Research Article |
Corresponding author: Ben Hanelt ( bhanelt@unm.edu ) Academic editor: Hans-Peter Fagerholm
© 2018 Rachel J. Swanteson-Franz, Destinie A. Marquez, Craig I. Goldstein, Andreas Schmidt-Rhaesa, Matthew G. Bolek, Ben Hanelt.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Swanteson-Franz RJ, Marquez DA, Goldstein CI, Schmidt-Rhaesa A, Bolek MG, Hanelt B (2018) New hairworm (Nematomorpha, Gordiida) species described from the Arizona Madrean Sky Islands. ZooKeys 733: 131-145. https://doi.org/10.3897/zookeys.733.22798
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Gordiids, or freshwater hairworms, are members of the phylum Nematomorpha that use terrestrial definitive hosts (arthropods) and live as adults in rivers, lakes, or streams. The genus Paragordius consists of 18 species, one of which was described from the Nearctic in 1851. More than 150 years later, we are describing a second Paragordius species from a unique habitat within the Nearctic; the Madrean Sky Island complex. The Madrean Sky Islands are a series of isolated high mountains in northern Mexico and the southwestern United States (Arizona and New Mexico), and are well known for their high diversity and endemicity. The new species is described based on both molecular data (COI barcoding) and morphological characters of the eggs, larvae, cysts, and adults. Adult females have unique small oblong mounds present on the interior of the trifurcating lobes with randomly dispersed long hairs extending from the furrows between the mounds. Marked genetic differences support observed morphological differences. This species represents the second new hairworm to be described from the Madrean Sky Islands, and it may represent the first endemic hairworm from this biodiversity hotspot.
Nematomorpha , Gordiid, hairworm, Gordius , Sky Islands, new species
Hairworms are in the phylum Nematomorpha, belonging to one of only 3 entirely parasitic metazoan phyla (
Hairworms have been chronically understudied. One estimate suggests that only 14% of species have so far been described (
First field collections occurred on 27 July, 2011, at a stream in the Huachuca Mountains, Sunnyside, Cochise Country, Arizona, USA (31.445, -110.402, elevation: 1770 m). Subsequent collections were made on 28 July, 2011, from stream puddles near mile marker 12 on Madera Canyon Road, Madera Canyon, Santa Rita Mountains, Santa Cruz County, Arizona, USA (31.713, -110.87, elevation: 1640 m). All specimens were collected as free-living adults and transported alive, in stream water, to the laboratory. In the laboratory and before adult worms were processed for morphological and molecular analyses, worms were allowed to mate and females were allowed to deposit egg strings.
Adults. Physical attributes of specimen length and color were recorded in the laboratory. Measurements were obtained by placing specimens on a metric ruler, taking precautions to not stretch specimen. Specimens were cut into four pieces using razor blades. Pieces from the anterior, posterior, and mid-section were preserved in 70 % ethanol at room temperature for future microscopy work. The remaining mid-section pieces were preserved in 100 % ethanol at -80 °C for future molecular analysis. Tissue samples preserved for microscopy were imaged using a Scanning Electron Microscope (SEM). Specimens were cleaned of debris using a previously-described method (
Eggs and larvae using light microscopy. For egg and larval measurements, pieces of egg string and hatched larvae were prepared as live wet mounts and observed using an Olympus BX-51 upright research microscope configured for bright field and DIC microscopy with plain fluorite objectives at 400× to 1000× total magnification. For egg measurements, the length and width was recorded for 30 eggs. For larvae, the length and width of the preseptum, postseptum, pseudointestine and stylets was measured for 30 larvae following the protocols of
Larval preparation for SEM and external larval characteristics. Poly-L-Lysine coated cover-slips were placed in 1.5 ml plastic well plates. Frozen and live larvae were then thawed, suspended in water, then pipetted onto the Poly-L-Lysine coated cover-slips and fixed in 10 % neutral buffered formalin. Poly-L-Lysine coated cover-slips with fixed larvae were dehydrated in a graded series of ethanol by first placing the Poly-L-Lysine coated cover-slip with fixed larvae in a 1.5 ml plastic well with 0.5 ml of 30 %, 50 % and 70 % ethanol for 30 min each. Next, 1 ml of 100 % ethanol was dripped into the well over a period of an hour, 1 ml of ethanol was then removed from the well and the process repeated 3 additional times. Finally, specimens were dried using HDMS, mounted on aluminum stubs, coated with gold palladium, and examined with a FEI Quanta 600 field emission gun ESEM (ThermoFisher Scientific, Hillsboro, OR) with Evex EDS and HKL EBSD as described previously (
Infection of snails to obtain cysts. Hatched larvae were collected with a Pasteur pipette and approximately 100 larvae were pipetted into 48 1.5 ml well plates filled with 1 mm of aged tap water. Four species of laboratory reared snails from three families maintained at Oklahoma State University following the protocol of
Infection of crickets to obtain adults. Since P. varius and P. obamai can be domesticated by use of Acheta domesticus crickets as definitive hosts (
A 1.0 cm mid-section piece, approximately 0.5–2.0 g, was cut into small pieces, dried at room temperature, and DNA was extracted using the E.Z.N.A.® Mollusc DNA Kit (Omega Bio-Tek, Norcross, Georgia), following manufacturer instructions. DNA yield was determined using a NANO DROP 2000c spectrophotometer (Thermo Scientific, Walthem, MA). The Paragordius specific cytochrome c oxidase I (CO1) gene was amplified using modified universal CO1 primers (
Partial CO1 sequences were aligned by eye; no sequences contained indels. As outgroups, two previously-published sequences from Paragordius spp. (Table
Huachuca Mountains, Sunnyside, Arizona, USA (31.445, -110.402, elevation: 1770 m).
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.
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.
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).
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.
Current known distribution is limited to the Madrean Sky Islands of southeastern Arizona in the Huachuca and Santa Rita Mountain Ranges.
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.
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.
Adults (n=3) were 198 mm, 216 mm, and 234 mm in length and medium brown in color. Trifurcated posterior end (Figs
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.
Larvae possessed a cylindrical body divided by a septum into two regions, the preseptum and a postseptum (Fig.
Externally, larvae were superficially annulated and the postseptum contained two pairs of terminal spines located ventrally (Fig.
Species/sample | Accession† | Collection location‡ | Lat. | Long. | Genbank accession |
---|---|---|---|---|---|
Paragordius amicus sp. n. | |||||
N289A | MSB:Para:26389 | Arizona | 31.713 | -110.874 | MG654049 |
N289B | MSB:Para:26390 | Arizona | 31.713 | -110.874 | MG654050 |
N291A | MSB:Para:26387 | Arizona | 31.445 | -110.402 | MG654047 |
N291B | MSB:Para:26388 | Arizona | 31.445 | -110.402 | MG654048 |
Paragordius varius | |||||
N000 | MSB:Para:26391 | Nebraska | 40.994 | -96.566 | MG654052 |
N138§ | MSB:Para:26392 | Montana | MG654053 | ||
N210 | MSB:Para:26393 | Missouri | 37.300 | -89.550 | MG654054 |
N256 | MSB:Para:26394 | Mississppi | 34.359 | -88.462 | MG654055 |
N364A | MSB:Para:26395 | New Mexico | 34.766 | -106.328 | MG654056 |
N364B | MSB:Para:26396 | New Mexico | 34.766 | -106.328 | MG654057 |
N398 | Oklahoma | KU721073 | |||
Outgroups | |||||
Paragordius sp.§ | South Africa | AY428843 | |||
Paragordius obamai | MSB:Para:26397 | Kenya | -0.152 | 34.446 | MG654059 |
Paragordius tricuspidatus | MSB:Para:26398 | France | 43.755 | 3.110 | MG654058 |
Paragordius amicus sp. n. adult male from the Huachuca Mountains. A Midbody cuticle showing lack of obvious surface structure; some superficial structure is seen (arrows) B Bifurcating posterior end exhibiting the characteristic male bifurcating lobes with small, circular pointed lobes extending from the base of the bifurcation up the interior side of the lobes. These mounds are also found on either side as well as below the cloacal opening, eventually merging into the border of the ventral smooth cuticle line. C Cloacal opening, oblong with a straight slit opening extending the length of the opening surrounded by spines.
Paragordius amicus sp. n. adult female from the Santa Rita Mountains A–B Midbody cuticle exhibiting transverse striations made of ridges separated by furrows A In some areas with hollow round to oblong indentations B In other areas with more oblong indentations containing structures C Trifurcating posterior. Note the cuticle features that can be seen on the interior surface of the lobes D Increased magnification of the lobes showing the oblong mounds (arrows). Note the long and thin hairs extending from some of the furrows between the mounds.
Paragordius amicus sp. n. adult female from the Santa Rita Mountains A Trifurcating posterior B–D Midbody cuticle surface of with transverse striations containing round to slightly oblong indentations B–C Areas with only hollow indentations D Area of cuticle with more oblong indentations housing structures.
Egg, larva and cyst characteristics of Paragordius amicus sp. n. A DIC photomicrograph of an egg with a fully developed larva. Note the stylets (arrow) B DIC photomicrograph of a live larva. Note the pseudointestine composed of two anterior granules (gr) and posterior mass (pm) C SEM photomicrograph of larva. Note the distinct preseptum (pre) with relatively long outer hooks, and postseptum (pos) with posterior spines D SEM photomicrograph of the anterior end larva. Note the partially everted proboscis (p) with distinct spines on the left and right lateral sides (white arrows), and the outer row of hooks containing two ventral outer hooks (voh) E SEM photomicrograph of larva. Note two posterior spines (ps) on the dorsal side of the postseptum (pos) F DIC photomicrograph of cyst (dorso-ventral view). Note the clear cyst wall (white arrow), distinct spines on the preseptum (black arrows), and the position of the posterior end of the postseptum (white arrow).
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.
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.
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 (
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 (
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.
Paragordius amicus sp. n. represents the first hairworm described as an endemic to the Madrean Sky Islands, and so far only the second new species to be documented form the Chiricahua Mountains (see also
The Madrean Sky Islands are considered a biodiversity hotspot, or “cradles of diversity”, and thus climate change may have a proportionally large impact on this ecosystem (
Like oceanic islands, Sky Islands are habitat surrounded by barriers to biological dispersal. These barriers lead to isolation and ultimately high rates of endemism. Although Paragordius amicus sp. n. is not isolated to a single island, we have tantalizing evidence that the populations on the Santa Rita Mountains and Huachuca Mountains, separated by just 53.5 kilometers, may have been temporally isolated. These two populations appear to vary morphologically, in their cuticle pattern, but also are separated genetically by about 1.1%. Comparatively, P. varius, collected from several locations separated by hundreds and up to 1,600 kilometers apart vary genetically by only an average of 0.72%. In the future, we hope to collect additional specimens to more thoroughly document geographical variation in morphology and genetics.
This work was supported by the National Science Foundation, award numbers DEB-0949951 to Matthew G. Bolek and DEB-0950066 to Ben Hanelt and Andreas Schmidt-Rhaesa. We thank Terry Colberg and Lisa Whitworth of the OSU microscopy facility and Michael Spilde of the UNM Earth and Planetary Sciences SEM facility for their invaluable help with SEM work during this study. In addition, we thank Ryan Shannon, Chelsie Pierce, and Kristen Enyart for help in infecting and maintaining snails. We would also like to thank the UNM Center for Evolutionary and Theoretical Immunology (CETI) and the UNM Molecular Biology Facility, supported by a National Institute of Health grant 1P20RR18754 from the Institute Development Award Program of the National Center for Research Resources. Finally, we would like to thank two Citizen Scientists who collected and sent samples to us. Ryan Elwell sent worms from Missouri, and Maggie Oswald, at age 11, sent us samples from Mississippi.