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Tarantobelus jeffdanielsi n. sp. (Figs. 1–8) Adult female diagnosis: Body straight to slightly curved ventrad when relaxed (Fig. 1B, E); 833–1,288 µm long. Anterior end and lip region low, rounded, continuous with body contour. Oral aperture opening triangular (Fig. 2B), surrounded by 6 lips each with small, slightly raised papilla and each ending in small flaps (Figs. 2A–C, 3B–D). Four cephalic papillae are also similar in appearance with labial papillae. Amphidial aperture oval, about 3 µm from cephalic papillae. Cuticle with fine (width) annules, lateral field bearing 3 transverse incisures (Fig. 2E). The females have a typical panagrolaimid stoma (Figs. 1A–C, 3B–D), with a length of 18.1 ± 0.6 (16.5–19) µm [presented as mean ± standard deviation (range)] with stegostom comprising half of its length. Cheilostom and gymnostom comprise the other half, of equal length, the former lacking refringent rhabdia; gymnostom well developed, with distinct and refringent rhabdia; stegostom short, funnel-shaped, with poorly refringent rhabdia (Fig. 3B–D). Typical panagrolaimid pharynx with cylindrical muscular corpus, slightly widened posteriorly but with no distinct procorpus and metacorpus; 2.3–3.4 times longer than isthmus (Figs. 1A, B, E; 3E). Isthmus about as long as the spheroid basal bulb. Pharyngeo-intestinal valve distinct, conoid. Nerve ring and excretory pore located 60–80% and 70–90%, respectively, of neck length from the anterior end (Figs. 1A, 3G). Deirid inconspicuous, and where observed, located just posterior to the excretory pore (Fig. 2D), at level with the posterior part of the basal bulb, 90% of neck length. Vulva located 46–68% of body length; vagina transverse, oblique where the body curvature is prominent, occupying more than a third of vulva body width, and often seen with secretion/copulation plug (Figs. 1B, 2F, 3A). Reproductive system monodelphic–prodelphic, with outstretched ovaries, oftentimes extending past anal aperture position in mature females and in few specimens with tip reflexed anteriorly (Figs. 1B, D; 3A). Uterus long, tubular, with eggs almost as wide as long (28–46 X 27–50 µm) in various developmental stages and oftentimes with hatched juveniles typical of endotokia matricida; spermatheca distinct, elongate, dextral relative to the intestine (Figs. 1D, 3H). Postvulval sac short, 0.4–0.7 times the vulva body width (Figs. 1D, 3H). Rectum length 1–1.6 times anal body with, with 3 prominent rectal glands. Tail conical with acute tip; phasmid inconspicuous, located less than half (mean ¼ 0.25; range 0.22– 0.35 µm) of tail length (Fig. 3H, I). Morphometrics of the holotype (female), female and male paratypes are presented in Table II. Adult male diagnosis: Similar body habitus to female when relaxed, but posterior prominently curved ventrad, appearing as J-shaped; 842–1,288 µm long. All other morphological features of the anterior region (stoma, pharynx) typical panagrolaimid. Reproductive system monorchic, its length occupying 60–70% of total body length; testis ventrad, anteriorly reflexed. Cloacal aperture with opposing papilla-like processes (Figs. 2J, 3J, K). Genital papillae 2/ 1 þ 1 þ 3 þ p, arranged as follows (Fig. 4H, I, K): 2 latero-ventral, precloacal pairs (GP1, GP2); 1 midventral papilla just anterior to the cloaca (MP); 1 pair, lateral, ad-cloacal (GP3); about halfway to the tail, 2 ventro-lateral pairs (G4 and G7); and 2 dorso-lateral pairs (G5, G6), with the phasmid positioned in between them, very posterior to the tail. Spicules typical panagrolaimid, curved ventrad with rounded manubrium, short calamus, ventrally curved lamina with very slight dorsal hump. Gubernaculum thick, curved, with small rounded manubrium and fine acute tip. Tail 61–78 µm long, conical, wider at more than halfway its length, and strongly curved ventrally in its tip; with very posterior phasmids, between GP6 and GP7. Entire body of the male can be found in Figure S4. Taxonomic summary Type host: Grammostola pulchra. Site of infection: Oral cavity, sternum, labium, various regions of the exoskeleton. Type locality: Captive-bred tarantulas in Virginia Beach, Virginia. Specimens deposited: Eight females (holotype and paratypes) and 8 males (paratypes) were deposited to the nematode collection at the University of California –Davis, Davis, California. ZooBank registration: urn:lsid:zoobank.org:act: 8DE3E8EB-9465-4436-B372-8E7BD63FBF7A. Etymology: Tarantobelus jeffdanielsi n. sp. is named after American actor Jeff Daniels, whose character in the 1990 film Arachnophobia kills the queen spider and saves the fictional town of Canaima from a deadly infestation of spiders. Remarks The new species conforms with taxonomic characters typical of the Panagrolaimomorpha, Panagrolaimoidea, family Panagrolaimidae (Thorne, 1937), subfamily Tarantobelinae (Abolafia and Vecchi, 2021). The genus Tarantobelus was proposed with a single species, T. arachnicida, a nematode isolated from mouthparts of a young adult, male greenbottle blue tarantula, Chromatopelma cyaneopubescens (Strand, 1907; Theraphosidae) that came from Venezuela and was bred in captivity in Poland. This new species is the second described in this genus and is proposed based on combined morphospecies (morphological and morphometrical data) and phylogenetic species concepts. Additionally, some biological features including its virulence to A. domesticus and G. pulchra, and species fecundity and mortality were characterized. Tarantobelus jeffdanielsi is characterized by a simple lip region, each ending in small flaps; papilliform sensilla; stoma stegostom comprising half of stoma length; cuticle without loose sheath; annules 1 µm wide, panagrolaimid pharynx with cylindrical muscular corpus, slightly widened posteriorly with no distinct metacorpus; monodelphic, prodelphic female reproductive system with axial spermatheca; postequatorial vulva, vulva–anus distance longer than its tail length; conical tail with acute tip; male spicules relatively broad with slightly rounded manubrium; papillae formula 2/ 1 þ 1 þ 3 þ p; life cycle with dauer stage. Tarantobelus jeffdanielsi closely resembles T. arachnicida morphologically and morphometrically, and a number of their other measurements are reflective of their main difference in size. Tarantobelus jeffdanielsi female and male are longer, with mean ± standard deviation (range) of 1,090.4 ± 127.3 (833–1,289) vs. 874.7 ± 50.4 (769–954) µm. In addition, the following are also longer: female ovary and uterus, rectum, tail, and testis. Interestingly, despite its longer body, vulva position is more anterior compared to T. arachnicida (46–68% vs. 72–77%, respectively). Other parameters that are shorter are: length of pharyngeal corpus (91.7 ± 4 [83–98.8 µm]) vs. 125.9 ± 4.9 (116– 133 µm) and postvulval sac (7.7 ± 0.9 [6.1–9.8 µm] vs. 32.7 ± 5.8 [23–45 µm]). The ranges of the lengths of stoma (female and male), spicules, and gubernaculum were similar for both species, suggesting that these taxonomic characters are relatively stable within the genus. Molecular characterization and phylogenetic analyses The near full-length sequences of T. arachnicida (MW559214) and T. jeffdanielsi (MW560268) 18S; and D2–D3 expansion segments of T. jeffdanielsi (MW560627) rDNA were obtained via Sanger sequencing. The nearly complete 18S sequences of both Tarantobelus species consisted of 1,736 base pairs (bp); and differed by 4 point mutations: 2 transversions and 2 insertions/deletions. However, between the 2 species, of 735 nucleotides, 28 point mutations consisting of 7 transitions, 11 transversions, and 10 insertions/ deletions in the D2–D3 domains of the 28S were observed. Virulence assays As with Aryal et al. (2019), house crickets were in poor health for the virulence assays for unknown reasons. All crickets died within 10 days of the experiment regardless of treatment type. No significant differences in mortality were observed between control crickets A. domesticus and those exposed to T. jeffdanielsi IJs (Fig. 6A). Virulence assays with waxworms showed a dose-dependent effect to exposure to T. jeffdanielsi IJs (Fig. 6B). Ten days after inoculation, wax worms showed a 47% average survival rate when exposed to 200 IJs, 49% when exposed to 40 IJs, and 78% when exposed to tap water (Fig. 6B), with significant differences among all treatments (P ¼ 0.0008 and 0.0044, respectively). Infected waxworms died and showed visible melanization (Fig. S5). Nematodes recovered from waxworm cadavers were confirmed as T. jeffdanielsi by D2–D 3 28S rDNA sequencing. The tarantula virulence assay resulted in 75% mortality of G. pulchra after 94 days when fed dead crickets inoculated with 1,000 IJs, 20% mortality when fed live crickets inoculated with 1,000 IJs, and 0% mortality when fed live uninoculated crickets (Fig. 6C). There was a significant difference in survival between G. pulchra fed with dead, infected crickets and control (P ¼ 0.0221). No significant differences were observed between G. pulchra fed with live, infected crickets and the control (P ¼ 0.3173); and G. pulchra fed with live, infected crickets and G. pulchra fed with dead, infected crickets (P ¼ 0.1877). Infected tarantulas showed large masses of mixed-stage nematodes in the mouth and around the sternum (Fig. 7). The most affected areas of the tarantulas included the upper section of the sternum, the labium, and the mouth (Fig. 7). Individual nematodes were also observed on the leg hairs and head of the tarantula. Infected tarantulas showed all signs of infection including lethargy, anorexia, itching, and tiptoe behavior throughout the infection period. Nematode fecundity and mortality assays Tarantobelus jeffdanielsi cultured at 20 C in 10 µl NGG droplet had an average life span of 11.2 days, where the longest-lived individual died on day 18 (Fig. 8A). Tarantobelus jeffdanielsi hermaphrodites began their reproductive period on day 3. A total of 474 offspring were produced among all 36 nematodes. The total fertility rate (TFR, i.e., the total number of offspring a hermaphrodite would have, on average, if individuals were to live to the maximum age) of a T. jeffdanielsi hermaphrodite was 158 progeny (Fig. 8B). |
