INTRODUCTION
The nematode family, Thelastomatoidea, are parasitic or commensal in arthropods. Thelastomatoids live in the hindgut of the host and usually feed upon the gut microfauna. Thelastomatoids have been studied in many countries and regions around the world [1–14]. Adamson and van Waerebeke [2] diagnosed 27 genera and provided a list of 174 species in the family, Thelastomatidae. Since then, 6 new genera have been described, including 21 species [8, 9, 13, 15]. However, few studies have been carried out on these nematodes in China and only one report has been published (Leidynemella shahi from wood-eating cockroaches in the Yunnan; [16]). Indeed, the thelastomatoid nematodes parasitizing the genus, Eupolyphaga Walker, 1868 (Insecta: Blattaria: Corydiidae), have not been described and reported.
Eupolyphaga is a group of wingless and therefore flightless cockroaches. Of the two most widely distributed species, E. sinensis (Walker, 1868) often inhabits in dark and humid earthen kitchen floors of traditional rural dwellings and mainly occurs in north and central China [17]. E. sinensis (Walker, 1868) is a medicinally important species used in traditional Chinese medicine for enhancing immune responses, promoting blood circulation, treating arthralgia, and inhibiting tumor growth [18–23]. E. sinensis (Walker, 1868) is also well-known as the Chinese medicinal cockroach (literally ‘Tubie Chong’) in Chinese. Generally, the dried cockroach from artificial breeding farms is consumed as a medicinal material. Raw or undercooked wild cockroach is occasionally ingested as treatment of waist pain in rural areas.
The present study arose from an investigation of the pathogenesis underlying E. sinensis (Walker, 1868) in a patient who had encephalopathy for several months. He ingested live E. sinensis captured from natural conditions to alleviate his waist pain. To identify the causative agent for his condition, the toxicity and unknown microorganisms and parasites of the cockroach were investigated. Four different species of nematodes and two species of protists, which have not been previously described, were found after inspecting the guts of 55 wild E. sinensis specimens. For nematodes the prevalence of E. sinensis was 94.5%. Infection intensity was up to 356 nematodes in a single insect. The most abundant nematode species (up to 325 in a single insect) was studied.
To understand the prevalence and distribution of this new nematode in Hunan, China we also collected 200 wild E. sinensis from other two rural areas (see supplementary geographic distribution map). When these samples were dissected and examined in the laboratory, six species of nematodes were identified in the hindgut of this insect. One of the species was morphologically identical to the species described in this study and exhibited a high infection rate.
We examined the morphologic characters of this new species, the egg development pattern, and genetic markers, including the 18S and 28S ribosomal DNA genes [24, 25]. Herein we describe and illustrate the morphologic and molecular characteristics of this new nematode living in wild E. sinensis. This study provides significant data for the development and research of thelastomatid nematodes in China. The microstructure of the sensillae on the male copulatory papillae in thelastomatid nematodes is described for the first time in this study.
MATERIALS AND METHODS
Samples and morphologically based taxonomic identification
Live E. sinensis specimens were collected from the soil (mixed with wood shavings) of an earthen kitchen floor in an old house in Lianhua village (Shitan township, Hengdong county, Hunan province, China [26°55.38’ N, 112°53.26’ E]) on 16 July 2015 and 5 August 2016. Insects were transported to the lab in a plastic container. The insects were rinsed with distilled water to remove the soil before inspection. The body was dissected for removal of abdominal contents, then placed in a clean glass Petri dish. The intestines were examined in 0.1M PBS solution (pH 7.0) using micro-dissecting needles. The nematodes were separated from the intestinal contents and collected into a sterile Petri dish under a 20 × stereoscope, then taxonomically identified according to the morphologic characteristics under a 100 × inverted microscope (Olympus (China) Co., Ltd. Beijing Branch, Beijing, China). The nematodes (both females and males) were then thrice-washed in PBS. The morphologic and morphometric characteristics were described from living and fixed specimens. The specimens were fixed with 80% ethanol and cleared in a solution containing 50% absolute ethyl alcohol and 50% glycerol for 5-10 days to perform morphologic studies. Measurements and photographs were obtained using a microscope with a Motic Images Advanced 3.2 system (Motic, city, China). Scanning electron microscopy was performed, as previously described [26] with minor modifications. The specimens have been deposited into the Parasitology Department of the Xiangya School of Medicine (Central South University [CSU], Hunan Province, China). Because the study did not involve vertebrates or regulated invertebrates, no special permits were required to retrieve and process the samples.
We continuously collected fresh fecal pellets (deposited by E. sinensis in a clean basin on the same day) every day for a total of 21 days to observe embryogenesis from the single cell egg stage to the mature egg, hatching larvae, or host infection. The fresh fecal pellets were placed in EP tubes and incubated at 28°C in 70% humidity, which are conditions suitable for breeding E. sinensis. The fecal particles from day 1-21 were mashed in a Petri dish and soaked in a 0.1M PBS solution (pH 7.0). After soaking, the embryonic developmental stages and egg morphology at different time points were observed in vitro using an inverted microscope. We also continuously observed egg development in the uterus. The morphologic characteristics of the embryos at different developmental egg stages of the nematode were recorded.
DNA extraction, sequencing, and phylogenetic analyses
We collected 20 female and 30 male specimens from the hindgut of the host, E. sinensis. The collected nematodes were first preserved in 95% ethanol, then processed for DNA isolation. Total genomic DNA was extracted from a pool of lysed females and males using an E. Z. N. A. Tissue DNA Kit (Omega Biotek, Shanghai Co., Ltd., Shanghai, China). The 18S and 28S sequences were amplified from total DNA material of the nematodes using primers and conditions of previous studies (18S-forward: 5′-AAACGGCTACCACATCCAAG-3′ and 18S-reverse: 5′-CCAAGCACATGAACCAAATG-3′ [28S-forward primer #391-5′-GCGGAGGAAAAGAAACTAA-3′ and 28S-reverse primer #501-5′-TCGGAAGGAACCAGCTACTA-3′]; [25, 27]). Amplicons of the 18S or 28S region were subsequently purified using a Purelink QuickGel Extraction Kit (Invitrogen, city, state, country) following the manufacturer’s instruction. Purified PCR products were sent to BGI (Guangzhou, China) for DNA sequencing. The newly generated 18S and 28S of nematode sequences were utilized to perform the DNA-based species identification using the online BLAST tool on the NCBI website. All the available 18S/28S sequence records (> 500 bp) of Thelastomatoidea were obtained from NCBI for phylogenetic analyses. After deleting repeated sequences, a single sequence record of each species was chosen for further analysis. Multiple sequence alignment between nematodes was performed using Clustal Omega 1.2.1 (company, city, state, country) with default parameters [28]. Bayesian analysis (BI) was performed to reconstruct a phylogenetic tree using Mrbayes [v3.2.7] [29]. Neighbor-joining (NJ) trees were reconstructed using MEGA5 (company, city, state, country). These trees were analyzed using Figtree (v1.4.3; company, city, state, country). The accession number of each sequence is provided on the tree figures. Pseudonymus islamabadi and P. spirotheca were chosen as outgroups for 18S and 28S trees under the family scale. Pseudonymus belongs to the family Pseudonymidae under superfamily Thelastomatoidea. The 28S tree outgroup under the superfamily scale was used based on a recent study [13], including Distolabrellus veechi and Meloidogyne incognita. The 18S tree outgroup under superfamily scale was Protozoophaga obesa and Wellcomia siamensis, which belong to the superfamily, Oxyuroidea.
RESULTS
Diagnosis
The first and second annules of female Thelastomatidae are simple. A short labial disc with a cephalic crown is formed by three heart-shaped lips surrounded by interlabia lined with a thick cordon. Lateral alae are absent. The pharynx occupies one-third of the body length, excluding the tail. The pharyngeal corpus is thin and cylindrical. The basal bulb is small and spherical. A nerve ring is present at the anterior one-third of the pharyngeal corpus. The excretory pore is situated in the anterior part of the intestine dilated region. The reproductive system is amphidelphic and didelphic, and there is a lack of seminal receptacle on two oviducts. The vulva is at the posterior one-third of the body, including the tail. The vagina is directed anteriorly. The egg is elliptical and the tail is filiform.
The first annule of male Thelastomatidae is elongated and the second annule is simple. The star-shaped oral opening is surrounded by a smooth and conical head capsule with six triangular toothed lips and a large buccal cavity. The lateral alae are absent. The pharynx length is approximately one-fourth of the body length. The pharyngeal corpus is cylindrical. The nerve ring is at the posterior one-third of the pharyngeal corpus. The excretory pore is situated in the middle of the intestine dilated region. Two ventral mamelons are near the tail region. Seven copulatory papillae are in the genital cone region, 5 of which bear a total of 10 sensillae. One pair of additional post-cloacal papillae are on caudal appendage. A spicule is present. The tail is short and filiform.
Etymology. The generic name, Corydiinema is derived from that of the host family, Corydiidae.
Composition of Corydiinema hunanensis sp. nov. (♂♀)
Distribution in China (Hunan)
Description
Twenty-eight gravid specimens were evaluated (Figs 1A–D and 2A-G). The body was small, stubby, fusiform, and wide with a rounded head end (approximately 55-60 μm in diameter). The length was 2,526 ± 198 μm and the width was 222.8 ± 38.6 μm. The maximum body width was at the vulva. Fine transverse striations of the cuticle and microtrichiae were present on the body (Fig 2G); lateral and caudal alae are absent. The tail comprises 23%-26% of the total body length and is filiform, tapering more sharply after the anus (Figs 1A and 2G). There is a short labial disc (in longitudinal length) with a unique crown formed by three heart-shaped lips with well-defined bilobes surrounded by interlabia lined with a thick cordon; each lip has two pyriform lobes. The base of each lip pair is joined at the stoma opening, expanding into two pyriform lobes. Cordons extend from the tripod slit-shaped stoma to the base of each lip and along the perimeter of each lip. Prominent cordons consisting of fine extensions extend on both sides along the surface and are joined by a central smooth region, tripod slit-shaped oral opening, and small buccal cavity (Fig 1C). Sectors (labial papilla) are absent and amphids are not observed at the head (Figs 1B, C and 2A–F). The pharynx consists of a long, thin, and cylindrical corpus, a short isthmus, and a spherical bulb with a valvular apparatus. The pharynx occupies 25%-28% of the total body length and the isthmus is relatively narrower than the corpus. The anterior part of the intestine is wider than the bulb. The nerve ring is located at the anterior one-third of the corpus. The excretory pore is situated in the anterior part of the intestine dilated region (Fig 1A). Three brown-yellow cyst-shaped bodies (a fused mass formed by arcade cells) are prominent, originating in the mouth and around the pharynx, and extending posteriorly 145-161 μm from the head region (Fig 1A, B). The vulva slightly protrudes from the posterior one-third of the body, including the tail (Fig 1A). The distance from the vulva to the anus is 318-326 μm. The reproductive system is well-developed, amphidelphic, and didelphic. The vagina is directed anteriorly, the length is 80-100 μm, then branches into two uteri. The anterior uterus is straight and short, extending anterior to the posterior one-third of the body (excluding tail) and joining the anterior ovary. The anterior ovary is directed and extended anteriorly to the mid-pharynx with no reflex. The posterior uterus is long and curved and joins the posterior ovary, then extends anteriorly to the mid-body after extending posteriorly between the vulva and anus. The posterior uterus is reflexed anteriorly, then the blind end terminates at the mid-body (Fig 1A). The posterior and anterior seminal receptacles are reduced or absent. Different numbers of eggs (n = 12-22) are visible in the uterus, which are arranged in a single row (Fig 1A). The eggs are elliptical with a thin and smooth shell (Fig 3).
Corydiinema hunanensis sp. nov. female. (A) Entire adult female, lateral view. (B) Head region. (C) Cephalic extremity, en face; male. (D) Entire adult male, lateral view. (E) Head region. (F) Cephalic extremely, en face. (G) Posterior region, showing the tail and mamelons. (H) Genital papillae of the cloacal region, ventral view. (I) Spicule.
Scale bars: 500 μm (A); 100 μm (B, E); 30 μm (C); 250 μm (D); 15 μm (F). 500 × 341 mm (300 × 300 DPI).
SEM micrographs. Adult females Corydiinema hunanensis sp. nov. (A-C) Cephalic region, showing the detailed structure of cordons, heart-shaped lips, and stoma. (D) En face, sublateral views. (E) En face. (F) Lateral view. (G) Entire worm with details of cuticular annulations.
Scale bars: 30 μm (A); 20 μm (B, E, F); 10 μm (C, D); 500 μm (G). 169 × 311 mm (500 × 500 DPI).
SEM micrographs. Adult males Corydiinema hunanensis sp. nov. (A) En face view of the cephalic region, buccal cavity, and teeth lips tlp. (B, C) Anterior and cephalic regions, lateral view showing the head capsule. (D) Cloacal region, ventral view of the posterior part of the body showing the filiform tail and genital cone with papillae. (E) One pair of post-cloacal papillae on the tail. (F) Genital and perianal regions, showing perianal papillae, black arrowheads pointing to the sensillae on prpa, adpa, and vppa. (G) Ventral view of the body, two mamelons (m) on the posterior part of the body.
adpa, adcloacal papillae; m, mamelon; popa, post-cloacal papillae; prpa, pre-cloacal papillae; vppa, mid-ventral post-cloacal papilla with two sensillae.
Scale bars: 10 μm (A, E); 30μm (B); 20 μm (C, F); 100 μm (D, G). 226 × 169 mm (500 × 500 DPI).
Fifteen males were evaluated (Figs 1F–I). The body size of males is smaller than females (Fig 1D); specifically, the length is 1,178 ± 164 μm and the width is 111 ± 25 μm. Fine transverse striations of the cuticle and microtrichiae are present (Fig 1G) and lateral alae are absent. The cephalic end consists of a smooth conical head capsule with six triangular toothed lips and a large buccal cavity with a star-shaped oral opening (Fig 1F). The pharynx occupies 21%-28% of the total body length. The pharynx corpus is thin and cylindrical, the isthmus is short, and the basal bulb is spherical and small. A cardia is not present. The pharyngo-intestinal junction is simple. The nerve ring is at the posterior one-third of the corpus. The anterior intestinal tract is greatly expanded at the post-bulb section. The excretory pore is located in the middle of the intestine dilated region. Three brown-yellow, cyst-shaped bodies (arcade cells) are prominent that originated in the mouth and around the pharynx and extend posteriorly (Fig 1D). There is a single testis that is directed to the anterior portion containing amoeboid spermatocytes and extending anteriorly to one-third of the total body length (Fig 1D). The genital cone is prominent (Fig 1H). Seven copulatory papillae are present on the genital cone, comprising one pair of large pre-cloacal papillae (prpa) with six sensillae, one pair of slightly smaller adcloacal papillae (adpa) with two sensillae, one pair of large post-cloacal papillae subventrally, one large mid-ventral post-cloacal papilla (vppa) just posterior to the cloaca, and two sensillae on the end of the vppa. The root diameter of one sensillae is approximately 1.5 μm. One duplex of additional post-cloacal papillae is on the ventral aspect in the middle of the filiform caudal appendage (Fig 1E). The spicule is sclerotized and nearly straight; gubernacular are absent (Fig 1H, I). The tail filiform is approximately one-seventh of the total body length (Fig 1D, G). Two prominent and tandem ventral mamelons are near the tail region, which are likely upwelling areas formed by cutaneous wrinkles on the posterior body (Fig 1D, G). The eggs and developmental stages are shown.
The eggs and developmental stages are shown in Fig 4A–K. The 1-cell stage egg is released from the female uterus and is oval, transparent, and colorless with a thin eggshell, measuring 85-89 μm × 63-69 μm in size. Eggs at different developmental stages (Fig 4) are observed in the fecal pellets of insects and the female uterus when cultured in vitro. Under suitable conditions (26-30°C and humidity = 70%), eggs take approximately 6-9 days to develop from a zygote into a linear first stage larva (L1) with a visible tail, which is the active vermiform stage. It then takes an additional 5-8 days for these eggs to develop into the mature stage, in which an egg contains an oval second stage larva (L2) with a tail spike, the resting stage. The mature stage of the egg also serves as the infective stage to the host. In the fresh fecal pellets excreted by the host, the eggs are primarily in the single-cell stage, with a few cleaved, and some in various embryonic stages. Upon maintaining the fecal pellets in vitro for 7 days, many eggs are observed to contain L1. By the 14th day, almost all eggs develop to the L2 stage. The hatching process of the L2 stage in mature eggs can also be observed in an in vitro culture solution (PBS at 28°C). Under suitable conditions, the developmental process from the 1-cell stage to the mature egg takes 12-16 days.
Images of different developmental stages of Corydiinema hunanensis sp. nov. eggs. The figure shows the developmental process and morphology from the 1-cell stage egg to the mature egg with larvae naturally hatching in vitro. (A) 1-cell stage. (B) 2-cell stage: the first cleavage of the embryo divided unequally to form a larger cell and a smaller cell to form a 2-cell stage. (C) Multicellular stage: early multiple cleavage. (D) Morula stage: embryonic cells divide and multiply. (E) Morphogenesis and organogenesis period: early embryonic development. (F) Early first stage: larva embryonic larval formation. (G, H) The active vermiform L1 stage: the types of eggs containing a linear L1 with a visible tail. (I, J) Resting stage egg: containing an oval or balloon-shaped L2 with a tail spike K. The state of L2 hatching from inside an egg.
Scale bars: 50 μm. 500 × 364 mm (300 × 300 DPI).
Diagnosis and relationships
Corydiinema gen. nov. resembles several genera of Thelastomatidae, as follows: Cordonicola, Robertia Travassos and Kloss, 1960; Galebia Chitwood, 1932; and Thelastoma Leidy, 1849. The general body shape is long and thin with a cylindrical pharyngeal corpus and the vulva is located in the the posterior of the body in females within many more species of these genera. Corydiinema hunanensis sp. nov. differs from Cordonicola, as follows: (i) females have a shorter labial disc with a crown formed by three heart-shaped lips surrounded by interlabia lined with thick cordons, a tripod slit-shaped stoma, an excretory pore situated in the anterior part of the intestine dilated region, a shorter vagina, an anterior ovary extending to the mid-pharynx that is not reflexed, and a longer tail rather than a very short tail; and (ii) males have a spicule, two ventral mamelons, and seven copulatory papillae on the genital cone. Corydiinema gen. nov. differs further from Robertia, which was transferred to the genus, Traklosia by Bernard & Phillips [30], based on a larger body shape and cephalic crown with three lips and a cordon, an amphidelphic and didelphic reproductive system, and a uterus containing > 10 eggs, rather than a small, monodelphic system and a single egg. Corydiinema gen. nov. can be distinguished from Galebia, which has features of a monodelphic reproductive system and elongated eggs. Corydiinema gen. nov. can be distinguished from Thelastoma, which has an enlarged second annule, a vulva near the midbody, a mouth surrounded by eight labial papillae and amphids present in the female and a ventral mamelon, which is absent in the male. Moreover, two ventral mamelons in male Corydiinema gen. nov. is one key diagnostic feature in this genus, but ventral mamelons also occur in the genus, Desmicola [1, 31]. The male of Corydiinema gen. nov. differs from males in the genus, Desmicola, in having two ventral mamelons rather than one mamelon. Specifically, females of six species in the genus, Desmicola, have a very long tail and shorter pharyngeal corpus and the males have a pair of caudal papillae pointed dorsally rather than ventrally.
Type species: Corydiinema hunanensis sp. nov. Figs 1, 2, 3.
Diagnosis. Corydiinema hunanensis sp. nov can be easily separated from all reported species of Thelastomatoidae by a wide and rounded head end and a thick vulva area in the posterior one-third of the body in females, two ventral mamelons in males, and three prominent brown-yellow, cystic-shaped bodies in both sexes under magnification (20X). However, this new species is similar to the type species, Cordonicola blaberi, because the female has a cephalic cordon, a long pharyngeal corpus, a vulva in the posterior one-third of the body, an amphidelphic and didelphic reproductive system, and body shape. However, the results of comparisons in Tables 1 and 2 show that Corydiinema hunanensis sp. nov. is also distinguishable from Cordonicola blaberi and Cordonicola gibsoni [15] in which the female has a triangular oral opening with three lips fused into one opening bearing eight sectors, the first annule is swollen, the second annule is enlarged, cephalic cordons are absent, lateral alae are present, and the tail is long and subulate. However, there are some species within the genus, Thelastoma, with a long, thin, cylindrical pharyngeal corpus (the length [L’] is > 30%) and the vulva in in the posterior one-third of the body or near the anus, which is similar to T. gueyei [33], T. gipetiti [34], T. attenuatum Leidys, 1849 [35], and T. indica Rao, 1958 [35]. T. gueyei is characterized by the vulva being in the posterior one-half of the body and near the anus (V’ = 75%-86%), whereas Corydiinema sp. nov. does not have this feature. Two key features of T. gipetiti are a long (L’ = 40%) and thin pharyngeal corpus and the posterior position of the vulva (V’ = 80%), but the anterior vulval lip has a prominent or flap or inflated extension to form a flap. T. attenuatum and T. indica belong to the long-tail type (C = 2.6 and 2.9). The pharyngeal corpus of the female T. attenuatum is shorter than Corydiinema sp. nov. T. indica (female length = 970-1,920 μm and width = 90-102 μm) is smaller overall than Corydiinema sp. nov. In addition, T. delphyhystera Dollfus, 1964 [36] with a long tail (C = 2.3) and T. retrovulvaris [37] with a short pharyngeal corpus are the species in the genus with a posteriorly located vulva. Corydiinema sp. nov. differs from T. delphyhystera and T. retrovulvaris based on having a cephalic crown with a cordon in the female and two mamelons in the male. The head end of the female of the species studied herein are similar to T. labiatum Leidy 1850, which was described in a millipede. However, T. labiatum has a small body size (female length = 1,058 μm and width = 127 μm). The cephalic annule is inflated, longer, and has eight lobes and two amphids, but does not have a cordon at the margin based on the drawing, original description, and an illustration from the subsequent description by Leidy [38] and Skrjabin et al. [35]. A re-description of T. labiatum by Basir [1] showed eight sectors (labial papillae) in the female anteriorly, including a maximum female length of 1,600 μm and a maximum width of 185 μm), which is overall considerably smaller than those given here for Corydiinema hunanensis sp. nov. Moreover, Geoscaphenema megaovum [8, 9] has also a very long pharyngeal corpus. the Corydiinema hunanensis sp. nov. female differs from G. megaovum, which has a monodelphic and opisthodelphic reproductive system, a vulva located in the anterior one-third of the body (not including the tail) near the mid-corpus, a single uterine egg, and a very large tail and subulate.
Morphometrics of female and male Corydiinema hunanensis sp. nov. and comparison with two species in Cordonicola. Measurements are in μm and in the form, mean ± standard deviation (range).
| Parameter |
Corydiinema hunanensis sp. nov. |
Cordonicola blaberi Ali and Farooqui [32]◆ |
Cordonicola gibsoni Jex et al. [15] | ||||
|---|---|---|---|---|---|---|---|
| Females | Males | Males | Females | Females | |||
| Holotype | Paratypes | Allotype | Paratypes | Paratypes | Paratypes | Paratypes | |
| N | - | 28 | - | 15 | No description | 10 | |
| L | 2,427 | 2,526 ± 198 (2,089-2,709) | 1,168 | 1,178 ± 164 (912-1,380) |
(1,050-1,180) |
(1,880-1,960) | 2,202 ± 124 (2,016-2,384) |
| L’ | 1,798 | 1,904 ± 206 (1,620-1,999) | 989 | 994 ± 115 (804-1,121) |
(940-1,060) |
(1,340-1,410) |
1,325-1,424 |
| A | 9.8 | 11.3 ± 2.1 (10.0-15.9) | 11.1 | 10.6 ± 1.5 (9.2-12.2) |
(7.6-7.9) |
(7.8-8.2) | 12.3 ± 1.2 (10.3-14.3) |
| B | 3.0 | 3.0 ± 0.2 (2.8-3.2) | 3.7 | 4.2 ± 0.3 (4.0-4.7) |
(3.8-4.2) |
(2.2-2.3) | 2.3 (2.0-2.3) |
| B’ | 4.1 | 4.1 ± 0.4 (3.6-4.3) | 4.4 | 4.5 ± 0.2 (4.0-4.9) |
(3.8-4.3) |
(2.0-3.1) | 3.8 ± 0.2 (3.5-4.1) |
| C | 3.9 | 4.1 ± 0.2 (3.8-4.4) | 6.5 | 6.4 ± 1.2 (5.3-8.4) |
(9.5-9.8) |
(3.5-3.6) | 2.5 ± 0.2 (2.2-2.7) |
| C’ | 2.9 | 3.1 ± 0.2 (2.8-3.5) | 5.5 | 5.4 ± 1.2 (4.3-7.4) |
8.6-8.8 |
(2.5-2.6) | 1.6 (1.5-1.7) |
| V | 54 | 54 ± 3 (51-60) | - | - | - |
(43-44) | 50 ± 2 (45-52) |
| V’ | 73 | 72.3 ± 6.8 (68.6-77.3) | - | - | - |
(60-61) | 83 ± 6 (71-91) |
| Body diameter (maximum width) | 247 | 222.8 ± 38.6 (131-271) | 106 | 111 ± 25 (75-150) |
140--150 |
(230-250) | 181 ± 22 (141-211) |
| First annule width | - | - | - | - | - | - | |
| Second annule width | - | - | - | - | - | - | |
| Pharynx length | 593 | 590 ± 29 (576-628) | 264 | 262 ± 26 (229-283) |
(250-310) |
(580-630) | 576 ± 23 (546-616) |
| Corpus length | 525 | 512 ± 66 (482-543) | 159 | 158 ± 9 (156-168) |
(180-230) |
(470-490) | 470 ± 21 (396-511) |
| Isthmus length | 53 | 56 ± 25 (46-78) | 54 | 56 ± 13 (50-72) |
(19-25) |
(43-47) | 39 ± 4 (32-45) |
| Bulb diameter | 72 | 70 ± 8 (68-74) | 45 | 44 ± 4 (42-48) |
(52-56) |
(70-90) | 64 ± 8 (55-77) |
| Tail length | 628 | 627 ± 62.9 (469-710) | 179 | 184 ± 42 (108-259) |
(110-120) |
(540-550) | 877 ± 69 (768-960) |
| Distance of nerve ring from the anterior end | 201 | 198 ± 22 (173-222) | 95 | 96 ± 4 (90-107) | (150-170) |
(210-230) | 458 ± 25 (407-497) |
| Distance of excretory pore from the anterior end | 625 | 647 ± 48 (598-695) | 317 | 335 ± 15 (318-360) | (300-310) |
(650-670) | 353 ± 9 (342-366) |
| Distance of vulva from the anterior end | 1,321 | 1,358 ± 58 (1,253-1,371) | - | - | - |
(800-860) | 1,090 ± 88 (912-1,216) |
| Egg length | - | 89 ± 6 (84.8-102.2) | - | - | - |
(110-120) | 89 ± 83 (71-100) |
| Egg diameter | - | 69 ± 5 (62.4-77.5) | - | - | - |
(80-83) | 56 ± 8 (48-67) |
| Spicule length ▽ | - | - | 54 | 52 ± 5(47-62) | - | - | - |
Morphologic comparison between Corydiinema hunanensis sp. nov. and two species in Cordonicola.
|
Corydiinema gen. nov. |
Cordonicola
| ||||
|---|---|---|---|---|---|
| Corydiinema hunanensis sp. nov. | C. blaberi | C. gibsoni | |||
| Host and locality | Eupolyphaga sinensis Walker in China | Blaberus sp. in India | Panesthia tryoni tryoni in Australia | ||
| Females | Males | Males | Females | Females | |
| Labial disc length | 27 | 52 | 39 | 38 | 76 |
| Base of cordons diameter | 56-60 | 34-30 | 56 | 55-56 | No description |
| Cephalic cordon | Present, thick | Absent | Present, thin | Present, thin | Absent |
| Amphid on the head | Not found | Not found | Present | Present | Present |
| Sectors on the lips of head | Absent | Absent | No description | Present 6. Each lip with 1 | Present 8 |
| Oral opening form and number of lip | Tripod slit-shaped Three heart-shaped lips | Star-shaped. Six triangular toothed lips | Roundness Six lips | Triangular Six lips | Triangular Three small fused lips |
| Lateral alae and caudal alae | Absent | Absent | Present | Present, confluent | Lateral alae present |
| Location of excretory pore | Anterior part of the intestine dilated region | Mid-intestine dilated region | Behind the basal bulb | Base of pharynx | Anterior to mid-corpus |
| Vagina length | 80-100 μm | 300-350 μm | 200-300 μm | ||
| Distribution of anterior ovary | Extending to the mid-pharynx, not reflexed | - | - | Extending to the mid-body, reflexed | Directed to mid-body then reflexed posteriorly at near the anus |
| Distribution of posterior uterus | Long, curving, extending at the mid-body reflexed | - | - | Short, no reflexed | Short, posteriorly directed to near the anus |
| Tail form of female | filiform | Filiform | Filiform | Filiform | Subulate |
| Two ventral mamelons | - | Present | Absent | - | - |
| One spicule | - | Present | Absent | - | - |
| Papillae on the genital cone | - | 7. | 9 | - | - |
| Sensillae on the papillae | - | 10 | Not recorded | ||
Note: No description of the mean ± standard deviation in the original description. ▽: n = 5.
Type materials. Holotype ♂♀ were from Eupolyphaga sinensis Walker (Insecta: Blattaria: Corydiidae) at Lianhua village (Shitan township, Hengdong county, Hunan province, China [26°55.38′ N, 112°53.26′ E]).
Type Locality: Lianhua village, Shitan township, Hengdong county, Hunan province, China
Etymology. The species name is derived from the source of the host distribution in Hengdong county (Hunan province, China).
Distribution. Eupolyphaga sinensis Walker is known only from the type locality.
Molecular evolutionary systematics
The 18S (1,273 bp) and 28S (1,075 bp) sequences of this nematode were successfully amplified and deposited in GenBank under accession numbers (MF580343 for 18S and MN181509 for 28S). Phylogenetic trees were reconstructed at the family and superfamily levels. The phylogenetic trees of family Thelastomatidae are provided in the main text and the trees of the superfamily Thelastomatoidea are provided (Supplementary file 2-3).
All inferred trees show that the genus, Thelastoma, is an apparent paraphyletic group consisting of separated branches. The support value on the top node of the Thelastoma group was low in each inferred tree, but high support values existed in the cluster of Corydiinema hunanensis sp. nov. with Thelastoma krausi (MG189597.1) in all trees.
The results between the BI and NJ trees were different at the low taxonomic level but similar at the higher taxon level in the 18S tree of the superfamily Thelastomatoidea (S2A, B Fig). Species of Thelastoma were largely clustered together and Corydiinema hunanensis sp. nov. were included in the main group of Thelastoma and clustered with Thelastoma sp. and T. krausi, although some nodes had low branch values. We further reconstructed the 18S tree of the family Thelastomatidae. Both BI (Fig 5A) and NJ trees (Fig 5B) were well-supported and consistent with each other. Specifically, the newly formed group of Corydiinema hunanensis sp. nov. was supported by a high branch value (100%).
Phylogenetic relationships of Corydiinema hunanensis sp. nov. with other species of Thelastomatidae based on 18S SSU rRNA sequences. (A) BI tree, four Markov chains, 500,000 generations, burn in = 3000. (B) NJ tree, 1000 bootstrap replicated. The bootstrap values are indicated on the branches. Bootstrap A and posterior probability values B are indicated on the branches.
Scale bars: 71 × 84 mm (600 × 600 DPI).
Trees of family and superfamily are both provided for the 28S analysis. The 28S tree results were largely consistent with 18S, although the former included more sequence records. Corydiinema hunanensis sp. nov. was also included in the large group of Thelastoma and clustered with Thelastoma krausi and Thelastoma sp., which formed a new branch in the family (Fig 6A, B) and superfamily trees (Supplementary file 3AB). Additionally, the genus, Cordonicola, included a subgroup of the genus, Thelastoma, which resulted in a more complicated phylogenetic relation. Additionally, a phylogenetic relationship existed between Stauratostoma shelleyi (MH016661.1) and Desmicola sp. (GQ368463.1) with Thelastoma was ambiguous, but largely separated from Thelastoma (Fig 6; Supplementary file 3).
Phylogenetic relationships of Corydiinema hunanensis sp. nov. with other species of Thelastomatidae based on 28S LSU rRNA sequences. (A) BI tree, four Markov chains, 500,000 generations, burn in = 3000. (B) NJ tree, 1000 bootstrap replicated. Bootstrap A and posterior probability values B are indicated on the branches.
Scale bars: 76 × 126 mm (600 × 600 DPI).
DISCUSSION
Thelastomatid nematodes have been extensively studied and reported by parasitologists from the North America, South America, Europe, Oceania, and countries in Asia and Africa [8, 9, 15, 35, 36]. In fact, Thelastomatid nematodes commonly parasitize arthropods, such as diplopods, orthopterans, cockroaches, and larvae of Scarabaeidae [13, 35, 39]. To date, approximately 33 genera and 200 species of these nematodes have been recorded within the family, Thelastomatidae [2–4, 6, 8, 9, 11, 13, 15, 35, 36, 39, 40].
Thelastomatoids have been reported in various species of cockroaches. The American cockroach, Periplaneta americana L., is the most extensively studied species as a host [5, 6, 12, 41–43]. However, records of these parasites in China are rare. Most notably, there have been no reports of parasitic nematodes in the Chinese medicinal cockroach, Eupolyphaga sinensis.
In terms of morphologic systematics, this study adhered to the taxonomy established by Adamson and van Waerebeke [2], Lee and Blaxter [7], and Shah et al. [11]. Based on the morphology of the studied nematode, which bears a cervical cuticle without spines, a vulva posterior to the base of the esophagus, eggs without filaments, and an embryogenesis pattern with an active vermiform stage (a linear first-stage larva [L1]) and a resting stage (an oval second-stage larva [L2]), clearly belongs to the Thelastomatidae family [11, 35, 42, 44]. To ensure reliable species identification, we consulted the original descriptions of all species of Thelastomatidea published in the literature and gathered information on morphology and biometrics from various specialized monographs [6, 8, 9, 13, 15, 31–37, 40, 42, 44–48]. Upon combining the basic morphologic characteristics with the embryogenetic pattern, we conclude that this nematode belongs to a genus within the Thelastomatidae (Thavassos, 1929), one of the four families in the Thelastomatoidea, as recognized by Lee and Blaxter [7]. Upon comparing the diagnostic features of each genus within Thelastomatidae, we discovered that the Corydiinema gen. nov. is distinguished from all other members by several unique characteristics, including a short labial disc, a cephalic structure that bears a crown with a three-slit-shaped stoma formed by three heart-shaped lips and a prominent cordon in the female, and two ventral mamelons in the male that serve as unique structural markers. Additionally, the presence of a straight anterior ovary in the female, directed anterior to the mid-pharynx, is a rare feature.
The morphologic features of Corydiinema gen. nov. clearly distinguish Corydiinema gen. nov. from all members of the Thelastomatidae. The female is characterized by a short labial disc bearing a unique crown consisting of three heart-shaped lips, interlabia lined with a thick cordon, a three-slit-shaped stomatal opening, a straight anteriorly directed ovary extending to the mid-pharynx that is not reflexed, and an excretory pore situated in the anterior part of the dilated region of the intestine. The male, in contrast, has 2 ventral mamelons, 7 copulatory papillae, 1 spicule, and 10 sensillae. Both sexes lack lateral alae. Based on the diagnosis of each genus in Thelastomatidae, we have demonstrated that none of these genera have the same combination of male and female characteristics.
Compared to each species in Thelastomatidae and based on existing records, Corydiinema hunanensis sp. nov. is very close to the type species [Cordonicola blaberi Ali and Farooqui [32] from Blaberus sp. in India]. The female of this species features a cephalic cordon, a long pharyngeal corpus, a vulva located in the posterior one-third of the body, and an amphidelphic and didelphic reproductive system. Both sexes of the genus, Cordonicola Ali and Farooqui [32], possess cephalics cordons. To date, only two species in the genus, Cordonicola, C. blaberi and C. gibsoni (from Panesthia tryoni tryoni in Australia), have been described. The diagnosis of Cordonicola was revised and determined by Adamson and van Waerebeke [2] and Jex et al. [15], which includes thelastomatoids without cordons. However, there are clear differences between Corydiinema hunanensis sp. nov. and the two species of Cordonicola, as outlined in Table 2. These differences led us to conclude that the current nematode does not belong to the genus, Cordonicola. Importantly, some descriptions of Cordonicola are lacking. For example, the presence of 2 ventral mamelons, a single spicule, and 10 sensillae in the male have not been shown or described in Cordonicola blaberi. This is particularly noticeable given the absence of SEM structures in C. blaberi and the unidentified male in C. gibsoni.
Of the species of Thelastoma Leidy, 1849, 51 were recorded in the revision of Adamson and van Waerebeke [2]. Seven new species were identified and described during 1995-2018. The type species of the genus Thelastoma is T. attenuatum (Leidy, 1849), which can be differentiated from Corydiinema hunanensis sp. nov. in having an enlarged second annule, a vulva near the midbody, a mouth surrounded by eight labial papillae, and amphids. Of the 58 species of this genus, 10 share some or a single similarity in morphology with Corydiinema hunanensis sp. nov. However, Corydiinema hunanensis sp. nov. can be differentiated from all species within Thelastoma due to the female having a cordon and the vulva present at the posterior one-third of the body. Specifically, the presence of two mamelons distinguishes the males of Corydiinema hunanensis sp. nov. from T. krausi and the other species. In addition, the male of Corydiinema gen. nov. differs from the genus, Desmicola, in having two ventral mamelons rather than one.
In addition, we observed that male Corydiinema hunanensis sp. nov. has 10 sensillae distributed on 5 different copulatory papillae situated around the anus when examined under SEM (Fig 4F). These tiny sensors have not been described previously in reported thelastomatid nematodes because most of the earlier results were derived from light microscopy or female samples. SEM photographs provided from other species failed to highlight the sensors. It is not possible to state that this is a unique or a new marker of Corydiinema hunanensis sp. nov. but could be used as one of the morphologic features for comparison of new species or re-described specimens. According to the above morphologic comparison, we conclude that the nematode described herein belongs to a new genus of Thelastomatidae.
The diagnostic tools of genetic markers provide the prospect for specific identification of members of Oxyuridea as a basis for systematic, ecological, and/or population genetic investigations based on molecular systematics. The 28S rRNA and 18S rRNA genes are useful markers for confirmation of the phylogenetic position of taxa and is commonly used in phylogenetic assessment of nematodes [8, 42, 49–55].
We performed a phylogenetic analysis of the 18S and 28S genetic markers. Because the Corydiinema hunanensis sp. nov. share some morphologic characteristics with the type species of Cordonicola and some species of Thelastoma, as described above, all available sequences (> 500 bp) of species from the superfamily, Thelastomatoidea, were used as candidates for generating a more robust topologic structure of phylogenetic evolutionary reconstruction. The phylogenetic topologic trees (Figs 5 and 6; S2 and S3 Figs) showed that the Corydiinema hunanensis sp. nov. was included into the clade consisting of most Thelastoma species plus Cordonicola. In addition, Corydiinema hunanensis sp. nov. has a closer evolutionary relationship with two species [Thelastoma krausi (EF180068.1) and T. sp. (KP715348.1)] and these three species form an independent group.
The reasons for the differences in the results of genetic molecular analysis between Corydiinema hunanensis sp. nov. and Cordonicola could be that the sequences of Cordonicola sp. 28S gene fragment in GenBank (GQ368464.1) was in fact C. gibsoni from Australia rather than C. blaberi. C. gibsoni is markedly different from C. blaberi and Corydiinema hunanensis sp. nov. based on morphologic features (Table 2). In addition, the 28S sequence records of C. gibsoni was too short (just > 200 bp) to clearly distinguish C. gibsoni from Thelastoma.
The results of phylogenetic analysis are also inconsistent with the morphologic study between Corydiinema hunanensis sp. nov. and T. krausi. One of reasons could be that the latter specimen (NCBI accession numbers, EF180068 for 18S and MG189597 for 28S) was misidentified. The other two sequence records of Thelastoma spp. (accession numbers, KX987865.1 and KP715348.1) that grouped with Corydiinema hunanensis sp. nov. in the phylogenetic tree cannot be further confirmed by the morphologic study herein. Based on the current analysis we concluded that the newly reported Corydiinema hunanensis sp.nov. might form a new group under the paraphyletic Thelastoma, which is defined as a new genus, Corydiinema gen. nov.
Our research group is currently examining the biological properties of Corydiinema hunanensis sp. nov., along with other species of entomogenous nematode fauna in E. sinensis. The diversity of pinworms within invertebrates remains largely unexplored and poorly understood in China, with most potential thelastomatoid hosts yet to be investigated. This report on Corydiinema hunanensis sp. nov. in E. sinensis contributes to documenting the thelastomatids of this region, which enhances our understanding of the systematics of this group. This is fundamental for further analysis of the relationship between the cockroach and its parasites, as well as their potential effects on humans.
Both female and male nematodes of the new species parasitizing in the hindgut of the cockroach E. sinensis exhibited high infection rates and intensities. These were observed and described in detail using light and SEM. A phylogenetic analysis based on both 28S and 18S genes of this species, together with those of other reported pinworms from the superfamily, Thelastomatidea, was conducted. The morphologic and molecular characteristics identified E. sinensis as a new genus and a new species. Additionally, this is the first record of a thelastomatid species parasitizing a new host.