06_Evfsaf'eva-1.indd UDC 595.132.6 MORPHOLOGICAL CHARACTERISTICS OF PARASITIC NEMA- TODES TRICHURIS SYLVILAGI (NEMATODA, TRICHURIDAE) V. Yevstafi eva1, V. Stybel2, V. Melnychuk1, L. Nagorna3, N. Dmitrenko1, O. Titarenko1, O. Dubova4, S. Makarets1, S. Filonenko1, M. Pishchalenko1, M. Kone1, A. Ilchenko1 1Poltava State Agrarian University, Skovorody st., 1/3, Poltava, 36003 Ukraine 2Lviv National University of Veterinary Medicine and Biotechnologies named aft er S. Z. Gzhytskyj, Pekarska st., 50, Lviv, 7900 Ukraine 3Sumy National Agrarian University, G. Kondrateva st., 160, Sumy, 40021 Ukraine 4Polissia National University, Staryi Blvd st., 7, Zhytomyr, 10008 Ukraine Е-mail: evstva@ukr.net V. Yevstafi eva (https://orcid.org/0000-0003-4809-2584) V. Stybel (https://orcid.org/0000-0002-0285-6182) V. Melnychuk (https://orcid.org/0000-0003-1927-1065) L. Nagorna (https://orcid.org/0000-0001-8307-183X) N. Dmitrenko (https://orcid.org/0000-0001-5336-2361) O. Titarenko (https://orcid.org/0000-0002-7370-8523) O. Dubova (https://orcid.org/0000-0002-8303-573X) S. Makarets (https://orcid.org/0000-0002-6844-261X) S. Filonenko (https://orcid.org/0000-0001-8360-8852) M. Pishchalenko (https://orcid.org/0000-0001-8954-8256) M. Kone (https://orcid.org/0000-0002-0828-6619) A. Ilchenko (https://orcid.org/0000-0003-1862-3639) Morphological Characteristics of Parasitic Nematodes Trichuris sylvilagi (Nematoda, Trichuridae). Yevstafi eva, V., Stybel, V., Melnychuk, V., Nagorna, L., Dmitrenko, N., Titarenko, O., Dubova, O., Makarets, S., Filonenko, S., Pishchalenko, M., Kone, M., Ilchenko, A. — Morphometric parameters are analyzed and systematized of parasitic nematodes of the species Trichuris sylvilagi Tiner, 1950, obtained from Lepus europaeus (Pallas, 1778). Th e following parameters are suggested for the exact identifi cation of female T. sylvilagi nematodes: the sizes of body, its anterior and posterior parts, width of body at the connection of esophagus and intestine, specifi c location of vesicular cuticular protrusions and vulva. Five metric parameters are established for the nematode eggs. Th e egg shell thickness, length and width of egg plug are suggested for use in addition to the length and width of eggs. Males of T. sylvilagi had strongly curved tail end, long spicule, and transparent spicule sheath which bears spines, and a bulbous infl ation in its proximal part. Th e thirteen metric parameters, which we found to be of identifi cation value for males, include sizes of body, length and width of its anterior and posterior parts, width of body at the connection of esophagus and intestine, sizes of spicule at various areas, and location of the vesicular cuticular protru- sions. Obtained data on the metric studies of T. sylvilagi nematodes are compared with the published data. K e y w o r d s : Trichuris sylvilagi, Lepus europaeus, parasitic nematodes, morphological features, metric parameters. Zoodiversity, 56(3): 233–242, 2022 DOI 10.15407/zoo2022.03.233 Morphology 234 V. Yevstafi eva, V. Stybel, V. Melnychuk, L. Nagorna, N. Dmitrenko, O. Titarenko, O. Dubova, S. Makarets et al. Introduction Parasitic nematodes are one of the most prevalent and rich in species groups of helminths. Among them, trichurids (Trichuridae Railliet, 1915) are an important group of pathogens which cause trichuroses. Th e patho- gens have a wide variety of hosts, are species-specifi c and diverse (Nechybová et al., 2018; Yevstafi eva et al., 2018, 2019; Yamamoto & Kalepouris, 2020; Montalbano Di Filippo et al., 2019). Scientifi c interest in parasitic nema- todes of the genus Trichuris Röderer, 1761 is confi rmed by the discovery and description of new species, which is possible by studying the peculiarities of their morphological structure, metric parameters, and genetic research (Robles et al., 2014; Hasegawa & Dewi, 2017; Eberhardt et al., 2019; Falcón-Ordaz et al., 2020; Zhou et al., 2021). Th e taxonomy of nematodes of the genus Trichuris is based mainly on morphological characters. A signif- icant amount of research has focused mainly on the study of individual organs and the reproductive system of males, rarely females (Skrjabin et al., 1957; Cutillas et al., 2009; Torres et al., 2011). Externally, all nematodes of the genus Trichuris share the characteristic general body structure, with the much thinner and longer anterior part of the body containing the esophagus, which is why trichurids are called whipworms (Skrjabin et al., 1957). At the same time, such features as the structure of the spicule and spicular sheath, the nature of its surface, fea- tures in the structure of the cloaca in males, the structure and location of the vulva in females are specifi c and serve as identifi cation criteria in species diff erentiation of Trichuris (Mahmoud, 2002; Robles, 2011). Determi- nation of the metric parameters of the morphological structures of nematodes should also be noted in species identifi cation (Panti-May & Robles, 2016; Xu et al., 2016). Scientists have found that nematodes of the genus Trichuris are among the most common helminths among wild species of lagomorphs. Th e authors note parasitism of the species Trichuris sylvilagi Tiner, 1950 in hares of the genus Lepus Linnaeus, 1758 (Czaplińska et al., 1965; Keith et al., 1986). To date, only some works are devoted to the study of morphological features of nematodes of this species. In particular, a study was conducted comparing males T. ovis Abildgaard, 1795, T. skrjabini Baskakov, 1924, T. sylvilagi, T. suis Schrank, 1788 and T. trichiura Linnaeus, 1771 on the basis of 15 morphometric features. When assessing the morpho- logical similarity of the studied species, it was found that the length and width of the spicule and the length of the body were the most characteristic diagnostic features for the studied male nematodes (Špakulová, 1994). According to the identifi cation keys, the main characteristics of morphological diff erentiation of T. sylvilagi were the length of body, the ratio of the length of anterior part of body to the length of its posterior part, the width of the anterior and posterior parts of body, the structure of the bacillary band, the structure and size of spicule and spicule sheath in males and vulva structure in females (Skrjabin et al., 1957; Gvozdev et al., 1970). According to other studies, T. sylvilagi is synonymous with T. leporis Zeder, 1803. Th e description of the spe- cies T. leporis given by Tiner (1905) was shown to refer to another species, for which the proposed name is T. tineri (Kutzer, 1978). Th e study of morphometric species characters of the parasitic nematodes of the genus Trichuris is an urgent task, because they parasitize a wide range of hosts and individual species are very similar, which leads to diffi culties in species diff erentiation. Th erefore, the aim of our work was to investigate the morphological characteristics and metric parameters of T. sylvilagi nematodes and to conduct a comparative analysis of the obtained and the published data. Material and methods During 2020–2021, 15 gray hares (Lepus europaeus Pallas, 1778) (Skriabyn, 1928) were examined by complete helminthological dissection of the large intestine. Wild animals were obtained by hunters during the hunting season in Sumy and Poltava regions. Th e type of isolated Trichuris was determined following the identifi cation key (Gvozdev et al., 1970). In total, 51 specimens of the species T. sylvilagi were collected, of which 17 were males and 34 females. Determination of morphometric parameters of adult males and females of T. sylvilagi nematodes was performed using ToupView soft ware version × 64, 4.10.17015.20200426 (Hangzhou ToupTek Photonics Co., Ltd, China) and AxioVision, Release SPS 4.8.2 (Carl Zeiss MicroImaging GmbH, Germany). Microphotography was performed us- ing a digital camera attached to a MICROmed 5 Mpix microscope (China) and SIGETA M3CMOS 14000 14.0 MP (China). Standard deviation (SD) and average values (М) were calculated. Results and discussion Morphologically, T. sylvilagi have a morphological structure of the body characteris- tic of all trichurids. In particular, the anterior part of body contains the esophagus and is much thinner and longer than the posterior. Also, the posterior end of the male nematode is spirally curved dorsally. In contrast, the posterior end of the female whipworm is slightly inclined laterally, yet has a straight shape (fi g. 1). Th e mouth is small, surrounded by weak lips. Th e esophagus goes through the entire anterior part of the body (fi g. 2, a) and has the muscular anterior part and the glandular 235Morphological Characteristics of Parasitic Nematodes Trichuris sylvilagi (Nematoda, Trichuridae) posterior one. On the surface of the cuticle of the anterior part of body there are vesicular protru- sions of diff erent sizes, visible both dorsally and laterally (fi g. 2, b). In female T. sylvilagi nema- todes, vulva is located directly be- hind the connection of the esopha- gus and the intestine. Th e opening of vulva is barely visible, without protrusions and cuticular forma- tions (fi g. 3, a). Th e uterus is lo- cated in the posterior, thicker part of the body. It has a sac-like part fi lled with eggs and a thinner loop- ing part (fi g. 3, b). Th e tail end has the form of a blunt cone. Th e anus is located terminally (fi g. 3, c). Fig. 1. External view of mature life stages of Trichuris sylvilagi nematodes. Fig.  2. Morphological characters of Trichuris sylvilagi nematodes: a — anterior end, mouth cavity (M), esophagus (Es); b — cuticular protrusions. Ten of analyzed metric parameters of T. sylvilagi females are valuable for identifi cation. At the same time, the number of parameters proposed by other authors for the identifi cation of females of this species ranged from three to eight (table 1). Th us, it has been proposed to determine the total body length of females, the length and width of the anterior and posterior parts of the body, and their ratio. We propose to use additional metric parameters to identify females of T. sylvilagi, which characterize the location of vesicular cuticular protrusions (2 parameters), the width of the body in the connection of esophagus and intestine, the location of the vulva relative to the head end. Metric parameters of eggs in the uterus of females of T. sylvilagi were also determined. Five features were identifi ed, two of which have been previously proposed for species identifi cation, namely: the length and width of eggs. We propose to additionally use the indicators of egg shell thickness, and the length and width of plugs. In males of T. sylvilagi, the tail end is strongly curved. Th e front of the cloaca has a well-defi ned zigzag-shaped bend. Spicule one, long and thin. Th e spicule sheath is covered with spines, which are denser in the proximal part and somewhat less oft en in the distal a b 236 V. Yevstafi eva, V. Stybel, V. Melnychuk, L. Nagorna, N. Dmitrenko, O. Titarenko, O. Dubova, S. Makarets et al. Fig.  3. Structure of } Trichuris sylvilagi: a — area of vulva; b — uterus fi lled with eggs; c — tail end; Vu — opening of vulva, Vg — vagina, U — uterus, E — eggs, An — anus. T a b l e   1 . Metric parameters of } Trichuris sylvilagi, n = 15 (х ± SD, min–max) Parameter Present specimens Skrjabin et al. (1957) Gvozdev et al. (1970) Hofi ng & Kraus (1994) Schoeb et al. (2007) Length of body, mm 43.41 ± 2.13 (40.0–48.0) 30.5–38.7 30.5–38.7 ‒ ‒ Length of the anterior part of body, mm 32.08 ± 2.36 (28.50–37.00) 19.3–28.7 ‒ 17.4–20.9 17.4–20.9 Distance from the head end to the vesicular cuticular protrusions, mm 0.72 ± 0.11 (0.53–0.93) ‒ ‒ ‒ ‒ Length of body in the area with the vesicular cuticular protrusions, mm 1.78 ± 0.12 (1.57–1.93) ‒ ‒ ‒ ‒ Maximum width of the anterior part of body, mm 0.25 ± 0.02 (0.22–0.29) 0.144–0.170 ‒ ‒ ‒ Width of body at the con- nection of esophagus and intestine, mm 0.41 ± 0.06 (0.29–0.49) ‒ ‒ ‒ ‒ Length of the posterior part of body, mm 11.33 ± 0.59 (10.50–12.50) 8.7–12.5 ‒ 4.2–5.5 ‒ a b c 237Morphological Characteristics of Parasitic Nematodes Trichuris sylvilagi (Nematoda, Trichuridae) Maximum length of the pos- terior part of body, mm 1.60 ± 0.13 (1.44–1.89) 0.75–1.0 ‒ ‒ ‒ Distance from the head end to vulva, mm 34.05 ± 2.04 (30.19–38.18) ‒ ‒ ‒ ‒ Anterior to posterior body parts ratio 2.84 : 1 (2.42 : 1–3.36 : 1) 1.8 : 1‒3.3 : 1 1.8 : 1‒3.3 : 1 ‒ ‒ Length of eggs in the gonads, μm 72.70 ± 2.83 (69.15–77.62) 0.068–0.073 0.068–0.073 60.0–65.0 60.0–65.0 Width of eggs in the gonads, μm 32.88 ± 1.83 (30.00–36.52) 0.035–0.037 0.035–0.037 29.0 29.0 Egg shell thickness, μm 1.22 ± 0.07 (1.10–1.33) ‒ ‒ ‒ ‒ Egg plugl ength, μm 9.76 ± 0.71 (8.58–11.05) ‒ ‒ ‒ ‒ Egg plug width, μm 11.90 ± 0.60 (11.08–12.99) ‒ ‒ ‒ ‒ N o t e . Рarameters were not defi ned. Fig. 4. Structure of { Trichuris sylvilagi: a — tail end; b — location of spicule and spicule sheath; Co — opening of cloaca, Рр — pericloacal papilla, Cz — zigzag-shaped opening of cloaca, S — spicule, Sp — proximal end of the spicule, Sd — distal end of the spicule, Ss — spicule sheath, Db — bulbous dilation. a b 238 V. Yevstafi eva, V. Stybel, V. Melnychuk, L. Nagorna, N. Dmitrenko, O. Titarenko, O. Dubova, S. Makarets et al. T ab le 2 . M et ri c pa ra m et er s o f { T ri ch ur is sy lv ila gi , n = 1 5 (х ± S D , m in –m ax ) Pa ra m et er Pr es en t s pe ci m en s Sk rj ab in et a l. (1 95 7) C za pl iń sk a et a l. (1 96 5) G vo zd ev et a l. (1 97 0) H ofi n g & K ra us (1 99 4) Šp ak ul ov á (1 99 4) Sc ho eb et a l. (2 00 7) Le ng th o f b od y, m m 40 .2 3 ± 1. 72 (3 7. 45 –4 2. 63 ) 27 .1 –3 5. 6 ‒ 27 .1 –3 5. 6 ‒ 37 .7 0 ± 3. 85 (2 5. 9– 44 .2 ) ‒ Le ng th o f t he a nt er io r p ar t of b od y, m m 25 .8 4 ± 1. 37 (2 3. 24 –2 8. 22 ) 13 .7 –2 0. 6 ‒ ‒ 19 .0 –2 1. 0 22 .4 4 ± 2. 54 (1 3. 3– 25 .8 ) 19 .0 –2 1. 0 Le ng th o f t he p os te ri or p ar t of b od y, m m 14 .3 9 ± 1. 46 (1 2. 19 –1 6. 69 ) 12 .5 –1 7. 0 ‒ ‒ 7. 4– 8. 7 ‒ ‒ A nt er io r t o po st er io r b od y pa rt s r at io 1. 81  :  1 (1 .3 9  : 1 – 2. 13  :  1) 1  : 1 –1  :  1. 6 ‒ 1  : 1 –1  :  1. 6 ‒ ‒ ‒ M ax im um w id th in th e an te ri or p ar t o f b od y, m m 0. 20 ± 0 .0 1 (0 .1 8– 0. 22 ) 0. 16 –0 .2 2 ‒ 0. 16 –0 .2 2 ‒ 0. 18 ± 0 .0 1 (0 .1 5– 0. 21 ) ‒ M ax im um w id th in th e po s- te ri or p ar t o f b od y, m m 0. 54 ± 0 .0 3 (0 .4 9– 0. 59 ) 0. 25 –0 .5 3 ‒ 0. 25 –0 .5 3 ‒ 0. 70 ± 0 .0 5 (0 .5 7– 0. 85 ) ‒ D is ta nc e fr om th e he ad e nd to th e ve si cu la r c ut ic ul ar pr ot ru si on s, m m 0. 76 ± 0 .0 6 (0 .6 5– 0. 83 ) ‒ ‒ ‒ ‒ ‒ ‒ Le ng th o f b od y in th e ar ea w ith th e ve si cu la r c ut ic ul ar pr ot ru si on s, m m 1. 97 ± 0 .1 4 (1 .7 7– 2. 18 ) ‒ ‒ ‒ ‒ ‒ ‒ W id th o f b od y at th e co n- ne ct io n of e so ph ag us a nd in te st in e, m m 0. 33 ± 0 .0 3 (0 .2 9– 0. 40 ) ‒ ‒ ‒ ‒ 0. 30 ± 0 .0 4 (0 .2 3 – 0. 26 ) ‒ Le ng th o f t he sp ic ul e, m m 7. 65 ± 0 .4 8 (7 .0 3– 8. 26 ) 6. 5– 8. 8 5. 6– 8. 2 6. 5– 8. 8 6. 0– 8. 0 7. 17 ± 0 .9 2 (3 .9 6– 9. 12 ) 6. 0– 8. 0 W id th o f t he p ro xi m al e nd of sp ic ul e, μ m 32 .4 5 ± 2. 77 (2 7. 55 –3 6. 71 ) ‒ ‒ ‒ ‒ 30 (1 0– 40 ) ‒ W id th o f s pi cu le in th e m id dl e, μ m 10 .9 5 ± 0. 98 (9 .1 4– 12 .8 0) 7. 2– 11 .4 ‒ ‒ ‒ ‒ ‒ W id th o f t he d is ta l e nd o f sp ic ul e, μ m 7. 80 ± 2 .0 1 (0 .6 9– 9. 12 ) ‒ ‒ ‒ ‒ ‒ ‒ N o te . Рa ra m et er s w er e no t d efi n ed . 239Morphological Characteristics of Parasitic Nematodes Trichuris sylvilagi (Nematoda, Trichuridae) part. Th e cloaca opens terminally. Th e periclocal papilla is also located in this area (fi g. 4, a). Th e distal end of spicule is narrow and pointed, the proximal end is slightly widened with uneven edges. Th e spicule sheath is long, transparent, and contains a bulbous dilation, which is clearly visible when the spicule is extended (fi g. 4, b). In males of T. sylvilagi, 13 features can be considered as identifi cation features. Th e number of parameters proposed by other authors for the diff erential diagnosis of nematodes of this species ranged from 1 to 8 (table 2). Th us, it has been proposed to determine the length of body, the length and width of the anterior and posterior parts of body and their ratio, the width of body at the connection of esophagus and intestine, the length of spicule and width in the middle of spicule and of its proximal end in males. We additionally propose to use metric parameters to identify T.  sylvilagi males, which characterize the distance from the head end to the vesicular cuticular protrusions, the length of body at their location, and the width of the distal end of the spicule. A signifi cant number of studies indicate the widespread prevalence of trichurosis caused by T. sylvilagi among the population of hares of the genus Lepus. Prevalence of this species range in those hosts ranged from 3.2 to 85 % (Czaplińska et al., 1965; Keith et al., 1986; Clemons et al., 2000; Tizzani et al., 2020). Th erefore, the issue of correct identifi cation of the pathogen, improving approaches to the diff erentiation of T. sylvilagi is an important area of research. Th is relevance is due to the fact that there are reports that indicate diffi culties in identifying species of T. sylvilagi and T. leporis (Skrjabin et al., 1957) or their relationship (Kutzer, 1978). Th e results of morphological studies of isolated nematodes of T. sylvilagi indicate that the general diff erential characters include the overall structure of the body (the presence of long thin anterior and short thick posterior parts, simple mouth opening, and the presence of vesicular cuticular protrusions in the anterior part of body). In males, the tail end is dorsally spirally curved, there is one long and thin spicule, wrapped in a transparent spicule sheath and covered in spines. Moreover, the spicule in the distal part forms a bulbous dilation, which is clearly visible when the spicule is extended. In females, the opening of the vulva is barely visible, the area around the opening of the vulva is smooth, without any cuticular formations. Th e data obtained on the important diff erential features of T. sylvilagi are consistent with most published studies (Skrjabin et al., 1957; Gvozdev et al., 1970; Hofi ng & Kraus, 1994). Metric studies of adult males and females of T.  sylvilagi proposed the defi nition of parameters that will provide increasing amount of data for the eff ective identifi cation of these parasitic nematodes. Th e obtained data were also compared with those data proposed by the authors for the identifi cation of T. sylvilagi. In males, we proposed to use 13 morphometric parameters for their identifi cation. Th e number of parameters proposed by other authors ranged from one to eight. Th us, scientists propose to determine the total length of body of males, the length and width of the anterior and posterior parts of the body, their ratio, the width of body at the connection of esophagus and intestine, the length of the spicule and width in the middle part, the width of the proximal end of the spicule. Moreover, the data obtained by us and the results of other authors have some diff erences (Skrjabin et al., 1957; Czaplińska et al., 1965; Gvozdev et al., 1970; Hofi ng & Kraus, 1994; Špakulová, 1994; Schoeb et al., 2007). Such diff erences have been explained by the infl uence of diff erent hosts and diff erent geographical distribution (Skrjabin et al., 1957). Th erefore, we additionally proposed to use metric parameters to identify males of T. sylvilagi, which characterize the distance from the main end to the vesicular cuticular protrusions and the length of body at their location, the width of the distal end of the spicule. Some researchers also note that although the determination of metric parameters of spicules is the best diagnostic feature for male Trichuris, these characteristics alone are not suffi cient to distinguish between species (Špakulová, 1994). 240 V. Yevstafi eva, V. Stybel, V. Melnychuk, L. Nagorna, N. Dmitrenko, O. Titarenko, O. Dubova, S. Makarets et al. To eff ectively identify females of T.  sylvilagi, we propose to use 10 morphometric parameters. At the same time, other authors for this purpose indicate from three to eight parameters. Th us, scientists propose to determine the length of body, the length and width of anterior and posterior parts of body, and their ratio in female nematodes. Th eir data on some features diff er from the results obtained by other authors (Skrjabin et al., 1957; Gvozdev et al., 1970; Hofi ng & Kraus, 1994; Schoeb, et al., 2007). Th erefore, we propose to use additional metric parameters to identify females of T. sylvilagi, which characterize the location of the vesicular cuticular protrusions, the width of the body in the connection of the esophagus and the intestine, the distance from the vulva to the head end. Th us, the authors propose to determine the total body length of females, the length and width of the anterior and posterior parts of the body, their ratio. We additionally propose to use metric parameters to identify females of T.  sylvilagi, which characterize the location of vesicular cuticular protrusions (two parameters), the width of the body in the connection of esophagus and intestine, the location of the vulva relative to the head end. Metric parameters of eggs in the uterus of females of T.  sylvilagi were also determined. Five parameters of eggs located in the uterine cavity have been identifi ed, of which scientists have previously proposed two (length and width of eggs) for species identifi cation. In addition, we proposed to use the indicators of shell thickness, length and width of egg plugs. Th erefore, the use of the proposed morphometric indicators for the identifi cation of T. sylvilagi will expand the existing data and facilitate the diff erential species diagnosis of these parasites. Conclusion In order to morphologically identify parasitic nematodes of the species Trichuris sylvilagi Tiner, 1950, isolated from the colon of Lepus europaeus Pallas, 1778, it is proposed to use additional morphometric parameters that will increase the effi ciency of diff erential diagnosis. Altogether 13 parameters were determined in male nematodes, and 10 in female nematodes, which characterize the length of body, the length and width of the anterior and posterior parts of body, body width in the connection of esophagus and intestine, location of vesicular cuticular protrusions. Parameters, specifi c for male nematodes, characterize the size of the spicule in diff erent parts. In females, the proposed indicators determine the location of the vulva. Metric parameters of uterine eggs of Trichuris were determined. It is proposed to use fi ve of those: length, width of eggs and egg plugs, and thickness of the egg shell. Th e obtained data will allow to improve the diff erential diagnosis of T. sylvilagi. References Clemons, C., Rickard, L. G., Keirans, J. E., Botzler, R. G. 2000. Evaluation of host preferences by helminths and ectoparasites among black-tailed jackrabbits in northern California. 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