Morphobiological analysis of Trichuris vulpis (Nematoda, Trichuridae), obtained from domestic dogs

The parasitic nematode Trichuris vulpis Frölich, 1789 is the pathogen of trichuriasis in domestic and wild carnivores, and humans. This helminth species is distributed world-wide in populations of domestic dog (Canis lupus familiaris). The prevalence of T. vulpis in dogs depends to a large degree on the morphofunctional and biological adaptations of the parasite which support its high survivability in various environmental conditions. The present study considers the species-specific peculiarities of morphology, and metric parameters of the mature and embryonic stages of Trichuris nematodes parasitizing in C. lupus familiaris. We studied the periods of stages of development and infectious egg formation, and their survivability under optimal conditions for T. vulpis in laboratory culture. The differential characteristics of both female and male adult T. vulpis nematodes include the metric characteristics of cuticular protrusions at the surface of the anterior body part. The male T. vulpis nematodes can be distinguished from males of other species by the specifics of spicule sheath ornamentation, the shape and size of spicule, and the width of spicule sheath at different sections. In identification of the female nematodes of this species, it is necessary to consider the presence and size of papillary processes in the vulval area and metric parameters of vulva location. Nine metric characters of sexual dimorphism are described for T. vulpis nematodes. In laboratory conditions, five embryonic stages were observed for T. vulpis: protoplast, blastomere cleavage, and formation of bean-like embryo, larva and mobile larva. These stages are characterized by specific morphological features. The egg develops to the infectious stage at 27 °С in 18 days of culture, and their survivability is up to 76.6%. The egg development is associated with changes in their metric characters, such as decreasing egg length and width of egg shell, and increasing egg width and egg plug width.


Introduction
The nematodes of the genus Trichuris (Nematoda, Trichuridae) are a group of the most common parasitic pathogens in various climatic and geographic regions (Soulsby, 1982;Anderson, 2000;Bethony et al., 2006;Ghai et al., 2014). The adult and larval parasites live in organisms of mammals such as ruminants, marsupials, carnivores, rodents and primates. The embryonic life stages of parasites develop in the environment (Hasegawa & Dewi, 2017;Xie et al., 2018;Eo et al., 2019). Currently, there are almost 80 species in the genus Trichuris, and most of them are parasites of specific host taxa (Callejón et al., 2016). Researchers in many countries are interested in this parasitic group because of the danger it poses to humans. According to certain statistics, in total 465,000,000 humans are infected with trichuriasis (Stephenson et al., 2000;Pullan et al., 2014;Betson et al., 2015;Adriko et al., 2018).
Nematode taxa are identified by several morphological and metric characters. The nematodes of the genus Trichuris are similar by their body structure: the anterior part is elongated to filiform, and the posterior part is thicker. Hence in species identification, the following characters are considered: the length of body in males and females; the spicule sheath surface, shape and ornamentation; the spicule length, the shape of its proximal and distal ends; the morphology of the vulva, the structural specifics of the cuticle (Spakulová, 1994;Robles et al., 2006;Ketzis, 2015). Coprological differentiation of the eggs of T. trichiura and Т. vulpis has epidemiological significance; hence, a more detailed morphometric study of embryonic stages of Т. vulpis is of interest to improve diagnosis of the disease caused by this Trichuris species (Kagei et al., 1986;Yoshikawa et al., 1989;Dunn et al., 2002).
The adaptations supporting the wide distribution of parasitic nematodes in the host populations are shaped to a large extent by several factors including the biological features of the parasite species such as the nature of their interactions with the environment at all stages of ontogenesis (Boyko & Brygadyrenko, 2016. One of the important biological adaptations which allows Trichuris nematodes to persist and disseminate is the exogenous egg developpment, i.e. the maturation of egg to the infectious stage in the environment (Fahmy et al., 1954;Lee, 2002;Stroehlein et al., 2017). There are reports of embryogenesis of Trichuris nematodes, according to which the features distinguishing egg development stages, egg viability and metric parameters are speciesspecific and can be used in species identification (Fataliev, 3013;Yevstafieva et al., 2015;Melnychuk & Berezovsky, 2018). Thus, a comprehensive study of morphobiological specifics of Т. vulpis parasitizing domestic dogs is not only of interest in itself but is of significant importance for the diagnostics of that zoonosis.
The objective of the present research was studying the species morphometric identification parameters of mature T. vulpis nematodes (Nematoda, Trichuridae) parasitizing in dogs, and to establish the specifics of the parasite's embryogenesis in laboratory culture.

Materials and methods
The parasitological analysis of T. vulpis nematodes was conducted in 2017-2018. Nematodes were collected during helminthological investigation of the large intestine of 63 dead domestic dogs (Skriabyn, 1928). The Trichuris species was identified using identification keys (Skriabyn et al., 1957). In morphological analysis, 376 adult specimens of Т. vulpis (172 males and 204 females) were used.
To study the biological specifics of Т. vulpis nematodes in laboratory culture, eggs were collected from gonads of female nematodes. Each separate culture was placed in a Petri dish and cultured in a thermostat at 27 °C to the point of mobile larva formation. The cultures were examined every three days under a light microscope. The stage of development was identified by morphology of the embryo, also eggs that had stopped developing or were destroyed were counted. Each experiment was performed in triplicate.
Morphometric parameters of adult and embryonic life stages of Т. vulpis were studied using the software ImageJ for Windows ® (version 2.00) in interactive mode using 10 × , 40 × , 100 × objective and 10 × photo eyepiece. To calibrate the image analyzer, the ruled scale of the ocular micrometer was aligned with the scale of stage micrometer included in MikroMed microscope kit. Microphotographs were taken using a digital camera of MikroMed 5 Mpix (China) microscope.
Statistical processing of the experimental results was carried out using standard deviation (SD) and average values (x) were calculated. Significance of difference between average values in the studied nematode groups was established using one-way analysis of variance and Ftest for 95% confidence level.

Results
It was established that the T. vulpis nematodes were morphologically similar to other Trichuris nematodes in the external body structure. Thus, their anterior body part was elongated, filiform, the posterior body part was strongly thickened and short. Notably, in males the posterior part was sharply bent and sometimes coiled in a spiral, in females the posterior part was slightly curved like a sickle (Fig. 1).
The mouth was terminal, with slightly protruding lips (Fig. 2а), opening into a very thin and long esophagus seemingly immersed in a well-defined stichocyte layer (Fig. 2b). The transition of esophagus to gut was located at the beginning of the thickened posterior body part. The cuticle was with slight transverse striation. Vesicular cuticular processes were characteristic for the surface of the anterior body part of these nematodes (Fig. 2с), both in males and in females.
The morphological study of T. vulpis males resulted in the following observations. The tail end was strongly curved and shaped like a bluntly rounded cone. The spicule was singular, long and filiform. Its distal end was thinner and rounded; its proximal end was widened into a funnel. The spicule sheath tightly covered the spicule (Fig. 3). Males of this species were distinguished by spikes at the proximal part of the spicule sheath (Fig. 4а) in contrast to the distal part of spicule sheath, without spikes (Fig. 4b). That character was easily seen on a maximally protruding spicule sheath. In T. vulpis females, the vulval opening was located posteriorly to the end of the esophagus and the proximal end of gut.   Papillary processes were characteristic near the vulval opening. The processes were of different sizes and shapes. The vagina was tubular, muscular, short and slightly bent (Fig. 5). The uterus had thin muscular walls, in mature females it was filled with several layers of eggs (Fig. 6а). The tail end was bluntly rounded. The anus was terminal (Fig. 6b).
Eggs obtained from uteri of the mature T. vulpis females insignificantly varied from barrel-shaped to lemon-shaped. Both symmetrical and somewhat oblate on one side eggs were observed. There were plugshaped protrusions on both egg ends. Egg shell was smooth and quite thick. Notably, the external shell layer covered egg plugs. Egg colour varied from dark yellow to dark brown (Fig. 7). T. vulpis males and females were measured, resulting in the following characters, useful for species identification. Also in the nematodes of this species, sexual dimorphism was easily observed (Table 1). Thus, the male and female T. vulpis nematodes were statistically different by nine metric parameters. By seven of them, female nematodes were larger than males. However by two characters male nematodes were significantly larger than females. It was established that females were longer by 12.7% (Р < 0.001) than males. Particularly, the female head end was longer by 21.3% (Р < 0.001). The body area with cuticular processes was longer in females by 11.0% (Р < 0.01) compared to males. The female nematode bodies were wider at: anterior edge of cuticular processes (by 7.9%, Р < 0.001); transition of esophagus to gut (by 8.5%, Р < 0.001); tail end (by 23.3%, Р < 0.001). The length ratio of head to tail body parts was 2.5 : 1 in females and 1.8 : 1 in males. At the same time the distance between head end and cuticular processes and the width of body at the middle of head body part were larger in males (Р < 0.001) by 19.1% and 11.5% compared to the respective values for females.
In males, 10 additional metric parameters were recommended for species identification (Table 2). It is most important to consider the spicule measurements, namely its length (8.6 ± 0.4 mm) and width, which gradually increased from minimum at the distal part (13.8 ± 1.1 μm) to the middle (22.3 ± 1.0 μm) and maximum at its proximal part (52.2 ± 2.2 μm). Also, the metric parameters of the spicule sheath width consistently repeated at various spicule sheath parts. The proximal part of the spicule sheath was the widest (128.8 ± 7.0 μm), gradually tapering to the anus area (to 40.1 ± 1.7 μm), after which the spicule sheath slightly widened at the distal end (47.2 ± 1.8 μm). Surface spikes of the spicule sheath were 1.6 ± 0.1 μm long. Fig. 7. Morphological structure of Trichuris vulpis egg Note: ** -Р < 0.01, *** -Р < 0.001 -compared to character values of ♂.  In female nematodes, nine metric parameters were recommended for species identification (Table 3). The most important of the aforementioned parameters were linked to the location of the vulva. The dis-tance from the vulva to the head end was 51.0 ± 3.1 mm, from the vulva to the transition of the esophagus to the gut 162.7 ± 11.3 μm, and from the vulva to the anus 14.2 ± 1.1 mm. The papillary processes near the vulva should also be measured and taken into account. The distal papillary process was large (19.9 ± 0.9 х 28.6 ± 1.3 μm) compared to the proximal papillary process (14.4 ± 1.1 х 17.5 ± 0.9 μm).
The embryogenesis of T. vulpis in laboratory culture at 27 °С occurred in five stages: protoplast, blastomere cleavage, and formation of bean-like embryo, larva and mobile larva. Each stage was morphologically distinct. Eggs developed to the infectious stage in 18 days, which is fast, and their survivability reached 76.6% (Table 4). All eggs obtained from female nematode gonads (100%) were at the protoplast stage. At the third day of culture, 35.0% of eggs were at the blastomere cleavage stage. At the sixth day of culture, maximum eggs at the blastomere cleavage stage were observed, 52.6%. At the same time, the morphological changes characteristic of bean-like embryo were noted in 21.0% of eggs. From the ninth day of culture, no eggs with protoplasts were seen, 20.6% of eggs remained at the blastomere cleavage stage and most of the rest (32.3%) were at the bean-like embryo stage. The larva formation was recorded from ninth to 12th day of culture (from 23.6 to 51.0% of all eggs). Later, at the 12th day of culture, 12.6% of eggs contained a mobile larva, at the 15th day 71.0% of eggs contained one, and on the 18th day, 76.6%. Overall, 23.3% of eggs did not develop and died in culture.
In morphometric study, it was established that measurements of T. vulpis eggs changed throughout their development in culture. Hence, the length and width of eggs obtained from the female nematode gonads at the protoplast stage were 86.4 ± 2.5 and 35.3 ± 0.7 μm, respectively. The length and width of the egg plug were 6.1 ± 0.5 and 10.0 ± 0.6 μm, and egg shell thickness was 1.5 ± 0.1 μm. The infectious eggs with mobile larvae were shorter by 11.7% (76.3 ± 2.3 μm) (Fig. 8а), and their egg shell was thinner by 14.5% (1.3 ± 0.1 μm) (Fig. 8е) compared to the initial values (Р < 0.001). Simultaneously, eggs widened by 18.9% (43.6 ± 2.6 μm) (Fig. 8b). Egg plug width in matured eggs increased by 11.4% (11.2 ± 0.6 μm) compared to protoplast egg values (Fig. 8d). The egg plug length (6.5 ± 0.4 μm) did not significantly change in culture (Fig. 8с). Those specifics in the metric parameters and morphological characteristics, and the periods of development of T. vulpis eggs should be considered in species identification.

Discussion
According to the scientific reports, Trichuris vulpis (Frölich, 1789) is a specific parasite of carnivore animals. However, it is also a zoonotic pathogen of certain danger to human health (Hall & Sonnenberg, 1956;Singh et al., 1993;Mohd-Shaharuddin et al., 2019). This species is also common in the domestic dog (Canis lupus familiaris) in most of the world (Ramos et al., 2015;Scaramozzino et al., 2018;Opazo et al., 2019). Thus, human infection with T. vulpis is a matter of considerable importance since dogs are not only domestic pets but helpers of humans (Kenney & Eveland, 1978;Kenney & Yermakov, 1980;Dunn et al., 2002). Hence, further investigations of morphobiological specifics are needed concerning the nematodes of this species.
The morphometric study of adult females and males of T. vulpis established the metrics of sexual dimorphism in the nematodes of this species. Significant differences were found by nine metric parameters. By seven of those, females are larger than males: length of body (by 12.7%, Р < 0.001); length of head body part (by 21.3%, Р < 0.001); length of body area with the cuticular processes (by 11.0%, Р < 0.01); width of body at the higher edge of cuticular processes (by 7.9%); width of body at the transition of esophagus to gut (by 8.5%); width of body at the tail body part (by 23.3%); head to tail length ratio (by 26.7%). Conversely, males are larger by two parameters: distance from head end to the cuticular processes (by 19.1%); width of body at the middle of the head end (by 11.5%). The size dimorphism is reportedly one of the most common manifestations of sexual dimorphism, and understanding the reasons for the latter is one of the important questions of the evolutionary biology. For example, the fact that females of parasitic helminths are significantly larger than males is explained by the sexual selection of females by fertility (Morand, 1996;Poulin, 1997).
It is well-known that the species identification of helminths necessitates studying their morphological specifics and metric parameters. There is a history of reports on identification of T. vulpis by morphological characters (Skriabyn et al., 1957;Taylor et al., 2007). We have analyzed the morphobiological specifics of nematodes of this species, and determined the characteristic morphological specifics of T. vulpis males and females. We also suggest additional morphometric parameters to identify the species. One of the characters is the vesicular cuticular processes at the head end of the nematode. They occupy 1060.3 ± 69.8 μm of body length in females and 943.6 ± 67.4 μm in males. The cuticular processes have been considered in species identification of the genus also for T. serrata, obtained from the domestic cat (Ketzis, 2015).
The spikes in the proximal part of spicule sheath but not in its distal part are characteristic of the T. vulpis males. That character is easily seen at maximum protrusion of the spicule sheath as reported previously (Skriabyn et al., 1957). Spicule size should also be considered. Its length is 8.6 ± 0.4 mm, its width depending on the measured area ranges 13.8 ± 1.1 μm (distal end) to 52.2 ± 2.2 μm (proximal end). Spicule sheath width is not constant: at the proximal part it is 128.8 ± 7.0 μm, near the anus it is 40.1 ± 1.7 μm, at the distal part it is 47.2 ± 1.8 μm. The males of T. vulpis can also be identified by the length of spikes on the spicule sheath. The females of T. vulpis are morphologically distinguished by two papillary processes near the vulval opening. Their metric parameters should also be considered, with the distal papillary process longer by 27.3% and wider by 38.7% compared to the proximal process. Parameters characterizing the location of the vulva are also important (distance to head end; to transition of esophagus to gut; to anus). Those additional parameters help to identify that species not only by males but by females too. Many researchers point out the necessity of species identification of Trichuris at the egg stages because helminthological dissection and obtaining mature nematodes are not always possible. In that case, coprological analysis is the only method of diagnostics. Eggs of nematodes of the genus Trichuris in different species are similar by many morphological characters and are not easily identified by those. It is even more problematic in the case of differentiating between T. trichiura and T. vulpis. Hence, metric parameters are used together with the morphological specifics (Vásquez et al., 1997;Conboy, 2009;Di Cesare et al., 2012). We have established that the measurements of T. vulpis eggs obtained from female nematode gonads depend on the stage of development of the egg. Hence, the egg length can range 76.3 to 86.4 μm, egg width 35.3 to 43.6 μm, egg plug length 10.0 to 11.2 μm, egg plug width 6.1 to 6.5 μm, egg shell thickness 1.5 to 1.3 μm. However, only two or three parameters were used previously in species identification: length (72-105 μm) and width (32-44 μm) of eggs and length of egg plugs (9.0-12.0 μm) (Skriabyn et al., 1957;Dunn et al., 2002;Márquez-Navarro et al., 2012).
In the embryogenesis of T. vulpis, five stages were observed in laboratory culture at 27 °С: protoplast, blastomere cleavage, and formation of bean-like embryo, larva and mobile larva. The eggs become infectious in 18 days, while their survivability in culture is 76.6 ± 2.1%. Maximum number of eggs with protoplasts was noted at the first day of culture (100%), that of eggs with cleaving blastomeres was seen at the sixth day (52.6 ± 2.1%). Maximum number of eggs with bean-like embryos was recorded at the ninth day of culture (32.3 ± 2.5%), that of larva-containing eggs was established at the 12th day of culture (51.0 ± 4.5%), and maximum of infectious eggs with mobile larvae was seen at the 18th day (76.6 ± 2.1%). Biology of Trichuris nematodes is a popular object of study. However, the embryogenesis is studied in the following species: T. ovis, T. skrjabini, T. globulosa (Thapar & Singh, 1954;Melnychuk & Berezovsky, 2018), Trichuris suis (Yevstafieva et al., 2015). The stages and periods of development in those species are significantly different from our data for T. vulpis. Our results add to the body of knowledge on the morphobiological specifics of T. vulpis nematodes, enhancing the species identification.

Conclusion
Our study revealed a significant sexual dimorphism in T. vulpis obtained from domestic dog. Differences were found in nine metric parameters. According to seven of those, female nematodes are larger than males, and by two males are conversely larger. In Trichuris species identification, the following should be considered: presence, location and metric parameters of the cuticular processes on the body surface. The presence of spikes in the proximal part of spicule sheath, in contrast to the lack of spikes on the distal part at maximum protrusion should be noted for the identification of adult males. In females, the most reliable morphological character is presence of papillary processes in the area of vulva. Ten morphometric characters for males and nine for females are proposed as additional parameters for species identification.
It was determined that the process of T. vulpis embryogenesis in laboratory conditions occurs in five stages: protoplast (1-6th day), blastomere cleavage (3rd to 9th day), bean-like embryo (6-12th day), larva (9-15th day), and mobile larva (12-18th day). The eggs become infectious in 18 days, and their average survivability is 76.6%. In identification of Т. vulpis eggs in coprological studies of samples obtained from humans and animals, the metric parameters of eggs should be considered, taking into account the egg stage of development. During egg development, the following characters change: length (decreases from 86.4 to 76.3 μm), shell width (decreases from 1.5 to 1.3 μm), width (increases from 35.3 to 43.6 μm), plug width (increases from 10.0 to 11.2 μm) and plug length (increases from 6.1 to 6.5 μm).