Chordodes kenyaensis, Bolek & Szmygiel & Kubat & Schmidt-Rhaesa & Hanelt, 2013

Bolek, Matthew G., Szmygiel, Cleo, Kubat, Austin, Schmidt-Rhaesa, Andreas & Hanelt, Ben, 2013, Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa, Zootaxa 3717 (1), pp. 23-38 : 26-33

Description of Chordodes kenyaensis n. sp.

( Figs 1–6 )

Holotype: 1 partial male from a population originally collected as a cyst stage in a Biomphalaria pfeifferi snail from Kasabong stream , Nyanza province, Kenya Africa and reared in an experimentally infected snail Physa gyrina and cricket Gryllus texensis , deposited at the Museum of Southwestern Biology-Parasitology Division, accession number MSB Para 18595 .

Allotype: 1 partial female from a population originally collected as a cyst in Biomphalaria pfeifferi snail from Kasabong stream , Nyanza province, Kenya Africa and reared in an experimentally infected snail Physa gyrina and cricket Gryllus texensis , deposited at the Museum of Southwestern Biology-Parasitology Division, accession number MSB Para 18596 .

Other material deposited: Larvae fixed in 95% ethanol from laboratory cultures, Museum of Southwestern Biology-Parasitology Division , accession number MSB Para 18597; 2 free-living male paratypes and 2 free-living female paratypes fixed in 95% ethanol, Museum of Southwestern Biology-Parasitology Division , accession number MSB Para 18598: 1 deformed worm paratype fixed in 100% ethanol, Museum of Southwestern Biology-Parasitology Division, accession number MSB Para 18599; 1 deformed worm paratype fixed in 10% formalin, Museum of Southwestern Biology-Parasitology Division, accession number MSB Para 18600; and 1 deformed worm paratype fixed in 95% ethanol, Museum of Southwestern Biology-Parasitology Division, accession number MSB Para 18601 .

Type locality: Kasabong stream, Nyanza province, Kenya (-0.1519°, 34.4455°, approx. 1,170 m altitude).

Other localities: Cysts were also collected from Asao stream, Nyanza province, Kenya (-0.3176°, 35.0065°, approx. 1250 m altitude). Samples MSB Para 18599 and 18600 were derived from cysts from this site .

Hosts: Definitive: unknown in nature but Acheta domesticus , Gryllus firmus and Gryllus texensis in the laboratory; paratenic: Biomphalaria pfeifferi in nature and Physa gyrina in the laboratory. Note that in the natural system, snails most likely represent a dead-end host since they are an unlikely part of the normal cricket diet.

Material examined: Free-living adult male holotype and female allotype, additional male and female adult worms including deformed individuals, egg strings, eggs, larvae and cysts. SEM of mid-body, anterior and posterior ends of free-living adult male and female, and larvae, and LM of mid-body, anterior and posterior ends of free-living adult male and female; egg strings, eggs, larvae, and cysts from laboratory infected snails.

Etymology: The species epithet is named for the country of origin ( Kenya) of this newly discovered species.

Description of male: Body color tan to dark brown with anterior end lighter in color, no dark collar present. The lighter coloration blends into the normal coloration of the remaining body. Body length 114.3±32.0 (38–162; N = 15) mm, with mid-body diameter 577.5±111.6 (432–800) µm. Anterior end distinctly tapering ( Fig. 1A ) with a degenerate mouth; posterior end round with indication of two lobes ( Fig. 1E ). Cloacal opening located ventrally, 120–130 µm from posterior end, oval in shape and surrounded by circumcloacal spines ( Figs. 1E, 1F ). Additionally, short bristles, (10–20 µm by 1–2 µm in length and width), distributed laterally and medially on the non-areolated ventral region surrounding cloacal opening ( Figs. 1E, 1F ). Ventral region posterior and anterior to the cloacal opening lacks distinct areoles, is smooth, and contains canals (furrows) running laterally separated by 4–5 µm.

The body cuticle contains six types of areoles ( Fig. 1D ). Simple areoles are the most abundant and except for the ventral region surrounding the cloacal opening and the anterior region surrounding the degenerate mouth, they are distributed on the entire cuticle. On the anterior region they are 3–5µm high, oval in shape (7–8 by 6–7 µm in length and width), and their surface contains short terminal bristles 1–3 µm in length and less than 1 µm in width ( Fig. 1B ). Simple areoles on the anterior region are separated by interareolar furrows, 3–4 µm apart and contain canals running laterally across the cuticle ( Fig. 1B ). In the mid-body and posterior regions simple areoles are 5–6 µm high and 12–13 by 6–7 µm in length and width, and their surface is also covered with short bristles 1–3 µm in length and less than 1 µm in width ( Fig. 1D ). Scattered among the simple areoles are tubercle, thorn, bulging, and crowned areoles surrounded by circumcluster areoles ( Figs. 1A–D ). Bulging areoles common in the mid-body region ( Fig. 1D ). Occur in groups of 2–4, have surface covered with short bristles ( Fig. 1D ), 8–10 µm high and 12– 15 by 10–12 µm in length and width respectively. Crowned areoles more common in the mid-body region, absent from approximately 0.5–0.7 mm of the anterior and posterior regions of the cuticle. Crowned areoles occur in pairs, 8–10 µm high and 12–15 µm in diameter, and contain filaments 15–30 µm in length on their apical end ( Figs. 1C, 1D ). Each pair is surrounded by 6–12 circumcluster areoles ( Fig. 1D ). These are similar in size to bulging areoles being 8–10 µm long and 12–15 µm in diameter with small bristles on top ( Fig. 1D ). Thorn areoles are 10–14 µm in length and 3.5–4 µm in width at their base. Two types are present; most have typical thorn-like shape, but some have a round apical end ( Fig. 1B ). Tubercle areoles are most like simple areoles in size and shape. They contain filaments 7–10 µm in length and approximately 1–2 µm wide and some contain short bristles 1–3 µm in length and less than 1 µm wide surrounding the filament ( Figs. 1B, 1D ).

Description of female: Body color light tan to dark brown with anterior and posterior ends lighter in color; no dark collar on the anterior end. The lighter coloration of the anterior and posterior ends blends into the normal coloration of the remaining body. Body length 118.3±28.1 (77–180; N = 15) mm with mid-body diameter 818.7±70.3 (600–880) µm. Anterior end distinctly tapered ( Fig. 2A ) with a degenerate mouth; posterior end distinctly swollen in most but not all individuals, and cloacal opening terminal ( Fig. 2G ).

As in males, the body cuticle of females has six types of areoles. Simple areoles are most abundant, found on most areas of the cuticle, but lacking in regions surrounding the terminal cloacal opening and the degenerate mouth ( Figs. 2A, 2G ). Separated by interareolar furrows, 3–5 µm apart and running laterally across the cuticle ( Figs. 2A, 2B, 2F ). Unlike in males, in females simple areoles are morphologically distinct in the posterior region of the cuticle from those on the anterior and mid-body regions of the cuticle ( Figs. 2B, 2F, 2H ). In the anterior and midbody regions, they are 3–4 µm high, oval to round in shape (5–8 by 4–7 µm) with a smooth surface. On the anterior region of the cuticle, simple areoles contain terminal bristles 1–3 µm in length and less than 1 µm in width; but the bristles are much smaller in the mid-body region ( Figs. 2B, 2F ). In contrast, on the posterior region of the cuticle simple areoles are not as high, less well developed and nested among groups of bulging areoles ( Fig. 2H ). Bulging areoles occur in higher densities on the posterior than the anterior and mid-body regions ( Fig. 2H ). They are scattered among simple areoles and most common on the mid-body and posterior regions of the cuticle. Occur in groups of three on the mid-body cuticle and groups of two to four on the posterior region of the cuticle ( Figs. 2E, 2H ). Height 7.6–10 µm high, oval to round in shape (6–8 µm) with a smooth surface containing small terminal bristles 1–3 µm in length and less than 1 µm in width ( Figs. 2E, 2H ). Scattered among the simple areoles are tubercle and thorn areoles ( Figs. 2A, 2F, 2H ). Thorn areoles 7–14 µm in length and 3–4 µm wide at their base with typical thorn shape. The filaments of tubercle areoles are 5–7 µm in length and approximately 1–2 µm in width. Crowned areoles occur on mid-body and posterior regions of the cuticle, much denser in distribution than in males (see Figs. 1C , 2C ). As in males, crowned areoles in females occur in pairs with long filaments on top 20–70 µm long ( Figs. 2D, 2E, 2H ). Surrounded by 8–12 circumcluster areoles with small bristles on top and morphological similar to bulging areoles ( Figs. 2D, 2E, 2H ).

Description of deformed individuals: All individuals of C. kenyaensis recovered from crickets infected with P. obamai were much smaller in length than C. kenyaensis recovered from crickets with single infections. Females (N =3) were light tan or white in color and 16±3.6 (13–20) mm in length and 986.7±162.9 (800–1100) µm in midbody diameter; whereas males (N = 2) were tan in color and 12–16 mm in length and 800–1000 µm in mid-body diameter ( Fig. 3A ). Deformed individuals of C. kenyaensis contained six types of areoles on the cuticle including simple areoles with terminal bristles, bulging, tubercle, thorn and crown areoles surrounded by 8–12 circumcluster areoles ( Figs. 3B, 3C, 3D ). Areole patterns in deformed male and female C. kenyaensis were indistinguishable from normal individuals recovered from crickets with single species infections of C. kenyaensis .

Description of mating, oviposition, egg strings, and eggs: Individual male and female worms initiated typical Gordian knots within hours to days of being placed together in Stender dishes ( Fig. 4A ). Males were observed to move up and down the female’s body with their coiled posterior end. Once a males’ cloaca was in proximity with the female cloaca, the male deposited a mass of sperm that remained on the female for up to a week. Females began oviposition within a week of copulating. During oviposition female C. kenyaensis attached egg strings in a continuous zigzag pattern on small branches or air-hoses ( Fig. 4B ) but never deposited free egg strings in the water column. Egg string width was 449 (220–580) µm; whereas individual undeveloped eggs ( Fig. 4C ) were 39 (32–45) µm in length and 30 (24–35) µm in width ( Fig. 4C ). Larvae developed in eggs and began hatching within 3–7 weeks at room temperature.

Description of larvae: Larvae of C. kenyaensis possessed a cylindrical body divided by a septum into two regions, the preseptum and a postseptum ( Figs. 5A, 5C ). The preseptum was 21.0 (17–24) µm in length and 15.7 (14–17) µm in width and contained an eversible proboscis supported by three internal stylets which were 14.9 (12– 18) µm in length and 3.6 (3–4) µm in width ( Figs. 5A , 3B ). The postseptum was 23.0 (14–26) µm in length and 13.0 (12–15) µm in width and contained a clearly visible pseudointestine. The pseudointestine was v-shaped with one smaller and one larger branch both positioned anteriorly ( Fig. 5A ). The pseudointestine was 12.2 (12–14) µm in length and 9.7 (8–12) µm in width ( Figs. 5D, 5E ). Three to four weeks after hatching free-living larvae secreted thread like projections by empting their pseudointestine and stopped moving.

Externally, larvae were superficially annulated and the postseptum contained two pairs of terminal spines located ventrally ( Fig. 5D ). The pseudointestine exterior opening was centrally located above the pair of anterior terminal spines ( Fig. 5D ). The preseptum contained three sets of cuticular hooks ( Figs. 5C, 5E, 5F ). The outer ring of hooks contained seven hooks, two of which are very close together and ventrally positioned, and there were six hooks in the middle and inner rings ( Figs. 5E, 5F ). The dorsal and ventral side of the anterior end of the laterally flattened and eversible proboscis each contained five spines (two aligned pairs and one single spine above); whereas the left lateral side of the proboscis contained four spines ( Figs. 5E, 5F, 5G ).

Development and descriptions of cysts: After being ingested by snails, larvae penetrated snail intestines and became distributed throughout the snail tissues. Once inside snail tissue, larvae emptied the content of their pseudointestine and began the process of cyst formation ( Fig. 6A ). Fully formed cysts of C. kenyaensis were recovered from laboratory-reared and exposed snails 14–24 DPE. Fully formed cysts of C. kenyaensis possessed a clear cyst wall of unknown composition 11.4 (5.5–18.5) µm in length and 11.9 (7–16.5) µm in width ( Fig. 6B ). Folded larvae inside of the cyst were folded only once and were 28.2 (22–35) µm in length and 20.2 (15–25) µm in width ( Fig. 6B ).

Diagnosis and comments: Male and female C. kenyaensis contain six type of areoles and exhibit minor differences in cuticular morphology (number of circumcluster areoles, length of filaments on crowned areoles, density and distribution of bulging and crowned areoles); thus C. kenyaensis is sexually dimorphic. Chordodes kenyaensis belongs to a large group of African Chordodes in which simple areoles are smooth or superficially structured less so than “blackberry” areoles but contain filamentous projections. Among the simple areoles are clusters of bulging areoles, crowned and circumcluster areoles along with thorn and tubercle areoles. Our observations on deformed individuals of C. kenyaensis clearly indicate that this areal pattern is consistent within this species but worm length appears not to be a good character for species differentiation within the genus Chordodes . Of the 19 other sufficiently described African Chordodes species , 14 species contain simple areoles that are not of the “blackberry” type ( Zanca et al. 2006a; 2006b; De Villalobos et al. 2007; Schmidt-Rhaesa et al. 2008; De Villalobos et al. 2009; Bolek et al. 2010). Of those, seven species contain smooth simple areoles with minute bristles or projections on their apical surface ( Chordodes capensis Camerano 1895 , Chordodes digitatus Linstow 1901 , C. janovyi , Chordodes kolensis Sciacchitano 1933 , Chordodes madagascariensis ( Camerano 1897) , Chordodes sandoensis Sciacchitano 1937 , and Chordodes tuberculatus Linstow 1901 ). However, they can be differentiated from C. kenyaensis by the arrangement and types of areoles on the cuticle. Additionally, simple areoles of these seven species never contain bristles or projections on their apical surface which are as pronounced as in C. kenyaensis . Finally, males of two species, C. digitatus and C. tuberculatus , are superficially similar to males of C. kenyaensis because they possess a smooth cuticle structured with fine canals (furrows) surrounding the cloacal opening similar to the cuticular region in males of C. kenyaensis . However, both these species differ from C. kenyaensis by the following characteristics: crown areoles of C. digitatus are arranged in groups of three versus groups of two in C. kenyaensis ; whereas C. tuberculatus only contains three types of areoles (simple, tubercle and crowned) versus six types of areoles in C. kenyaensis .

Observations on the oviposition behavior, egg strings, eggs, larvae and cysts of C. kenyaensis indicate that non-adult characteristics of this species are most similar to other species in the genus Chordodes , and are distinct from other genera and species such as Gordius Linnaeus 1758 and Paragordius for which such characteristics are available ( Inoue 1958; Bohall et al. 1997; Schmidt-Rhaesa 1997; Bolek & Coggins 2002; Hanelt & Janovy 2002; Marchiori et al. 2009; Bolek et al. 2010; Chiu et al. 2011). Females of both the African C. janovyi and the North American C. morgani deposit egg strings in the water on twigs and detritus in a zigzag pattern, whereas females of the Asian Chordodes formosanus Chiu 2011 deposit their egg strings on rocks in a similar zigzag pattern. In contrast, the attachment of eggs strings to substrate has never been reported for other gordiid genera such as Gordius and Paragordius , which simply release egg strings into water ( Hanelt & Janovy 2002; Hanelt et al. 2012). Larvae and cysts of C. kenyaensis are also similar in morphology to other Chordodes species ( C. formosanus , C. janovyi , C. morgani , and Chordodes japonensis Inoue 1952 ), but differ morphologically from larval and cyst stages of species of Paragordius and Gordius (see Schmidt-Rhaesa 1997; Bohall et al. 1997; Bolek & Coggins 2002; Hanelt & Janovy 2002; Marchiori, et al. 2009; Bolek et al. 2010; Chiu et al. 2011; Hanelt et al. 2012). This is particularly true for the arrangement of spines on the proboscis of larvae. As in our study, both Bolek et al. (2010) and Chiu et al. (2011) clearly showed that the proboscis in the African C. janovyi and Taiwanese C. formosanus was laterally compressed and contained spines on the dorsal, ventral and left lateral side, whereas Marhiori et al. (2009) indicated that the proboscis in the South American Gordius dimorphus Poinar 1991 was dorso-ventrally compressed with spines positioned on the left and right lateral sides and the dorsal side of the proboscis.

Phylogenetic analysis of Chordodes kenyaensis . Both fragments of 658 base pairs were amplified from the cox 1 region and sequenced. Sequences derived from the worms were identical. These sequences were placed into GenBank ( KF381359 ; KF381360 ). After addition of samples from GenBank, sequences were trimmed to 526 bp, and aligned. Addition of gaps was not required. The phylogenetic tree produced was based on 17 nucleotide sequences ( Fig. 7 ). After removal of the third base codon positions, the final dataset contained 351 positions .

Figures

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 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 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. 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. 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 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 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.