Leidynema bestium sp. n. (Oxyuridomorpha: Thelastomatidae) an intestinal parasite of blaberid cockroaches from Yaroslavl Zoo, Russia Author Spiridonov, Sergei E. Centre of Parasitology, A. N. Severtsov Institute of Ecology and Evolution, RAS, Moscow, Russia Author Kiselev, Sergei M. Municipal Autonomous Enterprise « Yaroslavl Zoo », Yaroslavl text Zootaxa 2022 2022-10-28 5200 3 271 280 journal article 174289 10.11646/zootaxa.5200.3.5 afb4f1c4-7a5a-41a1-85aa-c984c16b1186 1175-5326 7260530 7161C67B-C509-429D-B189-03AB5F02C51E Leidynema bestium sp. n. ( Figs. 1 – 3 ; Tables 1 – 2 ) Female . Cephalic capsule well defined, 22 – 23 µm long and 51–53 µm in diameter with anteriorly protruding circumoral disk of 25 µm in diameter ( Fig. 1B ; 2A ). First five rings of cuticle narrow, 12 – 14 to 20 µm wide with diameter increasing from 50 – 52 µm up to 75 µm ( Fig. 2A ). Cuticular rings posterior to first five ones much wider (25 µm and more) and keeping this more or less uniform width throughout the body. Stomatal opening round, encircled with eight trapezoidal pseudolabia ( Fig. 2A – C ). Circumoral disk of eight pseudolabia separated from remaining surface of anterior end with a deep seam. Cuticle on both sides of this seam groove with wrinkled surface ( Fig. 2B ). Amphidial opening on separate plate 1 x 0.6 µm in size, wedged between pseudolabia ( Fig. 2B – C ). Buccal cavity thin-walled at anterior (cheilostom), containing strongly cuticularised tubular part at level of cephalic capsule ( Fig. 1B ). Additional cuticularised structures at bottom of buccal cavity embedded into pharyngeal tissue (stegostom). Lateral alae 13–18 µm wide, starting from bulb level and terminating before anus level without sharpened spikes ( Fig. 2F ). Three elongated structures 4 x 1.5 µm in size situated at anterior of pharyngeal lumen ( Fig. 1B ). Anterior half of pharyngeal corpus narrower (26 – 27 µm) than its basal part (57 – 58 µm). Nerve ring situated at border between narrow and wider parts of pharynx, 157 – 168 µm from anterior. Basal bulb and isthmus separated from corpus by thick basal membrane ( Fig. 1C ). Bulb containing valves and separate from valvular cuticularised structures: triangular plates posterior to valves and round structures closer to cardia. Excretory pore located in 310 – 580 µm from anterior. Cuticularised excretory duct leading from pore to excretory vesicle ( Fig. 1D ). Four wide thin-walled excretory channels running into lateral chords (X-shaped excretory system). Intestine composed of numerous hexagonal-polygonal cells. Proventriculus wide with thick walls. Voluminous caecum starting from proventriculus and ending 150 – 230 µm from anterior border of muscular vagina. Didelphic amphidelphic reproductive system. Two uteri run in opposite direction from proximal end of vagina. Anterior uterus then turns back into posterior body part, with majority of eggs situated posterior to vulva. Egg-shell length 115 μm (112–119 μm), width 45 μm (41–47 μm). Muscular vagina about 120 – 180 µm long directed anteriorly from vulvar opening. Vulva equatorial (V% = 49–51). Posterior body tightly filled with gonadal tubes. Anterior ovary tip cell situated usually at level of intestinal caecum and posterior ovary tip cell midway between vulva and anus. Anal diameter four times less as mid-body diameter. Tail filament comparatively short, dagger-like ( Fig. 1A , 2D ). Female juvenile of fourth stage . Cuticle on ventral and dorsal sides of the body covered with regularly distributed bulges ( Fig. 2E ) with approx. size (length x width) 5 – 10 x 4 – 9 µm. Male . Body slender, with ventrally bent posterior end ( Fig. 1G , 3C ). Circumoral plate 10 µm wide. Cephalic capsule swollen, 15 µm in diameter. Body narrowing just posterior to capsule (11 – 12 µm in diameter). Fifteen anterior cuticular rings prominent, with longitudinal ridges (striation) increasing in length from 2 – 3 µm to 7 – 8 µm ( Fig. 3A ). Lateral alae starting at level of fifteenth ring or mid-corpus level (body diameter at this level about 30 µm) and terminating near rectum. Buccal cavity tubular with transparent walls and single short mid-length thickening of cuticular lining ( Fig. 1H ). Three refractive thickenings on stoma-pharynx junction. Nerve ring at posterior of corpus. Isthmus transparent, swollen at border with corpus bulb ( Fig. 1H ). Bulb containing three distinct valves with very thin cuticular folds. Excretory pore visible in some specimens at bulb level. Intestine anterior with wide lumen. Testis reflexing at mid-body ( Fig. 1G ). Posterior half of gonadal tube with transparent walls. A portion of gonadal tube before cloaca with vacuolated walls ( vas deferens ). Single rod-like spicule with slightly curved tip and thickened head. Four pairs of copulatory papillae: a pair of protruding subventral ones before cloacal opening ( Fig. 3C – F ); two pairs of subventral postcloacal papillae of different size: a pair of larger-sized situated at midtail on cuticle protrusion, and a pair of smallest ones close to tail mucron ( Fig. 3D – F ) and a subdorsal pair of papillae intermediate in size. Precloacal and subdorsal papillae of typical thelastomatid structure with central round protrusion and nine smaller tubercles around ( Fig. 3D – G ). Well defined mid-ventral opening of unknown function behind the cloaca. Deep lateral surface pit at mid-tail distinguished under SEM only (maybe due to an artefact of drying). Tail mucron 3 – 5 µm long, rod-like ( Fig. 3F ). Type host . Diploptera punctata (Eschscholtz, 1922) . Another host . Elliptorhina chopardi (Lefeuvre, 1966) Etymology . The specific name of the new species reflects its finding in cultured cockroaches from a zoo ( bestiarium ). Localization. Hind gut lumen (colon). Type specimens . Holotype male on the slide accession number 1333 ( IPEE RAS parasites) and paratypes ( five males on slides 14301 a–e and five females on slides 14300 a–e) are deposited in the Museum of the Helminthological Collections of the Centre of Parasitology , A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences. The voucher specimens, two females of L. bestium obtained from the dissections of Elliptorhina chopardi , are stored at the same collection (slides 14302 a–b). Bionomics . Intensity of the infection with Leidynema bestium of the Diploptera punctata : 1– 3 females and 1– 2 males per host. Molecular phylogenetic analysis . The LSU rDNA sequences of specimens obtained from both hosts were compared and found to be completely identical ( Table 2 ). The final dataset of the obtained alignment for new and related species contained 650 positions. The LSU rDNA sequence of the new species has demonstrated the 42 bp difference with L.portentosae Van Waerebeke, 1978 and 68–70 bp difference with the type species L. appendiculatum . The nucleotide difference between L. appendiculatum and L. portentosae was 63 – 64 bp. Remarkably, the nucleotide sequences of L. appendiculatum were splitting onto two clades ( Fig. 4 ), designated conditionally as clade A and clade B. The sequences of the clade A were obtained from Leidynema nematodes parasitizing in different cockroach genera ( Blaberus Serville, 1831 , Blatta Linnaeus, 1758 , Periplaneta Burmeister, 1838 , Shelfordella Adelung, 1910 . The sequences of clade B were obtained from L. appendiculatum nematodes originating nearly exclusively from Periplaneta cockroaches with only sequence deposited under KY057029 originating from nematode parasitizing Pycnoscelus surinamensis (Linnaeus, 1758) . The nucleotide difference between these two clades was in 10 bp. FIGURE 1. Leidynema bestium sp. n. A–F: female; A—total view; B—cephalic end; C—pharynx basal bulb; D—excretory pore and adjoining channels; E—vulvar opening and vagina; F—posterior end, ventral view; G–K: male; G—total view; H— cephalic end; I—oesophageal region; K—posterior end. All in lateral aspect if not otherwise specified. Scales in micrometers. FIGURE 2. Leidynema bestium sp. n. , females, scanning electron microscopy. A–C—anterior end, sub-apical view; D— posterior end, subventral view; E—female juvenile 4th stage, subdorsal view. The distances between of L. bestium sp. n. and species of other thelastomatid genera associated with cockroaches were more remarkable accounting for 109 bp of Buzionema validum Kloss, 1966 , 112 bp of Severianoia blapticola Guzeyeva, 2009 , 121 bp of Cranifera cranifera (Chitwood, 1932) , 138 bp of Aoruroides chubadaigaku Morffe, García, Hasegawa & Carreno, 2019 and 167 bp of Hammerschmidtiella keeneyi Carreno, 2017 . Differential diagnosis . Morphology of L. bestium sp. n. demonstrates several features to distinguish it from the previously described species ( Singh et al . 2014 ). The new species is characterised by the presence of lateral alae in both sexes with posterior margin of alae before the anal opening level, absence of a spike-like posterior termination of lateral alae in females, longitudinal striation of the anteriormost annuli in males; four pairs of male genital papillae, and the equatorial position of vulva. The lateral alae are present both in males and females of L. bestium sp. n. In this aspect the new species is similar to L. appendiculatum , L. delatorrei Chitwood, 1932 ; L. periplaneti Farooqui, 1967 ; L. orientalis Singh & Malti, 2004 ; L. saltense (Achinelly & Camino, 2008) and L. meerutensis Singh, Rastogi & Singh, 2014 . Leidynema bestium sp. n. is closest to L. delatorrei in the presence of longitudinal striation of the first 15 annuli at the male anterior end ( Leibersperger, 1960 ) and in the structure of a female tail filament (quite short, dagger-like), but differs in the total number of genital papillae in males (five in L. delatorrei vs four in L. bestium sp. n. ). Lateral alae in L. delatorrei males end at cloaca level vs in 20 µm anterior to cloaca in L. bestium sp. n. The new species also differs from L. delatorrei in having the longer average male body (above 900 µm vs 680 µm in L. delatorrei ). According to Singh et al . (2014) , the vulva in L. delatorrei is displaced to anterior part of the body (V% = 41 – 43) while in the new species vulva is located in mid-body. TABLE 1. Morphometric features of the Leidynema bestium sp. n. specimens from two cockroach hosts.

Character	Holotype (Male from the type host Diploptera	Males from the type host Diploptera punctata (paratypes, n=15)	Males from Elliptorhina cho- pardi (n=10)	Females from the type host Diploptera punctata	Females from Elliptorhina chopardi (n=10)
punctata )	(paratypes, n=15)
Body length	999	974±170 (655 – 1212)	947±186 (710 – 1345)	2671±555 (1665 – 3480)	2510±473 (1750 – 3200)
Maximum body diameter	52	53±9 (35 – 74)	63±11 (50 – 80)	236±64 (150 – 310)	241±54 (150 – 300)
Pharynx length	191	195±17 (161 – 222)	192±16 (156 – 210)	466±29 (420 – 520)	442±38 (380 – 510)
Stoma length	16	16±1.2 (15 – 18)	14±3.4 (8 – 17)	26±0.3 (23 – 27)	25±0.2 (24 – 26)
Basal bulb length	49	36±2.2 (30 – 58)	51±6 (42 – 59)	103±9 (90 – 115)	101±7 (89 – 112)
Tail length	12	6.6±3.2 (3 – 12)	4.5±0.7 (4 – 6)	535±101 (400 – 672)	540±101 (410 – 660)
Spicule length	31	31±5.5 (20 – 38)	34±3.5 (29 – 37)	–	–
a	19.2	18.8±4.9 (14 – 35)	15.1±1.4 (12.4 – 16.8)	11±1.3 (10.2 – 14.5)	11.0±1.1 (9.7 – 13.2)
b	5.2	5.0±4.9 (3.9 – 6.2)	4.9±0.8 (3.6 – 6.5)	5.7±1.0 (3.8 – 6.7)	5.7±0.9 (4.2 – 6.8)
c	83.3	195±86 (60 – 331)	215±55 (142 – 336)	5.1±0.8 (38 – 62)	4.8±0.6 (4.0 – 5.9)
V%	–	–	–	51±0.1 (46–53)	49±0.1 (47–52)
Egg-shell length	–	–	–	116±2.0 (113–119)	114±1.9 (112–118)
Egg-shell width	–	45.2±1.3	44.1±1.7
(43–47)	(41–47)

The new species can be distinguished from the type species L. appendiculatum by the presence of longitudinal striations of annuli at the male anterior end, presence of four vs five genital papillae and the wider and shorter female tail filament.All remaining species of Leidynema , both sexes of which possess lateral alae can be distinguished from L. bestium sp. n. by the following unique features ( Singh et al . 2014 ): five pairs of papillae in L. periplaneti and L. orientalis , anteriorly displaced excretory pore in L. saltense and asymmetrically disposed female ala posterior ends (spikes) in L. meerutensis . Thus, L. orientalis differs from the present species by the posterior position of vulva. L. saltense unlike L. bestium sp. n. n has more anterior position of the excretory pore. Leidynema meerutensis can be differentiated from L. bestium sp. n. in the presence of a terminal spike, i.e. pointed posterior end of female lateral alae vs not pointed.