Paragordius amicus, Swanteson-Franz, Rachel J., Marquez, Destinie A., Goldstein, Craig I., Andreas Schmidt-Rhaesa,, Bolek, Matthew G. & Hanelt, Ben, 2018

Swanteson-Franz, Rachel J., Marquez, Destinie A., Goldstein, Craig I., Andreas Schmidt-Rhaesa,, Bolek, Matthew G. & Hanelt, Ben, 2018, New hairworm (Nematomorpha, Gordiida) species described from the Arizona Madrean Sky Islands, ZooKeys 733, pp. 131-145 : 135-141

Paragordius amicus sp. n.

Type locality.

Huachuca Mountains, Sunnyside, Arizona, USA (31.445, -110.402, elevation: 1770 m).

Holotype.

Female collected on 27 July, 2011, from type locality (N291A). Deposited into the Museum of Southwestern Biology (MSB) Parasite Division, University of New Mexico (UNM), New Mexico, USA with accession number MSB:Para:26387.

Paratypes.

Allotype: male specimen collected on 27 July, 2011, from the type locality (N291B). Deposited into the MSB Parasite Division, accession number MSB:Para:26388. Paratypes: two females collected 28 July 2011, in the Santa Rita Mountains (N289A, and N289B). Deposited into the MSB Parasite Division, accession numbers MSB:Para:26389 and MSB:Para:26390.

Host.

Natural definitive insect host is unknown; in the laboratory, Acheta domesticus , crickets served as definitive hosts, but in nature are likely to be members within the family Gryllidae (crickets) or Tettigoniidae (bush-crickets or katydids).

Etymology.

The name is Latin for “friend”, referring to the fact this is the first description of another genus member for P. varius in the Nearctic.

Distribution.

Current known distribution is limited to the Madrean Sky Islands of southeastern Arizona in the Huachuca and Santa Rita Mountain Ranges.

Material examined.

Adults (n=5), eggs, larvae, and cysts. Tissue from field collected adult (N=4) midsections was utilized for CO1 analysis while adult posterior, anterior, and midsections were utilized for SEM. DNA was also extracted from a worm removed from a deceased, lab-infected Acheta domesticus 40 days post exposure to collected larvae. Egg, larvae, and cyst stages were imaged using SEM and/or DIC microscopy.

Description of male.

Adult (n=1) 205 mm long medium brown color. Bifurcating tail lobes on posterior roughly 400 µm in length, extending laterally away from the sagittal plane (Fig. 1B). Male cloacal opening oval with a vertical slit-like opening (30 × 60 µm, located anterior to point of tail lobe bifurcation (Fig. 1C). Post-cloacal spines present just above bifurcation but posterior to the cloaca extending onto the inside and ventral side of the tail lobes (Fig. 1B, C). Midbody cuticle lacks of obvious surface structures; some superficial structure is noted as dark-appearing areas (Fig. 1C). Cuticle on posterior end has wrinkled appearance made of grooves and circular pattern and is evenly dispersed across the cuticle surface with the exception of a 25 µm wide line running on the ventral surface, along the sagittal line, lacking grooves or circular patterning. Smooth ventral line of cuticle is bordered by small pointed mounds approximately the same size and shape as postcloacal spines but are mound rounded and mound-like (Fig. 1A, C).

Description of female.

Adults (n=3) were 198 mm, 216 mm, and 234 mm in length and medium brown in color. Trifurcated posterior end (Figs 2C, 3A, 4A), with varying degrees of openness. Distinctive oblong mounds, approximately 10µm in length, are found arranged in horizontal or vertical lines up and down the entire interior side of the trifurcating tail lobes (Figs 2C, D, 3A, B). Short, thick bristles (hair-like structures) randomly spaced between oval mounds (Figs 2D, 3B). Midbody cuticle geographically variable. Worms collected in the Santa Rita Mountains contained transverse striations consisting of raised ridges separated by narrow furrows (Figs 2A, B, 4B, C, D). In some areas, the cuticle also contains rounded indentations (Figs 2A, 4B, C), while in others the indentations were more oblong and housed structures (Figs 2B, 4D). The female collected from the Huachuca Mountains, just as the male collected from the Huachuca Mountains, lacked any obvious surface structure on the midbody cuticle (Figs 1A, 3C).

Description of egg strings, eggs, and larvae.

Females deposited continuous egg strings that were white in color and 1-3 times the length of the females. Eggs were elliptical to spherical in shape with a thin shell and were 36.6 (29.6-41.2) µm in length and 32.0 (25.9-43.8) µm in width. Over a period of 3-4 weeks, egg strings turned a light brown color at which time eggs contained fully developed larvae (Fig. 5A).

Larvae possessed a cylindrical body divided by a septum into two regions, the preseptum and a postseptum (Fig. 5B, C). The preseptum was 29.4 (24-38) µm in length and 15.4 (13-17) µm in width and contained an eversible proboscis supported by three internal stylets which were 13.8 (12- 16) µm in length and 4.5 (3-6) µm in width (Fig. 5A, B). The postseptum was 34.8 (29-39) µm in length and 12.6 (10-15) µm in width and contained a clearly visible pseudointestine. The pseudointestine contained two anterior granules and a large posterior mass and was 15.5 (11-18) µm in length and 10.1 (7-13) µm in width (Fig. 5B). The average preseptum to post septum ratio was 1:1.2 (1.1-1.5).

Externally, larvae were superficially annulated and the postseptum contained two pairs of terminal spines located ventrally (Fig. 5C, E). The preseptum contained three sets of cuticular hooks. The outer ring of hooks contained seven hooks, two of which are very close together and ventrally positioned (Fig. 5D), and there were six hooks in the middle and inner rings observed in live larvae. The length of the cuticular hooks on the outer ring was noticeably longer than the middle and inner cuticular hooks. Clearly visible spines on the proboscis could only be observed in a few individuals (Fig. 5D). The left and right side of the distal end of the dorsoventrally compressed and eversible proboscis each contained spines (at least four pairs arranged in tandem and one single spine above); whereas the distal end of the ventral side of the proboscis contained five spines (two pairs arranged in tandem and one single spine above; Fig. 5D).

Laboratory rearing of cysts and adults, and description of cyst.

Of the four snail species exposed to larvae of P. amicus sp. n. only Physa acuta and Biomphalaria glabrata became infected with cysts; however, not all individuals became infected. Seven of 10 (70 %) P. acuta were infected with a mean abundance of 2.8 ± 3.0 (range 0-8) cysts; and four of 10 (40 %) of B. glabrata were infected with a mean abundance of 1.0 ± 2.2 (0-7) cysts.

Fully developed cysts were recovered from laboratory-reared and exposed snails 14-21 DPE. They contained a clear cyst wall of unknown composition 16.1 (12-24) µm in length and 11.5 (9-13) µm in width (Fig. 5F). During cyst formation the content of the larval pseudointestine was emptied and the larva folded its postseptum twice around the preseptum. However, unlike cysts of other gordiid genera, the posterior end of the postseptum never reached the posterior end of the preseptum (Fig. 5F). The folded larva inside of the cyst was 28.1 (26-29) µm in length and 18.6 (18-20) µm in width.

Of approximately 40 A. domesticus crickets exposed to about 10-100 cysts each, approximately 7 worms developed in 4 cricket hosts. To establish that the parasite infection was P. amicus sp. n. one worm was extracted for DNA, amplified, and sequenced as described above. The sequence was 100 % identical to both worms sequenced from the Santa Rita Mountains, and was placed into Genbank as MG654051.

Diagnosis and taxonomic comments.

Paragordius amicus sp. n. has unique morphological features which warrant placing it as a new species and make it distinct from other New World Paragordius . First, the semi-oval raised cuticle structures and the short bristles (hair-like structures) on the inside of the female tail lobes have not been documented previously in Paragordius species. Second, despite the geographical variation in the cuticle structure of P. amicus sp. n., both variants have a cuticle pattern not seen in Nearctic and New World Paragordius species. Paragordius varius is the only species in the Americas also containing transverse striations separated by furrows. However, in P. varius the ridges within the striations are topped with round knobs ( Schmidt-Rhaesa et al. 2003). Paragordius flavescens Linstow, 1906, found in South America, and P. diversolobatus Heinze, 1935 from Costa Rica contain areoles. Paragordius esavianus Carvalho, 1942, from South America lacks areoles but the cuticle is covered by dispersed round tubercles, longer bristles, and irregular small cuticular elevations. Paragordius minusculus Carvalho, 1944, found in Brazil, lacks areoles but the midbody cuticle is fully covered by digit-like cuticular projections, like bristles, with blunt apexes. Finally, Paragordius andreasii , Zanca & de Villalobos, 2006, from Argentina, has a midbody cuticle with oval or rounded depressions arranged in pairs or forming perpendicular lines to the axis of the body. Finally, the female P. obamai Hanelt et al., 2012, does contain structures on the inside of the tail lobes. However, these structures are longitudinal, parallel ridges from which more narrow and longer bristles emerge ( Hanelt et al. 2012).

Morphological characteristics of egg stings, eggs, larvae, and cysts of Paragordius amicus sp. n. were indistinguishable from these non-adult stages of two other species of Paragordius ( P. obamai and P. varius ) for which non-adult descriptions exist ( Szmygiel et al. 2014). However, egg strings, larvae, and cysts of Paragordius amicus sp. n. were morphologically distinct from egg strings, larvae of other gordiid genera such as Acutogordius , Chordodes , Gordius and Neochordodes ( Chiu et al. 2017; Szmygiel et al. 2014).

Molecular data.

Genetic distances of the CO1 barcoding region supports our contention that P. amicus sp. n. is a new species and that it is distinct from P. varius . The intraspecific distances among P. varius samples from around the USA is 0.72 %, while among P. amicus sp. n. samples is 1.09 %. The interspecific distance between P. varius and P. amicus sp. n. is 25.33 %. The inferred phylogenetic relationship (Fig. 6) supports the clustering of P. varius from around the USA forming a monophyletic group.