Currently, only five species of the genus [
In the present paper, a cultured population of
The nematodes were initially obtained from rotting pears on the ground in Borgata Città, Bologna, Italy (44°34′42.7″N 11°10′26.6″E; 27th October 2019; leg. Matteo Vecchi). Other than nematodes, the fruits were heavily colonized by
The specimens were killed by heating, fixed in a 70% ethanol solution, transferred to pure glycerin following the Siddiqi’s (1964) technique, and mounted on glass microscope slides with the glycerine-paraffin method (de Maeseneer and d’Herde, 1963) somewhat modified using hot liquid paraffin.
Observations were made and measurements were taken using a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope with a drawing tube (
Specimens preserved in glycerine were selected and prepared for observation under SEM according to Abolafia (2015). They were cleaned in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).
Nematode DNA was extracted from single individuals using a modified DNA extraction and PCR assays described by Castillo et al. (2003) and Archidona-Yuste et al. (2016). The specimens were cut in small pieces using a sterilized dental needle on a clean slide with 18 ml of TE buffer (10 mM Tris-Cl + 0.5 mM EDTA; pH 9.0), transferred to a microtube and adding 2 μ l proteinase K (700 μ g/ml‒1) (Roche, Basel, Switzerland), and stored to –80°C within 15 min (for several days) until processing. The microtubes were incubated at 65°C (1 hr), then at 95°C (15 min). For DNA amplification, 3 μ l of the extracted DNA was transferred to a microtube containing: 0.6 μ l of each primer (10 mM), 3 μ l Master Mix Taq DNA Polymerase (5x Hot FirePol Blend Master Mix) and ddH2O to a final volume of 20 μ l. The primers used for amplification of the region of 18S rRNA gene were the forward primer SSU F_04 (5′-GCTTGTCTCCAAAGATTAAGCC-3′) and the reverse primer SSU R_26 (5′-CATTCTTGGCAAATGCTTTCG-3′) (Blaxter et al., 1998). The primers used for amplification of the D2-D3 region of 28S rRNA gene were the forward primer D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the reverse primer D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 1999; Nunn, 1992). PCR cycle conditions were as follows: one cycle of 94°C for 15 min, followed by 35 cycles of 94°C for 45 sec + annealing temperature of 55°C for 45 sec + 72°C for 45 sec, and finally one cycle of 72°C for 5 min. After DNA amplification, 5 μ l of product was loaded on a 1% agarose gel in 0.5% Tris-acetate-EDTA (40 mM Tris, 20 mM glacial acetic acid and 2 mM EDTA; pH = 8) to verify the amplification using an electrophoresis system (Labnet Gel XL Ultra V–2, Progen Scientific, London, UK). The bands were stained with 1.25 µl RedSafe (20,000x) previously added to the agarose gel solution (25 ml). The sequencing reactions of the PCR products were performed at Sistemas Genómicos (Paterna, Valencia, Spain) according the Sanger et al. (1977) method. The DNA sequences obtained for
For phylogenetic relationships, the analyses were based on 18S and 28S rDNA fragments. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with other 18S and 28S rRNA gene sequences representative of Panagrolaimomorpha and closely related taxa available in GenBank (accession numbers available in Supplementary Table 1) with MAFFT ver. 7 (Katoh and Toh, 2008; Katoh et al., 2002) with the G-INS-i method (thread = 4, threadtb = 5, threadit = 0, reorder, adjustdirection, anysymbol, maxiterate = 1,000, retree 1, globalpair input). Alignments ends were trimmed using MEGA7 (Kumar et al., 2016) up to 1,851 and 1,195 bp for 18S and 28S respectively. The best-fit models of nucleotide substitution used for the phylogenetic analysis were selected using jModelTest 2.1.10 (Darriba et al., 2012). Sequences were concatenated with the R package ‘concatipede’ v1.0.0 (Vecchi and Bruneaux, 2021). The phylogenetic tree was generated with Bayesian inference method using MrBayes 3.2.6 (Ronquist et al., 2012).
=
Material examined: 10 females and 10 males obtained from culture.
Measurements are provided in Table 1.
Morphometrics of
Sex | Female | Male |
---|---|---|
n | 10 | 10 |
Body length ( |
1,114.2 ± 103.5 (993–1258) | 1,022.0 ± 117.5 (931–1,322) |
a | 26 ± 2.0 (23.6–29.6) | 27 ± 1.6 (24.6–29.9) |
b | 6.9 ± 3.4 (5.3–16.5) | 5.4 ± 0.5 (4.6–6.5) |
c | 7.5 ± 0.4 (6.8–8.1) | 8.6 ± 0.9 (7.8–10.9) |
c' | 6.0 ± 0.6 (4.9–7.0) | 4.4 ± 0.5 (3.6–5.1) |
V | 66.5 ± 1.8 (63–69) | – |
Lip region width | 14.8 ± 1.2 (12–16) | 14.1 ± 0.7 (13–15) |
Stoma length | 13.8 ± 1.4 (12–16) | 12.7 ± 1.2 (10–14) |
Pharyngeal corpus length | 109.4 ± 4.9 (104–120) | 107.4 ± 6.8 (93–114) |
Isthmus length | 39.6 ± 5.7 (30–50) | 40.0 ± 5.7 (32–53) |
Bulbus length | 27.9 ± 2.0 (24–31) | 27.8 ± 2.5 (24–33) |
Pharynx length | 176.1 ± 6.6 (167–190) | 175.2 ± 11.8 (151–189) |
Nerve ring – anterior end | 148.3 ± 8.8 (137–160) | 139.7 ± 11.0 (122–161) |
Excretory pore – anterior end | 128.9 ± 7.5 (120–139) | 115.8 ± 11.0 (104–139) |
Deirid-anterior end | 153.3 ± 9.9 (142–160) | 163.0 ± 6.1 (156–167) |
Neck length | 189.9 ± 6.8 (181–203) | 187.9 ± 12.5 (161–202) |
Body diameter at neck base | 37.9 ± 3.1 (34–44) | 35.1 ± 2.7 (33–42) |
Body diameter at midbody | 42.4 ± 4.6 (37–52) | 37.6 ± 4.1 (32–47) |
Lateral field width | 5.7 ± 2.1 (4–8) | 6.3 ± 1.0 (5–8) |
Anterior ovary or testis length | 551.4 ± 49.5 (463–650) | 233.0 ± 41.7 (188–289) |
Anterior oviduct length | 56.1 ± 15.8 (40–91) | – |
Anterior uterus length | 377.4 ± 63.8 (287–496) | – |
Post-vulval uterine sac length | 133.4 ± 22.2 (99–162) | – |
Vagina length | 42.0 ± 8.5 (36–48) | – |
Vulva – anterior end | 740.9 ± 74.0 (668–863) | – |
Rectum or cloaca length | 28.2 ± 3.8 (20–32) | 9.8 ± 0.7 (9–11) |
Anal body diameter | 25.1 ± 2.8 (22–30) | 27.2 ± 3.4 (23–34) |
Tail length | 148.9 ± 11.9 (133–170) | 119.6 ± 10.1 (104–137) |
Phasmid - anus distance | 55.6 ± 6.1 (46–67) | 47.7 ± 6.7 (36–57) |
Spicules length (arc) | – | 77.4 ± 3.3 (70–81) |
Gubernaculum length | – | 30.0 ± 1.2 (29–32) |
Moderately slender to slender nematodes of small size, 0.93–1.32 mm long. Upon fixation, habitus nearly straight in females or somewhat curved ventral, J-shaped, in males. Cuticle 1–2 µm thick, bearing minute transverse striations, with annuli 1–2 µm wide. Lateral fields occupying 10–20% of mid-body diameter, with four longitudinal incisures or three alae. Lip region continuous with the adjacent body: lips six, separate, slightly rounded, with protruding, rounded labial and cephalic sensilla; primary and secondary axils with similar morphology, primary ones slightly deeper. Oral opening large, with smooth margin. Amphids very small, oval, located at middle length of lateral lips. Stoma panagrolaimoid, 0.7–1.1 times the lip region diameter long: cheilostom large, slightly wider than long, with strongly refringent, bar-shaped rhabdia, posteriorly thicker; gymnostom very reduced, with small rhabdia; stegostom funnel-shaped with poorly refringent rhabdia, metastegostom bearing dorsal acute rhabdia (dorsal tooth). Pharynx also panagrolaimoid: pharyngeal corpus robust, subcylindrical, 2.2–4.0 times the isthmus length, with procorpus and metacorpus not well discernible; isthmus comparatively thin; basal bulb ovoid, with both valvular apparatus and posterior haustrulum well developed. Cardia small, surrounded by intestinal tissue. Nerve ring at 70–85% of neck length from the anterior end, surrounding the anterior part of isthmus. Excretory pore at 52–75% of neck length, at level of the posterior part of metacorpus. Hemizonid located at level of isthmus. Deirids 78–90% of neck length, at level of isthmus-bulb junction. Intestine without distinct specializations, but with slightly thinner walls at cardiac part; intestinal lumen with rest of diatom frustules.
Reproductive system monodelphic-prodelphic. Ovary very long, lacking flexure at post-vulval region, having oocytes arranged in several rows at its distal part and then in only one rows at its proximal part. Oviduct short, slightly longer than the body diameter developing a scarcely discernible spermatheca at its proximal part. Uterus very long, 6.8–10.9 times as long as body diameter, tubular, frequently including uterine eggs (20–33 × 28–54 µm) inside in different stages of development. Post-vulval uterine sac well developed, 2.6–3.8 times as long as the body diameter, with very thin walls, frequently poorly discernible, with proximal part short, tubular, and distal part large, swollen. Vagina extending inwards to 31–43% of body diameter, sigmoid. Vulva slightly protruding. Rectum short, 0.9–1.3 times the anal body width; three small gland-like cells are distinguishable around the intestine-rectum junction. Tail conical-elongate with acute terminus. Phasmids located at 34–40% of tail length from anus.
Reproductive system monorchic, with testis reflexed ventrad anteriorly. Spicules paired and symmetrical: manubrium ventrally bent, angular hook-shaped, reduced calamus, and very curved ventrad lamina lacking dorsal hump, with well-developed ventral velum, narrower at its middle length, and spatulate tip in lateral view having a refringent forked axis. Gubernaculum well developed, slightly curved, about 0.4 times of the spicules length, with thin corpus. Three small gland-like cells are distinguishable around the beginning of the cloaca. Tail conical, slightly curved ventrad, conoid anterior to phasmid and them filiform. Genital papillae seven pairs, tree pre-cloacal (GP1 and GP2 subventral, GP3 lateral) and four post-cloacal: two pairs subventral (GP5, GP6) and one subdorsal (GP4) at the middle of tail length and one subdorsal (GP7) at beginning of the filiform part. One mid-ventral adcloacal papilla (MP) present. Phasmids at 32 to 50% of tail length from the cloacal aperture, close to genital papillae GP7.
The material examined in this study agrees well with the type population of
Comparative morphometrics of
Reference | Present paper | Thorne (1938) | Hechler (1971a) | Andrássy (2005)** | ||||
---|---|---|---|---|---|---|---|---|
Country | Italy | USA | USA | Hungary | ||||
Sex | Female | Male | Female | Male | Female | Male | Female | Male |
n | 10 | 10 | ? | ? | 12 | 15 | ? | ? |
Body length ( |
993–1,258 | 931–1,322 | 1,000–1,400 | 800–1,200 | 1,170–1,410 | 900–1,222 | 1,170–1,410 | 800–1,200 |
a | 23.6–29.6 | 24.6–29.9 | 18.0 | 21.0 | 16.0–21.5 | 20.6–26.9 | 16.0–22.0 | 21.0–27.0 |
b | 5.3–16.5 | 4.6–6.5 | 8.0 | 6.8 | 6.1–8.0 | 5.7–7.4 | 6.1–8.0 | 5.7–7.4 |
c | 6.8–8.1 | 7.8–10.9 | 8.5 | 9.0 | 7.6–10.4 | 7.9–10.4 | 8.0–10.0 | 8.0–10.0 |
c' | 4.9–7.0 | 3.6–5.1 | 4.7* | 4.2* | 5.8* | 4.7* | 6.0 | 3.5–4.0 |
V | 63–69 | – | 73 | – | 71–77 | – | 71–77 | – |
Stoma length | 12–16 | 10–14 | ? | ? | 11–14 | 10–13 | 12–14 | 12–14 |
Spicules length (arc) | – | 70–81 | – | ? | – | 71–81* | – | ? |
Spicules length (chord) | – | 56–61 | – | ? | – | 54–70 | – | 50–70 |
Gubernaculum length | – | 28–29 | – | ? | – | 25–27 | – | 25–27 |
On the other hand, the most similar species to
The
Length of the spicules (measured in arc) of the
References | Ceylonensis | Dubius | Filiformis | Leperisini | Pycnus | Redivivus | Redivivus as leucocephalus | Redivivus as parasiticus | Silusioides |
---|---|---|---|---|---|---|---|---|---|
Present paper | – | – | – | – | 70–81 | – | – | – | – |
Goodey (1922) | – | – | – | – | – | 70* | – | – | – |
Steiner (1936) | – | – | – | – | – | – | ? | – | – |
Thorne (1938) | – | – | – | – | 74*** | – | – | – | – |
Sandground (1939) | – | – | – | – | – | – | – | 65–68* | – |
Rühm (1956) | – | – | – | – | – | 70–90* |
– | – | – |
Tsalolikhin (1965) | – | – | – | – | – | – | – | – | 75–90** |
Sanwal (1960) | – | 60–62* | – | – | – | – | – | – | – |
Hechler (1970) | – | – | – | – | – | 77* | – | – | – |
Hechler (1971a) | – | 56–60* | – | – | 71–81* | 64–74* | 45* | – | – |
Hechler (1971b) | 81–89* | – | – | – | 77–83* | – | – | – | – |
Sukul (1971) | – | – | 27* | – | – | – | – | – | – |
Massey (1974) | – | – | – | 60* | – | – | – | – | – |
Stock and Nadler (2006) | 83* |
64* |
– | – | – | 65* |
– | – | – |
One 928 bp 18S rDNA sequence (GenBank accession number MZ656001) and one 766 bp 28S rDNA sequence (GenBank accession number MZ656000) were obtained for
The morphology and morphometry of the material examined now agree with the type population of
Other species have very similar spicules and could be confused with
On the other hand, Stock and Nadler (2006) characterized morphological and molecularly three species of the genus
The material examined now of
With respect to the subfamily Panagrellinae Andrássy, 1976 containing the genera
Other subfamilies of the family Panagrolaimidae as Medibullinae Siddiqi, 1993 [tranferred to Panagrolaimidae by Abolafia and Peña-Santiago (2018), including
Conversely, the subfamily Panagrolaiminae is shown as polyphyletic. Four genera belonging to this subfamily,
Nevertheless, the genus
On the other hand, the genera
Tarantobelinae n. subfam.
Diagnosis: small body with cuticle nearly smooth, lateral field inconspicuous, lip region with six separated lips, each bearing a small cuticular flap topping it, panagrolaimoid stoma with well-developed gymnostom having broad and refringent rhabdia, panagrolaimoid pharynx with isthmus robust and slightly longer than basal bulb, excretory pore situated at level of isthmus, female reproductive system monodelphic-prodelphic, length of post-vulval sac less than the corresponding body diameter, vulva post-equatorial and distinctly protruding, female tail conical with acute tip, male tail conical with a long and thin mucro, spicules curved ventrad with rounded manubrium, and thick gubernaculum.
GenBank accession numbers of the species used in the phylogenetic tree.
Species | 18S rDNA | 28S rDNA |
---|---|---|
|
EU196013 | EU195976 |
|
KU180671 | DQ145620 |
|
DQ102707 | DQ903076 |
|
HQ130163 | HQ130212 |
|
KJ869356 | KP835679 |
|
FJ665982 | KP204846 |
|
KX017492 | NA |
|
KX185606 | NA |
|
AY593913 | KC818620 |
|
AF547385 | NA |
|
NA | DQ077787 |
|
EU196018 | KU180677 |
|
EU196017 | EU195989 |
|
FJ040451 | NA |
|
AJ966477 | NA |
|
KP453998 | NA |
|
JN636068 | JN636068 |
|
AY284663 | AF143368 |
|
AF202152 | KU180683 |
|
EU196019 | EU195991 |
|
KJ636326 | NA |
|
KJ877216 | KJ877249 |
|
AY593921 | NA |
|
KJ636422 | MN307126 |
|
AY284665 | HM439768 |
|
KJ877235 | KJ877282 |
|
JX674039 | JX194163 |
|
NA | KU180686 |
|
MZ027493 | MZ027488 |
|
AB597232 | KJ877284 |
|
LC382049 | NA |
|
KX789708 | NA |
|
AF083013 | NA |
|
JX163973 | NA |
|
KP067833 | NA |
|
KU180665 | KU180676 |
|
NA | DQ145643 |
|
KR817916 | KR817917 |
|
AF083019 | EU195968 |
|
NA | DQ408251 |
|
NA | DQ145648 |
|
NA | DQ408252 |
|
KY126845 | NA |
|
MZ656001 | MZ656000 |
|
MH608262 | MH608297 |
|
MH608263 | MH608298 |
|
AF083007 | DQ408250 |
|
MK541674 | MK541658 |
|
NA | AF331910 |
|
MN082326 | NA |
|
MH608264 | MH608299 |
|
KP876562 | KM489128 |
|
LC382079 | NA |
|
FJ969134 | NA |
|
AJ567385 | AY878385 |
|
EU543176 | GU014547 |
|
KY119431 | NA |
|
KF011487 | NA |
|
EU003189 | EF990724 |
|
AY593920 | NA |
|
KJ636307 | NA |
|
AY284700 | EF417147 |
|
KJ877237 | KJ877273 |
|
EU543179 | NA |
|
KJ636392 | NA |
|
KJ434175 | NA |
|
AF083001 | AY602168 |
|
AF202150 | NA |
|
MK639403 | MW716284 |
|
NA | AY602177 |
|
JX674037 | JX674036 |
|
AF202151 | AY294185 |
|
KP718970 | KP718967 |
|
KM454872 | KM454873 |
|
KM454874 | KM454875 |
|
KM454870 | KM454871 |
|
FJ040419 | NA |
|
FJ040424 | AY172023 |
|
AB272231 | AB272231 |
|
AB272232 | NA |
|
KJ877232 | KJ877279 |
|
MG669658 | MF177710 |
|
MZ655998 | MZ656002 |
|
MZ655999 | MZ656003 |
|
EF990716 | EF990721 |
|
AF202165 | AY294184 |
|
KU180673 | KU180690 |
|
NA | DQ145662 |