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FIGURE 6 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 6. Developing and fully formed cysts of Chordodes kenyaensis n. sp. recovered from an experimentally infected Physa gyrina snail. (A) Larva in snail tissue 14 DPE in the process of folding (lateral view). Note the empty pseudointestine (black arrow). Scale bar = 12 µm. (B) Mature cysts (lateral view); note the clear halo surrounding the encysted larvae and single folding pattern. Scale bar = 15 µm.

FIGURE 3 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 3. Adult and areole pattern of deformed Chordodes kenyaensis n. sp. (A) Adult male removed from a cricket while trying to emerge. Note the larval cuticle (arrow). Scale bar = 1.0 mm. (B) DIC photomicrograph of the mid-body cuticle of a female showing tubercle (2), bulging (4), circumcluster (5) and crowned (6) areoles scattered among simple (1) areoles. Note that simple areoles are smooth but contain short bristles making them appear bumpy. Scale bar = 30 µm. (C) DIC photomicrograph of the same region of the cuticle in a higher focal plane of the mid-body cuticle showing tubercle (2), bulging (4), circumcluster (5) and crowned (6) areoles scattered among simple areoles. Scale bar = 30 µm. (D) DIC photomicrograph of a thorn (3) areole among simple areoles of the mid-body region of an adult deformed female worm. Scale bar = 7 µm.

FIGURE 4 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 4. Mating, oviposition behavior, egg strings, and eggs of Chordodes kenyaensis n. sp. (A) Male and female in a Gordian knot. Note the lighter color and thicker size of the female worm compared to the male. Scale bar = 2.0 mm. (B) Female in the process of depositing egg strings in a zigzag pattern on a stick. Scale bar = 5 mm. (C) Undeveloped egg. Scale bar = 15 µm.

FIGURE 5 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 5. Larval characteristics of Chordodes kenyaensis n. sp. (A) DIC photomicrograph of a live larva (lateral view). Note two of the three stylets (black arrow) and v shaped pseudointestine (white arrow). Scale bar = 15 µm. (B) DIC photomicrograph of anterior end of larva (lateral view), note the everted proboscis (arrow) with three stylets. Scale bar = 13 µm. (C) SEM photomicrograph of an entire larva (lateral view). Note the distinct preseptum (Pre) and postseptum (Pos) Scale bar = 8.0 µm. (D) SEM photomicrograph of the posterior end (ventral side). Note the two anterior spines (AS), two posterior spines (PS) and pseudointestine gland opening (PSGO). Scale bar = 3.0 µm. (E) SEM photomicrograph of the anterior end (en face view). Note the orientation of the proboscis (P) with left lateral spines (LLS) to the ventral hooks (VH). Scale bar = 3.0 µm. (F) SEM photomicrograph of the preseptum (ventral view). Note the orientation of the proboscis (P) to the ventral hooks (VH); median hooks (MH). Scale bar = 3.3 µm. (G) Close up SEM photomicrograph of a ventral view of the proboscis (P). Note the arrangement of the five ventral spines (VS). Scale bar = 1.5 µm.

FIGURE 2 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 2.Allotype of female Chordodes kenyaensis n. sp. (A) SEM photomicrograph showing the tapered anterior end and areole pattern. Note distinct thorn areoles (arrows) among simple areoles. Scale bar = 115 µm. (B) SEM photomicrograph showing higher magnification of the anterior region of the cuticle with simple (1) and tubercle (2) areoles. Note that interareolar furrows are arranged laterally across the cuticle. Scale bar = 10 µm. (C) SEM photomicrograph showing overview of the mid-body cuticle. Note the pattern of crowned areoles. Scale bar = 300 µm. (D) DIC photomicrograph showing two crowned (6) areoles surrounded by circumcluster (5) areoles in the mid-body region. Scale bar = 20 µm. (E) SEM photomicrograph showing simple (1), bulging (4), circumcluster (5) and crown (6) areoles in the mid-body region. Note the long filaments on crown areoles. Scale bar = 20 µm. (F) SEM photomicrograph showing higher magnification of simple (1), tubercle (2), and thorn (3) areoles in the mid-body region. Interareolar furrows are arranged laterally across the cuticle. Note the fine terminal bristles on the simple and tubercle areoles. Scale bar = 10 µm. (G) SEM photomicrograph showing posterior end and cloacal opening (arrow). Note the non-areolated region around the cloacal opening and bristles on the lateral sides of the non-areolated field. Scale bar = 150 µm. (H) SEM photomicrograph showing higher magnification of the posterior region of the cuticle with simple (1), tubercle (2), bulging (4), circumcluster (5) and crowned (6) areoles. Note the variation in simple areoles in this body region compared to the anterior and mid-body region of the cuticle. Scale bar = 40 µm.

FIGURE 1 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 1. Holotype of male Chordodes kenyaensis n. sp. (A) SEM photomicrograph showing the tapered anterior end and areole pattern. Scale bar = 170 µm. (B) SEM photomicrograph showing higher magnification of the anterior region of the cuticle with simple (1), tubercle (2), and two types of thorn (3 and 3*) areoles. Note that interareolar furrows are arranged laterally across the cuticle. Scale bar = 15 µm. (C) SEM photomicrograph showing an overview of the mid-body cuticle. Note the pattern of crowned areoles. Scale bar = 190 µm. (D) SEM photomicrograph showing a higher magnification of the midbody cuticle containing simple (1), tubercle (2), thorn (3), bulging (4), circumcluster (5) and crowned (6) areoles. Interareolar furrows are arranged laterally across the cuticle. Note the long filaments on crowned areoles and short bristles on top of simple smooth areoles which are separated between neighbors by interareolar furrows. Scale bar = 25 µm. (E) SEM photomicrograph showing the posterior end and cloacal opening (arrow). Note the indistinct lobes (thick arrows). Scale bar = 115 µm. (F) SEM photomicrograph showing a higher magnification of the posterior region of the cloacal opening surrounded by circumcloacal spines. Note the non-areolated region and bristles on the lateral regions of the non-areolated field. Scale bar = 40 µm.

FIGURE 7 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 7. Molecular phylogenetic analysis by maximum likelihood method based on partial cox1 gene from Chordodes kenyaensis n. sp. and 16 other nucleotide sequences from Genbank. Paragordius and Gordius were used as outgroups. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Only bootstrap values with a value of 85% or higher are shown. Note that Chordodes kenyaensis n. sp. is placed within other species of Chordodes.

FIGURE 5 in Two new Gordionus species (Nematomorpha: Gordiida) from the southern Rocky Mountains (USA)

FIGURE 5. Molecular phylogenetic analysis by maximum likelihood method of Gordionus bilaus n. sp. and Gordionus lokaaus n. sp. collected from the Santa Fe National Forest. Two unidentified Gordionus species from Washington State (WA) were used as outgroups. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Only bootstrap values with a value of 85% or higher are shown.

FIGURE 3. Gordionus bilaus n in Two new Gordionus species (Nematomorpha: Gordiida) from the southern Rocky Mountains (USA)

FIGURE 3. Gordionus bilaus n. sp. (A) Male posterior end showing thin row of precloacal bristles and postcloacal spines. (B) Postcloacal spines. Note mound-like appearance. (C) Rows of broad bristles with stout apexes surrounding the cloaca. Note that some bristles are branched (arrow). Cuticle showing flat (D) polygonal or (E) roundish areoles. Note the indistinct interareolar furrows, making areoles appear fused. (F). Cuticular tubercles in between areoles. (G) Adhesive warts are present anterior to the cloacal opening on the ventral sided. (H) Precloacal bristles are branched apically. Scale bars = 10 µm except where indicated.

FIGURE 2. Gordionus lokaaus n in Two new Gordionus species (Nematomorpha: Gordiida) from the southern Rocky Mountains (USA)

FIGURE 2. Gordionus lokaaus n. sp. cuticle of female. (A) Cuticle in anterior end. Note the roundish appearance of areoles. (B) Cuticle in posterior end. Note the polygonal shape of areoles with serrated edges. (C) Megaeroles with tubercles situated in hollows. Scale bars = 10 µm.

FIGURE 4. Gordionus bilaus n in Two new Gordionus species (Nematomorpha: Gordiida) from the southern Rocky Mountains (USA)

FIGURE 4. Gordionus bilaus n. sp. cuticle of female. (A and B) Cuticle contains both polygonal and more roundish areoles. Note the indistinct interareolar furrows giving neighboring areoles a fused appearance end. (C) Cuticle near the anterior end showing numerous tubercles. Scale bars = 10 µm.

FIGURE 1. Gordionus lokaaus n in Two new Gordionus species (Nematomorpha: Gordiida) from the southern Rocky Mountains (USA)

FIGURE 1. Gordionus lokaaus n. sp. (A) Male posterior end showing thin row of precloacal bristles and postcloacal spines. (B) Postcloacal spines. Note thin, triangular shape. (C) Cloacal spines surrounding cloacal opening are broad and stout, forming a tube-like structure. Note that several of the spines appear to be branching (arrow). (D and E) Cuticle with one type of flat, irregularly-sized areoles, with tubercles between areoles (arrows). Note that some areoles have (D) serrated edges, and others (E) smooth edges. (F) Adhesive warts showing canoe-shaped bulges. (G) Precloacal bristles are branched apically. (H) In some areas the areoles disappear and are replaced with grooves, which run perpendicular to the body axis. Scale bars = 10 µm, except where indicated.

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Supplementary material 2 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Fasta 2

Figure 6 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Figure 6 Immature stages of Gordius chiashanus sp. nov. A, B free-living larva (A) treated with hot water and a living larva showing the depression in the anterior end of the pseudointestine (arrow) C, D eggs with the inner membrane examined using an (C) SEM and (D) compound microscope E egg strings F–H cysts in the paratenic host with (F) a unfolded larva and (G) a folded larva, showing (H) a single posterior spine (arrow) after treatment with a 5% KOH solution. Abbreviations: Ho, hooklet; PostS, postseptum; PreS, preseptum; Pro, proboscis; PsI, pseudointestine. Scale bars: 50 µm (A–D, F–H), 1 mm (E).

Figure 7 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Figure 7 Phylogenetic relationship of Gordius/Acutogordius spp. restructured using COI partial sequences compared with C. formosanus, E. nigromaculatus, and P. diblastus as out groups. Numbers at the nodes represent the percentage of 1000 bootstrap replicates.

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Supplementary material 1 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Video S1

Figure 5 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Figure 5 Field observation of Gordius chiashanus sp. nov. A hazy appearance (arrows) surrounding the body surface in hot water B spermatophore (arrow) on a female collected on the surface of the soil C rainbow-like reflection on the body surface D free-living adult collected in wet soil E, F infected host, Spirobolus sp. nov. (Hsu and Chang, unpublished), harboring (E) three immature worms (arrow) and (F) an adult worm. Photographs courtesy of (D) Fang, Hua-Te and (F) Hung, Ming-Chin. Scale bars: 1 cm (E).

Figure 8 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Figure 8 Seasonal occurrence of free-living adults of Gordius chiashanus sp. nov. Numbers at the bottom indicate the actual number of each bar.

Figure 4 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Figure 4 Female Gordius chiashanus sp. nov. A, B anterior end examined using a (A) stereomicroscope and (B) SEMC–E posterior end with the terminal view examined using a (C) stereomicroscope and (D) SEM, and the (E) lateral view examined using a stereomicroscope F, G mid-body examined using a (F) stereomicroscope and (G) compound microscope. Co, cloacal opening. Scale bars: 1 mm (A, F, G), 200 μm (B–D).

Figure 2 from: Chiu M-C, Huang C-G, Wu W-J, Lin Z-H, Chen H-W, Shiao S-F (2020) A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida). ZooKeys 941: 25-48. https://doi.org/10.3897/zookeys.941.49100

Figure 2 Posterior end of male Gordius chiashanus sp. nov. A stereomicroscopic image of the posterior end B–DSEM images of (B) overview of the posterior end with bristles concentrated on the (C) lobe tips (arrow), and (D) inner side of the lobe tips and the formation of a bristle field on each tail lobe posterior to the tips of the postcloacal crescent (arrows) E cloacal opening with areoles on the inside wall. Scale bars: 1 mm (A), 500 μm (B), 200 μm (C–D), 50 μm (E).