Research Article |
Corresponding author: Christina Anaya ( christina.anaya@okstate.edu ) Academic editor: Yasen Mutafchiev
© 2019 Christina Anaya, Andreas Schmidt-Rhaesa, Ben Hanelt, Matthew G. Bolek.
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:
Anaya C, Schmidt-Rhaesa A, Hanelt B, Bolek MG (2019) A new species of Gordius (Phylum Nematomorpha) from terrestrial habitats in North America. ZooKeys 892: 59-75. https://doi.org/10.3897/zookeys.892.38868
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Freshwater hairworms (class Gordiida) are members of the phylum Nematomorpha that use terrestrial arthropods as definitive hosts but reside as free-living adult worms in rivers, lakes, or streams. The genus Gordius consists of 90 described species, of which three species were described from freshwater habitats in North America. In this paper we describe a new species of Gordius from terrestrial habitats in Oklahoma, Texas, and Louisiana, United States. Oddly, each year hundreds of adult free-living worms appear after bouts of heavy rain on streets, sidewalks, and lawns during the winter season, when terrestrial arthropod hosts are not active. The new species is described based on morphological characters of adults and non-adult stages including the egg strings, eggs, larvae, and cysts. Adult males have a unique row of bristles on the ventral inner side of each tail lobe and a circular pattern of bristles on the terminal end of each lobe, which distinguishes them from all other described North American species of Gordius. The egg string, larval, and cyst morphology of this new species conform to previous descriptions of non-adult hairworm stages for the genus Gordius. However, the eggs of this new species of hairworm are unique, as they contain an outer shell separated by distinct space from a thick inner membrane. The consistent occurrence of this gordiid in terrestrial habitats, along with its distinct egg morphology, suggests that this new species of hairworm has a terrestrial life cycle.
eggs, Gordiida, hairworm, life cycles, North America, Oklahoma, soil
The Phylum Nematomorpha, commonly known as hairworms or Gordian worms, or simply gordiids, are parasites of terrestrial arthropods with a complex life cycle that includes a free-living and parasitic phase with multiple hosts (
Although first described more than 300 years ago, gordiids have been identified as one of the most understudied groups of parasites (
At present, approximately 360 gordiid species have been described from across the world within 18 extant and two extinct genera (
The majority of Gordius diversity has been identified from the palearctic region which harbors 71% of Gordius diversity with the remaining 29% distributed throughout the world with the exception of Antarctica (
Based on genetic data, one of the eight species, identified as clade 7 by
A total of 39 female and 194 male free-living hairworms were collected from two suburban locations in the city of Stillwater, OK, USA (36.12091, -97.03669; 36.13653, -97.04266). All free-living worms were collected after bouts of heavy rain from streets, sidewalks, or lawns between November-December 2014 and January-March 2015. In addition, each location was searched for potential definitive arthropod hosts by visually scanning the locations when worms were present. All specimens were placed in 950 ml glass jars containing aged tap water and transported to the laboratory at Oklahoma State University. A subsample of adult worms was processed for morphological characters; whereas the remaining worms were allowed to mate to obtain non-adult life stages (see below). Additionally, two male specimens from a single location in Montgomery, Texas (30.38988, -95.69552) and one male from Baton Rouge, Louisiana (30.40661, -91.18734) were collected by citizen scientists and sent to us as per the instructions on our website (www.nematomorpha.net) and its Report-A-Worm feature.
Adults. Length, width, color, and color pattern (presence of a calotte, dark pigmented ring, and spots on the cuticle) were recorded for all male and female individuals collected from Stillwater, OK. Lengths of worms were obtained by placing individuals on a ruler without stretching the specimen and measured to the nearest 1 mm. The width of each worm was obtained using an Olympus SZ1145 Stereomicroscope and a calibrated ocular micrometer. Posterior ends of males were then photographed with a Sony Cybershot camera and the angle of postcloacal crescent was measured using ImageJ software (
For scanning electron microscopy (SEM), four female and six male worms collected from Oklahoma and two males collected from Texas were imaged as described by
Obtaining non-adult stages. A subset of single male and female worms from Stillwater, OK were paired and placed in 110 × 35 mm Stender dishes filled with filtered and aged tap-water (
Morphology of egg strings, eggs, and larvae. Photographs were taken of two-day old egg strings in Stender dishes and a plastic ruler as a reference using a Sony Cybershot camera and the length and width of 20 egg strings was measured using ImageJ software. Individual developed eggs, and two-day old larvae after hatching were prepared as live wet mounts and observed using an Olympus BX–51 upright research microscope (Olympus, Tokyo, Japan) configured for bright field and Nomarski differential interference contrast (DIC) microscopy with plain fluorite objectives at 400× to 1000× total magnification. Measurements of developed eggs with larvae were taken from captured digital images using an Olympus 5–megapixel digital camera and ImageJ software. Briefly, for developed eggs, 5 mm sections of egg strings were placed on microscope slides in a drop of water, covered with a coverslip without crushing, and observed for general morphology with an Olympus BX–51 upright research microscope and 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 individuals following the protocols of
Morphology of cysts. Laboratory infected and post frozen snails were processed for gordiid cysts following
Larval preparation for SEM and larval characters. Pieces of egg strings with developed larvae and hatched larvae suspended in water, were pipetted onto Poly-L-Lysine coated coverslips placed in 1.5 ml plastic well plates and fixed in a solution of alcohol, formalin, and acetic acid. Fixed larvae were dehydrated in a graded series of ethanol in each plastic well with 0.5 ml of 30 %, 50 % and 70 % ethanol for 30 min each, followed by dripping 1 ml of 100% ethanol into the well over a period of an hour, 1 ml of ethanol was then removed from the well and the process repeated three additional times (
Egg morphology of aquatic gordiids. To compare the egg morphology of the new species to eggs of aquatic gordiids, we examined egg photomicrographs from our personal collections for three species/genera of aquatic hairworms. All species examined were collected from streams as free-living adults or cysts that were reared in crickets in the laboratory. These included Gordius difficilis from Waukesha County, Wisconsin, USA (42.966229, -88.364328), Neochordodes occidentalis Montgomery, 1898 from Pima County, Arizona, USA (31.8655, -109.1905) and Paragordius obamai Hanelt, Bolek, and Schmidt-Rhaesa 2012 from Nyanza province, Kenya (-0.1519, 34.4455). Information on how eggs were obtained and processed is reported in
A suburban lawn in the City of Stillwater, Payne County, Oklahoma; USA (36.12091, -97.03669; approximate altitude: 276–296 m).
Holotype. Male collected on 5 December 2014. Deposited in the Museum of Southwestern Biology (MSB) Parasite Division, University of New Mexico (
Paratypes. Female specimen collected on 5 December 2014, from the type locality. Deposited into the MSB Parasite Division, accession number MSB:Para:29148. Paratypes: two males collected 14 January 2003 in Montgomery, Texas (30.38988, -95.69552). Deposited into the MSB Parasite Division, accession numbers MSB:Para:19257 and MSB:Para:19258.
Larvae and egg strings with hatching larvae on SEM stubs obtained from laboratory cultures from Oklahoma collected worms. Deposited into the Museum of Southwestern Biology (MSB) Parasite Division, http://grbio.org/institution/university-new-mexico (
Natural definitive host is unknown, and no arthropod hosts were found during times when adult free-living worms were present.
The new species is named after the terrestrial habitat from which all adult free-living individuals were collected.
Stillwater, Oklahoma (36.12091, -97.03669; 36.13653, -97.04266), Montgomery, Texas (30.38988, -95.69552) and Baton Rouge, Louisiana (30.40661, -91.18734).
GenBank accession numbers for mitochondrial (CO1 and cytb) and ribosomal (partial 28S, ITS1, 5.8S and ITS2) DNA sequences for the Louisiana (KM382307; KM382349; KM382400), Oklahoma (KM382308 and KM382309; KM382350) and Texas (KM382351; KM382401 and KM382402; KM382403 and KM382310; KM382352; KM382404) samples of G. terrestris sp. nov. were published in
Adults (N = 233), eggs, larvae, and cysts. Eight adult males, six from Oklahoma and two from Texas, and four adult females from Oklahoma were imaged using SEM; and other male and female individuals were examined using DIC and bright field microscopy for color pattern. Additionally, egg, larvae, and cyst stages were imaged using SEM and/or DIC microscopy.
Adult free-living males were creamy white to dark brown in color and contained distinct white spots throughout the length of the body (Fig.
Gordius terrestris sp. nov., adult male from Stillwater, Oklahoma, light (A, D, G) and SEM (B, C, E, F, H, I) photomicrographs A anterior body region showing typical color pattern, showing the distinct calotte (arrow) and dark ring B anterior end, dorsal view C areole pattern on the anterior body region. Note the weakly developed areoles (circle) and the presence of bristles (arrows) D midbody region, dorsal view, showing distinct white spots and medial line E Midbody region, dorsal view, showing typical cuticular pattern F areole pattern on the midbody region; note the weakly developed polygonal shaped areoles (circle) G posterior body region, ventral view, showing distinct coloration; note the darkly pigmented postcloacal crescent and dark pigmentation on inner sides of the tale lobes (TL) H ventral view of the posterior region, showing the cloaca (Cl) and postcloacal crescent (PCC) I areole pattern on the posterior body region; note the weakly developed polygonal shaped areoles (circle) and the bristles (arrows). Scale bars: 210 µm (A); 130 µm (B); 18 µm (C); 220 µm (D, G); 290 µm (E); 10 µm (F); 175 µm (H); 20 µm (I).
Gordius terrestris sp. nov., adult males from Stillwater, Oklahoma, SEM photomicrographs A–C posterior body region, ventral view, note the variation in the shape of tail lobes and postcloacal crescents (PCC) below the cloaca (CL) D tail lobe showing the distinct row of bristles beginning below the postcloacal crescent (PCC) and progressing on the ventral inner side (small arrows) of the tail lobe (TL); and bristles distributed in a circular pattern on the terminal end (large arrow) of the tail lobe E–G variation in the weakly developed polygonal shaped areoles (circles) on the posterior body region of different male individuals; note the branching bristles (arrows) in E. Scale bars: 175 µm (A–C); 75 µm (D); 8 µm (E–G).
Adult free-living females were creamy white to dark brown, and contained dark dorsal and ventral lines along the length of the body. Females were 246 ± 41 (211–336; N = 39) mm long by 1.0 ± 0.1 (0.7–1.3) mm wide. The anterior end was tapered and contained a white calotte followed by a dark collar (Fig.
Gordius terrestris sp. nov., adult females from Stillwater, Oklahoma, light (A, D, G) and SEM (B, C, E, F, H, I) photomicrographs A anterior body region showing typical color pattern, showing the distinct calotte (arrow) followed by a dark ring B anterior end, dorsal view C areole pattern on the anterior body region; note the weakly developed polygonal shaped areoles (circle) D midbody region, lateral view, showing typical color pattern E midbody region, dorsal view showing typical cuticular pattern F midbody region, dorsal view, showing finer details of the cuticle; note the branching bristles (arrow) G posterior body region, ventral view, showing typical coloration H posterior body region, ventral view showing the location of the cloaca (CL) I posterior body region, areole pattern on the posterior body region; note the weakly developed polygonal shaped areoles (circle). Scale bars: 160 µm (A); 150 µm (B); 10 µm (C); 440 µm (D, G); 330 µm (E); 15 µm (F, I); 190 µm (H).
When placed together, male and female worms immediately formed Gordian knots. Males moved up and down the female’s body with their coiled posterior end. Once the male’s bifurcated tail was in proximity of the female’s cloaca, the male deposited a mass of sperm on the female’s posterior end. Egg strings were deposited within 7–30 days after copulation. Newly deposited egg strings were white in color and deposited in a continuous string that broke as it emerged from the female’s cloaca into short segments (Fig.
Gordius terrestris sp. nov., eggs and egg strings, light photomicrographs A newly deposited egg strings B egg string segment showing tightly aggregated undeveloped eggs; note the eggshell (arrow) C segment of an egg string showing developing larvae within eggs D eggs with fully developed larvae; note the distinct space between the eggshell and the thick inner membrane. Scale bars: 4 mm (A); 40 µm (B); 25 µm (C); 20 µm (D).
Larvae of G. terrestris sp. nov. possessed a cylindrical body divided by a septum into two regions, the preseptum and a postseptum (Fig.
Externally, larvae were superficially annulated with a single spine located on the posterior region of the postseptum (Fig.
Gordius terrestris sp. nov., larvae, light (A, B) and SEM (C–F) photomicrographs A live larva, showing the preseptum (PRE), postseptum (POS) and pseudointestine (PI) B recently hatched larvae showing everted proboscis (arrow) C larva note the superficial annulations (small arrows) and a single terminal spine located (large arrow) on the posterior region of the postseptum (POS) D preseptum, showing the arrangement of three sets of cuticular hooks, including outer hooks (OH), middle hooks (MH) and inner hooks (IH); and fused ventral outer hooks (VOH) E anterior end with the eversible proboscis (P); note the distinct spines on the distal end of the left lateral side (LLS), right lateral side (RLS) and dorsal side (DS) in respect to the ventral outer hooks (VOH) F partially everted proboscis (P) showing pairs of small spines (numbers) and a larger terminal spine on the distal end of the left lateral (LLS), right lateral (RLS) and dorsal sides (DS). Scale bars: 12 µm (A); 13 µm (B); 8 µm (C); 2.5 µm (D); 6 µm (E) 0.8 µm (F).
After being ingested by snails, larvae develop into cysts and became distributed throughout the snail tissues. During cyst formation the content of the larval pseudointestine was emptied and larvae folded their postseptum twice around the preseptum (Fig.
Gordius terrestris sp. nov., cysts, light photomicrographs A–B fully formed cysts in experimentally infected Physa acuta snails; note the folded larva surrounded by a clear cyst wall of unknown composition with a distinct inner layer (IL) and outer layer (OL) C remaining cyst wall after the folded larvae was extruded under coverslip pressure. Note the opening where the larvae emerged (arrow) D–F different focal planes showing the distinct larvae folding pattern; note the location of the terminal spine (arrow) in F and that the larva folds twice within the fully formed cyst. Scale bars: 20 µm (A–F).
Gordius terrestris sp. nov. has unique morphological features which warrant placing it as a new species and make it distinct from the other three described Nearctic species of Gordius. Gordius terrestris sp. nov. differs morphologically from G. difficilis by lacking distinct pre-cloacal bristles which are present in males of G. difficilis (
Although, the distribution of the cuticular bristles on tail lobes of male G. terrestris sp. nov. distinguish it from all other described North American species of Gordius, several European species including Gordius helveticus Schmidt-Rhaesa, 2010, Gordius karwendeli Schmidt-Rhaesa, 2010, Gordius spiridonovi Spiridonovi, 1984, Gordius terminosetosus Schmidt-Rhaesa, 2010, and Gordius zwicki Schmidt-Rhaesa, 2010 also contain cuticular bristles on tail lobes (
The general morphology of the egg string, larvae, and larval folding pattern within the cysts of G. terrestris sp. nov. conform to previous descriptions of these non-adult stages for the genus Gordius. However, these non-adult stages are morphologically distinct from egg strings, larvae, and cysts of other gordiid genera such as Chordodes, Neochordodes and Paragordius (
Eggs of aquatic and terrestrial hairworms, light photomicrographs A egg of Gordius difficilis stained with Nile Blue; note the thin inner membrane (arrow) surrounding the larva B egg of Neochordodes occidentalis showing a thin inner membrane (arrow) surrounding the larva C egg of Paragordius obamai showing a developing larva surrounded by a thin inner membrane (arrow) D egg of Gordius terrestris sp. nov. showing a distinct thick inner membrane (arrow) surrounding the larva. Scale bars: 6 µm (A); 8 µm (B, C); 11 µm (D).
Gordius terrestris sp. nov. represents the first hairworm species consistently collected from a terrestrial habitat. Hundreds of adult free-living worms appeared after bouts of heavy rain on streets, sidewalks, and lawns during the winter season, where male and female worms were observed mating and some females were observed depositing egg strings (unpublished data). It is currently unclear what definitive host is used in the life cycle of G. terrestris sp. nov. However, over a two-year sampling period, no arthropod hosts were observed in the areas when adult worms appeared. More intriguing, free-living adult worms would disappear from these locations within days after the rains subsided.
Currently, there is only one other report of a European gordiid depositing egg strings in a terrestrial habitat.
One significant observation is the unique egg morphology of G. terrestris sp. nov. with a thick inner membrane surrounding the developing larval stage. Although few detailed hairworm egg descriptions or egg photographs exist in the literature, our evaluation of eggs for three aquatic gordiid species, clearly indicate that in aquatic Gordiids the developing larval stage is surrounded by a thin inner membrane (
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 and Oklahoma State Women’s Faculty Council Student Research Award to Christina Anaya. We thank Brent Johnson and Lisa Whitworth of the OSU microscopy facility for their invaluable help with SEM work during this study and undergraduate students who assisted us in the laboratory including Lila Trainor, Kenneth Rogers, Ayrianna Swanson, Madison Houghton, Claire Gallagher, and Jordyn Taylor. Finally, we would like to thank the citizen scientists who collected hairworms and sent them to us. The authors have declared that no competing interests exist.