGATTGGTGGTTTTGGTAATTG-3′) and COI-R2 (5′-GTAGCAGCAGTAAAATAAGCACG-3′) (Kanzaki and Futai, 2002). PCR amplifications were carried out in 100 µl volumes. PCR mix was added to each tube: 10 µl 10 x PCR buffer, 2 µl dNTP mixture (10 mM each), 2 µl of each primer 10 mM, 0.25 µl of Taq DNA polymerase (Roche), 73.5 µl of distilled water and 10 µl of crude DNA. Cycling conditions were 1 cycle of 94°C for 7 min followed by 35 cycles of 94°C for 50 sec, 55°C for 50 sec, and 72°C for 50 sec. The last step was 72°C for 10 min. PCR products were purified using the protocol listed by the manufacturers of Nucleospin Extract II (Macherey-Nagel, Duren) or QIAquick (Qiagen, USA) gel extraction kits and used for cloning or direct sequencing in both directions with the primers given above or M13 forward and M13 reverse primers. pGEM-T Vector System II kit (Promega) was used for cloning of PCR products. ITS-RFLP analyses were performed on PCR products from individual nematodes and digested with five units of the following restriction enzymes:
The newly obtained sequences were aligned together with publicly available homologous sequences of
Measurements and figures are reported in Tables 1, 2 and Figures 1-3.
Body length = 618 ± 45 (530-676); pharynx length = 156 ± 8.0 (145-169); anterior end to excretory pore = 121 ± 7.5 (111-135); tail length = 75 ± 5.0 (72-78);
Body is loosely sheathed by cuticle of second-stage juvenile, particularly visible by LM at mid-body and at anterior and posterior ends. The structure of the cuticle is barely visible under membrane and width of annuli 1.2 to 1.6 µm. Lateral fields are 5 to 6 µm wide at mid-body. Lip region is expanded, with incompletely merged lips, bearing sensillae (Fig. 2A). Metastomatal teeth are barely noticeable. Pharyngeal procorpus is 48 to 55% of total pharynx. Granular formations visible inside the body. Tails of second- and dauer stages long, and cone-shaped, tapering to pointed end (Figure 2C). Phasmids difficult to see.
Body is slightly longer and slender than that of dauer larva. Anterior body is end faintly annulated, width of five annuli at mid-body 9 to 12 µm; lateral fields are 7 to 8 µm wide at mid-body, extending from basal part of the stoma to the phasmids. Lips are merged (Fig. 2D); distal aperture is closed (Fig. 2F). Metastomatal teeth is not evident. Pharynx is longer than that of the dauer larva. Procorpus is 52 to 61% of pharyngeal length. Excretory pore is 75 to 98% of pharynx length. Intestinal cells are arranged to give a zebra-like appearance. Tail is conical (Fig. 2F), with finely rounded terminus. Phasmids are difficult to obseve by LM.
In male bursa, the standard number of papillae (10 papilla) and their location (1 + 1/5 + 3) display a range of variability similar to that reported for males of
Collected from the cadaver of a beetle,
The amplification of D2-D3 expansion domains of the 28S rDNA, the ITS containing region and the mitochondrial COI yielded single fragments of 556 bp, 611 bp, and 712 bp, respectively, based on sequencing.
Very low intra-individual and intra-population sequence variability in the D2 to D3 sequences have been observed (1-5 nucleotides). A BLAST search (Altschul et al., 1997) for D2 to D3 region showed a 100% identity with the homologous sequences of
The ITS sequence for Georgian isolate of
The ITS1 and ITS2 sizes were 186 bp and 217 bp, respectively, constituting the shortest ITS recorded for nematodes so far. PCR-ITS-RFLP patterns for
The COI gene of one specimen sequenced here, as apparent from BLAST searches against the GenBank database, was also the first such sequence deposited in that database for a
In the present study, an approach integrating morphological and molecular sequence analyses was used to characterize a Georgian population of
The morphology and morphometrics (Tables 1, 2) of the Georgian population of
Molecular characterization of the new isolate also improved identification of this species with respect to other
Second, the present phylogenetic analysis, using the D2-D3 expansion domains of the 28S rRNA gene, confirmed the grouping of the Georgian