Comparative embryonic development of nematodes of the genus Тrichuris ( Nematoda , Trichuridae ) obtained from sheep ( Ovis aries )

Biological specifics of Trichuris оvis Abildgaard, 1795 and T. globulosa Linstow, 1901 parasitizing domestic sheep were analyzed enhancing the species identification of these nematode species. The embryonic development of nematodes was timed, survival of embryonic stages was determined taking into account their morphological and metric specifics in laboratory culture. Trichuris eggs were isolated from gonads of adult female nematodes collected from caeca of dissected sheep. Then the eggs were cultured at optimal temperature to the formation of eggs with mobile larvae. Six morphologically distinct stages of embryogenesis were established in T. оvis and T. globulosa nematodes. The protoplast stage lasted from the 1st to the 12th day in T. оvis and to the 18th day in T. globulosa. Blastomeric formation occurred from the 3rd to the 18th day in T. оvis and from the 3rd to the 21st day in T. globulosa. Bean-shaped embryos formed from the 6th to the 21st day in T. оvis, and from the 9th to the 30th day in T. globulosa. Tadpole-like embryos developed from the 12th to the 24th day in T. оvis and from the 18th to the 33rd day in T. globulosa. Larvae formed in eggs of T. оvis from the 18th to the 27th day, and in eggs of T. globulosa from the 21st to the 36th day. Mobile larvae formed from the 21st to the 30th day in T. оvis, and from the 30th to the 39th day in T. globulosa. At 27 °С, mature eggs with mobile larvae developed in 30 days in T. оvis and in 39 in T. globulosa. The egg survival in laboratory culture was 84.3 ± 4.2 % and 76.3 ± 1.5%, respectively. Developmental changes of metric parameters in Trichuris nematode eggs (length and width of eggs, plug length, eggshell thickness) were species-specific.

The development of Тrichuris nematodes is direct, without an intermediate host.The main and deciding environmental factors in viability and durability of embryonic stages in these nematodes are the vegetation cover and also the temperature, the moisture of air and soil, landscape forms, rainfall, and other abiotic factors (Thapar & Singh, 1954;Mamedova & Fataliev, 2009).Other researchers observe that the development of infectious eggs of Тrichuris nematodes in the environment is principally influenced by temperature (Beer, 1788) or soil type (Brown, 1927).
It is known that the embryogenesis of Тrichuris nematodes occurs in several stages.Some authors recognize the following stages of development of Тrichuris eggs in the environment: nuclear fusion, first rest period, fission groove formation, second rest period, protoplasm clearing, larva formation, larva in one revolution, larva in two revolutions, formation of infectious larva (Fataliev, 2013).Other researchers note such embryonic periods in T. trichiura (Linnaeus, 1771): fertilized egg with two pronuclei, formation of 2 to 10 blastomeres, further cleavage, gastrulation, embryo with a closed blastopore, moving embryo, formed larva with a stylet (Malahov et al., 1984).
There are many studies of embryonic development of Тrichuris nematodes which infect humans (Bundy & Cooper, 1989;Skriabyn et al., 1957).At the same time, certain aspects of embryonic development in eggs of Тrichuris nematodes of sheep are still insufficiently known.There are also rather counterintuitive data on the periods of egg development in nematodes of this genus (Skriabyn et al., 1957).Thus, the aim of the present work is to study the specifics of embryogenesis in nematodes of the species T. оvis and T. globulosa in vitro, taking into account the morphological and metric changes in the eggs.

Materials and methods
Nematodes were collected in during helminthological investigation of the large intestine of dead or slaughtered sheep (Skriabyn, 1928).The nematode species were identified using keys (Skriabyn et al., 1957;Ivashkyn et al., 1989).To study the biological specifics of Т. оvis and Т. globulosa nematodes in laboratory culture, their 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 Т. оvis and Т. globulosa eggs (length and width of egg, length and width of egg plug, eggshell thickness) were studied in cultures, using the software Image J 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 coincided with the scale of stage micrometer included in the MikroMed microscope kit.Microphotographs were taken using a digital camera of a MikroMed 5 Mpix (China) microscope.
Statistical processing of the experimental results was carried out using Statistica 10 (StatSoft Inc., 2011) software.Standard deviation (SD) and average values (x) were calculated.Significance of difference between average values in the studied egg cultures of Тrichuris nematodes was established using one-way analysis of variance and F-test for 95% confidence level.

Results
It was established that embryogenesis of Т. оvis and Т. globulosa nematode parasites of sheep occurs in six stages in laboratory culture at 27 °С: protoplast, blastomeric formation, bean-like embryo, tadpole-like embryo, larva formation, and mobile larva formation.These stages are characterized by morphological changes in nematode eggs.At the protoplast stage, egg cytoplasm was filled with large granules which visually merged into one mass (Fig. 1а).Blastomeric formation was characterized by the cleavage and formation of two (Fig. 1b) and then three and more large cells, and the size of the blastomeres decreased in inverse proportion to their numbers (Fig. 1c).The bean-like embryo was morphologically distinct by the development of multicellular beanshaped embryo in the egg (Fig. 1d).Later, the embryo changed into a shape resembling a tadpole, which was hence the tadpole-like embryo stage (Fig. 1е).The next stage was characterized by formation of motionless larva, with granular, slightly contoured body (Fig. 1f).The embryogenesis of the studied nematodes, regardless of the species, ended in mobile larva formation in the egg.The larva was twisted, completely filled the internal volume of the egg and actively moved under the influence of warmth, and also had a clear morphological structure (Fig. 1g).
Although the process of embryogenesis did not differ by morphologic changes in eggs of Т. оvis and Т. globulosa nematodes, their periods of development and survival were dissimilar.For example, eggs obtained from T. оvis female gonads in vitro at 27 °С developed mobile larvae in 30 days, and their survival was 84.3 ± 4.2% (Table 1).
All of the eggs (100.0%),obtained from female nematode gonads were protoplast.From the third to 12th day of culture, the eggs progressed to the second stage of development, formation and cleaving of blastomeres.First, two blastomeres appeared (44.3 ± 3.1% at the third day), then three and more blastomeres (41.3 ± 1.2% at the sixth day, 41.7 ± 3.5% at the ninth day).From the sixth day, 8.0 ± 1.7% of all eggs were at the beanlike embryo stage.The maximum ratio of eggs on the third stage of development (54.7 ± 6.4 and 57.7 ± 8.1%) was recorded from the 12th to 15th day of culture.At the 12th day of experiment, 5.3 ± 3.5% of T. оvis eggs were observed to be developing from bean-like to tadpole-like stage.Maximum ratio of eggs at this stage was observed at the 21st day of culture (44.3 ± 4.0%).At the 18th day, 14.0 ± 4.4% of eggs contained formed, though motionless, larvae.The duration of that stage of development was until the 27th day of culture, with maximum ratio (50.3 ± 5.7%) on the 24th day of culture.Formation of mobile larvae started at the 21st day, when they comprised 10.3 ± 2.1% of all eggs.Subsequently, the ratio of eggs containing mobile larvae was 29.3 ± 2.5% at the 24th day, 81.3 ± 4.9% at the 27th day, and 84.3 ± 4.2% at the 30th day of experiment.Also, 15.7 ± 4.2% of all eggs did not continue developing and died.In the study of the embryonic specifics of Т. globulosa nematodes, their eggs were observed to mature to the mobile larvae stage more slowly compared to Т. оvis nematodes.That development occurred in 39 days.At the same time, Т. globulosa eggs were less viable: only 76.3 ± 1.5% developed into mature eggs, while 23.7 ± 1.5% of eggs did not develop and died (Table 2).The obtained cultures of Т. globulosa eggs were all at the protoplast stage at the beginning of experiment.Further, the ratio of protoplast egg decreased to 23.7 ± 1.5% at the 18th day.From the 3rd to 21st day of culture, blastomeric formation occurred in the eggs.Maximum ratio of eggs with two blastomeres was observed at the 3rd day (35.3 ± 2.1%), that of eggs with three and more blastomeres at the 6-9th days (39.7-41.3%).The bean-like stage of development was recorded at the 9th (23.0 ± 1.0% of all eggs) to 30th day of culture.Their ratio increased to its maximum at 15th to 18th day (from 52.0 ± 3.0 to 54.0 ± 2.7%).From the 21st to 30th day of experiment, the numbers of eggs containing beanlike embryos gradually decreased with their development to the next stage, the tadpole-like embryo.That stage occurred from the 18th to 33rd days of culture.Maximum ratio of eggs with that embryonic stage was recorded at the 24th to 27th day of culture (39.7-42.3%).The larval formation was observed from the 21st day of experiment (11.3 ± 1.5%) to 43.7 ± 3.5% at the 30th day, the stage was observed till the 36th day.Mobile larva formation occurred in Т. globulosa eggs from the 30th to 39th day of experiment, the ratio of the eggs with that stage increasing from 5.3 ± 2.1 to 76.3 ± 1.5%.Morphometric study revealed that the sizes of Т. оvis and Т. Globulosa eggs changed during embryonic development, and that there were species-specific differences.The length and width of eggs obtained from Т. оvis females at protoplast stage were 71.3 ± 1.7 and 32.7 ± 1.8 μm respectively, with egg plug length of 8.8 ± 1.1 μm and egg plug width of 10.7 ± 0.6 μm.Eggshell thickness of those eggs was 3.0 ± 0.2 μm, internal surface area was 1053 ± 82 μm 2 .Mature eggs containing mature larvae were statistically smaller (Р < 0.05) by 3.4% (68.9 ± 1.3 μm) of egg length (Fig. 2а), 16.8% (2.5 ± 0.3 μm) of eggshell thickness (Fig. 2е) and 13.7% (7.6 ± 0.4 μm) length of egg plugs (Fig. 2с).Simultaneously, egg width increased to 35.1 ± 1.2 μm (by 6.8%, Р < 0.05) (Fig. 2b).There were no statistically significant differences in egg plug width (11.1 ± 0.6 μm) and internal surface area (1107 ± 31 μm 2 ) during the studied embryogenesis (Fig. 2d, f).

Discussion
In the course of the present study, new data is collected on the embryogenesis of sheep nematode species T. оvis (Abildgaard, 1795) and T. globulosa (Linstow, 1901), on eggs obtained from nematode gonads.Knowledge of biological specifics, especially those of human parasites, factors into the regulation of their numbers, more so in the ecosystems experiencing anthropogenic pressure.Also in several cases, such data is taken into account in species or genus identification (Liang et al., 2007;Wong & Candolin, 2015;Zvinorova et al., 2016).According to scientific reports, trichurosis is very common in wild and domestic ruminants.The infection is recorded world-wide, with rates varying in different countries (Salaba et al., 2013;Yaro et al., 2015;Eichenberger et al., 2018).In our opinion, domination of one species over several other Trichuris species in a host population is to some degree dependent on the nematode specifics of embryogenesis which occurs outside the host.We observe that in vitro, at 27 °С the eggs of Т. оvis develop faster (in 30 days) and are more viable (84.3 ± 4.2%) compared to the eggs of Т. globulosa (which develop to mobile larva stage in 39 days with 76.3 ± 1.5% survival).This may be the basis of domination of Т. оvis over Т. globulosa in sheep in several regions of Ukraine (Yevstafieva et al., 2018).
Hence, we delineate the main morphological stages of development in eggs of Т. оvis and Т. globulosa, which occur in the same way.The embryogenesis in the nematode species, studied at 27 °С, occurs in six stages: protoplast, blastomeric formation, bean-like embryo, tadpolelike embryo, larva formation, mobile larva formation.These stages of development are quite morphologically distinct.Similar stages of development were found for T. suis (Schrank, 1788) nematodes obtained from pigs, and T. skrjabini (Baskakov 1924) roundworms, obtained from sheep (Yevstafieva et al., 2015(Yevstafieva et al., , 2018)).At the same time, quantitative indicators of changing embryonic stages in the development of Т. оvis and Т. globulosa nematodes were different.For example, protoplast stage in T. оvis occurs during the 1st to 12th days of culture, and from the 1st to 18th days of culture in T. globulosa.At the first day of culture, 100% of all eggs were at the protoplast stage.Blastomeric formation occurred from the 3rd to 18th days of culture in T. оvis nematodes, and from the 3rd to 21st days of culture in T. globulosa, with maximum ratio of eggs with protoplasts, respectively, 41.7 ± 3.5% to 44.3 ± 3.1% and 35.3 ± 2.1% to 41.3 ± 1.5%.The bean-like embryo developed from the 6th to 21st days of culture in the eggs of T. оvis, and from the 9th to 30th days of culture in the eggs of T. globulosa (the maximum ratios were respectively 57.7 ± 8.1% and 54.0 ± 2.7%).The tadpole-like embryo occurred in the eggs of T. оvis from the 12th to 24th days of culture and in the eggs of T. globulosa from the 18th to 33rd days of culture.The maximum ratio of eggs with tadpole-like embryos were, respectively, 44.3 ± 4.0% and 42.3 ± 0.6%.Larva formation occurred in the eggs of T. оvis from the 18th to 27th days of culture, and in the T. globulosa eggs from the 21st to 36th days of culture (maximum ratios were 50.3 ± 5.7 and 43.7 ± 3.5%, respectively).Mobile larva formation occurred in the eggs of T. оvis from the 21st to 30th day of culture, and in the eggs of T. globulosa from the 30th to 39th days of culture (maximum ratios were 84.3 ± 4.2 and 76.3 ± 1.5%).These data can be used in differential diagnostics of the studied Trichuris species.Most authors note that nematodes are identified by the morphology of adult specimens, especially the males (Bailey et al., 2009;Ghasemikhah et al., 2011).However, adult nematodes are not always available because they are obtained from dead hosts.In most cases, especially in diagnosing intestinal nematodes, coprologic samples are taken and only eggs are available.Some authors propose keys based on egg identification using various methods such as genetic analysis, electronic microscopy, dyeing and metric parameters (Palmer & McCombe, 1996;Sommer, 1996;Phosuk et al., 2018).Hence we propose new data on morphometric indicators of the eggs of Т. оvis and Т. globulosa and their changes in embryogenesis.We found that morphology of eggs obtained from gonads of the studied nematode species were not statistically different.The eggs were different by several metric parameters.For example, length and width of the eggs of Т. оvis were 71.3 ± 1.7 and 32.7 ± 1.8 μm, and those of Т. globulosa were 70.8 ± 3.6 and 38.9 ± 1.6 μm, and eggshell thickness was 3.03 ± 0.19 and 4.05 ± 0.25 μm respectively.Length and width of egg plug in Т. оvis eggs were 8.8 ± 1.1 and 10.7 ± 0.6 μm, and those of Т. globulosa were 3.3 ± 0.5 and 9.1 ± 0.4 μm, the internal surface areas were, respectively, 1053 ± 82 and 1306 ± 51 μm 2 .At the same time, embryogenesis in both species was characterized by speciesspecific changes of metric parameters.The mature eggs with mobile larva in Т. оvis eggs were accompanied by decreasing egg length (by 3.4%, Р < 0.05), eggshell thickness (by 16.8%, Р < 0.05), egg plug length (by 13.7%, Р < 0.05), and increasing egg width (by 4.3%, Р < 0.05).The eggs of T. globulosa grew and developed with decreasing length (by 4.9%, Р < 0.05), eggshell thickness (by 9.6%, Р < 0.05), and increasing egg width (by 4.3%, Р < 0.05).Our data increase the knowledge of biological features of Т. оvis and Т. globulosa nematodes.

Conclusion
The study showed that it is possible to use embryonic specifics (morphometric changes of eggs, periods of development, survival ratio) of nematodes of domestic sheep ( Ovis aries) of the genus Trichuris Roederer, 1761 in species identification.Six stages of embryonic development are recognized in Trichuris eggs: protoplast, blastomeric formation, bean-like embryo, tadpole-like embryo, larva formation and mobile larva formation.The stages are similar in T. оvis (Abildgaard, 1795) and T. globulosa (Linstow, 1901) nematodes.The differentiating species characters of T. оvis and T. globulosa are times of development and quantitative parameters of each embryonic stage.At 27 °С in vitro, maximum numbers of mature T. оvis eggs occur at the 30th day of culture with 84.3% survival.The embryonic development of T. globulosa in culture is longer, it takes place in 39 days, with lower survival of 76.3%.Metric changes of the eggs of T. оvis and T. globulosa during embryogenesis occur dissimilarly, pointing to their species differences.Formation of the mature eggs with mobile larva in T. оvis is accompanied by increasing egg width and decreasing eggshell thickness, egg length and egg plug length.In T. globulosa, the morphometric characters of eggs during embryonic development are characterized by decreasing length and eggshell thickness, with increasing egg width.These data have an application value in identifying the studied Trichuris species by collected eggs.

Fig. 1 .
Fig. 1.Embryonic stages of development in nematodes of the species Т. оvis and Т. globulosa parasitizing domestic sheep: а -protoplast, b -cleavage and formation of two blastomeres, с -cleavage and formation of three and more blastomeres, d -formation of bean-like embryo, е -formation of tadpole-like embryo, f -larva formation, g -mobile larva formation

Table 1
Parameters of embryonic development of eggs obtained from Trichuris оvis gonads, in experimental culture (х ± SD, n = 100) Table 2Parameters of embryonic development of eggs obtained from Trichuris globulosa gonads, in experimental culture (х ± SD, n = 100)